Lab Manual

General Microbiology (BIOL 251)

Spring 2015

TABLE OF CONTENTS FOR LAB MANUAL BIOL 251 Fall 2014

Lab #

Page numbers

Safety in the Microbiology Lab

3 5

1, 2

Ubiquity, Sampling, Handwashing and Media

6 --- 11

Microscopy

12 --- 17

Survey of Eukaryotes: Protista, Fungi, and Animalia

18 22

Aseptic Technique

23 --- 24

2, 3

Pure Culture Techniques: Streak Plate Method

25 --- 29

Structure and Classification of Bacteria

30 --- 32

Negative Stain: Structure of Bacteria

33 34

3, 4

Biochemical Identification of Gram Negative Enteric Bacteria

(Triple sugar iron agar, Citrate, Urea, SIM)

35 41

3, 4

Rapid Identification Using Differential Media (Chromagar)

42 --- 43

3, 4, 5

Smear Prep and Gram Stain

44 50

Acid Fast Stain

51 53

Capsule Stain

54 55

Endospore Stain

56 --- 57

Flagellar Stain

58 --- 59

Electrophoresis

60 --- 61

Viral Fingerprinting

62 65

7, 8

Growth factors: Temperature

66 --- 70

7, 8

Growth Factors: Water Activity and Osmosis

71 73

7, 8

Growth Factors: Ultraviolet Radiation

74 --- 76

7, 8

Growth Factors: Aerotolerance

77 79

7, 8, 9

Bacterial Conjugation

80 83

8, 9

Antibiotic Sensitivity (Kirby Bauer Technique)

84 89

9, 10, 11

Water Micro: Coliform test, Differential Media

90 94

9, 10

Identification of Staphylococci (Catalase, Beta---Hemolysis,

Name of Exercise

Mannitol Salt Fermentation, Coagulase)

95 100

9, 10

Synthetic Epidemic

101 104

10, 11

Handscrubbing Technique (Colony forming units/ml)

105 110

11

Gel Immunodiffusion: Precipitation

111 113

11

Latex Agglutination

114 --- 116

SAFETY IN THE MICROBIOLOGY LABORATORY

Microbiology lab is generally viewed by students as a great learning and fun experience. However, there are
unique safety issues in the microbiology laboratory environment. In addition to hazards common in other
labs such as chemicals, glassware, and fire hazard, microbiology laboratories also include working with
living infectious microorganisms. Although rare, some laboratory personnel have acquired infections from
working with some of these agents in the laboratory. Microbial cultures must be handled with appropriate
technique to maintain safety for laboratory personnel and the environment. Minimum standards, biosafety
levels (BSL), have been established for handling these organisms when working with them in the laboratory.
Our lab uses biosafety levels (BSL) 1 and 2 (see four levels below).
Table of Biosafety Levels (BSL 1--- 4) and practices and examples
BSL

Health Risk

Practices

Primary Barriers

Not known to cause

disease in healthy humans

Standard
open
bench microbiology

None required

Associated with human

disease in healthy but
easily contained.
Transmission through
percutaneous, ingestion,
mucous membrane.

BSL-1 practice plus:

Limited access
Biohazard warning
signs, Sharps
precautions,disposal
of waste and
surveillance
BSL-2 practice plus:
controlled access

Class II Biosafety
cabinet (BSC) and
containment devices
for aerosols or
splashes (goggles,
masks); lab coats
and gloves
BSL2 plus BSC use
for all procedures

BSL-3 practice plus

BSL3 plus complete

isolation

Disease may have serious

or lethal consequences

Agents with high risk of

life threatening disease

Secondary
barriers
Open benchsink; goggles
and gloves for
liquids
BSL-1
plus
autoclave
available

Organisms

BSL2 plus

Example: Mycobacterium
tuberculosis
None used
Example: Ebola Virus
None used

BSL-3 plus

Many used

Some used, indicated as

BSL-2. Staphylococcus sp.
Proteus sp., Bacillus cereus,
Pseudomonas, Enterococcus,
Klebsiella, Chromobacterium,
Salmonella,
Morganella,
Shigella

Here are some basic microbiology lab safety rules in addition to the lab safety sheet:
1. Personal Protection Requirements
a.
b.
c.
d.
e.

Wear a full---length lab coat buttoned up completely when worn. Leave lab coats in the lab stored in
plastic resealed bag. They are autoclaved and returned the last week of lab.
Wear disposable gloves while staining or working with microbial cultures. Remove by placing thumb
under cuff of other hand and turn inside out. Dispose of these in glove disposal.
Wear goggles and leave in lab (even if you wear glasses) when working with liquid cultures.
Use the Biosafety Cabinet when working with BSL---2 liquid cultures.
Closed toe shoes required. Tie back long hair.

2. Standard Lab practices
a. Wash hands before and after the lab session
Wet hands; apply soap to cover hand surfaces; wash for 10 seconds: rub hands palm to palm;
interlace fingers and thumbs; rub tops and bottoms of hands and fingernails, then rinse hands; dry
hands thoroughly, use towel to turn off water. http://www.cdc.gov/features/handhygiene/
b. Disinfect bench before and after lab session.
c. Do not bring food, gum, drinks (including water), or water bottles in the lab.
d. Do not touch face, apply cosmetics, adjust contact lenses or bite nails.
e. Do not bring out in lab or handle personal items (cell phones, electronic devises, cosmetics,
calculators, writing utensils). Use lab manual and writing utensils provided in the lab.
f. Never pipette or place anything in your mouth when in the microbiology lab.
g. Place back---pack and other non---essential books and papers in designated storage area.
h. Know the location of the biosafety manual.

3.

Procedures for note taking in lab

a. Place personal items in assigned location.
b. Remove note---taking papers from backpack. Carry note---taking papers only to lab bench during note
taking period in lab. Use lab provided lab manual and utensils. Copy results from experiments from
lab manual onto personal notepapers.
c. Keep notepaper in drawer during experimental work with microbes. Place in backpack at end of lab.

4.

Procedures for working with microbial cultures

a. Hold broth culture tubes upright or they may leak live microorganisms into the environment.
b. Carry broth culture tubes in a transport rack.
c. Inform the instructor of microbial spills. Cover culture spills with paper towels. Soak with
disinfectant immediately, let stand for 10 minutes, re---clean, dispose of towels in autoclave bag.
d. Dispose of cultures and materials used when working with cultures in appropriate locations for
autoclaving.
e. Parafilm agar plates with personal samples such as throat and skin.

5. If you are immune compromised, taking immunosuppressant drugs, living with an immunocompromised
individual, or pregnant, you must discuss this with the lab instructor.

QUESTIONS FOR DISCUSSION:
1. Describe the proper way to wear a lab coat. How are lab coats stored?

2. What is biosafety level 1 and 2?

3. When should goggles be worn?

4. What is the procedure if a culture spill occurs?

5. What is the procedure for a fire?

6. What is the last procedure that students should perform prior to leaving the lab?

7. A student used his cell phone while he was working with a culture of bacteria at the lab bench. He
had no gloves. What are risks of this procedure?

CSN requires students also review and sign knowledge of the CSN safety sheet

CSN SAFETY SHEET

Always follow the instructions in the lab book, and those given by the lab instructor. When in doubt,
ASK!

Personal belongings should be placed in the space designated by the instructor.

Smoking, eating, and drinking are prohibited in laboratories.

Children and/or guests will not be admitted to the laboratory.

No unauthorized laboratory experiments are permitted.

Students will NOT be permitted to work in labs without instructor supervision.

Students must wear/use all of the protective clothing and/or equipment designated by the laboratory
instructor. If the student does not, he/she will not be permitted to remain in the laboratory.

All laboratory materials must be cleaned up and put away at the end of the laboratory session. Students
must leave the work area clean and dry.

All waste materials MUST be disposed of in the manner designated by the instructor.

There will be no unauthorized removal of laboratory materials or equipment from the classroom.

The student is responsible for knowing the location of, and proper use of, all safety equipment in the
classroom and its immediate surroundings.

In case of fire, students should inform the instructor immediately, and evacuate the area. Pull the fire
alarm. Inform the CSN campus security officer on duty.

All classroom accidents/injuries must be reported to the instructor immediately.

The student is responsible for reading the labels of all laboratory materials and for reading and
understanding the posted safety information concerning those materials. The information will always be
available in a designated site within the department. Your instructor will inform you of the location.

A list of emergency procedures is available in Section XIII of the College of Southern Nevada Hazardous
Materials Right to Know Safety Training manual. The student is responsible for reading and
understanding these procedures. Your instructor will inform you of its location.

Lab coats are required in the microbiology lab (251 and 251G). Students leave the lab coats in plastic bags in the lab
during the semester and do not take them out. Lab coats and plastic bag have the students name on them. At the
end of the semester, the lab coats are autoclaved and returned to the student for washing. Goggles are stored with
the lab coat and cleaned with disinfectant at the end of the semester. Students that do not pick up their lab coat and
goggles by the last day of the semester (last day of finals week) will forfeit their lab coat. There is no additional space
for storing unclaimed lab coats for any time after the end of the semester.

UBIQUITY OF MICROBES/ENVIRONMENTAL SAMPLING/CULTURE MEDIA

REFERENCE: Differential Media - hemolysis patterns on blood agar http://www.wisconline.com/objects/ViewObject.aspx?ID=mby4307. Growth of Microbes on cell phones: http://www.youtube.com/watch?v=4lmwbBzClAc

INTRODUCTION: Microorganisms are ubiquitous, i.e., they can be found in every habitat on earth.
They are the most numerous life forms on earth and they are the most diverse. Microbes are in water,
soil and air, and are essential for all life to exist on earth. They inhabit internal and external surfaces of
other organisms, such as animals and plants. They are around us all the time, yet most are unaware of
their presence. Many microbes are free living in the external environment. Other microbes live in a
symbiotic relationship with other living organisms. Commensals are microbes that inhabit our
bodies in ways that may benefit both them and us. Some microbes however, are capable of causing
disease in the right circumstances. These types of organisms are called opportunists because when they
have the opportunity to cause disease they will often do so. It is important to use care and remember
that microorganisms take every opportunity to grow. Being cautious in the microbiology lab will help
prevent infections.
The purpose of this exercise is to demonstrate the ubiquitous nature of microorganisms by sampling
various sources, inoculating onto culture media, and observing for growth of microbes. A culture
medium is used as a nutrient source to allow microbes to grow. Culture media can be liquid, which is
liquid at room temperature, or solid, which is solid at room temperature. Solid media contains agar
(derived from seaweed) in addition to the nutrients. Media with agar becomes a liquid when it is
boiled. It is dispensed into round plastic culture plates with loose-fitting lids called Petri plates.
When the media cools to ~45o C it becomes a solid and provides a flat, solid surface for
inoculating and growing microorganisms. We will use only solid media in todays exercise.
Culture media is also classified as to the purpose (see Figure 1). General purpose media allow the
growth of many microorganisms that do not need special growth factors. Some examples of general
purpose media are such as nutrient broth, tryptic soy broth and tryptic soy agar. Enrichment media
contain special growth factors required by some microorganisms to grow. An example is blood agar
containing sheep red blood cells. These blood cells are broken open by some microbes to obtain
nutrients, such as heme. Selective media is used to inhibit the growth of some groups of
microorganisms and select for the growth of other microorganisms. An example is Sabourauds dextrose
agar which contains high concentrations of dextrose to inhibit the growth of most bacteria but encourages
the growth of fungi (mold and yeast). Differential media show differences between microbial colonies
based on their cultural characteristics (what they look like when growing in colonies). Blood agar is also
a differential medium, as different patterns of hemolysis (lysis of blood cells) can be observed and
are characteristic for certain bacteria. See hemolysis link above.

Figure 1. Examples of Culture Media for growing microorganisms.

After inoculation the media will be incubated at 25oC (room temperature) or at 37oC (body
temperature). Molds grow optimally at 25oC, whereas many bacteria grow optimally at 35-37oC. The
plates will typically remain in incubation for 24 - 48 hours but may remain incubated for up to one week.
As bacteria reproduce in broth their increased number produces turbidity, or cloudiness. As bacteria
and fungi reproduce on an agar surface, a visible mass of microbes forms. If it is distinct and separated
from other similar masses, it is called a colony (clones from one bacterium). Typically, each
different species produces colonies with different characteristics. The shape of the colony may be round,
irregular, or filamentous. Color varies, and the size may be small, intermediate or large.
In this exercise you will use cotton swabs, to collect microorganisms from the environment, inanimate
objects, and human tissue. Collected microorganisms will be inoculated in culture media (see figure 2)
for growth and later examination. In addition, you will access the effects of antiseptics (hand sanitizer or
hand soap) on the growth of skin microbes.

Materials, supplies and equipment:

Tryptic Soy Agar (TSA) plates (2 for each student)

Sabourauds agar plates (1 for each student)
Blood agar plates (1 for each student)
Sterile cotton swabs (3 for each student)
Sterile saline (0.7%) (2 for each student)
Marking pencils
Incubators, vortex machines

Hand sanitizers and hand soaps

Environmental Source Samples (TSA and SDA)
Food source: fruit and vegetables (washed and unwashed), deli meat, yogurt, and cheese (others)
External: door handle, floor, elevator button, water spigot, air, water fountain
Other: hair, phone, keyboard, coin, shoe, purse
Other approved personal item: cellphone (washed and unwashed), toothbrush, chapstick, hand cream,
mascara, credit card (washed and unwashed)
Human source samples (BAP and TSA): throat and fingers

Procedure: LAB 1. Inoculation of specimens collected

Environmental samples: Use same sample source for both TSA and SDA plates
TSA Plate: Use a sterile swab to swab an area of the assigned environmental source using
repetition. You may need to immerse the swab in sterile saline first. Then swab the TSA plate with the
cotton swab twirling as you swab down the plate (see figure 2). Do not press too hard or the agar will
split. Dispose of the cotton swab in the discard box. Label the bottom of the Petri plate with your

initials and specimen source, place on tray and incubate inverted at 37o C. Inverting the plate allows
you to see the plate label and prevents condensation from accumulating on the top of the plate lid and
falling into the media.
Sabourauds agar plate (SDA). Using the same environmental source as you did for the TSA plate.
Streak the swab across the surface of the SDA plate. Dispose of the cotton swab in the discard
cardboard box and swab wrapper goes in the trash. Label the bottom of the Petri plate with your initials
and specimen source. Invert and incubate on tray labeled 25 o C for the SDA plate.
Human samples:
Throat: Blood agar plate (BAP): Please do not cough in the plate. Use a sterile cotton swab to
gently swab the inside of your pharyngeal region. Lift the cover from the Petri plate and the inoculate
the surface of the blood agar plate, across and from top to bottom in a zigzag motion and twirling as
shown in figure 2. Replace the lid and seal the plate with parafilm. Label bottom of Petri plate,
invert and incubate on 37o C tray.
Fingers (before and after hand sanitizer or hand soap): TSA plate: Make a mark down the middle
of the TSA plate, label one side unwashed and the other washed. Gently touch your fingertips to the
side labeled unwashed. Then, take some hand sanitizer or soap of your choice and rub on or wash your
hands as you normally would and dry hands. Then, again, gently touch your fingertips to the other side
of the TSA plate labeled washed. Record your name and the product used on the plate. Invert and
incubate on 37o C tray.

Results Lab 2:
After incubation, collect your 4 plates and observe media for growth. Petri plate media will show
individual clustered groups of visible growth, called colonies. These may be different types of microbial
colonies or similar ones. The cultural characteristics of size, shape and color can help in the
identification of unknown microorganisms. Your instructor will help you observe the Petri plates.
Environmental samples (TSA and SDA): Examine each of the agar plates for the presence of
individual colonies. Observe number of different colonies, based on size, shape of colony, color, and
whether colonies are mold or not. Count bacterial colonies on TSA and count mold (hairy colonies) on
SDA. You may observe more mold colonies on the SDA plates since this media encourages fungal
growth. To determine if you have mold growing in your plates, observe the mycelia of the known mold
plates provided and compare to your plates. See how the front and back colors of these molds may not
always be the same. Your instructor will assist you in making observations. Do not open the plates unless
instructed to do so as mold colonies release spores and may be allergenic. Use demo fungal plates and
Figure 3 in this document for help differentiating bacteria and mold. Record results and complete Table 1.

Throat samples on Blood agar plate (BAP): It is normal to see many colonies in this plate. Also,
because BAP is differential media you can differentiate the types of hemolysis present. You should see
many small colonies with greenish areas around them (see hemolysis link above). This is called alpha
()-hemolysis; which is a partial hemolysis of red blood cells. You will also see many other colonies that
are not hemolytic called gamma ()-hemolysis, including small buff colored, round, white and other
colonies. These are all considered normal microbiota, which are present in healthy individuals.
The

pharyngeal cavity may also contain very tiny, white colonies with a large clear around them (beta-()hemolysis). The red blood cells have undergone complete hemolysis. Your instructor will advise you if
these may be Streptococcus pyogenes, which can cause strep throat and are considered pathogens.
Some individuals may have these bacteria in their throats temporarily but it is not generally considered
normal flora. Record results in Table 2. Do not open plates.
Finger Samples on TSA: Observe both sides of the TSA plate for numbers and types of colonies from
fingers. Record the number of colonies on the unwashed and washed sides of the plate in Table 2. Make
a note of any differences in colony types.
After reading and recording results, tape Petri plates shut and discard these plate cultures in the red
biohazard container.

Table 1. Results of Environmental sampling on various media.

TSA Plate Inoculation Source:

Number of
colonies

Classification
of media

Mold or Bacteria?

Size (small,

Color

medium or large)

Sabourauds Agar Plate Inoculation Source:

Number of
colonies

Classification
of media

Mold or Bacteria?

Size (small,

Color

medium or large)

Table 2. Results of Throat culture and Finger sampling on various media.

Blood Agar Plate Inoculation Source:

Classification of
media

Normal Flora Present?

Hemolysis present?
(type(s))

Tryptic Soy Agar plate Washing agent used:

Unwashed fingers
Colon
y
count

Types of colonies (similar or

different)

Colon
y
count

Washed fingers
Types of colonies (similar or different)

DISCUSSION AND INTERPRETATION

1.
What does culture media provide microorganisms?

2.

Discuss solid culture media. What makes it solid? What is the advantage for microbial growth.

3.

What is a colony on an agar plate?

4.

Explain four types of media by purpose and explain how blood agar can be both enrichment and
differential?

5.

Which environmental sample (s) do you expect to show the most growth? Did the expectation
fit your class results?

6.

Should one be concerned to find bacterial growth in the throat or on the skin? Explain your
answer.

7.

Explain the results of your finger sample counts both before and after washing. Which agent

was most effective in reducing colony counts in the class? Why were there growth of colonies
even after the washing was performed?

8.

Should one be concerned to find microorganisms on food samples? Was there a difference
between washed and unwashed fruit? Could this result in food infection? Explain your
answer.

9.

Explain microbial growth on personal specimens?

10.

Explain the difference between a bacterial colony and a microbial colony. (Hint: use the demo
fungal plates to compare with yours).

11.

Explain microbial diversity regarding the results you obtained from all of your samples.

12.

Based on your results and the results of the rest of the class, discuss the statement
microorganisms are ubiquitous.

Figure 3: Examples of Mold and bacterial colonies from environmental sampling plates.

MICROSCOPY
ADDITIONAL INFORMATION:

http://www.jove.com/science-education/5041/introduction-to-light-microscopy

INTRODUCTION:
Brightfield, light microscopy is commonly used by beginning microbiologists. This lab exercise will
acquaint you with a general understanding of the light microscopes component parts and their functions.
Knowledge of the microscope and its use is crucial to successfully viewing and studying microbial
images during the semester.
Brightfield microscopy produces an image by transmitting light through a specimen. The resulting image
appears dark against a light background. The light microscope has three lens systems. The microscopes
ocular and objective lenses work together to magnify the image from prepared slides. The ocular lenses
are in the eyepiece, closest to your eyes, and magnify 15x. The objective lenses are grouped in the
revolving nosepiece. They include the scanner (4x magnification), low power (10x), high dry (40x)
and oil immersion (100x). The third lens is the condenser which is located under the stage and is
therefore, not involved in magnification. The condenser focuses and concentrates the light reaching the
specimen. The iris diaphragm is within the condenser and can be opened and closed with a lever to
regulate the amount of light reaching the slide. Total magnification is determined by multiplying the
ocular lens magnification by the objective lens magnification. The light microscope magnifies to 1500x.
Magnification is a property of the resolution of the lens. Resolution is the ability to distinguish objects.
Short wavelengths of light such as blue light increase the resolution. Resolving power is the minimal
distance two objects must be apart in order to distinguish them as separate and distinct. The maximum
resolution of the light microscope is 0.2 micrometers (M), also called microns. This means that
objects on the slide which are less than 0.2 M cannot be resolved as separate and distinct objects. Most
bacteria, fungi, and protists have a size range greater than this and can be visualized by light microscopy.
Viruses generally cannot be visualized by the light microscope and require electron microscopes to study.
Maximum magnification and resolution can be achieved with the oil immersion objective lens. When
light passes from a material of one refractive index to material of another, as from glass to air (different
densities), it bends, causing images to become less distinct. The use of immersion oil with the oil
immersion objective (100x) reduces this bending and distortion. Oil has the same refractive index as
glass, so less light bends preserving good resolution when the oil immersion objective is sitting in the oil.
Our microscopes are equipped with parfocal design which is a property whereby an image in focus in a
lower power will remain in focus when changing to a higher power objective. The image is then brought
into sharp focus using fine adjustment. Working distance is the distance between the objective lens
and the slide when in focus. The table below shows these distances. The working distance is very small
when the 40x or the 100x lens is in place. Only the fine adjustment knob is used when focusing using
40x or 100x objectives.
TABLE OF OBJECTIVE LENSES AND THEIR WORKING DISTANCES
Objective Lens
Use
Magnification
Viewing large specimens or
150x (15 x 10)
10x (low power)
finding the specimen
40x (high dry)
Viewing living material
600x (15 x 40)
100x (oil immersion)
Viewing small stained materials
1500x (15 x 100)

Working distance (mm)

7.7
0.3
0.12

Focusing a slide
1. Sign up for a microscope that you will use all exclusively. Carry the microscope with two hands
by the arm and the base. Place the microscope on the lab bench, unwrap the electrical cord and
plug it in.
2. Turn the lamp switch to the on position to verify the light is working and adjust the intensity
with the rheostat knob.
3. Place the slide with a specimen on the stage directly over the light path and fasten with the stage
clips. The stage clips are juxtaposed to the sides of the slide not above or below. The
mechanical stage allows the slide to be moved back and forth or side to side without having to
physically move the slide itself.
4. Generally under low power, the iris diaphragm is adjusted to allow only a small amount of light
through. With higher magnification, the iris diaphragm can be opened to allow more light
through the condenser. Adjust the iris diaphragm for your light sensitivity. The condenser is
generally close to the highest position relative to the slide but can be lowered with the condenser
knob to change the contrast.
5. Begin with the 4x objective (scanner in position). Raise the stage all the way up, the automatic
stop will stop the process and prevent the stage from touching the objective lens. Looking
through the microscope, lower the stage with the course adjustment knob until the specimen
comes into focus.
6. Place the low power objective in place and adjust the focus with the fine adjustment knob.
7. Finally place the high dry objective in place for living material. Use only the fine adjustment
knob to bring the image into sharp focus using high dry or oil immersion objective lenses.
8. As you increase magnification, you will generally need to increase the light intensity.
Using Immersion Oil

1. To use an oil immersion lens for viewing stained bacteria on a slide, first focus on the area of
specimen to be observed with 10x objective (see above).

2. Without moving the stage, move the lower power lens out of place, and place a drop of
immersion oil directly on the slide or cover slip over that area.
3. Carefully swing the oil immersion lens (100x) into place so that it is sitting in the oil. Due to the
parfocal design, the objective will not touch the slide. Because of the small working distance, use
only the fine adjustment knob to gently adjust the specimen on the slide into sharp focus.
Caution: If you are focusing toward the specimen, you can drive the lens into the glass slide and
damage the lens.
4. NOTE: You will begin using the oil immersion objective in Lab 2.

CARE OF THE MICROSCOPE

When finished observing slides, lower the stage, remove the slide and dispose in the designated location.
Turn off the light switch and turn the rheostat to the lowest setting (left). Lower the stage to the lowest
position using the course adjustment knob. Using Kimwipes and cleaning agent at your station, wipe the
oil from the oil immersion objective. This may need to be done several times. Also verify that no oil is
on the high dry objective as it will damage the lens if allowed to dry. Wipe any oil from the stage and
the condenser. Lock the scanner objective in place. Wrap the cord loosely around the microscope as
instructed. Check your microscope with your instructor for the proper technique. Using two hands
return the microscope to the appropriate location in the microscope cabinet.

Microscope Parts
1. As your instructor reviews the parts of the microscope, complete the boxes in figures 1, 2 and 3
2. Study the functions for each part in Table 1.
Table 1. Parts of the Brightfield Microscope and the Functions
Part
Function
Ocular lens
Lens located in eye piece with 15x magnification
Objectives
Lenses on revolving nose piece, 4x, 10x, 40x, and 100x
magnification
Knobs that move the platform stage from side to side and
Mechanical stage
forward and backward
Condenser lens and
Lens system below the stage that concentrates the light
adjustment knob
reaching the specimen. The adjustment knob for raising and
lowering the condenser is on side below the stage.
Iris diaphragm
Lever on condenser that regulates amount of light entering
specimen
Lamp switch
On/off switch for light source
Rheostat (on/off light switch)
Knob that regulates the amount of light leaving the lamp
Large knob on both sides toward base, used with 4x and 10x
Coarse adjustment knob
for initial focusing
Small inner knob on both sides next to coarse adjustment
Fine adjustment knob
knob, used for fine focus with 40x and 100x objective
Stage clips
Clips that juxtapose the slide for holding in place on stage
Stage
Flat surface for placing slide
Arm
Metal area in back for holding microscope with hand
Base
Flat metal bottom for holding microscope when carrying

3. Calculate and complete the total magnification table below.

Ocular
magnification
15x
15x
15x
15x

Objective
magnification
4x
10x
40x
100x

Total Magnification

Questions for Discussion:

1.

Describe the proper method of carrying a microscope.

2.

Define parfocal and write an advantage of this design.

3.

Define resolution. A virus is 10 nanometers. Will the light microscope resolve this virus?

4.

What is the purpose of immersion oil? Why is it used only with the oil immersion objective?

5.

Venus is viewing pond water with living protozoa.

a. What is the appropriate objective to start with?_
b. What is the appropriate ojective to use for studying the protozoa?
c. Explain your answers.

6.

What is the relationship between working distance of an objective lens and its magnification?

NIKON E200 MICROSCOPE USED IN MICROBIOLOGY LABS

Microbial Eukaryotes (Protista, Fungi, and Animalia)

REFERENCES: Cell Models http://www.cellsalive.com/cells/3dcell.htm, links below, Talaro text chapters 4 and 5.
INTRODUCTION.
The tree of life classifies all organisms into three Domains: Archaea, Bacteria, and Eukarya. The
members of Domains Archaea and Bacteria are generally microscopic. The Domain Eukarya is
subdivided into the Kingdoms, Protists (includes protozoa and algae), Fungi (mold and yeast),
Plantae (plants), and Animalia (animals including helminths). Protists, Fungi and even Animalia
contain microscopic members we will study.
There are currently two cell types, prokaryotic and eukaryotic. Prokaryotic cells are relatively small and
simple in structure and have generally have no true nucleus. Archaea and Bacteria are prokaryotes.
Eukaryotic cells are typically larger and more complex than prokaryotic cells. They contain a nucleus
and other membrane bound organelles. Microorganisms in the domain Eukarya are eukaryotes. In
addition to cell type, organisms have a particular organization including number and arrangement of cells.
Although there are exceptions, Archaea and Bacteria organisms are generally unicellular, that is, they
consist of a single cell. In Kingdom Protista some microscopic organisms are unicellular and some are
multicellular, that is, consist of many cells. There are also intermediate kinds of organization such as
colonial and filamentous forms which are protists. Kingdom Fungi have unicellular microscopic
members called yeasts. Other organisms in Kingdom Fungi are multicellular and are called molds which
are microscopic and macroscopic. Kingdom Animalia, including worms and helminthic organisms, are
all multicellular. Some stages in the helminths life cycle are microscopic, while others are macroscopic
(visible with the naked eye).
Protista is a very large and diverse kingdom of eukaryotes. This kingdom is in the process of being
further classified. Protista includes protozoa which may be classified by their method of locomotion
(cilia, pseudopodia, flagella or non-motile). Protista also include certain types of algal protists which are
unicellular, free-living eukaryotes, although some algal protists are filamentous and colonial. These
organisms may be found in freshwater environments, such as ponds, and in marine environments.
Protists are important in the food web as producers (photosynthetic phytoplankton) and consumers.
There are several examples of protists to view using wet preps.
In comparison, there is one example of photosynthetic cyanobacteria, Anabaena, which is a bacterium. It
is a colonial prokaryote which generally grows in filaments. Compare this filamentous organism with
the photosynthetic eukaryote Spirogyra, which is a filamentous algal protist. Both of these types of
photosynthetic organisms produce significant amounts of oxygen and take up carbon dioxide. Notice the
size difference. Anabaena also has heterocysts which are locations of nitrogen fixation.
There are two members of the animalia kingdom which are also eukaryotic cells. These are Turbatrix
aceti (vinegar eels) that grow well in acetic acid and are motile, and Planaria, which are motile freeliving flatworms, with eyespots (prepared slide).
There are several examples of fungal eukaryotes. Fungi are non-photosynthetic heterotrophic (use
organics for carbon and energy) decomposers. They may be saprophytes, part of a symbiotic

relationship, or they may be parasites. Yeasts are best known for their role in fermentation processes of
food microbiology, such as making bread and beer. Molds are saprophytic and produce long hyphae
which absorb nutrients that have been digested extracellularly. When the hyphae become a large
macroscopic mass they are referred to as mycelium. The mycelia (plural) have characteristic colors and
appearance depending on the mold. Yeasts reproduce asexually by budding while many molds
reproduce with spores. The asexual spores of mold are unique to the mold and are another method of
identifying the fungus. There are two slides of mold and a live culture of yeast to observe.

Procedure:
Make a wet prep of the living microorganisms in culture that are available in the laboratory and observe
with the microscope. Since these are living, increase the contrast and adjust the light to obtain a better
view. To make a wet prep, you need a microscope slide, coverslip, transfer pipette, and liquid culture of
living microorganisms. Using the transfer pipette dispense one small drop of liquid material onto a
microscope slide in the center. Hold a coverslip at an angle with its edge touching the microscope slide
next to the culture medium. Carefully lower the coverslip. If protists are swimming too fast, you may
add Proto-Slo to thicken the water.
Depending upon the size of the specimen, the total magnification that provides the best view will vary.
For each specimen complete the row in the Table 1. The first column is the genus name of the organism.
In the second column enter the cell type, prokaryotic or eukaryotic. In the third column enter the
organization, unicellular or multicellular. Your instructor may also point out intermediate forms, such as
filamentous or colonial, if appropriate. In the fourth and fifth columns enter the biological classification:
Kingdom and Domain.

DESCRIPTION OF SPECIES TO VIEW:

Protists (Kingdom Protista): Kingdom Protista are generally microscopic, individual cells or in small
groupings of cells called colonial. Eukaryotic cells with one or more nuclei and organelles. In general,
they are larger and more complex than bacteria. Note magnifications listed in the links provided below.
Protozoans:
Amoeba http://www.microbelibrary.org/images/durr/images/amoeba%20proteus.jpg.. Trophozoites are motile forms. This
unicellular microorganism is classified sarcodina since it forms pseudopods for locomotion. The
nucleus may be visible. Cysts are round, non-motile forms with multiple nuclei.
Paramecium: http://protist.i.hosei.ac.jp/pdb/PCD0360/htmls/021.html. Ciliates are large unicellular organisms, cilia
around its entire border provides motility. A nucleus and consumed bacteria may be visible. Contractile
vacuoles regulate water concentrations inside the ciliate by expelling excess
water.
http://www.youtube.com/watch?v=iG6Dd3COug4

Algal Protists:
Euglena http://protist.i.hosei.ac.jp/PDB2/PCD2968/htmls/07.html These are flagellated algal protists. Photosynthetic,
small unicellular-organism with a greenish color due to photosynthetic pigments contained in packets
called chloroplasts. It swims using an anterior flagellum (classification is mastigophera).

Spirogyra http://protist.i.hosei.ac.jp/pdb/PCD1784/htmls/03.html These are filamentous algal protists. Photosynthetic,

non-motile and spiral shaped. Chloroplasts for photosynthesis are prominent within the individual cells
of this filamentous organism. A nucleus may also be seen.

Filamentous photosynthetic bacteria (Cyanobacteria):

Anabaena http://www-cyanosite.bio.purdue.edu/images/lgimages/AZOLLAE1.JPG.
These
are
filamentous
and
photosynthetic prokaryotes which resembles higher plants biochemically. Colonies are made up of
filaments. The bacteria typically show a blue-green coloration due to pigments associated with their
photosynthesis. Note individual cells and also see heterocysts which are clear, ovoid and larger cells
thought to conduct nitrogen fixation.

Kingdom Animalia: Worms are multicellular and are more complex in structure than archaea and
bacteria, protists, or fungi. They are generally, but not always, larger in size as well. Often a particular
stage within the life cycle of the organisms listed below is microscopic, and is thus studied in the field of
microbiology, or parasitology. Worms may be free-living or parasitic.
Planaria http://web.mit.edu/neuro/planaria2.jpg; http://www.youtube.com/watch?v=axBaCD4wYXE&feature=related. Nonparasitic flatworm adults are shown in these preparations. They live in fresh or marine water, under rocks
in streams or ponds. Some of their internal organs are visible as are the eyespots. These organisms can
regrow parts of their anatomy, if injured or lost.
Turbatrix aceti: http://www.youtube.com/watch?v=1Dd0wsxm_Go; http://www.youtube.com/watch?v=UnjwvtFvyeQ.
common name vinegar eel. Roundworm which lives in acetic acid (vinegar). The vinegar we consume has
been filtered to remove possible vinegar eels. The arrangement of muscles in this worm gives it its
characteristic whip-like movement.

Kingdom Fungi: View prepared slides of mold and prepare yeast wet prep
Aspergillus: http://microfungi.truman.edu/showThumbs.php?div=Anamorphic&gen=Aspergillus&spec=niger. Hyphae with
fruiting body with conidia. Common bread mold and cause of swimmers ear and serious hospital infection. Mycelia

are visible masses of hyphae with spore structures that look like fans at the tips. Observe culture plate provided.
Note the front and back color and the aerial mycelia.

Rhizopus : http://academic.pgcc.edu/~kroberts/web/eukary/rhizopus.jpe Hyphae and sporangium. Widespread fruit rot

mold which may cause opportunistic disease in humans and animals. They are very rapidly growing, multicellular
fungus, with threadlike hyphae and large spherical fruiting body structures with tiny spores inside. Observe culture
plate provided. Note the front and back color and the aerial mycelia.

Saccharomyces cerevisiae is common commercial yeast used in fermentation processes such as in making bread
and beer. These organisms are not considered pathogenic. They are unicellular, oval shaped cells that replicate
asexually by budding. Video non-motile Saccharomyces http://www.microbiologybytes.com/video/Scerevisiae.html;
scroll down to see bread rising. Prepare a wet prep of this yeast. Observe the creamy appearance of the culture
plate provided. Note the lack of aerial mycelia.

Table 1. Results of Microscopic Analysis of Protists, Fungi, Animalia and Cyanobacteria.

Name

Cell Type

Organization

Kingdom

Domain

Prokaryotic

Filamentous

Eubacteria

Bacteria

Drawing

Anabaena
(Cyanobacteria)

Aspergillus

Rhizopus

Euglena

Spirogyra

Paramecium
Amoeba
/Pond Water
Volvox

Planaria
slide
Turbatri
x aceti

Saccharomyce
s cerevisiae

Use the rest of the page for more drawings you wish to make.

Questions:
1. In which domains are protozoa, algae and cyanobacteria classified.

2. Describe the two cell types.

3. Name one similarity and one difference between algal protists (Euglena), plants, and
cyanobacteria (Nostoc).

4. Describe three different methods of locomotion you have seen today.

5. Name three differences between paramecium and vinegar eels.

6. List unique characteristics of the kingdom fungi.

7. Differentiate between microscopic appearance of mold and yeast. You may use a drawing with
labels. What microscopic property of mold is used to aid in identification?

ASEPTIC TECHNIQUE

Additional Information: http://www.jove.com/science-education/5036/an-introduction-to-working-in-the-hood and

http://www.jove.com/science-education/5035/introduction-to-the-bunsen-burner
Aseptic means free of living pathogens, ie., disease causing microbes. Aseptic technique is a way of
working with microbial cultures that insures the environment, personnel, and the microbial cultures are
NOT contaminated. Therefore, after you have worked with the cultures, no contamination remains and
the original culture is still pure. The following is a list of general techniques and practices used when
working with microbial cultures. These techniques are to be used on every occasion when working with
microbial cultures. They are not listed in the order used.
Work Area Disinfection. The work area (lab bench) is treated with disinfectant twice when working
in the lab. The first disinfection is performed when first entering the lab prior to any materials being
placed on the bench. The disinfection will destroy vegetative cells and viruses but generally will not
destroy endospores. Therefore it is not a method of sterilization. The physical process of scrubbing the
bench removes microorganisms. The work area is also disinfected after work is complete.
Bacteriological Loops and Needles. The transfers of culture material are achieved by using inoculating
loops and needles which are sterilized before and after coming in contact with the culture. The
incinerator is used to sterilize the loop or needle by placing in the incinerator opening until they glow
orange. The loop or needle must be cooled prior to use which is done by holding for at least 5 seconds.
The loop or needle is sterilized again after transferring cultures and before returning to the bench to
prevent contamination of the environment. Do not store the loop or needle in the incinerator as the
loop becomes so hot the rubber holder may melt. Return the loop or needle to the bench top location.
Culture Tube Handling, Flaming, and Inoculation. Hold culture tubes in your non-dominant hand.
Prior to inserting a sterile, cooled loop or needle into the culture, the cap is removed, and the mouth is
held next to the incinerator opening. Pass the culture next to the hot opening several times to produce
convection currents to prevent microbes from the air from entering the culture. Hold the broth tube at
an angle but still upright to minimize the chance of airborne contamination. When finished inoculating,
flame the culture mouth again using the same procedure, and replace the cap on the tube. The broth
tube is placed in a test tube rack and not laid on the bench top.
Petri Plate Inoculations. The plate lid is used as a shield to prevent airborne contamination by raising
the cover and holding it diagonally over the plate. The loop with the inoculum is streaked over the agar
surface gently to as the agar can be torn or gouged. The cover is replaced and loop is flamed.
Hand Washing (degerming). Hands are washed as the last act before students leave the lab and after
the bench top disinfection. Students also wash hands if they come into contact with bacterial cultures.
Minimize Potential of Contamination. Do not perform transfers of microbes over your books and
papers. Place materials safely away from the culture material.
Suspend Bacteria in a Broth. Mix broth cultures gently prior to obtaining inoculum by drumming your
fingers on the tube or using a vortex mixer. When using a vortex mixer, start the process slowly. Do not
allow broth to get into the cap, lose control of the tube, or allow broth to come out of the tube.

Disposal of Used Materials. Materials should be discarded in the appropriate areas for eventual
autoclaving (steam sterilization) or incineration (burning).
Be Organized and Take Your Time. Arrange all media in advance and label as instructed by
your instructor. Broth tubes are held upright and written on the glass portion. Petri plates are
labeled on the bottom and incubated inverted. Work efficiently but do not hurry. Keep your
books and papers away from the area in which you are working with cultures.
QUESTIONS for DISCUSSION
1. Define aseptic technique and explain why it is important to use when handling microbial cultures.

2. List the procedures used in making a streak plate that fulfill this technique.

3. What is the purpose of flaming culture tubes prior to entry with loops?

4. What is the proper method of labeling and incubating Petri plates?

5. What will disinfection of the bench kill? Not kill?

6. What is degerming? Sanitization? Disinfecting? Sterilizing?

7. Name the instrument commonly used in microbiological laboratories to sterilize materials prior to
disposal. How is sterilization accomplished?

PURE CULTURE TECHNIQUES - STREAK PLATE TECHNIQUE

REF: Streak plate animation http://learndat.tech.msu.edu/sites/default/files/showcase/Streak_plate/streak_plate.html
INTRODUCTION: Isolation of Pure cultures.
Most environmental samples contain many different microbial species and generally not just one single
microbe. Also, sample material introduced into a culture medium may produce a large number of
bacteria. A culture with many different species of bacteria is called a mixed culture. Isolation is the
separation of different species in a sample or culture medium from each other. Isolated bacteria can be
placed in new culture medium to have only one species of bacteria. Such a culture is called a pure
culture. Obtaining a pure culture is essential in order to study a single bacterial species. Definitive
results generally cannot be determined regarding identification, antimicrobial sensitivity patterns, or DNA
analysis on cultures of mixed microorganisms.
Two common methods of isolation are the streak plate and pour plate techniques and both utilize a solid
culture medium. In the streak plate method a bacteriological loop is used to obtain a sample or culture
medium using aseptic technique. The loop is then streaked across a nutrient agar surface in a specific
manner (see link above and figures 1 and 2). As the streaking progresses the bacterial cells are spread out
(thinned out), farther and farther apart from each other. The separated cells reproduce and form a colony
separated from other colonies. The colony consists of identical cells, which are called clones, because
they are all descended from a single, original cell. If the original sample contained more than one type of
bacteria, they should be isolated into separate single colonies. In the pour plate method a loop is used
similarly to sample material. Serial transfers are made to additional tubes so that each transfer results in a
greater dilution of the bacteria present in the original medium or sample. If the dilution has been
adequate, bacteria will be separated sufficiently in the solidified agar, so that individual colonies will
form. You will use this type of method in the handscrubbing exercise later in the course.
Regardless of which technique has been used, the colonies with different characteristics are made up of
different kinds of bacteria present in the original sample material.
Supplies, Reagents, Equipment
Bacteriological loops, marking pencils, Incinerators, gloves and goggles
Media: Tryptic soy agar (TSA) plates (2 for each student)
Bacterial Cultures:
Tryptic Soy Broth (TSB) mixed culture with three different bacteria: Chromobacterium volacium
(BSL-2), Serratia marcescens and Escherichia coli
Pure cultures in agar plates: Bacillus cereus or Staphylococcus epidermidis or your own TSA
environmental plate (check this with your instructor).

PROCEDURE : PREPARING STREAK PLATES using aseptic technique

The instructor will demonstrate the following methods.
Broth Culture Tube to Agar Plate:

1. Obtain a TSB mixed culture (3 species of bacteria), a TSA plate, and a bacteriological loop.
2. Hold the tube upright and disperse the bacteria in the culture tube by gently shaking side to side
or using a vortex mixer. Be careful not to tilt the tube or vortex too vigorously as the bacterial
culture will spill and contaminate the environment.

3. Sterilize the bacteriological loop in the incinerator for five seconds. The loop will be red hot, so
avoid skin contact with the loop. Allow the loop to cool for approximately five seconds.

4. Hold the tube upright and open the bacterial culture tube by removing the cap. Use the little
finger (on the same hand that you use to hold the loop) to hold the cap. If you place the cap on
the table, it may become contaminated. Hold the mouth of the culture tube next to the opening of
the incinerator for approximately five seconds. (This will remove organisms from the tube
opening, as well as heat the surrounding air, which causes the air to rise and prevent contaminants
from falling into the culture tube.)
5. Insert the loop into the culture tube and collect a loopful of culture medium with bacteria.
6. Heat the opening of the culture tube again, as before, and replace the cap.
7. Lift the cover of the agar plate and hold it slightly above and at angle over the bottom of the plate
in order to protect the plate from contamination.

8. Hold the loop so that the flat part gently touches the agar surface at a shallow angle. Move it
rapidly back and forth over the part of the plate designated as quadrant I.

9. Sterilize the loop again and streak quadrant II, slightly overlapping the previously streaked area in
QI. Note: Do not dip into the culture again. Cool the loop.

10. Repeat the process for quadrants III and IV. Use Figure 1 as a diagram for inoculating each
quadrant or view the animation link at the beginning.
11. Label the bottom of the plate with your initials, section, and date.

Invert and incubate.

Agar Plate to Agar Plate:

1 Next, transfer a colony from the bacterial culture plates provided in the lab or an approved colony
from your TSA environmental plate to prepare this streak plate.
2 Sterilize the bacteriological loop in the incinerator as described above.
3 Lift the cover of the agar plate and hold it slightly above and at angle over the bottom of the plate
in order to protect the plate from contamination.
4 Collect a small amount of bacteria from the bacterial growth on the plate with the loop. You
need only touch the colony to have enough to use.
5 Replace the plate cover.
6 Lift the cover of the receiving agar plate and hold it slightly above and at angle over the bottom
of the plate in order to protect the plate from contamination.
7 Perform the streak plate, preparing four quadrants as shown in Figure 1.
8 Replace the plate cover.
9 Sterilize the bacteriological loop before returning it to the tabletop or holder.
10 Label the plate. Invert and incubate.

Figure
.
co

stre

es

RESULTS : Next lab

Examine the streak plates for isolated colonies (see Figure 2). Observe the morphology of the colonies
using Figure 3 as a guide. Record the shape, elevation, color, and size of colonies in the appropriate
table. Figure 3 is a simple scheme that shows how these terms are applied. Use the adjectives small,
intermediate, and large to describe the size. Complete the last column with the identity of bacteria in the
mixed culture streak plate. Your instructor will assist you in describing colonies for help with accurate
identification.
Figure 3: Colony characteristics of bacterial isolated colonies.

RECORD RESULTS:

1.

Indicate on the two plate diagrams your level of isolation in quadrant 4.

2. Describe the colonies on each streak plate using the above drawing on shape, color, size and
elevation and record in Tables 1 and 2 below.

Table 1. Results for Mixed Culture Streak Plate (3 different species) from broth.
Shape

Color

Size / elevation

Identification

Table 2. Results for TSA Streak Plate from agar.

Shape

Color

Size

QUESTIONS FOR DISCUSSION:

Explain how a colony forms and the relationship to the word clone.

How are bacteria thinned out so that individual colonies are formed on the streak plate?

Name one major difference between the colonies of Chromobacterium violaceum, Serratia
marcescens and Escherichia coli?

4.

What is the purpose of describing isolated bacterial colonies?

5.

Did you achieve isolation on the mixed culture for all three species?

6.

Discuss among your table members, why is it important to isolate bacterial colonies from each
other.

STRUCTURE AND CLASSIFICATION OF BACTERIA

ADDITIONAL INFORMATION: https://www.youtube.com/watch?v=Xxw-JWDOiYQ
INTRODUCTION Bacteria are unicellular and much smaller than eukaryotic cells. Different species
of bacteria come in a variety of shapes (morphology) and arrangements (association with each other).
Many species have characteristic size, shape, and arrangement or grouping which are unique to that
species. In addition, members of a given species show some individual variation in these characteristics,
too. Determining the shape and arrangement are important first steps in classifying and identifying
unknown bacteria.
Bacterial cell shape may be spheres (cocci, singular coccus), rods (bacilli, singular bacillus), or rigid
spirals (spirilla, singular spirillum). Variations of these shapes include slightly curved rods (vibrios),
short rods (coccobacilli), and flexible spirals (spirochetes). Another term used for shapes is
pleomorphism, which means a variety of shapes seen in a given sample (slender, ellipsoidal, or ovoid
rods). These shapes are often accompanied with a variety of arrangements as well. See the illustration in
this document for examples of shapes.
Many bacteria also have a typical arrangement that is determined by the location and number of planes in
which division occurs and whether the cells separate after division. These arrangements are also helpful
in identification of unknown bacteria. If two daughter cells remain attached after a coccus or bacillus
divides, a diplococcus or diplobacillus, respectively, is formed. If the cells continue to divide in the
same plane, a streptococcus or streptobacillus arrangement is formed. In coccus, if division occurs
down a perpendicular plane, a tetrad is formed, a group of four cells. If an additional division of these
cells, perpendicular to this division occurs a cube shaped group of cells occurs, called sarcina. Also in
coccus, if the division planes are irregular, a cluster of cells is produced, called shaphylococcus. Refer
to the last page of this exercise for examples of these arrangements.
Not all bacteria and archaea fit conveniently into these shape and grouping categories. For example,
some bacteria are described as filamentous, having a thread-like and branching pattern of growth. Also,
sometimes microorganisms take on unusual shapes when growing in broth such as very long rods.
Sometimes cocci appear as singles, pairs and chains. Additionally, when bacilli are standing on their
heads or tails they appear as spheres. This is because cells are three-dimensional but when we stain
them on a slide, they are two dimensional.
Finally, bacteria can vary greatly in size in nature. A recently discovered bacterium, Epulopiscium, is
an endosymbiont within the gut of the sturgeonfish and can be up to 60 m in length (that is 0.6 mm).
However, many bacteria are small with dimensions of one to several micrometers and have to be
observed using oil immersion. Remember the size of the eukaryotic cells you viewed in Lab 1 and the
mold cells in this lab. Note the difference in size between prokaryotes and eukaryotes and the
magnifications used to study these different cell types.

View the models in lab and the drawings below to learn shape and arrangement.

QUESTIONS FOR DISCUSSION:

1. What is the purpose of determining the shape and arrangement of a bacterial culture?

2. A bacterium is viewed on a slide using the microscope. It is spherical and arranged in twos.
What is the classification of their bacteria?

3. Material from a bacterial culture is stained and viewed on a slide. Long rods and very short
coccobacillary rods are observed. Give an explanation.

4. What is one type of culture medium that may have an effect on the shape and arrangement?

5. What is the measurement used for most bacteria? What is this measurement?

6. Can rod shaped bacteria look like cocci in some cases?

NEGATIVE STAIN AND BACTERIAL SHAPES

REF: Negative stain procedure: http://www.microbelibrary.org/library/laboratory-test/2843-negative-stain (Click animation).

INTRODUCTION: Negative stains are useful in observing morphology, size, and arrangement of
bacterial cells. In addition, some bacteria cannot withstand heat-fixing which makes the negative stain a good
choice for microscopic analysis. The negative staining technique uses a dye solution in which the chromogen
(nigrosin) is acidic (gives up a hydrogen ion) and carries a negative charge. Since bacteria are negatively
charged as well (due to the teichoic acid on Gram positive bacteria, and the lipopolysaccharide on the Gram
negative bacteria), they will repel the negatively charged chromogen. As a result, the bacterial cells will not
take up the dye and will remain unstained. However, the background will be colored with the dye.

Materials, Supplies and Equipment:

Nigrosin stain, clean microscope slides, gloves, inoculating loop, incinerator, wax pencil, sterile
toothpicks, small biohazard disposal bag
Bacterial Cultures:
Agar: Corynebacterium pseudodiphtheriticum, Staphylococcus epidermidis, Bacillus cereus
Broth: Rhodospirillum rubrum

PROCEDURE:
1.

2.
3.
4.
5.
6.
7.
8.
9.

10.
11.

Work by table to examine all four bacteria using the negative stain. For example, your instructor
may allow one person to stain one bacterium and the other members of the table will view them. In
this way, all the bacteria will be viewed by all of the students at the table.
Allow the instructor to demonstrate the procedure and use the procedure demonstrated.
Obtain a clean glass slide and place a drop of nigrosine on one the end of the slide.
Using aseptic technique, obtain a loopful of the Rhodospirillum broth and mix in the nigrosine.
For plate cultures, obtain a very small amount of a bacterial colony and mix in the nigrosine.
For observing microorganisms around the gingival area of your teeth, use a sterile toothpick and
gently remove a small amount of material from your teeth and mix in the nigrosine.
Obtain another slide and touch the slide to the slide with the nigrosine at a 45 degree angle. Pull the
slide on top back to touch the drop of nigrosine.
Slide the glass slide on top in the direction away from the nigrosine drop, taking the nigrosine with
it. This will produce a smear of nigrosine covering most of the slide.
View the link above to demonstrate the procedure for the negative stain
NOTE: If this method is too technically difficult, the negative stain may be made by placing a
drop of nigrosine on the middle of the slide. Mix and smear a loopful of bacteria throughout the
slide covering much of the slide.
Allow the slide with the nigrosine to completely air dry at your desk.
Visualize the slide under 10x. When it is in focus, move the 10x objective and place a drop of oil
on the slide, directly on top of the nigrosine. Swing the 100x oil immersion objective in place and
into the oil. Focus with the fine focus knob.

Table 1. Record results of Negative stained images.

Name
Staphylococcus
aureus
Corynebacterium
pseudodiphtheriticum

Shape

Arrangement

Sketch

Bacillus megaterium
Rhodospirillum
rubrum

Tooth area

Questions for Discussion:

1. Why is it important to limit the quantity of cells used to prepare a negative stain?

2. What causes a stain to adhere to bacterial cells? Why did the cells not stain with this dye?

3. What are the practical uses of a negative stain for studying bacteria?

IDENTIFICATION OF UNKNOWN GRAM NEGATIVE ROD:

ENTEROBACTERIACEAE
REFERENCES: Talaro text chapters 8 (metabolism), 17 (diagnostics) and 20, 21 (gram negative rods)
and https://www.youtube.com/watch?v=GLwsj13AACs
INTRODUCTION: In this lab we will use traditional techniques to differentiate bacteria on the basis
of their biochemical characteristics. Bacteria have a plethora of enzymes that catalyze many
biochemical reactions in metabolic pathways, such as, fermentation and respiration. Enzymatic activity
can be observed by using differential media which often changes appearance, for example, color, when
an enzyme causes the pH to change (becomes acidic or basic). Indicators that detect pH change are
added to differential media to observe various, easily detectable, biochemical reactions. Other indicators
detect the presence of various other products.
Differential media will aid in the identification of unknown gram negative rods in the
Enterobacteriaceae family. This is a very large family that includes normal gastrointestinal
microbiota, opportunists, and pathogens. Salmonella and Shigella and even some strains of E. coli are
intestinal pathogens. These bacteria cause diarrhea, dysentery and hemolytic uremic syndrome. Some of
these infectious diseases involve bacterial toxins and can be fatal. Other Enterobacteriaceae members are
well known causes of urinary tract infection, such as E. coli and Proteus. Still others are known to cause
pneumonia in immune compromised individuals such as Serratia, Enterobacter, and Klebsiella. Other
infections caused by enterics include sepsis and meningitis.
Although many of the Enterobacteriaceae are normal flora, when they are found in hospitals, they are
often resistant to multiple antibiotics and cause nosocomial infections in both humans and animals. The
identification of these bacteria is an important feature of clinical microbiology. Various biochemical
tests will help differentiate these bacteria from each other as they each have unique metabolic pathways.
Rapid identification tests have been developed for these gram negative bacteria which you will see as a
demonstration.
In this exercise we will use standard differential tube media: Simmons Citrate Agar (SCA), Urea
agar, Sulfide-Indole-Motility (SIM) medium and Triple Sugar Iron (TSI) agar. The bacteria we use
include BSL2 types and aseptic technique is required when working with these microbes.
SIM MEDIA: This is a combined media that tests for motility, sulfur reduction to hydrogen sulfide
(H2S), and indole production from the amino acid tryptophan.
H2S production in SIM: Some bacterial enzymes remove the sulfur from the amino acid cysteine or
reduce sulfate directly. Both pathways produce H2S. The SIM media contains ferrous sulfate (iron
salts) that will turn black when they combine with H2S gas. Salmonella species often produce H2S.
Motility in SIM: Some bacteria are motile by using flagella. The SIM media is used to test for motility
by using a needle and inoculating SIM with a stab. If the bacteria swim away from the line of inoculation
(stab) they are motile. If the bacteria grow only along the stab line, they are non-motile.

Indole production in SIM: Some bacteria produce the enzyme tryptophanase and thus are able to
hydrolyze the amino acid tryptophan producing indole and pyruvate. Indole can be detected by the
addition of Kovacs reagent which contains dimethylaminobenzaldehyde (DMABA). When added to
the SIM tube, the DMABA reacts with indole and turns cherry red, indicating the presence of rosindole.

Simmons Citrate test: Simmons Citrate Agar is used to differentiate gram negative bacteria on the
basis of citric acid utilization. The agar medium contains sodium citrate as the only available carbon
source, and a single nitrogen source as well. If bacteria grow, it means they used the citrate as their
carbon source. The medium also contains the indicator, bromthymol blue, which is green at pH 6.9 and
blue at pH 7.6 and above. Organisms which have the enzyme citrase, are able to utilize the sodium
citrate and nitrogen sources and produce ammonia, which converts the agar to an alkaline pH and a blue
color. If they cannot use these carbon and nitrogen sources, the medium remains green. However, in
some cases, the bacteria may grow using the citrate but do not turn the medium blue.

TRIPLE SUGAR IRON AGAR: This medium contains three carbohydrates and differentiates bacteria
on fermentation of glucose, sucrose, and lactose, or not. The medium also contains peptones as a
carbon and nitrogen source. The indicator, phenol red, is added to monitor the pH. This indicator is
yellow in acidic pH and pink-red in alkaline pH. Finally, the medium contains ferrous sulfate to detect
sulfur reduction and H2S production. The agar is slanted in the tube and biochemical reactions can be
observed in the bottom (butt) and on the surface (slant).
When sugars are fermented, acid byproducts are produced and turn the medium yellow. Glucose is added
in small amounts (0.1%), which mean some bacteria may use all of the glucose for food and then require
another source of food. When the entire tube is yellow, the bacteria fermented the glucose and at least
one of the other sugars, lactose and/or sucrose. Many bacteria but not all, produce gas during
fermentation of sugars. This is observed by the presence of breaks or air spaces in the agar. When the
butt is yellow but the slant is pink, the bacteria fermented the glucose quickly and could not ferment the
sucrose or lactose. The bacteria utilized the peptones for an additional food source rather than run out of
food. When peptones (amino acids) are used for food (peptonization), ammonia is produced which
converts the pH to alkaline and turns the agar pink to red. In addition to fermentation and peptonization
reactions, H2S production is observed when the medium turns black. In some cases, bacteria may
ferment the sugars turning the medium yellow but use up all the sugar and turn to the peptones for food.
When this happens the medium turns back to alkaline and red/pink. This is called conversion.

UREA HYDROLYSIS: Some bacteria have the urease enzyme which hydrolyzes certain amino acids
and urea to ammonia and carbon dioxide. When ammonia is produced the pH becomes alkaline (basic).
Urea agar contains urea, peptones, glucose and phenol red. When urease positive bacteria are inoculated,
the medium becomes alkaline turning the phenol red indicator bright pink.

SUPPLIES, REAGENTS, AND MEDIA

Triple Sugar Iron agar tubes (2 students per group, 1 per group)
Sulfide-Indole-Motility (SIM) medium tubes (1 per group)
Simmons Citrate Agar tubes (1 per group)
Urea agar (1 per group)
Incinerators, inoculating loops and needles, marking pencils
Kovacs reagent
BACTERIAL CULTURES
Agar Plate cultures: A H. Each group will be assigned a culture by the instructor.

PROCEDURE FIRST LAB (Lab 3) :

1. Your instructor will demonstrate the inoculations of each tube using a loop or needle. You will
receive a plate with a letter to use in your group of 2 students.
2. Obtain a set of SIM medium, SCA, Urea, and TSI agar tubes for each unknown organism. Label
the tubes with your initials and the letter of your unknown. Use aseptic transfer technique and an
inoculating loop or needle for making the inoculations.
3. To inoculate SIM medium, obtain bacteria with the needle and stab straight down into the center
of the SIM medium, about two-thirds of the distance to the bottom.
4. To inoculate the SCA tube, scrape the bacteria from the agar plate with the loop and streak the
surface of the SCA slant and stab the butt.
5. To inoculate the TSI agar, scrape the bacteria from the agar plate with the needle, streak the
surface of the TSI slant and then stab straight down into the TSI agar butt.
6. To inoculate the Urea agar, scrape the bacteria from the agar plate with a loop and streak the

surface of the Urea agar slant. Do not stab the agar.

7. NOTE: Leave all caps on tubes loose.
8. Place the tubes in the designated test tube rack and incubate for at 37o C.

RESULTS: Next lab (LAB 4)

1. Collect all of your test tubes and place in order and record results in Table 1.
2. TSI agar tube: Examine the slants and butts of the TSI agar tubes. If the color is yellow, record
A for acidic and if the color is pink/red, record K for alkaline in the data table. If the agar is
cracked, has moved higher in the tube or has bubbles, record + as a positive result for gas
production; otherwise, a 0 for a negative result. Note and record the result for H2S production.
3. SIM tube: Add 3-4 drops of Kovacs reagent gently down the side of the tube. If a red color
appears, indole is present. H2S production is indicated by the presence of a black precipitate.
Motility is indicated by diffuse growth throughout the medium and non-motility by growth only
along the stab line.
4. SCA tube: A blue color indicates citrate has been utilized and is positive. A green color or no
growth is negative.
5. Urea agar tube: Observe for the presence of a bright pink color which is positive for the urease
enzyme (and production of ammonia). No color change from the uninoculated media or the
presence of yellow is negative for the urease enzyme.
6. When you have finished recording your results, use the key in lab to determine the identity of
your unknown bacterium. Check the results with the instructor and observe other groups results.
7. Share your results of your biochemical reactions with the class for all to see.

Table 1. Results of biochemical tests on unknown bacteria.

For the TSI slant, record A for acid and K for alkaline on the slant and butt. For the rest, record a positive
result (presence) with a + and a negative result (absence) with a 0 in the data table. Record the color
of the colonies growing on the chromagar.

TSI
Bacteria

Slant

Butt

SIM
Gas

H2S

Indole

H2S

Motility

Citrate

Chromagar
Urea

Color

Questions for Discussion

1. Why is the media in the test tubes referred to as differential media?

2. What is the identity of your bacterium? [View and compare to other unknowns as you will need to
be able to identify an unknown on your practical exam].

3. What is a bacterial exoenzyme and what is the function? Why are they important for bacteria?

4. What is the evidence that your bacteria did or did not have the citrase or urease enzymes?

5. Why are indicators used in the differential media tubes? Name the indicators in the urea agar tube
and the SCA tube.

6. Write the chemical reaction for indole production.

7. Explain the different types of metabolic processes that can be observed and read from the triple
sugar iron agar media.

8. Name a pathogen that produces H2S but does not ferment lactose or sucrose.

9. Name a pathogen that may inhabit the stomach and produces the urease enzyme (we can detect it
using that method; refer to chapter 21 and discuss with your table)?

10. Name a possible pathogen that is motile and produces indole.

11. Name a pathogen that is non-motile but ferments glucose.

12. Describe 3 properties of Enterobacteriaceae. Why is it important to identify them correctly?

DIFFERENTIAL MEDIA AND RAPID IDENTIFICATION

Introduction: In many situations in clinical microbiology, whether in the veterinary clinic lab or the
hospital lab, a rapid identification of an infectious agent is needed and important. New instruments are
being developed which will identify bacteria in a few short hours, some directly from the specimen
source. However, sometimes it is important to isolate bacteria and perform tests on them which take one
or more days. Differential media and multiple test systems are among methods of rapid identification.
One new media is Chromagar orientation which helps differentiate many of the members of the
enterobacteriacea rapidly based on the color of the colony. This media is often used for rapid
differentiation of Gram negative bacteria in urinary tract infections. It has the advantage of being able to
differentiate and detect more than one population and even small numbers of microorganisms in a clinical
specimen. Bacteria express genus or species-specific enzymes on this media causing the chromogenic
agar to release certain colors. For example, the -galactosidase enzyme system causes the release of a
dye turning the colonies pink, and blue colonies result from the -glucosidase enzyme. Proteus groups
produce tryptophan deaminase enzymes which turn the media beige-brown. In many cases, a genus can
be determined by simply observing the color of the colony. You will see how this media complements
your biochemical tests.
In addition to Chromagar Orientation media for rapid preliminary identification, some clinical
laboratories use miniaturized multitest strips. Some of these multitest strips contain more than 20 tests
that can be inoculated with the bacterium in question and all the results read at once. These biochemical
tests include a series of sugar fermentations, amino acid decarboxylations, and more, including the
biochemicals you set up for your unknown bacterium. Rapid identification is becoming more important
in clinical labs in order to begin treatment quickly.
Materials and Bacteria:
Unknown bacteria assigned in this lab (A H)
One Chromagar Orientation plate per group of 2.
Inoculating loop

Procedure Lab 1 Chromagar:

1.
2.
3.
4.

Using a small amount of your unknown bacteria, prepare a streak plate on chromogenic agar.
Use the same technique you used in the pure culture technique lab to achieve isolated colonies.
Label the plate on the agar side with your initials and your unknown letter.
Invert and incubate for 48 hours at 36 degrees C.

Procedure Lab 2 Chromagar:

1. Collect your Chromagar Orientation plate and observe the color. Record the results in Table 1 in the
biochemical results exercise.
2. Compare your results with the Figure 1 chart below.
3. Using the indole results and the chromagar only, determine a presumptive identification of your
bacterium. Compare the results with those you obtained from the biochemical analysis.
Figure 1. Examples of Chromagar Orientation media:

Questions for discussion.

1.

Record the color of your bacterial unknown on Chromagar in Table 1 of the biochemical exercise.

2.

Use the color and the results of indole and try to identify your unknown. How did the
identification of your bacterial unknown on the Chromagar/Indole compare to the tube
biochemicals identification? Was it 100% successful?

3.

Why is rapid identification important in a clinical setting?

4.

Are these tests definitive?

SMEAR PREP

REFERENCE:
Smear prep video: http://www.youtube.com/watch?v=Qi8KQw4514E&feature=related; Bacterial Pronunciation Guide:
http://www.atsu.edu/faculty/chamberlain/Website/studio.htm

Introduction: To view bacteria microscopically, the bacteria are applied and adhered to a microscope
slide so they can be colored by chemical reagents called stains. The application of bacteria to a slide for
the purpose of staining is called a smear. Smears can be made from bacteria growing in liquid and solid
culture media.
Good smear technique is important in order to obtain accurate staining results. A smear will stick the
bacterial cells to the slide so they will not wash off or shrink inappropriately during the staining process.
When preparing a smear, it is essential to prepare thin smears so the cells are not piled on top of each
other. Thick smears may affect the staining results and even distort the shape and arrangement
determinations.

Materials and Equipment :

Gram stain set: Crystal violet, Grams Iodine, Grams Alcohol and Safranin
Microscope slides
Microscope slide holders (wire or clothespin)
Bacteriological loops and needles
Sterile saline
Staining trays and pads
Bottle with distilled water
Bibulous (blotting) paper
Incinerator
Goggles; gloves
BACTERIAL CULTURES
Corynebacteriumpseudodiphtheriticum
Staphylococcus aureus
Bacillus cereus
E. coli
Enterococcus faecalis
Neisseria sicca

PROCEDURE: Aseptic Transfer Technique and Smear Preparation

1. Acquire the bacterial cultures to prepare smears.
2. Label a slide (s) with a wax marker with the name or names of the bacteria you are preparing on
the slide. You may label several slides at one time.
3. Draw a circle on the underside of the slide using a wax marker. Place a drop of saline from the
dropper bottle in your drawer on the slide in the location you drew the circle and want to place

4.
5.
6.

7.
8.
9.
10.
11.

12.

the smear. Do not use too much saline as the slide will take too long to dry.
Sterilize the bacteriological loop by placing the loop in the incinerator for five (5) seconds.
Withdraw the loop and allow it to cool for at least five (5) seconds.
Lift the lid of the plate and hold it over the agar portion.
Using your sterilized loop, acquire a very small amount of a bacterial colony and smear it into
the saline making a smooth dispersed emulsion of bacteria. Do not use too much bacteria
make the suspension barely cloudy.
Return the plate lid to the agar portion of the petri plate.
Sterilize the loop between acquiring bacteria or before returning it to the bench top.
Allow the slide to completely air dry. Your instructor may allow you to dry the slide by placing
the slide on the heated platform on top of your incinerator.
If air dried, you must heat fix the slide by attaching a clothespin to the end of the slide. Pass the
slide in front of the opening of the incinerator several times being careful not to overheat.
NOTE: If you are taking bacteria from a tube of liquid culture media, do not add a drop of saline
to the slide. Simply use aseptic technique to obtain a loopful of the broth culture and smear this
on the slide. You might need to add two loopfuls to get adequate bacteria for successful stains.
Use these smears for the Gram stain procedure in this lab.

QUESTIONS FOR DISCUSSION:

1. What is the purpose of the smear preparation?

2. What is the purpose of heat fixation of a smear?

3. Discuss three errors that may occur when preparing the smear.

GRAM STAIN PROCEDURE

REFERENCES: Gram stain procedure and quiz link: http://highered.mcgrawhill.com/sites/007337525x/student_view0/exercise9/gram_stain.html; See Figure 2 below.

Online images of Gram stained bacteria

Grampositivecoccus:
http://www.microbelibrary.org/library/gram-stain/2859-gram-stain-gram-positive-cocci
Gram positive bacillus: http://www.microbelibrary.org/component/resource/gram-stain/2864-gram-stain-gram-positive-rods
Gram negative bacillus: http://www.microbelibrary.org/library/gram-stain/2860-gram-stain-gram-negative-rods
Gramnegativecoccus:
http://www.microbelibrary.org/library/gram-stain/2865-gram-stain-gram-negative-cocci

INTRODUCTION: The Gram stain is a differential stain which uses various dyes to differentiate two
large groups of bacteria based on their cell walls (peptidoglycan layer). In general, bacteria have one of
two types of cell walls which determine the Gram stain result. When the Gram stain is completed, if the
bacteria are purple, the bacteria are called Gram positive. If the color is pink to red, the bacteria are
called Gram negative.
Gram positive bacteria have a thick peptidoglycan layer (up to 25 layers), whereas, Gram negative
bacteria have a thin layer of peptidoglycan and an outer membrane. This difference in structure explains
how the dyes interact with these different cell walls.

In the first step of the Gram stain procedure crystal violet stain is applied and initially stains both Gram
positive and Gram negative peptidoglycan layers a purple color. Next, Grams iodine is added. It is
called a mordant and forms an insoluble chemical complex with crystal violet. Grams alcohol is
applied and removes the crystal violet-iodine complex from Gram negative bacteria, rendering the
pepetidoglycan layer in these bacteria colorless. However, the alcohol shrinks the pores of the
peptidoglycan layer in the Gram positive bacteria, trapping the dye and leaving the Gram positive bacteria
peptidoglycan layer purple. Removal of color with alcohol is called decolorization. The difference in
structure of the cell wall explains the different result obtained in the alcohol step. Finally, the
counterstain safranin is applied and stains the decolorized, Gram negative bacteria pink to red, whereas
the purple, Gram positive bacteria are unchanged and still purple. See Table 1.
The critical step in the Gram stain is the decolorization step. Incorrectly performing this step can result
in false positive and false negative results. Very thick smears and old cultures also yield incorrect results.

Table 1. Color results after each step of the Gram Stain procedure

Reagent
Gram Positive
Gram Negative

Crystal violet
Primary dye

Grams Iodine
Mordant
Alcohol

Decolorizer

Safranin

Counterstain

Gram stains are very common tests in identification of unknown bacteria and are the most commonly
performed lab test in clinical microbiology labs even today. These stains provide a great deal of clinical
information in a short period of time. Knowing the type of cell wall of a bacterium will aid in the choice
of antibiotic and allow chemotherapy to begin quickly.

PROCEDURE Gram stain each smear by carrying out the following steps.
1.
Place the smears on the stain tray (with or without slide holders attached).
2.
Apply a generous amount of crystal violet stain; let stand for 30 seconds.
3.
Hold the slide at a 45 angle and wash with distilled water from the wash bottle for several
seconds to remove the dye.
4.
Apply a generous amount of Grams iodine; let stand for one (1) minute.
5.
Hold the slide at a 45 angle and wash with distilled water for several seconds.
6.
Hold the slide at a 45 angle; apply Grams alcohol for 5 - 10 seconds or until alcohol flows
from the slide until very little or no color is coming off. Do this step carefully. It is possible
to decolorize too much or too little and create false Gram negative results or false Gram positive
results, respectively.
7.
Hold the slide at a 45 angle and wash with distilled water from the wash bottle for several
seconds. This stops the reaction with the decolorizer.
8.
Apply a generous amount of safranin stain for 30 seconds to 1 minute.
9.
Hold the slide at a 45 angle and wash with distilled water for two (2) seconds.
10.
Gently blot the slide between sheets of bibulous paper to remove excess water.
11.
Prepare Gram stains of the 6 bacteria as directed by your instructor.
12.
Using the microscope find the smear with 10x objective, add immersion oil, and observe under
100x oil immersion objective. Remember to use only the fine adjustment knob to focus under the
oil immersion objective.
13.
Note and record the color, shape, arrangement, and Gram reaction (positive or negative) in Table
2. Make a sketch of a few bacteria from a representative section.

SAFETY AND DISPOSAL

1
Avoid skin/eye contact and ingestion of biological stains.
2
Discard used stains according to the instructor. Place stain pads in regular trash or pour stain
from tray in beaker. Do not discard used stains in the sink.
3
Discard microscope slides in appropriate location.
4
Use kimwipes to remove all oil from the objectives. Lower the stage, turn down the rheostat, and
gently wrap the electrical cord. Check your microscope with your instructor before returning to
the shelf.

Table 2: Record Results of Gram stains. Use table in LABS 3, 4, AND 5 as needed.
Name
Staphylococcus
aureus

Shape

Arrangement

Gram
reactio
n

Sketch

Neisseria sicca

Corynebacterium
pseudodiphtheriticum

Enterococcus faecalis

E. coli

Bacillus cereus

Figure 2. Representative Images of certain Gram stain results.

QUESTIONS FOR DISCUSSION:

List the Gram stain reagents and the purpose of each in the Gram stain procedure.

Explain how bacterial cell wall structures account for the two different Gram reactions.

3.

Explain why the Gram stain is a differential stain.

4.

Explain why the decolorization step is the most critical step in the procedure.

5.

Why is the Gram stain an important first step in identification of an unknown bacterium?

6.

What clinical information does a Gram stain provide?

7.

Why does culture age affect the results of a Gram stain?

8.

Discuss two causes each for false positive and false negative stain reactions

ACID FAST STAIN

Ref: Acid fast stain examples: http://www.microbelibrary.org/component/resource/laboratory-test/3133-acid-fast-stain (see

Mycobacterium smegmatis and Staphylococcus epidermidis)

INTRODUCTION:
The Acid fast stain is a differential stain that detects the presence or absence of mycolic acids in the
bacterial cell wall. Mycolic acid is a waxy material that is found in the genera Mycobacterium and some
Nocardia in lesser amounts. The mycolic acid is 50% of the dry weight of the Mycobacterium cells and
is sometimes referred to as cord factor as it results in clumping and aggregating of the bacterial cells. It
is a virulence factor, allowing survival of Mycobacterium in phagocytes, and contributing to resistance to
disinfectants and antimicrobial therapy and may make a mycobacterial infection more difficult to treat.
The Kinyoun method is a cold acid fast stain method using a phenolic compound, carbolfuchsin as the
primary dye which is a lipid soluble, concentrated stain. This stain penetrates the waxy mycolic acid in
acid fast bacteria and is retained as a fuchsia pink complex. Mycolic acid gives lipids a higher affinity
for the primary dye, carbolfuchsin, and therefore resists decolorization with acid alcohol. Bacteria that
are not acid-fast are easily decolorized by the acid fast decolorization step and stain with the counterstain
methylene blue.
The acid fast stain is used as a presumptive test for the presence of acid fast bacteria in clinical
specimens where infections with Mycobacterium are suspected. Mycobacterium tuberculosis causes
tuberculosis, infecting about 1/3 of the earths human population. However, only those with the disease
are producing the bacterium and are infectious to others. Tuberculosis is a re-emerging infection. The
bacterium has gained resistance to a number of commonly used antibiotics and now is classified as multidrug resistant and extensively drug resistant. Mycobacterium leprae causes leprosy and is also acid fast
but is much less common than tuberculosis.
Materials, Supplies and Equipment and:
Kinyoun stain kit: carbolfuchsin, acid alcohol, methylene blue
Glass slides, slide staining tray and pad, clothespin, bibulous paper, wash bottle
Incinerator, bacteriological loop,
Goggles, Gloves
Bacterial cultures
Agar plate cultures of Mycobacterium smegmatis and Staphylococcus aureus

PROCEDURE:
1. Prepare a bacterial smear using aseptic technique as follows:
a. Draw a circle on the underside of a glass slide and add a drop of saline to the center of the
circle.

2.
3.
4.
5.

6.
7.
8.
9.
10.
11.
12.

b. Flame a loop and let it cool. Obtain a small amount of a Mycobacterium smegmatis with
the loop and spread out into the saline on the slide. Flame the loop.
c. In the same smear, mix a small amount of Staphylococcus aureus obtained with a
sterilized loop. Re-flame the loop and return it to the lab bench.
d. NOTE: the smear now contains two different bacteria.
Let the slide air dry or dry on the slide tray above the incinerator.
Place the slide on the slide staining tray and cover the smear area with carbolfuchsin. Let it stay
on the smear for at least five minutes.
Wash the stain off the slide with the wash water bottle.
Decolorize with the acid alcohol. Add the decolorizer drop by drop for 5 seconds or longer until
there is no more dye being removed. NOTE: This is the critical step. You can over-decolorize
the smear if you add too much alcohol.
Immediately add water to the smear to stop the decolorization step.
Add the methylene blue counterstain and let it stand for 30 seconds.
Wash the counterstain off and blot the slide dry with bibulous paper.
Observe under oil immersion.
Acid fast Mycobacterium will appear fuchsia pink. They are sometimes pleomorphic, long rods
and coccoid shapes. You may see only a few clumps of acid-fast shapes in the entire slide.
Non-acid fast Staphylococcus will appear as light blue cocci throughout the slide.
False positive and false negative results may also be observed.

RESULTS
1. Read pink rods as acid fast positive. Read blue cocci as acid fast negative.
2. Draw your results in the circle provided below.
3. Note: If you have endospores or free spores, they will appear as false positive acid fast.

QUESTIONS FOR DISCUSSION:

1. What is the cellular target of the acid fast stain?
2. What genera of bacteria is typically acid fast?
3. Discuss two diseases caused by acid fast bacteria.

4. Explain how the acid fast property of the bacterium is a virulence factor?

5. If a patient has acid fast bacteria in the sputum specimen they provided, does this mean they have
tuberculosis? Why or why not?

6. Explain a screening test and a more definitive test for diagnosis of tuberculosis.

7. Do animals get tuberculosis? Discuss this with your instructor.

8. Explain the meaning of tuberculosis as a re-emerging infection.

Capsule Stain
Reference: Capsule stain results: http://www.microbelibrary.org/component/resource/laboratory-test/3040-capsule-stain
INTRODUCTION: Some bacteria are surrounded by a glycocalyx which consist mostly of
polysaccharides and some polypeptides. Slime layers are a type of glycocalyx that are loosely bound. If
the glycocalyx is well organized and tightly bound, it is referred to as a capsule. Capsules can impart a
mucoid (wet, slimy) appearance to a bacterial colony containing encapsulated bacterial cells. Capsules
are considered virulence factors because their presence on bacterial cells increases the chance of serious
disease. Capsules are slippery to phagocytes (cell eaters, such as macrophages and neutrophils). Due
to this, phagocytes cannot easily perform phagocytosis. This results in shutting down the non-specific
defense which protects our body from the encapsulated bacterium. Our immune system can overcome
this phagocyte problem with opsonization, coating the bacteria with complement and antibodies. Some
pathogens with capsules are Streptococcus pneumonia (a major cause of pneumonia worldwide),
Klebsiella pneumonia (important cause of hospital acquired pneumonia) and Bacillus anthracis (cause of
anthrax).
In addition to the role in inhibiting phagocytosis, capsules are very important in aiding attachment and
formation of biofilms (community of bacteria stuck on a surface). Good examples of encapsulated
bacteria living in a biofilm are those in your oral cavity forming plaque. When biofilms form on surfaces
such as prostheses, they often have increased virulence and antibiotic resistance. They are difficult to
remove and a new prosthesis may be needed.
The capsule stain is a special stain. The polysaccharide and polypeptides of capsules do not take up dyes
and therefore appear a halo around a bacterial cell. The cell is stained with one dye, the background is
stained with a different dye and the capsule is clear. This is a negative stain. View prepared capsular
stains.
RESULTS
1.
2.

View capsular slides with the oil immersion objective.

Draw your results in the circle.

QUESTIONS FOR DISCUSSION:

1. Describe a capsule and how it surrounds the cell.

2. Explain why capsules are considered virulence factors?

3. Describe a biofilm and the role of capsules in their formation. Give an example of a biofilm that
could form in an infection.

4. Use your book to give an example of an oral bacterium with capsules which aids in biofilm
formation.

5. Name an encapsulated bacterial pathogen that is a common cause of pneumonia.

ENDOSPORE STAIN

REFERENCE: Talaro Ch 4; http://www.microbelibrary.org/component/resource/laboratory-test/3134-endospore-stain
Note: some endospores are unstained.

INTRODUCTION: Endospores are structures that form inside some bacteria. They are a dormant
form that survives harsh environmental conditions. Bacillus and Clostridium are among bacteria that
commonly produce endospores when their environmental conditions become unfavorable, most notably
due to lack of nutrients. These two genera of bacteria are soil bacteria, but also can be found as normal
flora and some may be pathogens. The formation of an endospore begins when a bacterial cell receives a
signal to turn off normal housekeeping functions and to turn on the spore forming genes. A sporulation
cycle may be complete in ten hours and does not reverse once it begins. Although the spore forms inside
the cell and is called an endospore, eventually the cell itself will disintegrate and only a free spore is left.
The spore consists of a core of DNA surrounded by a very thick cortex of peptidoglycan and spore coat
which protect the DNA. Dipicolinic acid complexed with calcium is found in high concentrations in the
endospore and water exits, leaving the spore very dehydrated. The DNA is in the core and also
surrounded by acid soluble proteins that protect the DNA. All of these components allow the DNA to
survive extreme conditions such as very high or low temperatures and severe dehydration. In addition,
spores serve as a barrier to the damaging rays of ultraviolet light. Spores are a dormant form of the
bacteria and not a reproductive form. When the conditions become favorable (such as more nutrients),
the spores undergo the germination cycle and form new vegetative bacteria.
Bacterial spores are important from a clinical standpoint as they are very hard to kill and require high
category agents for sterilization. Autoclaves, sterilizing gases, liquids, and ionizing radiation are some of
the methods used in hospitals to insure that spores are killed. Surgical supplies and other clinically
relevant materials are not considered sterile unless the bacterial spores are removed. Spores may also be
the infectious form of the bacteria and cause a new infectious disease. An example common in hospitals
are the spores of Clostridium difficile which can spread to individuals that have taken certain antibiotics
disrupting the normal GI flora. The spores germinate in the colon and easily spread due to the lack of
normal flora. The C diff causes severe diarrhea and may lead to pseudomembranous colitis, a
potentially fatal disease. Another disease spread by spores is anthrax caused by Bacillus anthracis and
an important pathogen of animals.
You will view prepared slides with endospore stains. The spores may be endospores inside the bacterial
cell, or free spores outside the bacterial cell. Spores will appear a different color than the vegetative
bacteria or they will not be stained at all and appear clear.

RESULTS:
1. Obtain the assigned slides of endospore stains and view under oil immersion.
2. Observe the endospores, free spores and the vegetative bacteria.
3. Record the results in the area below.

QUESTIONS FOR DISCUSSION

1. Explain the cycle in some bacteria of making spores and germinating them. What is the function of
spores? Why do bacteria make spores?

2. Name two genera of bacteria that produce endospores. Name an important pathogen in each genera.

3. What properties does an endospore provide the bacterium? Why for each?

4. Explain the importance of spores from a clinical standpoint.

FLAGELLAR STAIN

References: Video of bacterial locomotion and flagella: http://www.youtube.com/watch?v=6p9e0oolbmE
Link to flagellar stains http://www.microbelibrary.org/library/laboratory---test/3159---bacterial---flagella---stain

INTRODUCTION: Bacterial flagella are appendages that provide motility. The bacteria may have a
single polar flagellum (monotrichous) or be completely covered with flagella (peritrichous). Your
textbook shows different types of flagellation in bacteria. Flagella are composed of the protein flagellin.
The flagella traverse the membrane (s) and cell wall and function much like a propeller. Flagella are very
thin and below the level of resolution of our light microscope (0.2 m). Therefore, in order to see the
flagella, they must be thickened with a mordant. This is a stain that deposits on the flagella to a
thickness that allows them to become visible using the light microscope. The number and arrangement
of flagella are helpful in identifying bacteria but do not necessarily correlate with the speed of movement
of the bacterium.

Procedure and Results:

1. View flagellar stains as assigned by your instructor using oil and the 100x objective.
2. Observe for type of flagellation on the bacterial cells and draw results.

QUESTIONS FOR DISCUSSION

1. What is the flagellation pattern in the bacteria in your slide?

2. How do flagella cause motility (see text)? Why cant we observe flagella as they are moving?

3. What must be done to flagella to view them with the microscope? Why?

4. What is chemotaxis?

Do all bacteria exhibit chemotaxis?

5. Are flagella virulence factors (discuss with your instructor and classmates)?

Using Electrophoresis to Obtain a DNA Fingerprint of an Unknown Microbe

There are a number of methods used to identify unknown microorganisms. You have grown bacteria and
observed unique colony characteristics, and observed bacteria microscopically, identifying shape and
arrangement. You have stained bacteria to identify specific types of cell walls. Additionally, you have
used selective and differential media and performed biochemical analysis to determine metabolic
pathways that are unique to certain bacteria. DNA can also be used to identify unknown
microorganisms.
DNA generally exists as two long strands of complementary nucleotides. Each nucleotide is composed of
a phosphate sugar backbone and the nitrogenous bases, adenine, guanine, cytosine and thymine. These
bases exist in certain orders, some of which compose genes coding for proteins. In bacteria, the DNA
usually exists in a double helix circular form in the cytoplasm. In protozoa and fungi, the nuclear DNA
may exist in structures similar to ours since they are also eukaryotes. In viruses, the nucleic acids can be
DNA or RNA, single or double stranded, circular or linear segments.
DNA can be analyzed by cutting it into pieces called fragments, and separating these fragments by size.
DNA can be cut with enzymes called restriction enzymes. These enzymes occur naturally in some
bacteria, and are used to protect from viral invasion by destroying the viral DNA. The enzymes
recognize specific nucleotide sequences on the DNA, bind to the DNA and cleave, or cut, the DNA
between the bases (see below). RNA may also be cut by some enzymes and the RNA fragments
analyzed. There are many restriction enzymes that recognize and cleave different nucleotide sequences.
This may result in varied numbers of fragments or sizes of fragments of DNA. In addition, if the DNA
sequences between two sources of DNA are different, such as in different bacteria or viruses, then the
same restriction enzyme will generally result in different numbers and sizes of fragments between the two
microbial DNAs.
To determine the number and size of the DNA fragments, they must be separated from each other. This is
usually accomplished by using agarose gel electrophoresis which separates the negatively charged DNA
fragments using an electric current. The agarose gel is a porous material that provides a platform for
DNA to move through. The cut DNA moves from the negative pole to the positive pole and is separated
by size. The result is a series of bands that appear in patterns. These patterns of fragments are often
referred to as a fingerprint and are unique to a particular microorganism. We can visualize these
fragments by staining the gel with a dye, such as ethidium bromide, which binds to double stranded DNA.
The DNA-ethidium bromide complex emits a visible fluorescent orange light which can be visualized.
When the fingerprint of an unknown microbial DNA is compared to known microbial DNA and the
fingerprints are the same, then the unknown organism is likely the same or a similar microbe as the
known. If an unknown microbial DNA fingerprint is different from a known microbial DNA fingerprint
then the organisms are usually not the same. However, mutations in the DNA may change the DNA
fingerprint, even if the DNA is from a known microorganism. This fingerprint analysis can be used to
help identify an unknown microorganism, for example, one causing an outbreak of infectious disease. In
humans, DNA fingerprints may be used to determine if a particular person is related to another or to
determine if a person was present at a crime scene.

In the example today, we will perform electrophoresis on simulated viral nucleic acid from different
viruses involved in infectious disease outbreaks in order to obtain viral fingerprints. This is will provide
information on whether the different sources of DNA are from the same or different viruses.
QUESTIONS FOR DISCUSSION:

1. What causes the separation of DNA fragments during agarose gel electrophoresis?

2. How can Syber Green be used to detect DNA in agarose gels?

3. How is a DNA fingerprint prepared? How can it be used to identify an unknown microorganism?

4. How can a mutation change the DNA fingerprint?

Additional information on electrophoresis and DNA electrophoresis.

Nucleotide sequences are recognized by restriction enzymes. Example of how one restriction enzyme
cuts DNA. EcoR1 recognizes and cleaves between guanine and adenine forming a staggered cut in the
two DNA strands as follows:

. Now there are two fragments of DNA.

REFERENCES:

Electrophoresis (View the introduction. You do not have to make the gel as it is already made for you
in our lab.) http://learn.genetics.utah.edu/content/labs/gel/

DNA fingerprinting of DNA samples http://www.youtube.com/watch?v=PSwlCk_Z02c

*Please note here that a different and sterile pipette tip should be used between samples*.

Image of stained gel http://www.microbelibrary.org/images/hyatt/gel_image.jpg (note: copy this link into your browser
rather than click on it for best results).

OUTBREAK! FINGERPRINTING VIRUS DNA.

ADDITIONAL INFORMATION: http://www.pbs.org/wgbh/nova/body/herd-immunity.html
Introduction: An infectious disease epidemic occurs when the number of new cases of a disease in a
certain time and location are considered more than normally expected. Epidemics of an infectious
nature may be caused by bacteria, viruses or other microorganisms. Epidemiologists use various
methods to determine if an epidemic is occurring, that is, if the microorganism is the same in various
instances of the outbreak area. One common method used to definitively identify the microbe causing an
outbreak is to analyze the microbes nucleic acid using DNA (or RNA) fingerprint analysis. DNA
fingerprinting has been used in E. coli outbreaks frequently.
The following is a scenario example of DNA fingerprinting for an outbreak of viral infections.
It is the year 2015. You are a molecular epidemiologist working for the Centers for Disease Control and
Prevention (CDC). Your job is to help track epidemics and to monitor emerging diseases. Five years
ago, a cluster of cases of hemorrhagic fever occurred in an isolated town in northeastern Alabama. The
disease killed approximately 30% of those who got it. The most alarming aspect the new disease was that
it was highly contagious from human to human, thus posing the threat of an epidemic. Medical
authorities, including your office, believe the only reason the outbreak did not erupt into a major epidemic
was that the Alabama town was so small and isolated. A CDC team traced the disease to a virus carried
by the numerous local squirrels. An extensive campaign was carried out in an attempt to eliminate the
virus by capturing the squirrels.
Three years ago a suspicious outbreak occurred in Pennsylvania. Several people fell ill with a
hemorrhagic fever. The symptoms of the disease were similar as those with the Alabama fever, but no
one died. The Pennsylvania fever was apparently less contagious than the Alabama fever since many
people in Pennsylvania were exposed to the Pennsylvania patients but did not come down with the
disease. However, the Pennsylvania virus was also traced to the local squirrel population.
You were asked to compare the viruses that caused the two outbreaks. You found that the virus particles
looked similar. Both viruses had a DNA genome, but the nucleotide nitrogenous base - sequences of
the genomes were different in many places.
Now, three in Missouri have fallen ill with a hemorrhagic fever. Their symptoms are similar to the
symptoms of the Alabama and Pennsylvania fevers. Local medical personnel were alarmed and called the
CDC to determine if the dangerous Alabama virus had reappeared. The patients have been placed in
quarantine. You are flown to the scene. You must immediately determine whether the Missouri patients
are infected with the highly contagious and deadly Alabama virus, the Pennsylvania virus, or some other
agent.
One of the techniques you decide to use is examination of viral DNA by restriction analysis. Wearing
protective clothing, you enter the patients isolation rooms in the Missouri hospital, carefully draw blood
samples, place them on ice, and rush back to the biological containment laboratory at the CDC for a
variety of tests. You isolate virus particles, extract viral nucleic acids, and subject the samples to
restriction enzyme digestion in order to perform agarose gel electrophoresis. In your gel you include
samples of DNA from the Pennsylvania and Alabama viruses for comparison.

Procedure: Practice
1. Please wear gloves during the entire electrophoresis procedure. Ethidium bromide is a mutagen
so do not touch the gel.
2. Practice using the micropipettes and tips by loading the practice loading dye into wells in gels
provided as instructed by your instructor.

Procedure: Load the gel

1. Follow the guidance of your lab instructor. The gel is already prepared for you.
2. You will be given small plastic tubes containing simulated viral DNA with a loading dye (blue
color).
a. You will use a micropipet to place each DNA sample into separate wells on the agarose
gel. This device uses plastic tips and draws up a known amount of liquid containing
DNA to be placed in an agarose gel well.
b. There are eight wells. The path that each sample will take as it travels from the well
across the gel is called a lane.
c. Three samples must be loaded in three separate lanes in each gel. Load the DNA
samples into the well as directed by your instructor, for example, wells 2, 3, and 4.
Avoid using lane 1 or lane 8. Follow your instructors directions.
3. Place a sterile pipet tip on the micropipette.
4. Draw the entire sample in the plastic tube into the pipet tip as demonstrated by the instructor
(about 5 10 microliters).
5. Steady the pipet over the well in the agarose gel using two hands.
6. Be careful to expel any air in the micropipette tip end before loading the well in the gel. (If an air
bubble forms a cap over the well, DNA/loading dye will flow into buffer around the edges of the
well. You may also lose some of the DNA sample if you have bubbles. If you lose DNA in this
way, you may not see the bands you should see.)
7. Dip the pipet tip through the surface of the buffer (liquid covering the gel), position it over the
well, and slowly expel the mixture. Sucrose in the blue loading dye weighs down the sample,
causing it to sink to the bottom of the well. Be careful not to punch the tip of the pipet through
the bottom of the gel. Also, do not overfill the wells.
8. Use a new pipette tip and repeat steps 2 7.
9. Discard the pipette tips in the beaker provided.

Procedure: Electrophoresis
1. Close the top of the electrophoresis chamber, and connect electrical leads to the power supply,
anode to anode (red-red) and cathode to cathode (black-black). Make sure both electrodes are
connected to the same power supply.
2. Turn the power supply on and set voltage as directed by your instructor. Shortly after current is
applied, electrolysis should begin (bubbles rising from each electrode at bottom ends of each
apparatus).
3. Loading dye can be seen moving through the gel. Since the DNA is negatively charged it will
move to the positive pole.
4. If the loading dye contains bromophenol blue. Bromophenol blue migrates through the gel at the
same rate as a DNA fragment approximately 300 base pairs long which means it will reach the

5.

6.
7.
8.
9.

bottom of the gel before the DNA fragments do. Larger fragments (more base pairs) will not
migrate through the agarose gel as fast.
Allow the DNA to electrophorese until the bromophenol blue band is about 2 cm from the end of
the gel. This will take an hour or more. Your instructor will monitor the progress of the
electrophoresis.
To avoid electric shock, turn off the power supply. Then disconnect the leads from the inputs,
and remove the top of the electrophoresis chamber.
Your instructor will carefully remove the casting tray, and slide the gel into a plastic staining tray.
The instructor will assist your group in viewing the gel and taking a picture. You will use the
picture, and not the gel, for analysis.
Allow the instructor to dispose of the gels.

QUESTIONS FOR DISCUSSION:

1.

What is epidemiology?

2.

Give an example of a communicable infectious disease and an example of a non-communicable

infectious disease.

3.

Explain why it is important to definitively identify the microbe causing the outbreak.

4.

What is herd immunity? How could it affect an epidemic?

5.

Draw the results from your picture below. Compare the fingerprints of Pennsylvania, Alabama,
and Missouri virus isolates. What can you conclude about the virus infecting the Missouri
patients?

6.

Your instructor will provide you with two different DNA sequences. If you used EcoR1 which
cuts between A and G, what are the number of fragments in each of the two sequences?

7.

Why would the restriction fragment patterns from two viruses be different?

8.

Give one advantage and one disadvantage of using DNA for identifying unknowns vs. using
biochemical analysis.

GROWTH FACTORS: TEMPERATURE

Reference: Talaro textbook chapter 7.
INTRODUCTION: Bacteria grow at diverse temperatures, from below freezing 0oC, to over 110oC
(230oF). Bacteria living at extreme temperatures must have enzymes whose activity functions adequately
at these temperatures or they would not survive. In addition, some bacteria and archaea have specialized
membranes to withstand very hot temperatures and some have anti-freeze like proteins that survive very
cold temperatures. The temperature at which the organism grows best over time is called the optimum.
The temperature, below which the organism cannot grow, is called the minimum. The temperature, above
which the organism cannot grow, is called the maximum.
The optimum temperature of growth may be observed in broth cultures as measures of turbidity
(cloudiness). The turbidity is measured by absorbance using a
spectrophotometer.
A
spectrophotometer is designed to shine a beam of single wavelength light on a sample and measure the
absorbance (in this case) of that light. The higher the absorbance of light, the higher the growth, i.e., they
are proportional. As cells grow (increase in number), more light is absorbed, and the higher the
absorbance reading (also referred to as optical density).

Other phenotypic characteristics may also be moderated by temperature if they are controlled by
enzymatic activity. For many organisms a change in temperature away from the optimum, while not
resulting in growth inhibition or cellular death, may result in eliminating certain characteristics. One such
characteristic is color of the bacterial colony due to alteration of the enzyme activity controlling pigment
production.
Organisms can be classified by their growth characteristics in various temperatures. Organisms that grow
only below 20o C are classified as psychrophiles. They are commonly found in the ocean and the Arctic
and Antarctic where temperatures are always cold. The enzymatic activity of these microorganisms
functions best in the cold. Organisms that are adapted to the cold but can survive up to 35oC and slightly
higher, are classified as psychrotrophs. Mold and some bacteria such as Pseudomonas
are
o
psychrotrophs. In general they have an optimal temperature range of 15 30 C but some psychrotrophs

may be pathogens to humans. One example is Listeria, causing food infection, but also more serious and
fatal diseases. Microbes that are adapted to temperatures between 15oC and 45oC are classified as
mesophiles. Most bacteria living in a symbiotic relationship with humans or are pathogenic to humans
are mesophiles since body temperature is 37oC (See figure 1).
Organisms growing above 45oC - 80oC are classified as thermophiles and are found in hot springs and
other hot areas. Microbes growing above 80oC up to 110oC are extreme thermophiles such as those
bacteria that live in ocean floor ridges and thermal vents. While these microorganisms do not infect
humans, they are important ecologically. Another classification of temperature growth is themoduric.
These bacteria generally are mesophiles that endure high temperatures (up to 70o C or higher) for short
periods of time. These bacteria may survive standard pasteurization process and eventually spoil milk.
SUPPLIES, REAGENTS, EQUIPMENT, AND MEDIA
Inoculating loops, marking pencils, incinerators,
Nutrient agar slants (4 groups, 2 per group)
Nutrient broth tubes (4 groups, 8 per group)
BACTERIA
E. coli broth culture (hold tube upright)
Bacillus (Geobacillus) sterothermophilus broth culture (hold tube upright)
Serratia marcescens agar plate

PROCEDURE first lab (Lab 7):

1. Obtain the media listed and return to your lab bench.
2. Label four separate broth tubes as 4o C, 25o C, 36o C, and 55o C. Also label them as E. coli.
3. Label the other four broth tubes with the above temperatures but with the name B.
sterothermophilus. NOTE: You must keep all broth tubes upright or broth will spill out.
4. Inoculate the broth tubes for E. coli using aseptic technique. Use one loopful for each tube.
NOTE: It is important to use the same inoculum size for each tube or your will not get accurate
results.
5. Inoculate broth tubes for B. sterothermophilus using aseptic technique as described for E. coli.
6. Label the two nutrient agar slants, Serratia marcescens, 25o C and 36o C.
7. Inoculate the two nutrient agar slants with S. marcescens using aseptic technique. Obtain a
portion of a colony and inoculate the surface of the slant. Do not stab the agar.
8. Place all the inoculated tubes in the appropriate temperature rack for incubation.
9. Develop hypotheses about what your results will be for temperature growth.
RESULTS LAB 8: Pigment production
1. Collect your two nutrient agar slants.
2. Observe the slants for an orange red pigment. Record the results in the table below.
Table 1. Results of Serratia pigment production at various temperatures.
S. marcescens Pigment production
Temperature
Record intensity as ++ or +++
o
25 C

37o C

Comments

RESULTS LAB 8: Temperature and absorbance (spectrophotometer)

Growth results in broth will be determined with a spectrophotometer (SpectroVis) and the logger pro
program. As discussed in the introduction, turbidity in the nutrient broth indicates bacterial growth. The
objective is to quantify the amount of turbidity and, therefore, the amount of bacterial growth, by
obtaining an absorbance (A) value for each nutrient broth tube using the wavelength of light at 550 nm.
The A value can be read directly from the logger pro program connected to the SpectroVis.
1. Collect the nutrient broth tubes and place them in order from 4o C to 55o C by bacteria.
2. Gently vortex the broth tubes to suspend the bacterial cells. NOTE: Always keep the tubes
upright and do not vortex too vigorously as you may spill broth containing bacteria.
3. Open the Logger Pro program.
4. You need to blank the readings to remove background broth absorbance in an uninoculated broth
tube since you are interested in the absorbance value only from the bacterial growth and not the
culture broth. Follow these steps.
a.

Calibrate (blank):

choose experiment from the top menu, choose calibrate, choose

spectrophotometer 1, click enter. NOTE: Do not warm-up.

b.

Insert the nutrient broth in the test tube in the SpectroVis opening.

c.

Click finish calibrate. Click ok.

5. Now you are ready to collect the absorbance in your E. coli test tubes using the loggerpro
program.

6.

a.

Follow your instructors instructions.

b.

Insert the 4 degree test tube in the SpectroVis opening

c.

Find the 550 nm wavelength and record the absorbance reading. Ignore the other wavelengths.

d.

Record it in table 2 below.

e.

Remove the 4 degree test tube.

f.

Insert the 25 degree test tube.

g.

Find 550 nm and reading the absorbance measured at 550 nm.

h.

Record it in table 2.

i.

Remove the 25 degree test tube.

j.

Repeat these steps for 37 and 55 degrees. Record readings in table 2.

Repeat the steps to check the absorbance readings of the B. sterothermophilus

7. IMPORTANT NOTE: Do not shut down the computer. Log off only.
8. Dispose of the test tubes in the rack on the discard cart at the back of the lab.
Table 2: Absorbance and appearance of broth tubes at various temperatures.
E. coli
Temperature C
4
25
37
55

Absorbance

B. stearothermophilus
Visual

Absorbance

Visual

QUESTIONS FOR DISCUSSION

1. Using the data from the chart, determine the growth temperature classification for
a. E. coli
b. Bacillus sterothermophilus
2. Is it advisable to connect the data points from your graph? Why or why not?

3. Complete the absorbance axis and graph the results for each bacterium.

4. Why do different temperatures produce different growth rates?

5. Make a statement about the effects of temperature on the enzyme controlling pigment production
in Serratia marcescens.

6. Based just on your results, and regardless whether each bacteria is a pathogen, could any of the 3
organisms you tested infect humans or animals? If so, which one? Why or why not?

7. Differentiate psychrophile and psychrotroph.

they are pathogens.

Give some examples of psychrotrophs and state if

8. Why are thermoduric bacteria important to consider after pasteurization?

GROWTH FACTORS: WATER ACTIVITY AND OSMOTIC PRESSURE

INTRODUCTION: Water is essential for life and a major component of bacterial cytoplasm. It is used
to maintain turgor pressure (as also with plants), as well as pH, and metabolic processes. The availability
of water is called the water activity, Aw, and the values vary between 0 and 1.0. The closer the value is
to 1 (pure water, i.e., no solutes), the more water is available to the cell. Water activity decreases with
increasing solute concentration. These solutes can be salts or other solutes, such as glucose or amino
acids. In these conditions, water is less available to the cell.
Bacteria often maintain relatively high cytoplasmic solute concentration which promotes inward diffusion
of water. This may be relatively easy or a constant effort depending on the type of environment in which
bacteria live. Osmosis, the movement of water across a membrane, occurs as a result of the different
solute concentrations on either side of the membrane. If the solute concentrations are equal or similar on
either side of the membrane, it is an isotonic environment and water does not move favorably in either
direction. If the environment has very few solutes compared to the cytoplasm, this is called hypotonic.
Because water moves toward the higher solute concentration, water will move into the cell due to osmotic
pressure. As mentioned, bacterial cells with cell walls will swell and have increased turgor pressure.
The cell wall will protect the cell from bursting and, in general, this type of environment may be
favorable to some bacterial cells. Eukaryotic cells with no cell walls may burst in this type of hypotonic
environment. Hypertonic environments are those in which the solute concentration is higher in the
environment as compared to the cytoplasm. Since water moves toward higher solute concentrations,
water will leave the cell. As the cell loses water from its cytoplasm the cell membrane shrinks away
from the cell wall. This process is called plasmolysis. Most cells, unless they are adapted to this type of
environment, will not respond favorably for any length of time in a hypertonic solution.
Many bacteria are not tolerant to salinities of greater that 3-5%. We can find some of these bacteria living
with us in our bodies, such as E. coli in our colon. However, prokaryotes (both bacteria and archaea) are
extremely diverse and are adapted to many types of osmotic environments. Halophiles are adapted to
live and grow only in high salinity (very low water activity). Most are Archaea but some are bacteria.
Some halophiles such as Vibrio species can infect humans and cause gastrointestinal disease and sepsis.
Others are extreme halophiles because they can only grow in 15% - 30% saline environment.
Environments like this include the Dead Sea, the Great Salt Lake, and some ocean areas. These Archaea
have specialized membranes and enzymes which function best in these environments. Other bacteria are
halotolerant, in that they can survive and grow in slightly higher saline environments than nontolerant
bacteria, but the salt is not required for growth. The halotolerant bacteria, grow up to 11% salinity, and
include bacteria that grow on our skin as it is slightly hypertonic. One example is Staphylococcus which
normally lives on our skin. Staphylococcus can also tolerate the slightly acidic nature of our skin as well.

As mentioned water activity has to do with the solute concentration and is not always determined by
NaCl. Some microbes tolerate very high sugar concentrations and are referred to as osmophiles.
Examples of these microbes are yeast and some mold. Perhaps you have seen them growing in jelly jars.

Media: Nutrient agar plates with salt: 0%, 5%, 10%, 15%, 20% and 25% (2 groups per lab section)
Bacteria: Agar plates: Staphylococcus aureus, E. coli, Halobacterium salinarium

PROCEDURE Lab 8:
1. Obtain a set of six (6) salt plates for your group (assigned by the instructor). Make sure the
concentration of NaCl is written on the plate, 0%, 5%, 10%, 15%, 20%, and 25%.
2. Using a sterile loop or swabs inoculate each bacteria in the appropriate location for each salt
plate.
3. Invert and label the plate with your group initials. Place at 37o C.

RESULTS LAB 9:
1. Place the salt plates in order of increasing salt concentration and observe for growth.
2. Record the growth patterns in Table 1 below as + for growth and 0 for no growth.
3. Record the classification for each bacterium with help from class discussion.

Table 1. Growth results for various bacteria on salt plates. Record + for growth and 0 for no growth.

NaCl (Salt) Concentration

0%

Organism
Escherichia coli
Staphylococcus aureus
Halobacterium salinarium

5%

10%

15%

20%

25%

Classification

QUESTIONS FOR DISCUSSION

1.

Give an example of a hypotonic environment and explain the effects of such an environment on
bacterial cells? Animal cells?

2.

How do hypertonic environments affect bacterial cells? Animal cells?

3.

Differentiate halophile, halotolerant, osmophile.

or animals?

4.

Give examples of different environments where one might find bacteria classified as halophilic,
halotolerant, osmophilic, or nontolerant.

Which of these organisms could infect humans

CONTROL OF MICROBIAL GROWTH: LETHAL EFFECTS OF

ULTRAVIOLETRADIATION
Ref: Effects of UV light on DNA. http://www.dnalc.org/resources/3d/18-dna-damage.html Talaro chapter 11.
INTRODUCTION: Ultraviolet (UV) is nonionizing, short, high energy wavelength radiation
between 4 and 400 nm on the electromagnetic spectrum (see textbook). There are three types of UV
radiation, UV-A (longest wavelength), UV-B (280 315 nm), and UV-C (100 280 nm which are the
most damaging wavelengths). Prolonged exposure to UV radiation can be lethal to cells. UV
wavelengths at 254 260 nm result in DNA damage by the formation of covalent bonds between
adjacent pyrimidines. These are referred to as pyrimidine dimers (thymine-thymine the most common).
When these structures form, the DNA becomes kinked and the polymerase can no longer replicate the
DNA, resulting in cell death. This can occur in any cell type as long as direct exposure of the cell to the
UV light occurs. Human and other animal cells are also susceptible to the DNA damage by UV light.
Bacterial cells (and human cells) have several systems to repair damage to DNA caused by UV light. A
common system is the SOS system which is activated when large amounts of DNA damage occurs. In
this system, the damaged pyrimidine dimer is removed and replaced with new pyrimidine molecules. A
number of enzymes are involved in recognizing the distorted DNA and actively repair the damage.
However, excessive DNA damage caused by UV light overwhelms the repair system and cell death
usually results.
Several variables are important in the lethal effects of UV light. These include time of exposure, with
increasing time of continuous exposure eventually reaching lethal effects. Wavelength of approximately
260 nm is most damaging and lethal. The distance from the UV light source is also a limiting factor. In
addition, barriers are also important. Nonionizing UV light does not penetrate barriers as does the
ionizing gamma radiation. Plastic petri plate lids will deflect all of the UV light. Endospores also serve
as a type of barrier up to a point in time. In addition, DNA in endospores is bound with acid soluble
proteins which aid in protecting it from the damaging effects of UV light. Enzymatic repair proteins in
spores may also contribute to rapid repair of damaged DNA. Biofilms in nature also provide some UV
protection.
In this lab, you will expose two different bacteria to various times of UV light and observe the effects on
growth.

MATERIALS, SUPPLIES, EQUIPMENT AND MEDIA:

Sterile swabs, incinerators, markers
TSA plates (8 per group, 2 groups per lab as assigned by instructor)
Ultraviolet sources for bacteria at 260 nm, 3 x 5 cardboard covers for plate

BACTERIAL CULTURES: Broth cultures of Staphylococcus aureus (BSL2) and Bacillus megaterium

PROCEDURE first lab (LAB 7)

The lab is divided into two groups. One group will test the effects of UV light on Staphylococcus aureus
and the other group will test Bacillus megaterium as follows:
1. Each group will prepare a bacterial lawn on eight TSA plates with the assigned bacteria.
2. Label the bottom of S. aureus plates with 10, 20, 40, 80 seconds and 2.5, 5, 10 and 20 minutes.
Also label the 20 minutes plate control.
3. Label the bottom of B. megaterium plates with 1, 2, 4, 8, 15, 30, 60 and 60 minutes. Also label
one of the 60 minutes plates control.
4. Uncover the plates in your set (but not the one labeled control), cover one-half of the plate with
a 3 X 5 card and place under the ultraviolet light for the amount of time indicated by the label.
NOTE: Wear goggles and do not look directly at the UV light.
5. Allow the Petri plate cover to remain in place on the last plate of the set (20 min control for S.
aureus and 60 min control for B. megaterium).
6. After exposure, invert the plates, and incubate at 36o C for 48 hours.
7. Develop a hypothesis about your prediction for which bacterium is sensitive to UV light.

RESULTS next lab (LAB 8)

1. Collect the plates and place in order by time and bacteria.
2. Examine the set of plates exposed to ultraviolet light. Compare the exposed side to the covered
half of the first seven plates. The covered half did not receive the UV light and the uncovered
half should show a range of growth depending on the exposure time. The entire agar surface of
the eighth plate should show growth, since the plastic Petri plate cover blocked penetration by
ultraviolet light.
3. Compare growth on uncovered halves to covered halves.
4. For the uncovered halves, record 0 for no growth, + for few colonies, ++ for moderate
growth and +++ for the most growth in the Table 1.
5. Leave the plates in order on the student lab bench that all the members of the lab can view the
results.

Table 1. Growth results for Ultraviolet light exposure of various times.

Organism
S. aureus
Growth
B. megaterium
Growth

Duration of Exposure (* = cover left in place)

10 sec
20 sec
40 sec
80 sec 2.5 min
5 min 10 min 20 min*








30 min
1 min
2 min
4 min
8 min
15 min
60 min 60 min*

QUESTIONS FOR DISCUSSION:

1. Explain the effects of UV light on living cells. Name the most germicidal wavelength.

2. Which of the two bacteria is more resistant to UV light? Explain your answer. Did your results
concur with your hypothesis?

3. Explain the important variables in the UV light exercise. Also state the purpose of the last plate
with the lid in place and the use of the cardboard covers.

4. If cells are starved for nutrients, would you hypothesize they are more and less susceptible to UV
effects? Explain your answer?

5. Why might UV light exposure result in skin cancer and not liver or lung cancer?

GROWTH FACTORS: OXYGEN REQUIREMENTS (Aerotolerance)

References: Talaro book chapter 7

Introduction. In addition to temperature, osmotic pressure, and ultraviolet light tolerance, oxygen
requirements are also important in growing and identifying microbes. In nature, in soils, and even in
human and other animal bodies, oxygen concentrations may be very diverse. For example, in the GI tract
many areas are completely anaerobic, whereas on the surface of the skin, oxygen concentrations are much
higher. Microbial metabolism can also alter or deplete the oxygen concentrations as some
microorganisms, like humans, break down food molecules using aerobic cellular respiration. They do this
by using oxygen as the final electron acceptor in the electron transport system. Although oxygen is the
most efficient method and produces the most ATP, oxygen can produce toxic metabolites that must be
inactivated by enzymes. Microbes without these enzymes cannot tolerate oxygen and use anaerobic
respiration or fermentation with other final electron acceptors such as nitrates or sulfates or pyruvates. In
the absence of oxygen less ATP is produced, however, bacteria may survive in times of low oxygen
concentrations by changing their metabolism to fermentation or anaerobic respiration. When oxygen
levels increase, unless the bacteria are strict anaerobes, bacteria can switch their metabolism back to
cellular aerobic respiration and make much more ATP.
Microbes that rely solely on oxygen as the final electron acceptor to produce ATP are termed strict
aerobes. Microbes that can use oxygen but do not require it and grow in its absence are referred to as
facultative anaerobes. Microbes that cannot grow in the presence of oxygen due to the buildup of toxic
metabolites, are strict anaerobes. These organisms lack the enzymes necessary to inactivate toxic
oxygen metabolites (reactive oxygen species, ROS). Strict anaerobes use final electron acceptors other
than oxygen, including nitrates and sulfates in the process of anaerobic respiration. They do not produce
as much as ATP as aerobes. Some microbes are referred to as aerotolerant as they can tolerate living in
oxygen but do not require it and do not use it for energy production. Microaeropilic bacteria are those
that tolerate only small amounts of oxygen. The media fluid thioglycolate which has a gradient of
oxygen, highest concentrations at the top and none at the bottom shows how these groups of microbes
grow in different oxygen concentrations.

As well as serving important roles in soil and sewage treatment, strict anaerobes may also cause disease in
humans and animals. Your textbook and instructor will discuss environmental importance and disease
caused by microbes with various oxygen requirements. In this exercise, you will grow three unknown
organisms in various oxygen concentrations and observe for growth after incubation.

MATERIALS, SUPPLIES, EQUIPMENT AND MEDIA:

Sterile swabs, incinerators, markers
TSA plates (2 per group, 4 groups per lab as assigned by instructor)
1 - Anaerobe bag with CO2 vials per group
BACTERIAL CULTURES:
Unknown agar cultures A, B and C.

PROCEDURE: First lab (lab 7)

1.
2.

Each group of 4 obtains 2 TSA plates and the unknown agar cultures A, B, and C.
Divide the TSA plates into thirds. Label each third, A, B, and C, respectively. Label your groups
initials. Label one plate with O2 and the other no O2.
Use a loop and aseptic technique to inoculate one streak for growth in each third with the
appropriate bacterium.
Invert one plate and place on tray for incubation in oxygen
Place the other plate in a bag and allow the instructor to break a CO2 vial. This will remove all of
the oxygen and allow anaerobic bacteria to grow.

3.
4.
5.

RESULTS: Next lab (lab 8)

1.
2.
3.

Obtain your plates and observe for growth.

Record a + for growth and 0 for no growth in Table 1 below.
Classify the bacteria for oxygen tolerance accordingly.

Table 1. Results of Oxygen Requirements for Unknown Bacteria.

Bacterium
A

Growth in
Oxygen

Growth in
no Oxygen

Aerotolerance Classification

QUESTIONS FOR DISCUSSION:

1.

Explain why oxygen is ideal for producing the most ATP during respiration but lethal for some
bacteria.

Explain what is meant by the final electron acceptor in the electron transport chain.

Give examples of the final electron acceptor in fermentation and in anaerobic respiration?

In soil, organisms live in symbiotic relationships. In these environments, aerobes use up all the
oxygen during high metabolic activity and anaerobic microbes then begin to grow in the low

oxygen tension. Give examples of various aerobic and anaerobic areas in the human body.

Refer to your textbook and lab class discussion, examples of diseases caused by strict
anaerobes.

Bacterial Conjugation: The Transfer of Antibiotic---Resistance Plasmids

Reference:Conjugation animation quiz
https://www.youtube.com/watch?v=0o5bBLcGNWo

INTRODUCTION: Bacteria transfer genetic information using vertical and horizontal transfer. Horizontal
gene transfer includes conjugation, transformation, transduction and use of other mobile genetic
elements, such as transposons. Conjugation is a process of transferring genetic information (often in the
form of a plasmid, which also contains the information allowing it to be transferred) from a donor
bacterium to a recipient bacterium through a sex pilus. Plasmids are small, circular, double---stranded
DNA molecules that usually exist extra---chromosomally. Plasmids do not carry essential genes. They
carry genes that may provide an advantage in their environment, such as antibiotic resistance genes.
Transfer of antibiotic resistance genes is commonplace in the bacterial world and is greatly increased
when bacteria exist in a biofilm, which is common in infectious diseases. With the help of natural
selection, this genetic transfer is partly responsible for the serious problem with bacterial antibiotic
resistance that exists in health care settings today. In this lab, a bacterial plasmid, containing a
resistance gene, will be transferred to a recipient bacterium by conjugation. Escherichia coli strain I
contains a chromosomal gene coding for resistance to the antibiotic streptomycin (str---r). E. coli strain II
carries a plasmid gene coding for resistance to the antibiotic ampicillin (amp---r). You will incubate both
strains together and determine if conjugation (i.e., mating) has occurred.

Record which bacterium is the donor

.

and which bacterium is the recipient

Chromosome

SUPPLIES, EQUIPMENT: Incinerator, 2---1 ml pipettes, pipetteman

MEDIA AND BACTERIA: Luria Broth Agar (LBA) plates
Without antibiotics (1 per group)
With streptomycin (1 per group)
With ampicillin (1 per group)
With ampicillin and streptomycin (1 per group)
E. coli strains I, II in 3 ml LB tubes
Sterile LB broth to prepare the mated strain


PROCEDURE: This is a three part lab. Students will work as a group as assigned by the instructor for all
three parts.
Lab 7: Work in groups of 4. Cultures containing LB broth of E. coli Strain I and Strain II will be provided.
Each group will mate the bacterial strains as follows:

a. Label 3 test tubes of sterile LB broth as Strain I, Strain II and Mated and your group name.
b. Follow your instructors instructions to transfer the bacteria using sterile pipettes and
aseptic technique.
a. 5 drops of Strain I into LB broth labeled Strain I
b. 5 drops of Strain II into LB broth labeled Strain II
c. 3 drops of strain I and 3 drops of strain II into LB broth labeled mated

Gently mix, and incubate in test tube rack at 36o C for 48 hours.

c.

Lab 8: Each group will inoculate a set of agar plates with three bacterial cultures (I, II, Mated) on each
plate.
1. Collect one set of plates: one plate of LBA with no antibiotics, one plate of LBA with
streptomycin, one plate of LBA with ampicillin, and one plate of LBA with streptomycin and
ampicillin. Label them according to Figure 1.
2. Collect the bacterial Strain I, Strain II and your mated strain.
3. Using a sterile loop, inoculate three lines of bacteria. Note: sterilize between bacterial
strains as shown in Figure 1 below.
4. Label the bottom of the plates, invert and incubate at 36oC.
5. Complete the expected growth results in Table 1 below.

Strain I

Strain II
Mated

Lab 9: RESULTS:

Collect your set of LBA petri plates and observe growth for each strain on each plate. Record a + for
growth and a 0 for no growth in the actual growth columns of Table 1. Also see Figure 2 below.

Table 1: Complete the following table of your expected and actual results of growth for each strain on
the LBA plates.

No antibiotics

Strain

Expected

Streptomycin

Actual

Expected

Ampicillin

Actual

Expected

Streptomycin and
Ampicillin

Actual

Expected

Actual

II

Mated

DISCUSSION QUESTIONS:

1.

Do your actual results agree with your the expected results?

2.

Explain the bacterial growth for each of the three bacterial strains on each plate.

3.

Do bacteria reproduce sexually?

4.

Explain horizontal gene transfer and the process of conjugation.

Enterobacter or Serratia?

Can E. coli conjugate with

5.

Define biofilm and explain the relationship of biofilms to horizontal gene transfer. Give an
example of an infectious disease where biofilms may form and antibiotic resistance may be
transferred through horizontal gene transfer.

6.

Did any group in the class have a negative conjugation result? If not, what does that tell you
about the frequency of conjugation when the conditions are right and bacteria become close to
each other such as in a biofilm. Is conjugation a random event?

7.

Why is conjugation important in the health care setting?

Figure 2: Actual results for growth of Strain I, II and Mated on the LBA plates.

ANTIMICROBIC SENSITIVITY TESTS: KIRBY BAUER

INTRODUCTION: Antibiotics are chemical metabolites produced naturally by some fungi and
bacteria. They are antimicrobials specifically used in treating bacterial infections. The first antibiotic
used commercially was penicillin derived from the Penicillium mold. Natural antibiotics produced in
nature inhibit growth of other microbes and thereby provide an environmental advantage for those
microbes producing and resistant to the effects of the antibiotic. More recently however, evidence
strongly suggests that antibiotics serve other roles, such as growth factors for bacteria at various
concentrations, and active metabolites in metabolic pathways. Semi-synthetic antibiotics are those
produced chemically by keeping the active moiety intact but modifying an R group. There are a plethora
of these antibiotics that serve to increase the bacterial groups the parent antibiotics will target, particularly
those that are resistant to the effects of the parent molecule. One example is the very large family of
semi-synthetic penicillins derived from penicillin, all of which have the beta-lactam moiety. Synthetic
antibiotics, such as sulfonamides, are entirely produced in laboratories.
Antibiotics that kill bacteria are bactericidal and those that only inhibit the growth are bacteriostatic.
Bacteriostatic antibiotics work in concert with the immune system to eliminate an infectious disease.
Antibiotics that target single groups are narrow spectrum. Examples of these are vancomycin which is
active against Gram positive bacteria only and polymyxin B which is active against Gram negative
bacteria only. Broad spectrum antibiotics target more than one group of bacteria such as tetracycline,
which targets gram positive and gram negative bacteria, as well as, rickettesias and chlamydias. Although
broad spectrum antibiotics may have the advantage of inhibiting many types of bacteria when the
etiologic agent in the infection is not known, they have the disadvantage of removing normal microbiota,
particularly in the colon. This may allow a superinfection to become established.
Antibiotics work through several pathways including inhibiting cell wall synthesis, penicillin being the
most studied of this group. Other antibiotics target proteins synthesis usually working at the level of the
bacterial ribosome, either the 50S (large) subunit or 30S (small) subunit. Many antibiotics use this
mechanism of action. Another cellular component targeted by antibiotics is disruption of the cell
membrane. Polymyxin B is an example of this and is a highly toxic antibiotic for external use in humans.
Another group of antibiotics are those that inhibit DNA and RNA synthesis generally by inhibiting
enzymes necessary for replication or transcription. Ciprofloxacin inhibits bacterial DNA gyrase and
rifampicins inhibit bacterial RNA polymerase. Despite the fact, that antibiotics target bacterial cellular
components, they can be toxic to humans. High selective toxicity means the drug targets the bacteria and
not the humans. An example of this is penicillin which inhibits bacterial cell wall synthesis but not
humans, as humans do not have a cell wall. Humans may be allergic, however. Some antibiotics with
low selective toxicity cause more and serious side effects in humans.
If a bacterium is susceptible to the effects of an antibiotic, and is either killed or has its growth inhibited
when exposed to the antibiotic, that bacteria is referred to as sensitive. If a bacterium is resistant to the
antibiotic, it is not inhibited by the drug and therefore the drug should not be used to treat an infection
with that resistant bacterium. In some cases, bacteria are classified as intermediate. This means the
bacteria may be gaining resistance or the drug may be used at another concentration. The concentration
of an antibiotic in a patients serum can be determined to ascertain the appropriate serum-cidal level to
inhibit the bacteria but not make the patient toxic due to the effects of the treatment.

It is very important to treat bacterial infections with the appropriate and correct concentration of
antibiotic. One commonly used qualitative test used to determine the in vitro sensitivity pattern of a
particular bacterium to various antibiotics is the Kirby-Bauer method. This is a standardized method
used in many clinical laboratories and it is easy and reproducible. The U.S. FDA and the Subcommittee
on Antimicrobial Susceptibility Testing of the National Committee for Clinical Laboratory Standards
has approved this test for clinical labs and determined the standard conditions. The standardized
variables for this test include the use of Mueller-Hinton II agar at pH 7.2-7.4 and agar thickness of 4 mm
in a petri plate. Bacteria are grown in broth to a density standard indicating 107 bacterial cells/ml
followed by application of the bacteria with a cotton swab to prepare a bacterial lawn. Disks impregnated
with known concentrations of the antibiotic are applied and adhered to the agar with sterile forceps. The
plates are allowed to incubate for 16 18 hours. The zone of inhibition is the area around the disk in
which no bacteria grow. To determine the susceptibility rating of a bacterium to a particular antibiotic,
the diameter of the zone of inhibition is measured in millimeters and compared to a standardized chart to
determine the rating.

You will perform the Kirby-Bauer test on gram positive and gram negative bacteria and determine
susceptibility ratings for each.
MATERIALS, SUPPLIES, EQUIPMENT:
Sterile cotton swabs, metric rulers, antibiotic disk dispenser A and antibiotic disk dispenser B, bacteriological loops,
incinerator, goggles, gloves, vortex machine.
MEDIA AND BACTERIAL CULTURES:
Mueller-Hinton II agar (1 per group of 2 students).
18 hour broth cultures of E. coli, Pseudomonas aeruginosa, Proteus vulgaris, Staphylococcus aureus

PROCEDURE: LAB 8
1. You will work in pairs. Your instructor will assign your table a bacterial culture and your group
the antibiotics to use. One pair will use Disks A, the other pair will use Disks B.
2. Each group will obtain the assigned broth culture and one Mueller-Hinton II plate. Label the
bottom of the plate (agar side) with name, date and bacteria.
3. Use aseptic technique to prepare a bacterial lawn using a sterile cotton swab. Wet the swab with
the broth culture, squeeze the excess out on the side of the tube, and prepare the lawn. Swab the
entire surface of the plate, top to bottom and side to side, not missing any areas. Turn the plate
1/4 or the way in your hand and repeat the swabbing, turn the plate again, and repeat the
swabbing. The plate will have been completely covered three times in order to prepare a thick
lawn with no uninoculated areas.
4. Place the plate on the lab bench prepared for the antibiotic dispensers. Lay the petri plate agar
side down. Remove the lid. Position the assigned antibiotic dispenser over the plate and push
down. The disks will drop down into position.
5. Remove the dispenser. Use sterile forceps to gently touch the disks in place. Be careful not to
use too much pressure. Replace the petri plate lid.
6. Incubate the plate inverted at 36oC for 18 hours.

RESULTS: LAB 9
1. Obtain the Mueller Hinton plate. Measure the zone of inhibition (diameter in mm from edge of
growth to opposite edge of growth). If there is no zone of inhibition around a disk, record the
susceptibility rating as resistant (R) for that particular antibiotic.
2. Ask your instructor for help if necessary.
3. Refer to susceptibility table 2 provided in the lab. Find the antibiotic column on the left and the
code and potency in the next two columns. Find the bacteria if there is more than one. Move to
the three zones of inhibition columns and find the zone size that fits what you measured for your
bacteria. Record the size in mm and the susceptibility in the table below.

Record zone of inhibition (mm) and susceptibility rating for each bacterium
antibiotic.

ANTIBIOTIC

E. coli

ZONE
DIAMETER

Proteus
vulgari
s

Pseudomonas
aeruginosa

Staphylococcu
s aureus

Table 1.

RATIN
G (R, I,
S)

ANTIBIOTIC

ZONE
DIAMETER

and

RATIN
G (R, I,
S)

Table1. Zones of Inhibition in Kirby---Bauer Method of Antibiotic Sensitivity Testing

CODE
NAME
INTERPRETATIVE STANDARDS FOR ZONE DIAMETERS

RESISTANT

INTERMEDIATE

SENSITIVE

AN30

Amikacin

<14

15 --- 16

>17

ATM15
CFP75
CIP5
CCC2

Aztreonam
Cefaperozone
Ciprofloxacin
Clindamycin

<13

14 --- 15

>16

<15
<15
<14

16 --- 20
16 --- 20
15 --- 20

>21
>21
>21

E15
FOX30

Erythromycin
Cefoxitin

<13
<14

14 --- 22
15 --- 17

>23
>18

IMP10
N5

Imepenum
Neomycin

<13
<12

14 --- 15
13 --- 16

>16
>17

OX1

Oxacillin

<10

11 --- 12

>13

P10
PB300

Penicillin
Polymyxin B

<28
<8

9 --- 11

>29
>12

S10
TE30

Streptomycin
Tetracycline

<11
<14

12 --- 14
15 --- 18

>15
>19

VA30

Vancomycin

<14

>15

QUESTIONS FOR DISCUSSION:

1. Define and differentiate natural antibiotic, semi-synthetic antibiotic and synthetic antibiotic.

2. Differentiate broad and narrow spectrum antibiotics. Provide advantages and disadvantages of
each use in treating infections.

3. Explain selective toxicity.

4. What is a standardized test? Name 5 standardized variables for the Kirby Bauer test.

5. Were any antibiotics used in this lab narrow spectrum? If so, name them and state their action.

6. Were any antibiotics used broad spectrum? If so, name 2 of them and state their action. Did they
work similarly on both Gram positive and Gram negative?

7. State factors that influence the size of the zone of inhibition for an antibiotic? What type of error
could result for factors not controlled in this test.

8. Explain how two different antibiotics might have the same size zones of inhibition but not both be
effective on Bacteria A (i.e., Bacteria A is sensitive to one antibiotic and resistant to the other
antibiotic).

9. If you saw differences in susceptibility ratings for Gram positive and Gram negative bacteria,
name the antibiotics. Explain why a gram negative bacteria might be resistant to those that gram
positive are susceptible, and vice versa.

WATER MICROBIOLOGY: Coliform Testing and Differential Media

References: Figure 2 images on differential media at end of this lab exercise.

INTRODUCTION: Public drinking water supplies are tested and treated daily in order to
ensure they are safe to drink (potable) and free from contamination with disease agents. This
regulation is according to the Safe Drinking Water Acts of 1974, 1986 and 1996. Drinking
water supplies are an important nonliving reservoir for gastrointestinal disease agents.
Pathogens that may be present in drinking water include, the parasites, Cryptosporidium and
Giardia, the virus Norovirus, and coliform and non-coliform pathogens, Salmonella, Shigella,
Campylobacter, E. coli and Vibrio cholerae. These pathogens may cause diarrhea and/or
dysentery and possibly sepsis and death. Fecal contamination of water (from humans and some
animals) is one potential source of some of these pathogens. Some of these pathogens are
chlorine resistant, particularly the parasites, which are the most common causes of recreational
and drinking water gastrointestinal outbreaks in the U.S.
Many pathogens that enter the water supply via fecal contamination may be short-lived and in
low concentration. In addition, some are difficult to isolate and culture. Therefore, water
quality tests are directed toward the simpler and less expensive task of determining the presence
of coliform bacteria. Coliform bacteria are members of the Enterobacteriaceae. Members of
this large family are facultative anaerobic, gram negative rods, which produce acid and gas from
the fermentation of lactose. Coliform bacteria live in gastrointestinal tracts and typically enter a
body of water with the introduction of fecal material. Therefore, the presence of coliforms
indicates the presence of fecal contamination and the possible presence of pathogens. Common
screening tests detect total coliforms and also differentiate E. coli The media contains rich broth
with buffers to allow from rapidly growing coliforms. Gram positives are inhibited. A
chromogenic substrate, X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) will turn
the medium blue-green if coliforms are present due to cleavage of the galactose by galactosidase. MUG (4-methylumbelliferyl--D-glucuronide) which can be cleaved by the
enzyme -glucuronidase contained by 97% of E. coli strains. When this happens a fluorescent
chromogen can be detected by 365 nm UV light.
In this exercise, you will perform a screening of water with the above media and also and screen
the water with selective and differential media. These media typically select for the growth of
gram negative bacteria, inhibiting most gram positive bacteria, and differentiate lactose
fermenters (coliforms) from those gram negatives that do not ferment lactose (non-coliforms).
These media include Eosin Methylene Blue (EMB), MacConkey Agar (MCA), and Hektoen
Enteric Agar (HEA). Further verification for the colonies growing on the selective and
differential media may be performed in the completed test but we will not include that test here.
EMB contains dyes eosin Y and methylene blue which inhibit gram positive bacteria. These
dyes also react with the acid produced by lactose fermenters. E. coli produces a striking
metallic green sheen due to the excessive acid production from lactose fermentation. Other
coliforms produce less acid and turn the medium pink to dark purple. Non-fermenters are nonpigmented or the color of the medium. HEA aims at differentiating Salmonella and Shigella

from other enterics. Bile salts inhibit the growth of most gram positive bacteria. Enterics
fermenting any of the three available sugars such as E. coli are typically yellow to pinkish orange
and non-fermenters, such as Shigella, are typically blue-green. The media also has ferric
ammonium citrate which will react with any H2S produced to form a black ferrous sulfide.
Salmonella typically produces H2S and colonies will have some black. MCA is also used to
differentiate Salmonella and Shigella from other enterics. The media contains bile salts and
crystal violet which inhibit gram positives. The neutral red indictor dye turns lactose fermenters
pink and the non-fermenters are colorless.
MATERIALS, SUPPLIES AND EQUIPMENT:
Sterile loops, sterile 1 pipettes and pipettors
MEDIA AND WATER:
Water samples (1 per group)
Sterile bottles to contain 100 mls (1 per group)
Readycult Coliform 100 packets (1 per group)
EMB, HEA AND MCA plates (1 set per group)

PROCEDURE: This is a 2 lab exercise.

LAB 1: Screening test
1. Work by tables with the water sample you brought or were assigned by your instructor.
2. Remove 0.5 ml of sample and place on each plate, HEA, MAC and MSA.
3. Streak in zigzag and incubate these plates at 37 degrees for 18 hours.
4. Aseptically remove 100 ml of the water and place in screw cap bottle.
5. Add the contents of the entire packet to the water and shake to dissolve the granules.
6. Incubate the bottles at 37 degrees for up to 24 hours.
RESULTS:
Lab 2. Screening test Readycult.
1. Obtain your bottle and observe the color reaction.
2. Use goggles and a UV light source and observe for a blue fluorescence.
3. Record results in Table 1 below.
Table 1. Test results using Readycult coliform 100 screening media.
Water Source

Readycult color
reaction

Fluorescence

Conclusions

E. coli spiked

Pond water

DI water

Other water

Lab 2. Selective and Differential media Results.

1. Collect your group confirmed test plates.
2. Observe the agar plates for colonies that represent coliforms or non-coliforms as
described in the introduction for each type of media and in Figure 2 below.
3. Record results in table 2 below as follows:
a. Record the appearance of known coliforms (E. coli) and known non-coliforms
(Salmonella) on each type of media, HEA, EMB and MAC.
b. For your water sample,
i. indicate presence of E. coli and other lactose fermenters (coliforms)
ii. indicate presence of non-lactose fermenters (non-coliforms)
iii. indicate presence of H2S producers, black colonies (presumptive
Salmonella)

Table 2. Confirmed Test results using Selective and Differential Media

Media and water

Positive Coliform
appearance
(record E. coli if present;
NG for no growth)

Positive Non-coliform
appearance
(record possible Salmonella if
present; NG if no growth)

Conclusion

HEA control

EMB control

MAC control

Your Water Sample

Figure

Im e of Selec

e Differen

Me

Wa er

cr

g co firmed te

QUESTIONS FOR DISCUSSION:

1. Define and list characteristics of coliforms. Why is water tested for coliforms?

2. Name two bacterial pathogens, one viral pathogen, and two protozoan pathogens that
may be found in water.

3. Name three diseases spread through water.

4. Explain the tests used in the readycult coliform 100 media?

5. Did your growth results on selective and differential media confirm the readycult results?

6. Is the water you tested potable?

7. Explain the selective and differential aspects of the media used. Describe the appearance
of E. coli on each media and describe the appearance of Salmonella on each media.

Identification of Gram Positive Cocci: Staphylococcus

References: Catalase test results: http://www.microbelibrary.org/images/atlas_catalase/slide%20catalase%20test%20results.jpg
buubbles are positive (oxygen), no bubbles are negative. Coagulase test results:
http://www.microbelibrary.org/library/laboratory%20test/3243- coagulase-test (top tube is coagulase positive, second tube is
coagulase negative)

INTRODUCTION: Gram positive cocci include opportunistic pathogens such as Staphylococcus

aureus (typically clusters in a Gram stain) and Enterococcus fecalis (typically chains in a Gram stain).
These bacteria may be normal flora of the human nares and skin, and gastrointestinal tract, respectively.
However, they may cause disease when given the opportunity. When found in hospitals, these bacteria
may gain virulence factors and resistance genes. Both are responsible for a significant number of hospital
acquired infections each year. Staphylococcus aureus causes abscesses and has the ability to invade and
cause systemic diseases such as, acute endocarditis, necrolytic pneumonia, and toxic shock syndrome.
Methicillin resistant Staphylococcus aureus (MRSA) causes thousands of deaths per year in the U.S.
Enterococcus fecalis and other species of Enterococcus are generally not as serious as S. aureus but are
also associated with nosocomial infections. Enterococcus is a common cause of blood stream infections,
as well as being resistant to antibiotics, such as vancomycin resistant Enterococcus (VRE).
In this exercise you will identify a Staphyloccus species. They are gram positive cocci, typically in
clusters, and are catalase positive. Microbes that live in oxygen often produce toxic oxygen metabolites
such as peroxides and superoxide radicals, which must be broken down into less toxic materials such as
water and oxygen. Catalase is one enzyme used by aerobic and facultative bacteria that catalyzes this
reaction as follows: 2 H2O2 2 H2O + O2. Anaerobic and microaerophilic bacteria do not have this
enzyme and are thus destroyed by the build-up of toxic peroxides. The catalase test is performed by
placing a loopful of the bacterial colony into a drop of hydrogen peroxide and watching for bubble
formation. These are oxygen bubbles, the same oxygen bubbles produced by the catalase in your cells
when you place hydrogen peroxide on a cut. Staphylococcus species typically produce catalase but
Enterococcus, which is microaerophilic, does not produce catalase.
Once an unknown bacterium is presumed to be Staphylococcus a species determination may be made. S.
aureus is an important pathogen as mentioned. It is typically hemolytic on blood agar as it produces
several hemolysins. S. aureus is halotolerant allowing it to grow on salt plates and it ferments mannitol.
Finally S. aureus produces the coagulase enzyme which causes coagulation of plasma by converting
fibrinogen to a fibrin clot. S. saprophyticus may be normal flora of the vaginal tract and is associated
with urinary tract infections. It is typically not hemolytic on blood agar, is halotolerant, and generally
ferments mannitol. It is coagulase negative. Staphylococcus epidermidis is a common member of skin
flora but may be associated with hospital acquired blood stream infections. It is non-hemolytic on blood,
is halotolerant, but does not ferment mannitol, and is coagulase negative.
In this exercise we will differentiate the Staphylococci, S. epidermidis, S. saprophyticus, and S. aureus.
Rapid tests can be used for these identifications as with Gram negative rods but we will use the methods
in the flow chart below. In addition, you will sample your skin and nares for the presence of
Staphylococcus normal flora.

MATERIALS, SUPPLIES AND EQUIPMENT:

Sterile swabs, bacteriological loops, incinerators, markers, gloves, hydrogen peroxide, loop and
slides for instructor demo of catalase.
MEDIA AND BACTERIA:
Media: Blood agar plates (BAP) (1 for each for each group of 4)
Mannitol salt agar plates (MSA) (1 for each groups of 4 AND 1 for each student)
Sterile saline (1 for each student)
Bacterial cultures: Unknown plate cultures of Staphylococcus

PROCEDURE LAB 10: Identification of Staphylococcus

1. The catalase test is performed in the biosafety cabinet (see Figure 1).
a. Place one drop of hydrogen peroxide on two separate glass slides.
b. Using a sterile loop, obtain some of the Staphylococcus colony and place it in the
hydrogen peroxide.
c. Repeat this for the Enterococcus colony.
d. Positive tests show bubbles and are assumed to be Staphylococcus (or Micrococcus).
Negative tests show no bubbles and are not Staphylococcus. Record the results in table 1.

2. Complete the identification by working in groups of 4 (table). Obtain the bacteria, blood agar
plate and mannitol salt agar plate.
3. Use a marker to divide the blood agar plate and the mannitol salt agar plate into thirds.
4. Label the thirds as follows: A, B, C. Also mark your group name.
5. Use aseptic technique to inoculate A on each labeled area for A. Streak for growth in a zigzag
fashion as shown by your instructor.
6. Use aseptic technique to inoculate B on each labeled area for B.
7. Use aseptic technique to inoculate C for each labeled area for C.
8. Invert the plates and incubate for 24 hours.

PROCEDURE LAB 10: Sampling skin and nares for Staphylococcus normal flora.
1.
2.
3.
4.
5.
6.
7.
8.

Each student collects a mannitol salt agar plate and a sterile saline tube.
Use a marker to divide the plate in half.
Label one side skin and label the other side nares.
Use a sterile cotton swab and soak in saline. Squeeze the excess on the side. Collect a skin
sample.
Inoculate half of the MSA plate labeled skin in a zigzag fashion.
Use another swab to collect a nares sample and inoculate the other of the MSA
Seal the plate with parafilm.
Record your initials and section number. Invert the plates and incubate for 48 hours.

RESULTS LAB 11:

1. Collect your groups mannitol salt plate and the blood agar plate.
2. Observe for beta ()-hemolysis on the blood agar plate. S. aureus is hemolytic and shows a
complete clearing around the growth due to lysis of the red cells. Record results in Table 1.
3. Observe the mannitol salt plate for growth and fermentation of mannitol. Yellow is positive due
to acid formation, pink is negative due to no acid formation. Record results in Table 1.

4. Observe the demonstration tubes for the coagulase test. A clot in the plasma is positive for the
coagulase enzyme. If the plasma is still liquid, the coagulase enzyme is not present.
Table 1: Record results of Unknown Staphylococcus species
Test
Enterococcus
A

B

C

Catalase

Hemolysis

Fermentation
of mannitol
0

Coagulase

Identification

Enterococcus

5. Collect your own mannitol salt agar plate from skin and nares samples. Observe for the
presence of yellow or pink colonies. Ask your instructor for help. Many individuals have S.

epidermidis as normal skin flora. About 30% of individuals have S. aureus as normal flora in the
nares. Other microbiota may be present as well.
6. When finished, tape all the plates shut and discard in the autoclave bucket.

QUESTIONS FOR DISCUSSION:

1. Describe one biochemical test to differentiate Staphylococcus from Enterococcus.

2. Explain two biochemical tests for differentiating S. aureus from S. epidermidis.

3. Explain your results on your own mannitol salt plate for Staphylococcus species.

4. Why is it important to differentiate S. aureus from S. epidermidis and S. saprophyticus.

5. If Staphylococcus aureus is identified in a clinical specimen, what other important test should be
done?
6. Discuss the role of S. aureus as a cause of opportunistic infections and as a cause of nosocomial
infections.

7. At your table, discuss one virulence factor produced by methicillin resistant S. aureus (MRSA).

8. If you saw other microbes on your skin or nares plate, give possible identifications.

SYNTHETIC EPIDEMIC

INTRODUCTION:
Epidemics are diseases/disorders that are widespread in a particular community. Infectious
disease epidemiologists (people who study infectious diseases) are charged with determining
how, when, where, what, and who are involved in the source and/or spread of infectious
diseases in populations. They also must determine how to stop the spread of epidemics.
Infectious diseases, such as food infections, may be of a point source ( also called a common
source) nature or of a propagated nature. In a point source outbreak, all individuals in the
outbreak acquire the infectious disease at one location. The number may be large depending
on the number infected at that time and place. An example of this type of infectious disease
is Salmonella, which is not communicable (i.e., not spread from person to person) but several
people could become sick from one infected source. A graphical representation of this type of
outbreak shows a tall and narrow curve since the infection is acquired only at one location
and not spread from person to person. Propagated epidemics are infections that spread from
one person to another because the disease is communicable (spread from person to person, a
good example of this is influenza). Until they are brought under control, communicable
epidemics typically show an increase in the number of cases due to the spread of the infection.
A graphical representation of the case number curve continues to increase over time. A
pandemic, such as, H1N1 Swine influenza, tuberculosis, or HIV/AIDS, occurs when an
epidemic spreads to more than one continent.
In this exercise, the class will simulate a propagated epidemic in which the infectious agent is
spread from person to person. The infectious agent is a bacterium, Serratia marcescens, spread
through candy and handshaking. After the handshaking, you will inoculate media and observe
for the growth of bacteria after incubation. Your job will be to determine the index case, the
individual that was originally infected and spread the infection throughout the class.

Materials:
Petri plate with number and piece of candy
One TSA plate (divide in half with a marker)
Gloves, Swabs
Sterile saline

Procedure Lab 9:
Round 1
1.
2.

Obtain a Petri plate with a piece of candy.

Obtain a TSA plate and divide it in half with a marker. Label one side Round 1 and the
other side Round 2.
Record the number of your Petri plate in Table 1.
Place a glove on your non-dominant hand.
Open the Petri plate and grasp the candy in the gloved hand. Roll it around the palm,
covering the surface well. Discard the candy into the petri plate.
Your instructor will tell you who to shake hands with, but do not do so until told to shake.
You will also be told who to shake with.
Then shake once with another person. You are the shaker and the person you shake
hands with is the shakee.
Then swab the surface of your gloved palm and inoculate the surface of the TSA plate on
the Round 1 side. Discard the swab. Do not touch your glove to anything.
Record the number of the person (the shakee) you shook hands with on the table below.

3.
4.
5.
6.
7.
8.
9.

Round 2
1. Your instructor will again tell you who and when to shake hands with someone. Please
do not shake until told to do so.
2. Using a new swab, swab the surface of your gloved palm and inoculate the surface of the
TSA plate on the Round 2 side. You may need to immerse the swab in sterile saline
prior to swabbing the glove and inoculating the plate.
3. Record the number of the person (the shake) you shook hands with on the table below.
4. Remove and discard the glove appropriately and wash your hands.
5. Invert and incubate the TSA plate.
Procedure and Results: Lab 10
1.
2.
3.
4.
5.
6.

Obtain your plate and look for the growth of red colonies on either side of the plate.
Record the results as positive (+) or negative (0) on the table below.
Your instructor may transfer the table to the board.
Your job is to determine the index case.
Eliminate those that are not infected by crossing out those numbers.
Work backwards to determine the index case.

Table 1. Results for Synthetic Epidemic and bacterial growth.

The presence of any red colonies is positive and no red colonies is negative.
Shaker
Round 1
1

Result
+ or 0

Shakee
Round 1

Result
Shaker
Shakee
Round 2 Round 2 + or 0


1

10

10

11

11

12

12

13

13

14

14

15

15

16

16

Questions for Discussion:

1.

Define and differentiate a point source (common source) epidemic and propagated epidemic.

2.

State microorganisms causing pandemics that are occurring now or have recently occurred.
are they pandemics?

3.

Who is the index case (patient zero) in your class? What does this designation mean?

4.

How many were infected after Round 1?

Why

How many were infected after Round 2?

6.

How could this infectious cycle been stopped? What if it was a food infection?

7.

Explain any abnormal or unexpected growth (not red).

EFFECTIVENESS OF HAND SCRUBBING

INTRODUCTION: Handwashing is the most important procedure to prevent the spread of

infectious disease. The process is important for health care professionals, food handlers and also
for the general public. Health care professionals are exposed to pathogens frequently and can
spread these to patients (human and other animals) via their hands. The skin and hands have
normal microbiota, both transient flora, which are temporarily present, and resident flora,
which are entrenched in layers of the skin and always present. Ignaz Semmelweis was the first
to link an infectious disease, childbed fever, with microorganisms spread through contact with
contaminated and unwashed hands. An infectious disease acquired while in the health care
setting is termed a nosocomial infection. There are over two million of these infections yearly
in the U.S. with ~100,000 deaths attributed to these infections. Veterinary hospitals also have
nosocomial infections. The simple act of consistent handwashing can greatly reduce the spread
of nosocomial infections. See safety exercise, lab 1, for proper handwashing technique.
Although the human microbiome project is documenting the variation in normal flora between
individuals, the hands of all humans have some similar inhabitants. Microbes that inhabit the
skin must be able to survive slightly hypertonic (NaCl) conditions and slightly acidic conditions
(as low as pH 4.0, but more generally 5 6). In addition, certain areas of the skin have
sebaceous glands which produce oil that not all microbes can survive. Most of the outer surface
of the epidermis is composed of keratinocytes which have little nutrient. These conditions
explain why certain microbiota, particularly Gram positive bacteria, are commonly found on the
skin. Microbiota include Staphylococcus epidermididis, a common skin inhabitant, and
Staphylococcus aureus, much less common on the skin but, present in the nares of up to 30% of
individuals. Diphtheroids are species of Corynebacterium common on the skin which may be
aerobic or the anaerobic Propionobacterium living in sebaceous glands. Various mold and yeast,
Bacillus species, and anaerobic cocci are also common on the skin in addition to various less
commonly found microbes.
In addition to the normal resident microbiota, transient microbiota can be easily acquired and
transferred in a health care setting. These may include methicillin resistant Staphylococcus
aureus (MRSA) which commonly lives in many hospitals, antibiotic resistant gram negative
rods, vancomycin resistant Enterococcus (VRE) and Clostridium difficile (C. diff) a common
nosocomial pathogen which may be spread through spores. Other organisms are found in
veterinary hospitals. Surgeons scrub hands prior to surgery. They use germicidal soap and
remove much of the transient flora and resident flora during the scrubbing process but still need
to wear gloves as they do not achieve sterility.
In this exercise you will see a progressive removal of transient and resident flora through hand
scrubbing. You will determine the number of bacteria as colony forming units (CFU) / ml in
each original hand scrubbing basin. CFU/ml is a common unit of measure for determining the
number of microbes in samples. It is used for milk, water and other specimen sources.

MATERIALS, SUPPLIES AND EQUIPMENT:

5 basins of sterile water (A-E), 5 sterile scrub brushes, non-germicidal soap, 6 sterile petri plates,
sterile 1-ml pipettes, pipettors
MEDIA: Molten brain heart infusion agar in tubes (6 tubes per basin)

PROCEDURE LAB 10:

1. The instructor will choose volunteers for hand scrubbing and a hand scrubbers helper.
The helper will keep the scrubber supplied with scrubbers and keep the time.
2. The handscrubber will wash hands as follows:
a. Scrubber begins with a sterile scrub brush and scrubs hands without soap in
Basin A, 30 seconds on each hand. Note: scrub top and bottom of hands and
underneath the fingernails as well.
b. Scrubber will then scrub hands with soap under running water, 1 minute each
hand. NOTE: this is the only time soap is used.
c. Scrubber uses a new scrub brush to scrub each hand into Basin B without soap,
30 seconds each hand.
d. Scrubber then scrubs hands under running water only, 1 minute each hand.
e. Scrubber uses a new scrub brush and scrubs hands into Basin C, 30 seconds
each hand.
f. Scrubber then scrubs hands under running water only, 1 minute each hand.
g. Scrubber uses a new scrub brush and scrubs hands into Basin D, 30 seconds
each hand.
h. Scrubber then scrubs hands under running water only, 1 minute each hand.
i. Scrubber uses a new scrub brush and scrubs hands into Basin E, 30 seconds each
hand.
j. The hand scrubbing is complete. The basins are distributed to the appropriate
groups.
3. The class is divided into 5 groups
a. Each group labels 6 empty petri plates as follows: (Refer to Figure 1)
i. 2 plates are labeled with 0.1ml, their assigned Basin letter and group
name.
ii. 2 plates are labeled with 0.2 ml and the Basin letter.
iii. 2 plates are labeled with 0.4 ml and the Basin letter.
b. Each group obtains 6 molten agar tubes from the hot water bath and allows these
to cool at the lab bench until they are about 50 55o C.
c. While the agar is cooling, aliquots are added to the empty petri plates as follows:
i. The water in the water basin is stirred up with a sterile pipette.
ii. The same pipette is used to deliver 0.1 ml aliquots to these 2 petri plates.

iii. The same pipette is used to deliver 0.2 ml aliquots and 0.4 ml aliquots of
wash water to the correct petri plates.
d. When the melted agar is cool (cool to touch on wrist or ask the instructor), the
petri plate lid is removed and the liquid agar is gently and slowly poured into the
petri plate. The liquid agar is gently swirled to mix the water sample into a
homogeneous mixture throughout the petri plate. The petri lid is replaced and
cocked of the side. The agar is allowed to cool and solidified.
e. After all the agar has been mixed into the water in the petri plates and all the
plates have cooled and solidified, the plates are inverted and incubated at 35o C
for 48 hours.

DILUTION FACTOR DETERMINATION:

1. Since we will not add 1.0 ml to any of the petri plates, we have effectively performed a
dilution (delivered less than 1 ml).
2. We want to determine CFU/ml but we put in less than 1.0 ml, therefore, we must use a
dilution factor to get accurate results of the true CFU/ml in the original water basin.
3. Briefly, since 0.1ml is 1/10 of one ml, the dilution is 10. Since 0.2 ml is 1/5 of one ml,
the dilution factor is 5. Since 0.4 ml is 1/2.5 ml of one ml, the dilution factor is 2.5.
These are the dilution factors that you will use as multipliers to determine CFU/ml.

RESULTS LAB 11:

1. Collect the six petri plates from your handwashing basin. Place the plates in order by
dilution, 0.1 ml, 0.2 ml and 0.4 ml.
2. Combine the 0.1 ml plates together, the 0.2 ml plates together and the 0.4 ml plates
together.
3. In order to calculate CFU/ml you need to include the dilution factor. See above
explanation.
4. Count the plates according to the following rules:
a. Accurate plate counts are those in which the colony count is between 30 300
colonies. A plate with less than 30 colonies is not representative of the true
number of colonies and a plate greater than 300 is not accurately counted. If a
plate is between these numbers then it may be counted.
b. Count both plates of the particular dilution that you choose. Average the two
numbers to arrive at the count. You will probably not be able to count all the
plates as they will not fit the criteria of 30 300 colonies.
c. Multiply the average count times the particular dilution factor for that plate.
d. Use the following formula to determine cfu/ml:
Cfu/ml = average colony count X dilution factor
e. For example, if your average count is 125 from the 0.2 ml plates, then multiple
the count times 5. The CFU/ml in this example is 625 CFU/ml. This means that
the original basin of water had this number of microorganisms in each ml of
water.
f. If the colony count is obviously greater than 300, then record too numerous to
count in the table as TNC.
5. Complete table 1 below according to your instructions directions.
Table 1. Plate count results from handwashing basins and CFU/ml determinations
Bas
in

0.1 ml plates
Dilution Factor:

1st
plate
A
B
C
D
E

Avg.

2nd
plate

0.2 ml plates
Dilution Factor:

1st
plate

Avg.

2nd
plate

0.4 ml plates
Dilution Factor:

1st
plate

Avg.

2nd
plate

CFU/ml for plates

counted by your
group

Calculations: Choose one dilution, determine the average count and calculate the CFU/ml

QUESTIONS FOR DISCUSSION:

1. Complete the table above for all handscrubbing basins and plot the results on the graph in
this document. Draw a line following instructions from your instructor.
2. Explain the graph by Basin. Explain any increases or decreases between basins in terms of
transient and resident flora.

3. Differentiate between transient and resident flora. Give an example of each. Could transient
or resident flora be transferred from the hands of health care workers to a patient? If this
happened how would you classify this infection?

4. Name opportunistic pathogen (s) normally associated with the skin.

5. What type of soap did you use (germicidal or non-germicidal)? Do you expect a difference
between these two types of soap? Explain.

6. List three professions where lack of handwashing could lead to infectious disease outbreaks.

7. Why do surgeons still have to wear gloves even after they have scrubbed their hands prior to
surgery?

Line graph of handscrubbing results by basin in CFU/ml

asin

asin

asin C

asin D

asin E

Gel Immunodiffusion (Precipitation)

INTRODUCTION:

Antigen---antibody reactions are highly specific reactions in which the antigen binding site of an antibody
binds to one antigenic determinant (epitope). Antigen---antibody reactions include precipitation,
agglutination, neutralization of viruses and toxins, immobilization of bacteria, complement
fixation, antibody---dependent cytotoxicity, and opsonization. Antibodies circulate in the blood and
can be studied by collecting a sample of serum and using the serum for testing for the presence
of specific antibodies (or antigens). Serology is the discipline that studies antigen---antibody reactions
using serum and in vitro diagnostic tools.

This exercise uses gel immunodiffusion to study precipitation, a type of antibody---antigen reaction that
occurs between soluble antigens and their respective specific antibody. Gel immunodiffusion uses
saline agarose, a porous media that allows antibodies and antigens to diffuse through easily.
Antiserum (antibody in the serum) is placed in one well and an antigen is placed in the other well.
The two diffuse out of their respective wells toward each other in the gel. This is referred to as
double diffusion since both solutions diffuse out of the well. If the serum has a specific antibody that
recognizes the soluble antigen, then a white precipitin line will form at the region of optimal
proportions as their diffusion paths cross (see Figure 1). If no precipitation line forms, the test is
considered negative and the serum has no antibody that reacts with the known antigen. This test may
be modified in a number of ways and may be used to identify the presence of antibody with a known
antigen or an unknown antigen with a known antibody.

In this experiment, we will test an unknown simulated serum sample for the presence of specific
antibody and the ability to react with various simulated soluble microbial antigens. The simulated
soluble antigens are from HIV virus, Borrelia burgdorferi (the bacterial agent of Lyme disease), and
H1N1 influenza virus. The antigens are placed in outer wells and the unknown antiserum is placed in the
center well and they both diffuse outward. If there is a line between a particular soluble antigen and
the unknown serum, then the serum contains an antibody that binds to the soluble microbial antigen.

Materials per group of 2 students:

One saline agar plate
Plastic Agar punch devices
Unknown antiserum (containing simulated human antibodies).
Known simulated soluble antigens (HIV virus, Borrelia burgdorferi, H1N1 Influenza virus)

Procedure:

1. Cut four wells in the agar diffusion plate using the agar punch devices as shown in Figure 2.
2. Mark the wells to indicate which sample goes in each well.
3. Place two drops (0.1 ml) of serum in the center well using the dropper attached to the antibody
bottle. Be careful not to overfill the wells.
4. Place two drops of each antigen in the appropriate well using the dropper attached to the antigen
bottles (HIV, Borrelia burgdorferi, H1N1 Influenza Virus).
5. Cover the plates and incubate the plate at 25---37oC for one hour or longer.
6. Remove the plate and examine the area between the wells for white precipitation lines.
7. Record the results in Table 1 below.

Observation and Interpretation:

Table 1. Record your results in the table below and draw the location of the line on Figure 1.

Well

Precipitation line (yes or no)

HIV viral antigen

Borrelia burgdorferi bacterial antigen

H1N1 (Influenza virus)

QESTIONS FOR DISCUSSION:

1.

Define and describe a precipitation reaction? What is an antigenic determinant?

2.

a.

Explain the results for this individual?

b. Is this a definitive diagnosis? Why or why not

3.

Why is it important to use positive and negative controls?

Slide Agglutination Test (Latex Beads)

Ref: Example of slide agglutination. http://www.youtube.com/watch?v=oXL1rH11MTg

Introduction: Microorganisms possess molecular
structures
known
as
antigenic
determinants (epitopes). Antibodies recognize these structures and bind using their antigen
binding sites. When antigenic determinants (which are part of the microbial cell) combine with
specific antibodies, visible clumps (aggregates) are produced. This process is called
agglutination, a specific reaction between antibodies and the epitopes on the cells). The
clumps represent crosslinking of antigens with various antibodies.
To enhance the observation of clumps in an agglutination reaction, latex beads are often used.
These beads have specific antibodies adsorbed to the latex particles. When the latex particles
with the attached antibodies contact the antigenic determinants and specific binding occurs,
crosslinking occurs between cells and the agglutinates (clumps) are easy to see. Commercial kits
are available that will allow specific identification of an unknown bacterium using the antibodies
attached to latex beads. This is referred to as indirect agglutination and can be performed using a
slide. It is also always necessary to use a positive and negative control to verify that the results
obtained are not false positive or false negative.

In the gel immunodiffusion exercise we used unknown serum and known soluble antigens to
determine if an individual had an antibody to the antigen. In this exercise, we will use latex
beads coated with antibodies that bind to the cell wall of Salmonella species to determine if an
unknown bacterial culture is Salmonella. Salmonella is a bacterial intestinal pathogen which
causes diarrhea. It is a zoonosis and often transmitted through food and water. Slide
agglutination can be used for a rapid, specific, and definitive identification of this pathogen.

In this lab exercise, an unknown bacterium is mixed with an antibody specific for Salmonella
which has been adsorbed to latex beads. If agglutination occurs, the unknown bacterium is
Salmonella. If no agglutination occurs, the unknown bacterium is not identified as Salmonella.
Materials per group of 2 students:
Test Latex Antisera (antibodies to Salmonella)
Control Latex Antisera (antibodies not specific to Salmonella acts as negative control),
Salmonella control (positive control)
Unknown bacterial plate cultures A and B
Gloves, goggles

Agglutination card and toothpicks for mixing

Procedure: Students work by table using the biosafety cabinet

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.

Each group receives a card with 6 wells.

Each group receives bottles of test latex, control latex, and salmonella control
Each group receives culture A and B.
Add one drop of Test latex to wells A, B, and C.
Add one drop of control latex in wells D, E and F.
Use a sterile loop to obtain some of the unknown A colony and mix into well A for about 10
15 seconds.
Use a different loop and mix unknown A bacteria into well D.
Repeat the procedure with unknown B into wells B and E.
Add one drop of the positive control wells C and F and mix. Agglutination should occur in
well C but not in well F.
Rock the card back and forth under the biosafety cabinet and observe all the wells for
agglutination. If the reaction has not occurred in 15 seconds then let it go for 2 minutes.
Record the results in the table 1 below.

Table 1. Results of Latex Agglutination Test (Record + for clumps and 0 for
no clumps.
Well

Late
x Serum Latex
(+)
Control

Serum (0)
Test

Unknown B

Unknown A

Salmonella
Control

QUESTIONS FOR DISCUSSION:

1.

Explain the difference between precipitation and agglutination, include reaction components
and appearance.

2.

What role do latex beads play in the agglutination reaction?

3.

What is the result of your unknown bacteria? Is this definitive? Why or why not?

4.

Why are controls important?

5.

What is one advantage of using latex agglutination for identification of an unknown

bacterium over a more traditional method of biochemical identification?

6.

What disease does Salmonella commonly cause in the

transmission source.

U.S.?

Give an example of