LAB NOTES FOR EXAM 1 SECTION

EX. 2-1: DIVERSITY AND UBIQUITY OF MICROOGANISMS

Purpose: Microorganisms are found every where in the environment around us. To demonstrate this and to
get a taste of the different types of organisms in our environment, we can culture these microorganisms by
collecting them on a swab and transferring them to an agar plate.
Media & Materials: 1 Tryptic Soy Agar (TSA) plate, 1 sterile swab and 1 tube of sterile saline per person
Procedure:
1. We will use a simplified version of the lab manual protocol. The lab manual describes several
methods for sample collection. Confer with your lab partners at your table so that each person
uses a different method. Your table should have a total of 4 plates (5 if you have an extra
person at your table).
2. Label the plate with your name, today's date, and the source of inoculum to be collected (for
example, desktop, doorknob, faucet, hair, skin, etc.). You may also use any public body surface
except for your mouth (this means any areas of skin that you might normally expose in polite
company). Transfer any microbes you may have picked up to the agar plate by gently
swabbing the surface of the plate with the swab. The instructor will demonstrate the method
for spreading the cells on the plate.
3. The plates will be incubated at 37C until next lab period.
4. Observe your plates during our next lab meeting. On your data sheet, make note of the various
colors, textures, and shapes that are produced when microscopic organisms are allowed to
reproduce in large numbers. The individual areas of growth you see are colonies and consist of
millions of identical cells that all arose from a single parent cell.
5. Be sure to dispose of your cultures in the Biohazard waste containers when your observations
are completed.
EX. 1-3: ASEPTIC TRANSFER AND INOCULATION TECHNIQUES
Since this is the first time you will be working with bacterial cultures, the procedure for handling and
transferring microorganisms will be described in detail. Aseptic technique, the procedure used to prevent
contamination, is carried out so that you, your neighbors, and your belongings are not contaminated by the
microbial culture and so that microbes from the environment do not contaminate your bacterial culture.
Purpose: In this experiment you will be transferring living cells grown on two different types of media:
broth and slant. You will use these cells to inoculate a fresh slant and a fresh broth culture.
Cultures: a broth culture of Serratia marcescens and an agar slant culture of Serratia marcescens
Media: 1 tryptic soy agar (TSA) slant and 1 tryptic soy broth (TSB) per student
Procedure:
1. Before beginning, label all tubes of sterile media with the name of the organism, the date, and your
names or initials. ALWAYS label before inoculating.
2. Follow the protocol described in the manual. We will be using only one organism today, Serratia
marcescens. This organism produces a red pigment when incubated under the appropriate
conditions. This red or pink color will help you to determine if your aseptic transfer has been
successful when you look for growth next lab period. You should not see any pink or red color in
your freshly inoculated tubes today.
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3. Use the broth culture of Serratia marcescens to inoculate the slant. Use the slant culture to
inoculate the broth.
4. Your instructor may demonstrate some techniques that are slightly different from the ones described
in the lab manual. These variations are acceptable as long as they are safe and produce the desired
results. If you feel confused by any differences, ask for clarification.
5. After you replace the screw top on a culture tube, back off the top about 1/2 turn before placing
the tube in the incubator in order to allow air to circulate. Do not incubate cultures with the top
screwed down tightly. Those bugs need to breath, just like you!
6. Incubate all cultures at 37C until next lab period.
7. During the next lab meeting, make your observations on the data sheet located near the back of
your lab manual. Dispose of your cultures in the appropriate Biohazard container as directed by
your instructor.
EX. 1-4: STREAK PLATE ISOLATION OF PURE CULTURE (PART 1)
Purpose: A pure culture is one that contains a single type of organism. You must first isolate single
colonies in order to cultivate a pure culture. Single colonies contain identical cells that have arisen from a
single cell and are genetic clones of each other. Single colonies can only be obtained by spreading single
cells far apart from each other on the surface of an agar plate, then allowing them to grow. There are
several ways to separate single cells, but we will only be using the streak plate method using an inoculating
loop.
Cultures: a mixed broth culture containing both Serratia marcescens and Staphylococcus aureus and a
mixed broth culture containing both Escherichia coli and Micrococcus luteus.
Media: 2 TSA plates per pair of students
Procedure: The Streak Plate Method
1. Each student will inoculate a mixed broth culture onto a separate agar plates according to the
streak plate procedure described by the lab manual and demonstrated by the instructor. Work with
your lab partner so that each of you streaks a different mixed culture.
2. When you make the streaks across the plate, do them gently so that you do not dig into the agar
with the loop. This will cause growth to occur in streaks, rather than in single colonies.
3. Incubate your plates upside down (lid on the bottom, agar on top) in a wire basket at 37C for until
next lab period. Baskets can be shared between lab partners. These plates are considered mixed
cultures because they will contain two different types of colonies.
4. Next lab meeting: make your observations on the data sheet pages. Use blank paper if you need
additional space. Include a drawing and use the information in Ex. 2-2 to write a description of a
colony from each different type of organism. Observe the plates for visible differences in colony
morphology (size, color, shape, elevation, margin, texture, optical characteristics). Did you get nice
isolated single colonies? They should be spaced far enough apart so that you can pick up an
without touching more than one colony using a loop. Serratia marcescens colonies should be pink
or reddish. Staphylococcus aureus form smaller, white colonies. Escherichia coli produces beige
colonies 2 to 3 mm in diameter, while Micrococcus luteus will produce smaller yellow colonies.
EX. 1-4 (and EX. 2-2): STREAK PLATE ISOLATION OF PURE CULTURE (PART 2)
Purpose: Isolate a pure culture from mixed culture plates by restreaking a fresh streak plate from a single
isolated colony
Organisms: Last periods mixed culture plates with isolated colonies from Ex. 1-4.
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Media: 4 TSA plates per pair of students

Procedure:
1. Label fresh plates with the names of the four organisms found on your mixed culture plates, your
initials, and the date. Look at the two streak plates that you and your lab partner prepared from
mixed cultures the last lab period. Choose single colonies that are well separated from the others so
that they are easy to pick up.
2. From a mixed culture plate, aseptically touch the edge of the sterile loop to the colony. It is not
necessary to scoop up the entire colony. You should not transfer a visible quantity of inoculum to
the fresh plate. Using the streak plate dilution method that you used last week, streak a fresh,
labeled plate with the appropriate colony. Remember to flame the loop in between streaking each
quadrant of the plate. If the colonies are very small and close together, you can use a needle to pick
up cells. Note: Make sure that you pick up your inoculum from only one colony.
3. Use the same method to streak plates of all four organisms.
4. Place the plates inverted in a basket. Incubate the four plates at 37C until next lab period.
5. Next lab period: You will use these plates as part of Ex. 2-2, Colony Morphology. Record your
observations on your data sheet. Use additional paper for drawings or other information. Each
plate should have only one type of colony.
EX. 2-2: COLONY MORPHOLOGY
Purpose: Observe possible difference in colony morphology between different bacterial species grown on
solid culture media.
Organisms: broth cultures of Bacillus subtilis and Mycobacterium smegmatis
Media: Two TSA plates per pair
Procedure:
1. With a sterile loop, inoculate Bacillus subtilis and Mycobacterium smegmatis onto separate TSA
plates using the streak plate method. NOTE: Be careful to avoid cross-contamination between
plates. Be especially conscientious in flaming your loop after transferring any Bacillus species,
which produces spores. Make sure that the loops are flamed long enough to glow orange. Take
the time to flame the loops and go through the procedures slowly and carefully. Report any
spills to the instructor so that proper cleanup can occur.
2. You have already streaked plates with the other organisms from Ex. 1-4. Incubate all the plates
37C until next lab period. The Mycobacterium and Micrococcus cultures may require additional
incubation time. Therefore, if the growth is scant and the colonies are very small, incubate these
cultures for 1 to 2 more periods.
3. Next lab period: make observations on the 6 cultures on your data sheets. Use the terms we
covered in class and on the study guide in your descriptions. There may be demonstration plates of
other organisms to view as well. Be sure to include observations on these cultures in your results.
WHEN YOU ARE FINISHED MAKING ALL YOUR OBSERVATIONS, CULTURES SHOULD BE
DISPOSED OF IN YOUR RED AUTOCLAVE BAGS AND THOSE BAGS PLACED IN THE LARGE
GRAY BIOHAZARD WASTE CAN.
EX. 2-3: GROWTH PATTERNS ON SLANTS
Purpose: Observe possible differences in morphology between different bacterial species grown on slant
media
Organisms:
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Bacillus subtilis and Mycobacterium smegmatis

Last periods mixed culture plates with isolated colonies from Ex. 1-4

Media: 6 TSA slants

Procedure:
1. Label each slant tube with the date, organism name, and your initials.
2. Using the aseptic technique for inoculating slants that we learned last week, inoculate each tube.
For Serratia marcescens, Micrococcus luteus, Escherichia coli, Staphylococcus aureus,
carefully pick up cells of a single colony from your mixed plates. Normally, pure cultures would be
used for transferring organisms to slants or broths, but we are using the mixed plates due to time
considerations. If you do not have sufficient single colonies of each organism, there will be some
pure culture plates available as well.
3. Next lab period: make observations on the 6 cultures on your data sheets. Use the terms we
covered in class and on the study guide in your descriptions. For your uninoculated control, use a
fresh TSA slant, but return it to the cart when finished.
EX. 2-4: GROWTH PATTERNS IN BROTH
Purpose: Observe possible differences in morphology between different bacterial species grown in broth
media
Organisms:

Bacillus subtilis and Mycobacterium smegmatis

Last periods mixed culture plates with isolated colonies from Ex. 1-4

Media: 6 TSB broths

Procedure:
1. Label each broth tube with the date, organism name, and your initials.
2. Inoculate these tubes with the same cultures used in Ex. 2-3. Again, be especially careful when
transferring cells from your mixed culture plates.
3. Next lab period: make observations on the 6 cultures on your data sheets. Use the terms we
covered in class and on the study guide in your descriptions. For your uninoculated control, use a
fresh TSB broth tube, but return it to the cart when finished.
EX. 2-7: FLUID THIOGLYCOLLATE: ATMOSPHERIC OXYGEN REQUIREMENTS
Purpose: Observe the growth patterns of different organisms according to their oxygen requirements
Organisms: Broth cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa,
Neisseria sicca, and Clostridium butyricum
Media: Four tubes of thioglycollate broths and one tube of supplemented thioglycollate per pair
Procedure:
1.

Thioglycollate is a reducing agent that removes oxygen from the broth. Label the tube of
SUPPLEMENTED thioglycollate for the organism Clostridium butyricum (don't forget your initials
and the date). Label the other tubes for each of the remaining organisms.

2.

After observing the instructor's demonstration, inoculate each broth using a sterile disposable bulb
pipette: Transfer 0.25 ml (the first mark on the sterile pipette above the joint) of the appropriate
culture by gently squeezing the bulb to expel the inoculum into the broth from the bottom of the tube
to the top. DO NOT ALLOW ANY AIR TO BUBBLE INTO THE BROTH.
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3.

Discard the pipette in your autoclave bag.

4.

Repeat the procedure for the other four organisms. Incubate the tubes at 37C until next lab period.

5.

Next lab period: DO NOT SHAKE or disturb the broth cultures before you have the opportunity to
make your observations. Make observations on the 6 cultures on your data sheets. Use the terms we
covered in class and on the study guide in your descriptions.

EX. 7-3: THE KIRBY-BAUER ANTIBIOTIC SENSITIVITY TEST PROCEDURE

Purpose: Observe the effects of a variety of antibiotics on a Gram positive and a Gram negative organism
using the Kirby Bauer antibiotic sensitivity test. We will be using a procedure similar to the one described
in the lab manual, with some modifications. Follow the procedure described below:
Organisms: broth cultures of Escherichia coli and Staphylococcus aureus
Media and Materials:
2 tubes sterile TSB, 2 disposable 1 ml bulb pipets, 2 large Mueller-Hinton agar plates, 2 sterile
cotton swabs, Antibiotic disks for streptomycin, tetracycline, penicillin, chloramphenicol,
cephalothin, erythromycin, novobiocin, vancomycin
Procedure:
1) Dilute each stock culture 1:50 by adding 0.25 ml of culture to 5 ml sterile TSB. Be sure to label
the TSB tubes before adding the stock culture. Mix well.
2) Label each plate with name of an organism. Inoculate each plate with a single organism by dipping
a sterile swab into the diluted broth culture so that the swab is saturated. Remove excess inoculum
by gently rolling the swab against the inner surface of the tube. The swab should be moist, but not
dripping.
3) Use the swab to evenly cover the entire surface of the agar in a horizontal direction. Remoisten the
swab, turn the plate 90 and streak the surface in the other direction. Make sure that you cover the
entire plate and that the bacteria are spread all the way to the plate edge.
4) Dispose of the used swab in the autoclave bag.
5) Allow the plate to absorb the liquid for a few minutes, with the top on and right side up.
6) Inoculate the second plate with the other organism using the same procedure. Make sure to use a
fresh swab for the second culture.
7) We will NOT be using a disk dispenser. Instead, use a pair of flamed and cooled of forceps to
remove the antibiotic paper disk from the end of the cartridge. Place disks on the agar surface of
the plate, using the laminated guide to space them evenly around the plate. Gently touch the disk
with the tip of forceps so that it adheres to the surface of the agar. Do not push the disk into the
agar. Each disk is already marked, so you do not need to label the plate with the antibiotic names.
Symbol

Amount

Antibiotic

Intermediate
Range (mm)

S10

10 g

Streptomycin

12-14

TE 30

30 g

Tetracycline

15-18

P10

10 g

Penicillin

28-29

C 30

30 g

Chloramphenicol

13-17

CF 30

30 g

Cephalothin

15-17

E 15

15 g

Erythromycin

14-22

NB 30

30 g

Novobiocin

18-21

VA 30

30 g

Vancomycin

10-11

8) Invert the plates and incubate them 37C until next lab period. Since these plates should not
incubate more than 48 hours, be sure to view them the next period. IF they are incubated too long,
colonies may begin to grow in the zone of inhibition, making the interpretation of your results
difficult.
9) Next lab period: examine the plates for the presence or absence of a zone of inhibition around each
disk.
10) Measure the diameter of the zone of inhibition around each disk and determine the susceptibility
(resistant, intermediate, or sensitive) of the organisms to the antibiotics. Consult the chart in the lab
manual for interpretation of zone size. Consult the chart above for the intermediate range for the
eight antibiotics. A measurement that is smaller than the intermediate range indicates resistance
while a measurement larger than the intermediate range indicates susceptibility.

DISINFECTANTS AND ANTISEPTICS: AGAR PLATE SENSITIVITY METHOD

Purpose: Evaluate the effectiveness of some antiseptics and disinfectants on a Gram positive and on a
Gram negative bacterial species
Organisms: broth cultures of Escherichia coli and Staphylococcus aureus
Media and Materials:
2 large TSA plates, 2 sterile swabs, 6 small beakers, blank sterile disks, forceps
6 chemical agents: HP = Hydrogen Peroxide, L = Listerine, I = Betadyne (iodine), A = Rubbing
alcohol, B = Bleach, and W = water (negative control)
Procedure:
1.

Using the UNDILUTED stock culture for inoculating and TSA plates, follow steps from the previous
exercise (Kirby Bauer technique) for plate preparation and inoculation.

2.

Divide the plate into 6 pie wedges by drawing 3 lines diagonally across the plate (see demo).

3.

Using a flamed and cooled forceps, place two sterile disks into a beaker containing a small quantity
(5 drops) of one of the chemical agents. Drain the excess solution from the disks on paper toweling.
Place one disk on each plate within a marked wedge about 2 cm from the edge of the plate. Do not
drip any disinfectant on to the surface of the agar. Gently tap the disk with the forceps so that it
adheres to the agar. On the back of the plate write the code letter(s) for the agent used.

4.

Repeat the procedure for each agent. When you are finished you should have six different disks
arranged on the agar surface.

5.

Invert the plates and incubate them at 37C until next lab period.

6.

Next period: observe the plates for a zone of inhibition around each disk. Compare the difference in
effectiveness of each disinfectant or antiseptic against a Gram negative and a Gram positive
organism. Since the amount of each disinfectant or antiseptic is not standardized, the zone size does
not get measured in this exercise.
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EX. 2-9: TEMPERATURE

These experiments will be done on plates, rather than broths, to reduce the amount of media required
for each experiment. The instructor will demonstrate the changes in the procedure as written below.
Purpose: Determine the optimum growth temperature of different bacterial species
Organisms:
Escherichia coli, Staphylococcus aureus, Bacillus stearothermophilus, Pseudomonas fluorescens
Media: Four TSA plates per pair
Procedure:
1.

Draw two perpendicular lines on the bottom of the plate to divide the plates into fourths. Write the
incubation temperature on each plate: 4 C, 25 C, 40 C and 60 C. Label each sector of each plate
with the name of one of the four organisms listed above. Label the plates with your name and date as
usual.

2.

Make a straight line inoculation of each organism on each of the four plates. The instructor will
demonstrate.

3.

Put your plates in the appropriate baskets on the cart. These baskets will be incubated at the
appropriate temperature. The 4 incubator is the refrigerator. The plates are incubated at the
appropriate temperature until next lab period.

4.

Next lab period: examine the plates for the presence or absence of growth and record your
observations. Use a scale from 0-3 (0 being no growth and 3 being heavy growth) to describe the
degree of growth. Note the temperature or temperature range that supports the growth of each
organism. Is the organism a psychrophile, a mesophile, or a thermophile? In what possible
environments would you find these organisms?

EX. 2-11: OSMOTIC PRESSURE

Purpose: Demonstrate the effect of osmotic pressure on microbial growth
Organisms:

Escherichia coli, Staphylococcus aureus, Bacillus subtilis

Media: 5 beef heart infusion (BHI) plates, one with each of the following salt concentrations: 0.85%, 5%,
7.5%, 10% and 20% NaCl
Procedure: Work in pairs
1.
2.

Divide the plates into thirds. Label each sector with the name of one of the three organisms listed
above. Label the plates with your name and date as usual.
Make a straight line inoculation of each organism on each of the five plates.

3.

Incubate your plates at 37C until next lab period.

4.

Next lab period: examine the plates for the presence or absence of growth and record your
observations. Note the salt concentration that supports the growth of each organism. Which organism
or organisms are halophiles? In what possible environments would you find these organisms?

EX. 3-5: PREPARATION OF BACTERIAL SMEARS

We will work with the live cultures first and prepare our bacterial smears for staining before using the
microscopes. All live cultures should be disposed of before getting your microscope out.
Purpose: Learn to prepare bacterial smears from both liquid and solid media
Organisms: agar slant culture of Staphylococcus aureus, agar slant culture of Escherichia coli, broth
culture of Bacillus subtilis, agar slant culture of Rhodospirillum rubrum
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Equipment:
Glass slides cleaned with water, inoculating loop, bunsen burner, lens paper and bibulous paper,
lab marker, heating block
YOU AND YOUR LAB PARTNER WILL BE PREPARING A TOTAL OF 10 SLIDES:
2 slides each of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Rhodospirillum rubrum ,
and 2 slides of Staphylococcus aureus and Escherichia coli mixed together.
Notes:

Always make two smears of each specimen, one for staining and one for back up. Stain one at a
time. If you make a mistake, you can repeat the procedure with the back up smear. DO NOT
STAIN BOTH SMEARS SIMULTANEOUSLY!

Set up all the slides at the beginning of lab so they have time to dry. Make sure you label the slides
with the intended stain and organism.

There are many things to think about in this lab, however, the most important one is following
aseptic procedure and handling cultures appropriately.

Always flame your loop before setting it down.

Preparation of a bacterial smear from an agar (solid) culture:

1) Turn on your heating block to the lowest heat setting.
2) Wash a slide with tap water and dry it well. With the lab marker, make a small circle on the slide.
The circle will help you locate your specimen. Turn the slide over. You will place your culture on
the unmarked side of the slide so that the markings do not get washed away during the staining
process. Use the lab marker and label the slide with your initials and the initials of the organism.
3) Place a single, small drop of water from the dropper bottle onto the end of your inoculating loop.
Smear this drop onto the slide within the circle drawn on the opposite side.
4) Sterilize the loop, pick up a small amount of inoculum from an agar slant or agar plate and spread
it around in the water. Flame your loop when you have finished. Note: Water is used when the
inoculum is from a culture grown on solid medium (agar) to separate the cells. Beginning
students usually pick up much more inoculum than is needed. You only need a barely visible
amount on the loop to get a good specimen. Using a large inoculum will make it difficult to
observe the morphology of individual cells.
5) Heat-fix your preparation by placing the slide on a warm heating block until specimen is
completely dry. When it is dry, the specimen may be slightly visible or it may not be visible at all. If
visible, it will be dull in appearance, not shiny.
Preparation of a bacterial smear from a broth (liquid) culture:
1) Clean and label a microscope slide as described above. Following the aseptic procedure for picking
up an inoculum described previously, place a loopful of inoculum from a broth culture onto the
slide on the opposite side from the circle. Gently spread the inoculum around within the circled
area. Note that NO water is placed on the slide when the inoculum is from a broth culture.
2) Repeat the procedure and apply two more loopfuls to the slide. Make sure that you flame your loop
and cool it between each application.
3) Heat-fix your preparation by placing the slide on a warm heating block until specimen is
completely dry. When it is dry, the specimen may be slightly visible or it may not be visible at all. If
visible, it will be dull in appearance, not shiny. NOTE: Once a specimen has been heat-fixed, it
can be stored indefinitely until you are ready to perform your stain. Even though the majority
of cells have been heat-killed, you should still handle your slides with care. Store them in an
empty microscope slide box and keep them in your drawer.
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Preparation of mixed culture smear:

Place a small drop of water on your slide if you are using agar slats cultures. Aseptically transfer a loopful
of Staphylococcus aureus to a clean microscope slide, suspending the culture evenly in the drop of water.
Flame your loop. Aseptically transfer a loopful of Escherichia coli to the same slide. Mix the two cultures
together with your loop before sterilizing your loop. Heat-fix slide as directed above.

Ex. 3-1: USING THE LIGHT MICROSCOPE

Objectives: Identify the parts of the compound microscope and describe their function
Demonstrate the correct use of the microscope for observing stained slide preparations with
the dry and oil immersion lenses
Identify the basic morphologies and structures of bacterial cells
Observe your smears stained with simple stains
Look at the prepared slides that are available while you are waiting for your smears to dry. Look at the
specimens with all three of the dry objectives, beginning with the lowest power objective, and then go up to
the oil immersion objective. Your best view of the cells will be with the oil immersion; however, you must
find and focus your specimen with the three dry objectives before this is possible. If you have not had
experience using the oil immersion objective, make certain that you have the instructor explain how to do
this before you begin. Remember that once you have looked at a specimen with the oil immersion
objective, you cannot go back to the dry objectives!
PREPARED SPECIMENS
All bacterial cells are prokaryotes and, in general, they are considerably smaller than eukaryotic cells.
For this reason, they are usually harder to find and observe. Bacillus megaterium, however, is an
exceptionally large specimen, chosen to make your viewing easier. Always begin your observations with
the low power dry objective and then gradually work up to the oil immersion. All of the following
bacterial preparations must be observed with the oil immersion objective because it is only with this
objective that you can accurately observe the shape, arrangement, and stained color of the cells.

Bacillus megaterium. This stained preparation contains large rod-shaped bacterial cells that
remain attached to each other in long chains after cell division. The term for this arrangement of
cells is "streptobacilli" in which the term "strepto" refers to cells in a chain and "bacilli" refers to
the rod shape of the bacterial cells. One rod-shaped cell is a bacillus and two or more are bacilli.
The word bacillus is used in two ways: it describes the shape of some bacterial cells (eg.
bacillus) and it also refers to a genus (a category used in classification) of rod-shaped bacteria (e.g.
Bacillus). There are many, many rod-shaped bacterial cells (bacilli) that are not classified in the
genus Bacillus.

Coccus or Staphylococcus aureus. Observe the stained bacterial cells that are spherical in shape
and arranged in clusters. The term for one spherical bacterial cell is "coccus" and many spherical
cells are referred to as "cocci" (pl.). When spherical cells are arranged in clusters (often called
"grapelike"), the arrangement is known as "staphylococci". In this example, as in the previous, the
arrangement is also the genus designation of one group of bacteria, the genus, Staphylococcus.
What term would describe the arrangement of spherical bacterial cells arranged in chains?

When you are done, your microscope MUST be put away properly:

Rotate the nose piece so that the lowest power objective lens is directly over the stage

Lower the microscope stage to the lowest position

Remove the specimen slide from the microscope stage

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Wipe immersion oil from the lens using lens paper

Wrap the power cord around the base of the microscope

Cover the microscope with its protective cover

Return the microscope to microscope cabinet, matching the microscope number with the shelf
number

EX. 3-5: SIMPLE STAINING

Purpose: Stain bacterial specimens using simple staining technique. Observe the shape and arrangement of
stained bacterial cells.
Staining solutions:
Methylene Blue, Crystal Violet, and Carbol Fuchsin stains
Procedure:
1. YOU AND YOUR LAB PARTNER SHOULD HAVE 10 SLIDES THAT YOU PREPARED
LAST PERIOD: 2 slides each of Staphylococcus aureus, Escherichia coli, Bacillus subtilis,
Rhodospirillum rubrum , and 2 slides of Staphylococcus aureus and Escherichia coli mixed
together. Use one set for Simple Staining and save the second set for Gram Staining.
2. Do a simple stain of each organism according to the procedure in your lab manual using a different
stain for each slide. Note that the staining times could be different for each stain.
3. Observe your specimens under the microscope. Always begin your microscope observations with
the low dry objective and work up progressively through the dry objectives to the oil immersion
objective. The oil is applied directly on the specimen. Do NOT use coverslip with the stained
smear.
4. You MUST use immersion oil any time you look at a specimen using the 100X objective lens. Do
not get oil on any of the objective lenses other than the oil immersion objective. Once you have
looked at a slide with oil on it, you cannot go back to any of the lower power objectives. Do not
attempt to do so. Once you have used oil on a slide you have prepared, the slide is disposed of in
the special SHARPS BIOHAZARD container.
EX. 3-7: GRAM STAIN
Purpose: Understand the chemical and biological basis of the Gram stain; perform and interpret the Gram
stain
The Gram stain is a differential stain that separates bacteria based on structural differences in their cell
walls. Gram positive cells retain the primary stain, crystal violet, and appear purple blue in color after
completion of the procedure. Gram negative cells lose the crystal violet-stain after decolorizing and absorb
the counterstain, safranin, and will, therefore, appear pink.
Materials:

YOU AND YOUR LAB PARTNER SHOULD HAVE 10 SLIDES THAT YOU PREPARED
LAST PERIOD: 2 slides each of Staphylococcus aureus, Escherichia coli, Bacillus subtilis,
Rhodospirillum rubrum , and 2 slides of Staphylococcus aureus and Escherichia coli mixed
together.

Gram stain reagents: Crystal Violet, Gram's Iodine, 95% Ethanol, Safranin

Procedure:
1. Place a few drops of crystal violet on your specimen. Let the stain remain on the slide for one (1)
minute. Do not allow the stain to dry, as this will cause the formation of stain crystals.
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2. Gently rinse with a small stream of deionized water from a squeeze bottle by holding the slide at a
slight angle with the stream of water hitting the slide ABOVE the area with the bacterial smear. Do
not allow the water stream to hit the specimen directly. You can also use a stream of tap water if it
is VERY gentle.
3. Cover the smear with Grams iodine and let it stand for one (1) minute.
4. Gently rinse off the Grams iodine with water.
5. Decolorize with 95% ethanol using the same technique you used to rinse with water: hold the slide
at an angle and allow the decolorizer to flow over the stained area of the slide, without it directly
hitting the smear. The decolorizer should run clear within a few seconds. This is the critical step!
Do not over-decolorize.
6. Gently rinse with water.
7. Counterstain with safranin for one (1) minute.
8. Gently rinse off the safranin with water, blot with bibulous paper and observe your specimen under
the microscope.
NOTE:
Remember to put your microscope away correctly. The lowest power objective should be in position over the
stage opening, the slide should be removed, no oil should be present on the stage or on any of the dry lenses,
and the microscope should be placed into the correct box. NEVER use Kimwipes, paper towels or Kleenex to
clean microscope lenses - only the lens paper in your lab kit. Wrap the electrical cord around the base of the
microscope.

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