Microbiology Lab Manual'15 Batch PDF

KARPAGAM UNIVERSITY
Karpagam Academy of Higher Education

(Deemed to be University, Established under Section 3 of UGC Act 1956)
COIMBATORE-641 021.
Faculty of Engineering
Department of Biotechnology
15BTBT312
MICROBIOLOGYLAB
PREPARED BY
Dr. R. Thilagavathi & Ms. P. Sandhya
KARPAGAM UNIVERSITY
COIMBATORE-641 021.
MICROBIOLOGY
LABORATORY RECORD BOOK
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KARPAGAM UNIVERSITY
COIMBATORE- 641 021.
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by Mr./Ms/Mrs.......
for the course B.Tech ...
(branch) during..(year/semester) of
Academic year 2016 2017 is bonafide.
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B.Tech, Practical Examination of Karpagam University conducted on .....
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INDEX
MARKS
EXP
NO.
DATE
NAME OF THE
PAGE
EXPERIMENT
NO.
Laboratory safety and aseptic

techniques
Microscopy light microscopy
Culture media Types,

Preparation of nutrient broth and
nutrient agar
Culturing of microorganisms in
Broth and in plates
(Spread plate, Pour plate, Streak
plate)
TOTAL
PREP
OBS
REC
VIVA
(30)
(30)
(30)
(10)
Staining techniques
5
Motility test
6
Quantitation of microorganisms
Chemical control of
microorganisms
Antibiotic sensitivity assay
Bacterial growth curve
Effect of different parameters on

bacterial growth
(Temperature and UV
irradiation)
AVG:
FACULTY
SIGN
(100)
EXP. NO: 1
LABORATORY SAFETY AND ASEPTIC TECHNIQUES
DATE:
AIM:
To understand the lab safety guidelines, bio-safety rules and aseptic techniques to be followed
in the microbiology lab.
SAFETY GUIDELINES:
Safety in a microbiology laboratory is important in the prevention of infection that might be
caused by the microorganisms being studied. This laboratory does not require the use of virulent
human pathogens.
However, many types of microorganisms are potentially pathogenic.
This
means that, although they would not cause disease in a normal healthy host, they might possibly do
so if a large enough quantity of the microbes came into contact with a compromised host, such as by
wounds and cuts.
In addition to microorganisms, there are some chemicals used in this laboratory that
are potentially harmful. Many procedures involve glasswares, open flames, and sharp objects that
can cause damage if used improperly.
Specific lab safety guidelines are designed to address each of these potential routes of
exposure.
Microbiology Laboratory Safety Rules
1. Never work alone in the laboratory without permission and prior knowledge of the instructor.
2. Do not engage in rowdy, playful, or unprofessional activities in the laboratory.
3. Work surfaces must be disinfected at the beginning and at the end of every laboratory period.
4. All students must wash their hands at the beginning of the lab and at the end of every laboratory
period.
5. No eating or drinking is permitted in the laboratory. No open food or beverage can be taken into
the laboratory.
6. Lab benches are to be kept free of extraneous items while conducting experiments. This includes
personal items such as backpacks, cell phones, and unnecessary books.
7. Students must wear closed-toe shoes that cover the top of the foot, and appropriate clothing, at all
times in the laboratory.
8. Students must wear gloves when handling microorganisms. Wear lab aprons or lab coats as
advised by your instructor. Wear safety glasses when handling bacterial broth cultures, doing
Gram stains, and as otherwise advised by your instructor.
9. Keep hands away from your face, eyes, and mouth when working with chemicals or
microorganisms. This includes not applying cosmetics, not adjusting contact lenses, and not
biting your finger nails.
10. If any chemicals or other agents splash into your eyes, immediately go to the nearest sink and
flush your eyes with water.
11. Take special precautions when working with open flames. Loose hair, clothing, dangling jewelry,
and nearby paper must be secured. Do not leave a heat source (hot plate or Bunsen burner)
unattended. Keep containers of alcohol, acetone, or other flammable liquids at a safe distance
from flames.
12. Report ANY and ALL accidents, spills, BREAKAGES, or injuries to the instructor, no matter
how trivial they appear.
13. Any pregnant or immunocompromised student must notify the instructor of the course. A
pregnant student is required to wear safety glasses and 2 sets of examination gloves when
handling any bacterial broths or cultures.
14. Do not remove cultures, reagents, or other materials from the laboratory unless specific
permission from the instructor has been granted.
15. Do not use any lab equipment without instruction and authorization from the instructor. Report
any damaged or broken equipment to your instructor immediately.
16. Students must assume that all of the organisms that they work with in this laboratory are potential
pathogens (disease-producing microbes). However, this laboratory does not require the use of any
highly infectious human pathogens. Bacterial culture material used in this lab can include the
following organisms: Bacillus subtilis, Corynebacterium xerosis, Enterobacter cloacae,
Escherichia coli, Klebsiella pneumoniae, Lactobacillus casei, Micrococcus luteus, Proteus
vulgaris,
Pseudomonoas
aeruginosa,
Salmonella
typhimurium,
Serratia
Staphylococcus aureus, Staphylococcus epidermis, and Streptococcus mutans.
marcescens,
ASEPTIC TECHNIQUES
Only non-pathogenic cultures should be used in schools obtained from a recognised educational
supplier. Sterile equipment and media should be used in the transfer and culture of microorganisms.
Aseptic technique should be observed whenever microorganisms are transferred from one container
to another.
It is wise to treat all cultures as potentially pathogenic, because cultures may have been
contaminated, and because mutations to disease-causing forms may occur. The aseptic techniques
described here control the opportunities for contamination of cultures by microorganisms from the
environment, or contamination of the environment by the microorganisms being handled.
There are some general rules to follow for any aseptic technique.
1.
Close windows and doors to reduce draughts and prevent sudden movements which might
disturb the air.
2.
Make transfers over a disinfected surface. Ethanol disinfection is recommended because of its
rapid action. If the bench surface is difficult to clean, cover the bench with a sheet of tough
material which is more easily disinfected.
3.
Start the operations only when all apparatus and materials are within immediate reach.
4.
Complete all operations as quickly as possible, but without any hurry.
5.
Vessels must be open for the minimum amount of time possible.
6.
While vessels are open, all work must be done close to a Bunsen burner flame where air
currents are drawn upwards.
7.
On opening a test tube or bottle, the neck must be immediately warmed by flaming (see
below) with the vessel held as near to horizontal as possible and so that any movement of air
is outwards from the vessel.
8.
During manipulations involving a Petri dish, limit exposure of the sterile inner surfaces to
contamination from the air.
9.
The parts of sterile pipettes which will be put into cultures or sterile vessels must not be
touched or allowed to come into contact with other non-sterile surfaces, such as clothing, the
surface of the working area, or the outside of bottles/ test tubes.
10.
All items which come into contact with microorganisms must be sterilised before and after
each such exposure. This could be either by the technical team preparing for and clearing up
6
after a piece of practical work (for example, in the case of glassware to be used), or by the
worker during the course of the practical (for example, in flaming a wire loop).
Wire loops
For transferring fungal cultures which grow by producing a mycelium of hyphae, an inoculation wire
with the end bent into a small hook is better than a loop. Use the hook to gouge into the agar at the
edge of the culture and pick up a small piece of agar plus hyphae. Transfer this to agar plate or slope,
and invert the piece of fungus agar so that the fungus is in contact with the agar in the dish or tube.
Ensure that the culture adheres firmly to the new agar. You may decide not to invert agar plates
immediately in case the transferred culture falls off the agar.
Pipettes
A leaking pipette is caused either by a faulty or ill-fitting teat, or by fibres from the cotton wool plug
between the teat and the pipette.
A dropping (Pasteur) pipette can be converted to deliver measured volumes by attaching it by rubber
tubing to a non-sterile syringe barrel.
Cotton wool plugs
Cotton wool stoppers are easier for students to handle than screw caps this makes complex
manipulations more straightforward.
If a plug accidentally catches fire, douse the flames immediately by covering with a dry cloth, not by
blowing or soaking in water.
Inoculating agar plates, slopes and cultures
a Carry out the transfer of cultures as quickly as possible, with tubes and plates open to the air for the
minimum length of time.
b Normal practice is to open agar plates away from the body and without removing the lid
completely from the base.
c When the lid of the Petri dish is removed for longer periods than normal, work very close to the
Bunsen burner flame to reduce the chances of contamination.
d If you experience frequent contamination of plates with fungal spores, reduce the chance of
draughts further, and consider inoculating plates from below with the agar surface facing downwards.
In this way there is perhaps less chance of spores settling onto the plate from the air.
Using a wire loop

a Clean the loop by heating to red hot as described below, allow to cool, then reshape with forceps
before beginning again. Do not use your fingers because of the possibility of puncturing your skin.
b Hold the handle of the wire loop close to the top, as you would hold a pen, at an angle that is
almost vertical. This leaves the little finger free to take hold of the screw cap/ cotton wool plug of the
bottle/ test tube. It also ensures that any liquid culture on the loop will run down into the flame.
c Sterilize the wire loop by heating to red hot in a roaring blue Bunsen burner flame before and after
use. This ensures that contaminating bacterial spores are destroyed.
d The flaming procedure should heat the tip of the loop gradually. This is because after use it will
contain culture, which may splutter on rapid heating and possibly release small particles of culture,
forming an aerosol.
i Position the handle end of the wire in the light blue cone of the flame. This is the coolest area of
the flame.
ii Draw the rest of the wire upwards slowly into the hottest region of the flame immediately above
the blue cone.
iii Hold there until it is red hot.
iv Ensure the full length of the wire receives adequate heating.
v Allow to cool for a few seconds in the air, then use immediately.
vi Do not put the loop down, or wave it around.
vii Re-sterilise the loop immediately after use.
Using a pipette
Sterile graduated or dropping (Pasteur) pipettes are used to transfer cultures, sterile media and sterile
solutions.
a Remove the pipette from its container/ wrapper by the end containing a cotton wool plug, taking
care to touch as little of the pipette as you need to take a firm hold.
b Fit the teat. It is sometimes helpful to dip the teat first in sterile liquid to lubricate it.
c Hold the pipette barrel as you would a pen, but do not grasp the teat. This leaves your little finger
free to take hold of the cap/ cotton wool plug of a bottle/ test tube and your thumb free to control the
teat.
d Depress the teat cautiously and take up an amount of fluid which is adequate for the amount
required, but does not reach and wet the cotton wool plug. Squeezing the teat with the pipette tip
beneath the liquid surface introduces air bubbles which may cause spitting and, consequently,
aerosol formation. Avoid this by squeezing the teat before placing the tip into the liquid. Then gently
release the pressure until the required amount of liquid is drawn up, and lift the pipette tip out of the
liquid.
e Return any excess gently.
f Immediately after use put the contaminated pipette into a nearby discard pot of disinfectant.
g Remove the teat only once the pipette is within the discard pot otherwise drops of culture will
contaminate the working surface.
Flaming the neck of bottles and test tubes

9
This ensures that no microorganisms enter the mouth of the vessel to contaminate the culture or the
medium. Passing the mouth of the bottle through a flame produces a convection current away from
the opening, and helps to prevent contamination. The hot part of the flame is above the inner bright
blue cone and the vessel needs to be moved through the flame, not held in place.
a Loosen the cap of the bottle so that it can be removed easily.
b Lift the bottle/ test tube with your left hand.
c Remove the cap/ cotton wool plug of the bottle/ test tube with the little finger curled towards the
palm of your right hand. (Turn the bottle, not the cap.)
d Do not put down the cap/ cotton wool plug.
e Flame the neck of the bottle/ test tube by passing the neck forwards and back through a hot Bunsen
burner flame.
f After carrying out the procedure required, for example, withdrawing culture, replace the cap/ cotton
wool plug on the bottle/ test tube using your little finger. Take care! The bottle will be hot. (Turn the
bottle, not the cap.)
g If cotton wool plugs have partly lost their shape, they can be more easily guided back into the neck
of the vessel by slowly twisting the mouth of the vessel as the plug is pushed down.
Disinfecting surfaces
a For technicians, ethanol disinfection is recommended because of its rapid action (around 5
minutes). Technicians will be more experienced and able to deal with the associated fire hazards of
working with ethanol.
b For disinfection by students, 1% Virkon solution is safer and cheaper, but the surface must be left
wet for 10 minutes.
10
STERILIZATION TECHNIQUES
Sterilization is the killing or removal of all microorganisms, including bacterial spores which
are highly resistant. Sterilization is an absolute term, i.e. the article must be sterile meaning the
absence of all microorganisms.Disinfection is the killing of many, but not all microorganisms. It is a
process of reduction of number of contaminating organisms to a level that cannot cause infection, i.e.
pathogens must be killed. Some organisms and bacterial spores may survive.Disinfectants are
chemicals that are used for disinfection. Disinfectants should be used only on inanimate
objects.Antiseptics are mild forms of disinfectants that are used externally on living tissues to kill
microorganisms, e.g. on the surface of skin and mucous membranes.
Uses of Sterilization:
1. Sterilization for Surgical Procedures: Gloves, aprons, surgical instruments, syringes etc. are to be
sterilized.
2. Sterilization in Microbiological works like preparation of culture media, reagents and equipments
where a sterile condition is to be maintained.
Classification of Methods:
Sterilization and disinfection are done by :
(A). Physical Agents
1. Heat
2. Radiation
3. Filtration
(B). Chemical Agents
In practice, certain methods are placed under sterilization which in fact do not fulfill the definition of
sterilization such as boiling for 1/2 hr and pasteurization which will not kill spores.
STERILIZATION BY HEAT
Heat is most effective and a rapid method of sterilization and disinfection. Excessive heat acts by
coagulation of cell proteins. Less heat interferes metabolic reactions. Sterilization occurs by heating
above 100C which ensure lolling of bacterial spores. Sterilization by hot air in hot air oven and
sterilization by autoclaving are the two most common method used in the laboratory.
Types of Heat :
A. Sterilization by moist heat
B. Sterilization by dry heat
11
MOIST HEAT:
Moist heat acts by denaturation and coagulation of protein, breakage of DNA strands, and loss of
functional integrity of cell membrane.
Sterilization at 100C:
1.
Boiling. Boiling at 100C for 30 minutes is done in a water bath. Syringes, rubber goods and
surgical instruments may be sterilized by this method. All bacteria and certain spores are killed. It
leads to disinfection.
2. Steaming. Steam (100C) is more effective than dry heat at the same temperature as: (a) Bacteria
are more susceptible to moist heat, (b) Steam has more penetrating power, and (c) Steam has more
sterilizing power as more heat is given up during condensation.
Steam Sterilizer. It works at 100C under normal atmospheric pressure i.e. without extra pressure. It
is ideally suitable for sterilizing media which may be damaged at a temperature higher than 100C.
It is a metallic vessel having 2 perforated diaphragms (Shelves), one above boiling water, and the
other about 4" above the floor. Water is boiled by electricity, gas or stove. Steam passes up. There is
a small opening on the roof of the instrument for the escape of steam. Sterilization is done by two
methods :
(a) Single Exposure for 11/2 hours. It leads to disinfection.
(b) Tyndallization (Fractional Sterilization). Heat labile media like those containing sugar, milk,
gelatin can be sterilized by this method. Steaming at 100C is done in steam sterilizer for 20 minutes
followed by incubation at 37C overnight. This procedure is repeated for another 2 successive days.
That is 'steaming' is done for 3 successive days. Spores, if any, germinate to vegetative bacteria
during incubation and are destroyed during steaming on second and third day. It leads to sterilization.
Sterilization by Dry Heat:
Mechanisms.(1) Protein denaturation, (2) Oxidative damage, (3) Toxic effect of elevated electrolyte
(in absence of water).
Dry heat at 160C (holding temperature for one hour is required to kill the most resistant spores). The
articles remain dry. It is unsuitable for clothing which may be spoiled.
1. Red Heat. Wire loops used in microbiology laboratory are sterilized by heating to 'red' in bunsen
burner or spirit lamp flame. Temperature is above 100C. It leads to sterilization.
2. Flaming. The article is passed through flame without allowing it to become red hot, e.g. scalpel.
Temperature is not high to cause sterilization.
12
3. Incineration : It is an excellent method for destroying the materials rapidly. Eg: Animal dead
bodies, beddings, pathologic materials.
STERILIZATION BY AUTOCLAVE
Sterilization above 100C: Autoclaving
Autoclaving is one of the most common methods of sterilization. Principle: In this method
sterilization is done by steam under pressure. Steaming at temperature higher than 100C is used in
autoclaving. The temperature of boiling depends on the surrounding atmospheric pressure. A higher
temperature of steaming is obtained by employing a higher pressure. When the autoclave is closed
and made air-tight, and water starts boiling, the inside pressures increases and now the water boils
above 100C. At 15 ib per sq. inch pressure, 121C temperatures is obtained. This is kept for 15
minutes for sterilization to kill spores. It works like a pressure cooker.'Sterilization holding time' is
the time for which the entire load in the autoclave requires to be exposed.Autoclave is a metallic
cylindrical vessel. On the lid, there are : (1) A gauge for indicating the pressure, (2) A safety valve,
which can be set to blow off at any desired pressure, and (3) A stopcock to release the pressure. It is
provided with a perforated diaphragm. Water is placed below the diaphragm and heated from below
by electricity, gas or stove. Working of Autoclave. (a) Place materials inside, (b) Close the lid. Leave
stopcock open, (c) Set the safety valve at the desired pressure, (d) Heat the autoclave. Air is forced
out and eventually steam ensures out through the tap, (e) close the tap. The inside pressure now rises
until it reaches the set level (i.e. 15 Win), when the safety valve opens and the excess steam escapes,
(f) Keep it for 15 minutes (holding time), (g) Stop heating, (h) Cool the autoclave below 100C, (i)
Open the stopcock slowly to allow air to enter the autoclave002E
Checking of Autoclave for Efficiency.Methods :
(i) Spores of Bacillus stearothermophilus are used. Spores withstand 121C heat for up to 12 min.
Strips containing this bacteria are included with the material being autoclaved. Strips are cultured
between 50C and 60C for surviving spores. If the spores are killed the autoclave is functioning
properly.
(ii)Automatic Monitoring System.
STERILIZATION BY HOT AIR OVEN
Hot Air Oven (Sterilizer). It Is one of the most common method used for sterilization. Glass wares,
swab sticks, all-glass syringes, powder and oily substances are sterilized in hot air oven. For
13
sterilization, a temperature of 160C is maintained (holding) for one hour. Spores are killed at this
temperature. It leads to sterilization.
Hot Air Oven is an apparatus with double metallic walls and a door. There is an air space between
these walls. The apparatus is heated by electricity or gas at the bottom. On heating, the air at the
bottom becomes hot and passes between the two walls from below upwards, and then passes in the
inner chamber through the holes on Me top of the apparatus. A thermostat is fitted to maintain a
constant temperature of 160C.
STERILIZATION BY RADIATION
ULTRAVIOLET LIGHT:
This is commonly employed to aid the sterilization of air and surfaces in the processing
environment. UV light penetrates clean air and pure water. When UV light passes through matter,
energy is liberated to the orbital electrons within the constituent atom. This absorbed energy causes
highly energized state of atoms and alters their reactivity. UV lamps are used for their germicidal
effects on the surfaces or their penetrating effect through clean air and water
IONIZING RADIATIONS:
Ionizing radiations are high energy radiations emitted from radioactive isotopes such as Cobalt 60 (
rays) or produced by mechanical acceleration of electrons to verify velocity and energy ( rays).
Ionizing radiations destroy the microorganism by stopping the reproduction as a result of lethal
mutations. These ionizing radiations are particularly used for sterilization of medical classic devices.
Vitamins, antibiotics, hormones in dry state are also sterilized by this method.
STERILIZATION BY FILTRATION:
It is the method used for the removal of particles including microorganisms from solutions and gases
without the application of heat. Membrane filters function primarilyby screening particles from a
solution or gas thus retaining them on filter surface. Membrane filters also function in some instances
by electrostatic attraction. This would apply particularly to the filtration of dry gases.
LAMINAR FLOW CHAMBERS:
They are used to maintain specific aseptic work area. They are of two types:
14
Horizontal flow type
Vertical flow type
It consists of a HEPA filter (High Efficiency Particulate Air) to which air is forced to move at a
slow rate of 0.45 m/sec across the working space. Usually HEPA filters are capable of removing
99.97 to 99.99% of particles and bacteria of 0.3m and above. A pre filter which is made of a
glass fiber that has the ability to retain 99% of particles of size 5m or above is included in the
system. In such cabinets, the risk of contaminations during aseptic processing condition is very
low. To work with pathogenic microorganisms, cabinet with vertical flow is necessary to protect
the operator.
CHEMICAL METHODS OF STERILIZATION:
ETHYLENE OXIDE:
It penetrates rapidly through materials such as plastics, powders and paper boards. It exerts its effect
upon the microorganism by alkylating the essential metabolites affecting primarily the reproductive
process. It has extensive application in sterilizing plastic materials, rubber boots and delicate optic
instruments.
- PROPIOLACTONE:
These are bacteriocidal used agents against wide variety of microorganisms at relatively low
concentration. It is an alkylating agent and has mode of action similar to that of ethylene oxide. This
is used for sterilization of large surfaces such as an entire room.
PHENOL:
Phenol is a powerful bacteriocidal agent used for disinfection. The mode of action may be selfprecipitation, inactivation of enzymes present in the membrane. Eg: Lysol, Cresol and thymol. These
compounds are used for sterilizing surgical instruments.
ALCOHOLS:
70% ethanol and isopropyl alcohol are used for disinfections. It denatures the cellular proteins and
make them inactive.
15
HALOGENS:
The most widely used halogens for disinfection are iodine and chlorine compounds. They are used as
disinfectants for skin against both Gram Positive and Gram negative bacteria. It acts by oxidizing the
proteins.
Cl2 + H2O
HCl + HOCl
HOCl
HCl + [O]
HEAVY METALS AND THEIR COMPOUNDS:

Mercuric Chloride and silver nitrate prevent the growth of most bacteria. Copper salts give fungicidal
effect. These heavy metals combine with enzymes and make them inactive, disrupting the
metabolism of microbes.
SH
Enzyme
S
+ HgCl2
Enzyme
SH
Hg + 2 HCl
S
ALDEHYDES:
5-10% formaldehyde solution kills most bacteria. It also acts as bactericidal, sporicidal and are lethal
to viruses. It combines with Organic Nitrogen present in Nucleic acids and Proteins. Glutaraldehyde
is an excellent disinfectant for tuberculbacilli, fungi and viruses. It is used in operation theatres, face
mask, wounds and viral inoculating chambers.
DYES:
Aniline dyes like Malachite green, brilliant green and crystal violet are active against G+ve bacteria
and are also used as antiseptic for skin and wounds. Acridine dyes are also used as bacteristatic agent
ACIDS:
Inorganic acids, boric acid act as a bacteriocidal and anti-fungal agent. Formic acid is a powerful
germicide. Organic acid, benzoic acid and salicylic acid are anti-fungal and bacteriostatic agents
16
RESULT:
17
VIVA QUESTIONS:
1. What are the basic routes of exposure to microorganisms?
2. How will you avoid the exposure to microorganisms?
3. How will you dispose the microbiological waste?
4. Why the aseptic technique needs to be followed?
5. If any accidents take place what will you do?
18
EXPERIMENT NO: 2
MICROSCOPY LIGHT MICROSCOPY
19
MICROSCOPY LIGHT MICROSCOPY
EXP. NO: 2
DATE:
AIM:
To study the different parts of the light microscope and the principles of microscopy.
To prepare a wet mount.
PRINCIPLE:
The purpose of a microscope is to magnify an object, which often cannot be seen
without a microscope, so that it can be seen with the naked eye. The light microscope is an
important tool in the study of microorganisms.
The compound light microscope uses visible
light to directly illuminate specimens in a two lens system, resulting in the illuminated specimen
appearing dark against a bright background. The two lenses present in a compound microscope
are the ocular lens in the eyepiece and the objective lens located in the revolving nosepiece.
Compound light microscopes typically have the following components
1. Illuminator: the light source in the base of the microscope.
2. Abbe Condensor: a two lens system that collects and concentrates light from the
illuminatorand directs it to the iris diaphragm.
3. Iris Diaphragm: regulates the amount of light entering the lens system.
4. Mechanical Stage: a platform used to place the slide on which has a hole in the center to
letlight from the illuminator pass through. Often contains stage clips to hold the slide in
place.
5. Body tube: Houses the lens system that magnifies the specimens.
6. Upper end of the body tube --Oculars/Eye pieces: what you view through.
7. Lower end of the body tube --Nose-piece: revolves and contains the objectives
20
Figure 1: Light Microscope.

PRINCIPLE
Basically, a light microscope magnifies small objects and makes them visible.
The
science of microscopy is based on the following concepts and principles:

Magnification is simply the enlargement of the specimen. In a compound lens system, each
lens sequentially enlarges or magnifies the specimen. The objective lens magnifies the specimen,
producing a real image that is then magnified by the ocular lens resulting in the final image. The
total magnification can be calculated by multiplying the objective lens value by the ocular
lensvalue.
Resolving power is the ability of a lens to show two adjacent objects as discrete
entities. In general, the shorter the wavelength of light, the better the resolution, which is why a blue
filter is usually connected to the condenser to produce short light waves for optimum resolution.
Resolving power is also dependent on the refractive index or the bending power of light. Because air
has a lower refractive index than glass, light waves have a tendency to bend and scatter as
they pass through the air from the glass slide to the objective lens. Addition of immersion oil, which
has the same refractive index as glass, diminishes the loss of refracted light and improves resolution.
21
Contrast is the ability to distinguish an object from its background. Since most microbes are
relatively transparent when viewed under a standard light microscope they are difficult to identify.
Using a stain (labs 2-5) that will bind to the microorganism and not the glass slide
dramatically enhances their contrast enabling them to be observed more clearly.
Depth-of-focus is the thickness of the sample that appears in focus at a particular
magnification. As the magnification increases the depth-of-focus decreases, or the slice of
the sample thatappears in focus gets thinner. Many of the newer compound microscopes are par
focal, which means that if one objective lens has the object in focus, and you go to the next objective
lens, only minor adjustment (fine focus) is needed to bring the image back into focus. This is due to
the fact that as you increase the magnification, and thus the slice of the sample that appears
in focus becomes thinner, the correct plane-of-focus will always be within the depth of
focus of the previous objective. After you get the sample into focus at scanning or low power using
the course adjustment knob, you should only have to use the fine focus knob at the higher
magnifications.
Field-of-View is the area of the slide that you are observing through the microscope.
As you increase the magnification the actual area of the slide that you are looking at is getting
smaller. You can think of the field-of-view as a dartboard. At low magnification you are able to see
the entire dartboard, but as you increase the magnification you are only observing the bullseye, a much smaller portion of the dartboard.
These microscopes are also par central, which refers to the ability to keep an object in the
middle of your field-of-view when changing from one objective to another. It is useful to remember
this as you are increasing magnification. Always keeping your sample in the center of your field-ofview will avoid unnecessary searching of the slide for your sample.
Working distance is the distance between the objective and the slide. As you increase
magnification (by using more powerful objective lenses) the working distance decreases. So much so
that by the time you are using the oil-immersion objective (100X) the objective is almost
touching the slide, allowing the immersion oil to connect the slide and objective. It is important to
consider working distance in a number of applications, but practically there are two reasons you
should be aware of your working distance. The first is so that you do not inadvertently push the
objective through the slide, causing damage to the objective and your sample slide. The second isto
estimate whether you are in the correct plane-of-focus.
22
Materials:
Microscope
Newsprint
Stage micrometer
Slides
Coverslips
Transfer pipettes
Prepared slides of bacteria
Hay infusion
Protoslow
Immersion oil
Lens paper
PROCEDURE:
1.Place a piece of newsprint on a microscope slide and cover with a coverslip. ALWAYS USE A
COVERSLIP!
2.Turn the microscope on and set the light source on its highest setting.
3.Use the coarse adjustment knob to obtain maximum working distance.
4.Place the slide on the stage. The slide should fit into the slide holder but is not placed under the
slide holder. Use the stage adjustment knob to move the slide the edge of the coverslip bisects the
hole in the stage.
5.Rotate the scanning objective (4X) into place.
6.Use the coarse adjustment knob to obtain the minimum working distance. Develop the habit of
watching this process to be sure the objective does not crash into the slide.
7.Look through the oculars. Adjust the light with the iris diaphragm lever on the condenser if
necessary. Slowly turn the coarse adjustment knob until the edge of the coverslip comes into focus.
Use the fine adjustment knob to sharpen the focus.
8.Use the stage adjustment knob to locate the letter e in the newsprint. Note the orientation of the
letter e in the newsprint.
9.Rotate a higher power objective (10X) into place. Use the fine adjustment knob to sharpen the
focus. Do not use the coarse adjustment knob. Adjust the light using the iris diaphragm lever if
23
necessary. The image is now magnified 100X (10X ocular x 10X objective = 100X magnification).
Draw the letter e as it appears in the microscope on the lab report sheet.
10.Place a stage micrometer on the stage and determine the diameter of the field of view for all four
objectives. The micrometer is 2 mm in length. The ruler is divided into tenths. Record the distances
on the lab report sheets.
11.When using the high power objective (100X) use the following procedure. Rotate the turret
halfway between the 40X and 100X objective. Place a drop of immersion oil on the slide and rotate
the oil immersion objective (100X) into place. The objective should be immersed in the oil on the
slide. Use the fine adjustment knob to sharpen the focus. Adjust the light using the iris diaphragm
lever if necessary. Never use the coarse adjustment knob with high power.
12.Place a drop of water from the hay infusion on a microscope slide. Cover with a coverslip and
view under all four objectives. Sketch two (2) of the organisms at 400X magnification.
13.Obtain a prepared slide for two bacterial species. View slides under the 1000X objective and
sketch the bacteria. Dont forget the immersion oil!
14.When you are finished with the microscope clean the microscope, as described below, and return
it to storage.
PROCEDURE FOR CLEANING A MICROSCOPE:

1.Turn off the light and unplug the cord. Store the cord appropriately.
2.Using the coarse adjustment knob to obtain maximum working distance and
remove the slide from the stage.
3.Using lens paper clean all the lenses starting with the cleanest firstoculars,
4X through 100X objectives.
4.Clean any oil off of the stage using Kimwipes or paper towels.
5.Rotate the scanning objective into place. Use the coarse adjustment knob to
obtain minimum working distance.
6.Return the microscope to the appropriate storage area.
24
OBSERVATION:
RESULT:
25
VIVA QUEST IONS:

1. State the purpose of each of the following microscope components:
a. Condenser
b. Fine-adjustment knob
c. Coarse-adjustment knob
d. Iris Diaphragm
26
e. Mechanical stage control
2. What is the purpose of adding immersion oil when using the 100X objective?
3. If the ocular lens has a magnification of 10X and the objective lens has a magnification
of40X, what is the total magnification?
27
4. What is refractive index?
5. Name the types of microscopy techniques
28
EXPERIMENT NO: 3
CULTURE MEDIA TYPES, PREPARATION OF
NUTRIENT BROTH AND NUTRIENT AGAR
29
EXP. NO: 3
DATE:
CULTURE MEDIA TYPES, PREPARATION OF

NUTRIENT BROTH AND NUTRIENT AGAR
AIM:
To study the different types of culture media, preparation of nutrient broth and nutrient agar.
PRINCIPLE:
All microorganisms have certain nutritional requirements for their growth. All organisms
require an energy source, an electron source, carbon, nitrogen, oxygen, sulphur, phosphorous, trace
elements and water. To study the morphological, cultural and biochemical characteristics of various
organisms, it is essential to culture the media. Medium is a substance that provides nutrients for
growth and multiplication of the organism. In addition to nutrients, microorganisms need various
environmental factors such as temperature, pH etc. Many special purpose media are needed to
facilitate recognition, enumeration and isolation of certain traits of bacteria. Based on the accuracy of
nutritional values, media are of two types, Complex or Chemically undefined media which is
composed of compounds whose exact chemical composition are not known. Undefined or synthetic
media has known quantity of specific compounds.
According to the consistency three types of media are used: liquid, or broth, media; semisolid
media; and solid media. The major difference among them is that solid and semisolid media
contain a solidifying or gelling agent [such as agar, gelatin], whereas a liquid medium does not.
Liquid media, such as nutrient broth, tryptic soy broth or glucose broth can be used
in studies of growth and metabolism in which it is necessary to have homogenous media
conditions, to follow optical density, and to allow early sampling for analysis of substrates and
metabolic products. Tubes and flasks with liquid cultures can be incubated with either static or
shaken incubation.
Semisolid media (0.1-0.2% agar) can also be used in fermentation studies, in determining
bacterial motility, and in promoting anaerobic growth.
Solid media (1.5-2% nutrient agar), such as nutrient agar, are used 1) for the surface growth
of microorganisms in order toobserve colony morphology, 2) for pure culture isolation, 3) often in
the enumeration and isolation ofbacteria from a mixed population by diluting the original bacteria
suspension and spreading a small inoculum over the surface of the solidified medium and 4) to
30
observe specific biochemical reactions(extracellular enzymes diffusing away from the colony can be
detected as a result of their action on insoluble substrates present in the agar medium).
Solid media can be poured into either a test tube or Petridish. If the medium in the test
tube is allowed to harden in a slanted position, the tube is designated an agar slant; if the tube
is allowed to harden in an upright position, the tube is designated an agar deep tube; and if the agar is
poured into a Petri dish, the plate is designated an agar plate.
Media categorized based on their application:
An all-purpose medium, such as Tryptic Soy Agar, supports the growth of most bacteria
cultured in the laboratory. They do not contain any special additives.
Selective media enhance the growth of certain organisms while inhibiting the growth of
others due to the inclusion of particular substrate. (Eg: Mac Conkey agar)
Differential media allow identification of microorganisms usually through the (visible)
physiological reactions unique to those bacteria. The most practical media are those that both select
for and differentiate common pathogens. (Eg: Mac Conkey agar)
Enrichment media allow metabolically fastidious microorganisms to grow because of the
addition of specific growth factors. Enrichment culture is one obtained with the use of selected media
and incubation conditions to isolate the desired microorganisms from natural samples.
NON-SELECTIVE MEDIA:
Nutrient agar media:
It is the basic bacteriological media used for the isolation of total bacteria. The major components of
media are: Peptone, meat extract, glucose, NaCl and agar.
Potato dextrose agar:
PDA is used for the isolation of total fungi. Major components of the medium are potato, glucose,
agar and acidic pH
Oat meal agar medium:
OMA is used for the isolation of fungi. Commercially available OMA are pre-mixed. These are
dehydrated medium. 3.5g of oat meal agar is added and sterilized.
SELECTIVE MEDIA:
Some media have compounds that favour the growth and/or detection of specific microorganisms and
are inhibitory to others.
31
Mac conkey agar:

It is the differential plating medium used for the selection and recovery of Enterobacteriaceae and
related Enteric G-ve rods. It contains bile salts, neutral pH, lactose, neutralized crystal violet which
helps to inhibit the growth of G+ve bacteria and this medium helps to differentiate coli from bacilli,
typhi and paratyphi. Coli form bacteria forms acid as a result of lactose fermentation. When this
occurs, the medium surrounding the growth of the bacteria also becomes red because of the action of
the acid that precipitates the bile salts and is followed by absorption of neutral red. Typhoid and
Paratyphoid bacteria are non-lactose fermenters and therefore do not produce acid. Hence, colonies
are colorless or transparent.
DIFFERNTIAL MEDIA:
Differential medium contain substances that permit the detection of microorganisms with specific
metabolic activity. This can distinguish morphologically and biochemically related groups of
organisms. They contain chemicals and other compounds that produce a characteristic change in the
appearance of bacterial growth and of surrounding areas following inoculation and incubation.
Mannitol salt agar:

This medium inhibits the growth of all other bacteria except the Staphylococcus. It contains Mannitol
as Carbon souce which some Staphylococci are capable of fermenting. This also has a pH indicator
for detecting acid produced by mannitol fermenting Staphylococci which exhibits a yellow zone
surrounding their growth and non-fermenters will not produce the colour change.
MATERIALS REQUIRED
Nutrient agar
Nutrient broth
Distilled water
Water bath
2 L Erlenmeyer flask
Autoclave
32
COMPOSITION OF NUTRIENT BROTH:
Peptone
- 0.5 g
Yeast extract
- 0.3 g
NaCl
Distilled water 100 ml
- 0.5 g
pH -7 0.2
COMPOSITION OF NUTRIENT AGAR:
Peptone
- 0.5 g
Yeast extract
- 0.3 g
NaCl
Distilled water 100 ml
Agar
- 0.5 g
-2g
pH -7 0.2
PROCEDURE:
1. Dissolve the ingredients in 100 ml of distilled water.

2. Determine the pH and adjust with 0.1N HCl or 0.1N NaOH if necessary.
3. Take a conical flask with one-third free space and close the mouth with a cotton plug. Cover the
cotton plug with a stuff paper using a rubber band.
4. For solid medium, add the agar and boil to dissolve it.
5. Sterilize the medium by autoclaving at 15Lb pressure for 20 mins.
33
RESULT:
34
VIVA QUESTIONS:
1. What are the types of culture media?
2. What is the role of agar in culture media?
3. How will you prepare nutrient broth?
4. How will you prepare nutrient agar?
5. Why do we need to prepare culture media?
35
EXPERIMENT NO: 4
CULTURING OF MICROORGANISMS IN
BROTH AND IN PLATES (SPREAD PLATE, POUR
PLATE, STREAK PLATE)
36
EXP. NO: 4 CULTURING OF MICROORGANISMS - IN BROTH AND IN PLATES

DATE:
(SPREAD PLATE, POUR PLATE, STREAK PLATE)
AIM:
To learn the culturing of microorganism, isolation and preservation of bacterial culture.
PRINCIPLE:
Organisms grown in broth cultures cause turbidity, or cloudiness, in the broth. On agar,
masses of cells, known as colonies, appear after a period of incubation. Certain techniques will allow
bacterial cells to be widely separated on agar so that as the cell divides and produces a visible mass
(colony), the colony will be isolated from other colonies. Since the colony came from a single
bacterial cell, all cells in the colony should be the same species. Isolated colonies are assumed to be
pure cultures. Colony morphology is described in terms of shape, margin or edge, elevation and color
(Fig. 1).
Figure 1: Bacterial colony morphology description
37
MATERIALS REQUIRED:
Bunsen burner
Inoculating loop and needle
Glassware marking pencil
Culture media
INOCULATION AND OTHER ASEPTIC PROCEDURES
Essential points
There are several essential precautions that must be taken during inoculation procedures to
control the opportunities for the contamination of cultures, people or the environment.
Operations must not be started until all requirements are within immediate reach and must
be completed as quickly as possible.
Vessels must be open for the minimum amount of time possible and while they are open
all work must be done close to the Bunsen burner flame where air currents are drawn
upwards.
On being opened, the neck of a test tube or bottle must be immediately warmed by
flaming so that any air movement is outwards and the vessel held as near as possible to the
horizontal.
During manipulations involving a Petri dish, exposure of the sterile inner surfaces to
contamination from the air must be limited to the absolute minimum.
The parts of sterile pipettes that will be put into cultures or sterile vessels must not be
touched or allowed to come in contact with other non-sterile surfaces, e.g. clothing, the
surface of the working area, outside of test tubes/bottles.
Using a wire loop
Wire loops are sterilized using red heat in a Bunsen flame before and after use. They must
be heated to red hot to make sure that any contaminating bacterial spores are destroyed. The handle
of the wire loop is held close to the top, as you would a pen, at an angle that is almost vertical. This
leaves the little finger free to take hold of the cotton wool plug/ screw cap of a test tube/bottle.
Flaming procedure
The flaming procedure is designed to heat the end of the loop gradually because after use it will
contain culture, which may splutter on rapid heating with the possibility of releasing small particles of
culture and aerosol formation.

38
1. Position the handle end of the wire in the light blue cone of the flame. This is the cool
area of the flame.
2. Draw the rest of the wire upwards slowly up into the hottest region of the flame,
(immediately above the light blue cone).
3. Hold there until it is red hot.
4. Ensure the full length of the wire receives adequate heating.
5. Allow to cool then use immediately.
6. Do not put the loop down or wave it around.
7. Re-sterilize the loop immediately after use.
Using a pipette
Sterile graduated or dropping (Pasteur) pipettes are used to transfer cultures, sterile media
and sterile solutions.
1. Remove the pipette from its container/ wrapper by the end that contains a cotton wool plug,
taking care to touch no more than the amount necessary to take a firm hold.
2. Fit the teat.
3. Hold the pipette barrel as you would a pen but do not grasp the teat.
4. The little finger is left free to take hold of the cotton wool plug/lid of a test tube/bottle and
the thumb to control the teat.
5. Depress the teat cautiously and take up an amount of fluid that is adequate for the amount
required but does not reach and wet the cotton wool plug.
6. Return any excess gently if a measured volume is required.
7. The pipette tip must remain beneath the liquid surface while taking up liquid to avoid the
introduction of air bubbles which may cause spitting and, consequently, aerosol formation
when liquid is expelled.
8. Immediately put the now contaminated pipette into a nearby discard pot of disinfectant.
9. The teat must not be removed until the pipette is within the discard pot otherwise drops of
culture will contaminate the working surface.
39
Figure 2: Flaming a loop

Flaming the neck of bottles and test tubes
1. Loosen the lid of the bottle so that it can be removed easily.
2. Lift the bottle/test tube with the left hand.
3. Remove the lid of the bottle/cotton wool plug with the little finger of the right hand (Turn
the bottle, not the lid).
4. Do not put down the lid/cotton wool plug.
5. Flame the neck of the bottle/test tube by passing the neck forwards and back through a hot
Bunsen flame.
6. Replace the lid on the bottle/cotton wool plug using the little finger (Turn the bottle, not the
lid).
40
Figure 3: Flaming the neck of the bottles.

Culturing bacteria in broth
A broth culture is a bacterial culture in which desired bacteria are suspended in liquid broth; a
nutrient medium. The liquid broth is inoculated with the bacteria and left overnight for the bacteria to
grow. It is also referred to as liquid culture. The only difference between broth and agar media is that
broths do not contain an agar component. We use broth tubes primarily for specific assays, or (rarely)
for bacteria that will not form colonies on a solid surface. In broth a species may display motility
and/or a characteristic pattern of association among individual cells, such as chains or clusters, which
is not as obvious in agar cultures.
To prepare broth a dry medium is layered onto the surface of a measured volume of water as
with agar media, mixed, and distributed into individual loosely capped or vented capped tubes in
racks. Heating to dissolve components is sometimes required, but not always. Racks are steam
sterilized and then allowed to cool, and caps tightened to preventevaporation. Unlike preparation of
agar plates, tubes are prepared with media already in the incubation vessel. A large volume syringe
can facilitate distribution of media into individual tubes.
PROCEDURE
1. Light the Bunsen burner.
2. Place the culture you wish to transfer (tube A) near the tube of sterile broth that you will
inoculate (tube B).
41
3. Label the tube B with the name of the microorganism and the date
4. Hold the inoculating loop with your thumb and first two fingers. Heat the inoculating loop in
the Bunsen burner until it is red hot. Heat several inches of loop since that much of it will
contact the inside of the tubes. Allow the loop to cool for a few seconds while you hold it in
your hand. Do not put it down or allow the loop to touch any surface after it is sterile.
5. While continuing to hold the inoculating loop with your thumb and first two fingers, pick up
the tube A in your left hand and remove the cap with your thump and first two fingers, pick
up tube A in your left hand and remove the cap with the last two fingers of your right hand.
Keep the cap in your right hand and do not allow it to touch any surface.
6. Using your left hand, draw the open top of tube A gently through the flame of the Bunsen
burner. Do not hold it in the flame for more than a second.
7. Place the sterile loop out of the tube and continue to hold it in your hand.
8. Draw the open top of tube A gently through the flame of the Bunsen burner and then replace
the cap on the culture tube A. Replace tube A in its rack.
9. While still holding the loop with inoculums in your right hand, pick up the sterile tube of
broth (tube B) with your left hand. Remove the cap with the last two fingers of your right
hand. Keep the cap in your right hand and do not allow it to touch any surface.
10. Draw the open top of the sterile tube B gently through the flame. Do not hold it in the flame
for more than a second.
11. Place the loop containing the droplet of culture into the tube B and gently swirl it to transfer
the microorganisms to the sterile broth.
12. Remove the loop from the broth and continue to hold it in your hand.
13. Draw the open top of the sterile tube B gently through the flame and then replace the cap,
which should still be in your right hand, on tube B. Place tube B back in the test tube rack.
14. Heat the inoculating loop in the Bunsen burner until it is red hot. You can now place it on the
bench or in a rack.
15. At no time should the inoculating loop, the top of the tubes or the inside of the cap have
touched any non-sterile surface (especially your fingers or hand).
42
Culturing bacteria in plate

Spread Plate
The spread plate technique is an easy and direct way of attaining pure culture.When a mixture of
cells is spread on an agar surface,
every cell grows into a complete separate colony. A
microscopically visible growth or cluster of microbes on a solid medium can be visualized. Each
colony represents a pure culture. A small volume of dilute microbial mixture containing around 30 to
300 cells is transferred to the centre of an agar plate and spread evenly over the surface with a sterile
bent glass rod. Spread plates can be used to count the microbial population.
Procedure:
1. Pipette 0.1 ml of the sample on the centre of the plate containing agar medium.
2. Dip the L-rod into a beaker of ethanol and show it to a flame and allow it to cool.
3. Spread the cells evenly over the solid agar surface with the sterilized L-rod.
4. Incubate the plates at the desired temperature for overnight.
5. Note the results the next day morning.
Streak plate
The loop is used for preparing a streak plate. This involves the progressive dilution of an
inoculum of bacteria or yeast over the surface of solidified agar medium in a Petri dish in such a way
that colonies grow well separated from each other.
The aim of the procedure is to obtain single isolated pure colonies.
1. Loosen the top of the bottle containing the inoculum.
2. Hold the loop in the right hand.
3. Flame the loop and allow to cool.
4. Lift the bottle/test tube containing the inoculum with the left hand.
5. Remove the lid/cotton wool plug of the bottle/test tube with the little finger of the left hand.
6. Flame the neck of the bottle/test tube.
7. Insert the loop into the culture broth and withdraw.
At all times, hold the loop as still as possible.
8. Flame neck of the bottle/test tube.
9. Replace the lid/cotton wool plug on the bottle/test tube using the little finger. Place
43
bottle/test tube on bench.

10. Partially lift the lid of the Petri dish containing the solid medium.
11. Hold the charged loop parallel with the surface of the agar; smear the inoculum backwards
and forwards across a small area of the medium (see figure4 streaked area =A).
12. Remove the loop and close the Petri dish.
13. Flame the loop and allow it to cool. Turn the dish through 90 anticlockwise.
14. With the cooled loop streak the plate from area A across the surface of the agar in three
parallel lines (to B see figure 4). Make sure that a small amount of culture is carried over.
16. Flame the loop and allow to cool. Turn the dish through 90 anticlockwise again and streak
from B across
17. The surface of the agar in three parallel lines (to C see figure 3).
19. Flame the loop and allow to cool. Turn the dish through 90 anticlockwise and streak loop
across the surface of the agar from C into the centre of the plate (to D see figure 3).
20. Remove the loop and close the Petri dish. Flame the loop.
21. Seal and incubate the plate in an inverted position.
44
Figure 4: Streaking of Microorganisms.

Inoculating the cooled molten nutrient agar bottle
1. Pick up the cooled molten nutrient agar bottle.
2.Remove the lid from the molten nutrient agar bottle with the little finger of the right hand
which still holds the charged pipette. Do not put down the lid.
3.Flame the neck of the bottle.
4.Insert the pipette into the bottle and gently release the required number of drops of
inoculum onto the agar.
5.Flame the neck of the bottle and replace the lid.
6.Put the pipette into a discard pot. Remove the teat while the pipette is pointing into the
disinfectant.
Pour plate
A pour plate is one in which a small amount of inoculum from broth culture is added by
pipette to a molten, cooled agar medium in a test tube or bottle, distributed evenly throughout the
medium, thoroughly mixed and then poured into a Petri dish to solidify. Pour plates allow microorganisms to grow both on the surface and within the medium.
Most of the colonies grow within the medium and are small in size; the few that grow on
the surface are of the same size and appearance as those on a streak plate.
If the dilution and volume of the inoculum, usually 1 cm, are known, the viable count of
the sample i.e. the number of bacteria or clumps of bacteria, per cm can be determined.
Inoculation using a Pasteur pipette
45
1. Loosen the top of the bottle containing the inoculum.

2. Remove the sterile Pasteur pipette from its container, attach the bulb and hold in the right
hand.
3. Lift the bottle/test tube containing the inoculum with the left hand.
4. Remove the lid/cotton wool plug with the little finger of the right hand.
5. Flame the bottle/test tube neck.
6. Squeeze the teat bulb of the pipette very slightly, put the pipette into the bottle/test tube and
draw up a little of the culture. Do not squeeze the teat bulb of the pipette after it is in the
broth as this could cause bubbles and possible aerosols.
7. Remove the pipette and flame the neck of the bottle/test tube again, before replacing the
lid/cotton wool plug.
8. Place bottle/test tube on bench.
At all times hold the pipette as still as possible.
Pouring the pour plate
1. Roll the bottle gently between the hands to mix the culture and the medium thoroughly.
Avoid making air bubbles.
2. Hold the bottle in the left hand; remove the lid with the little finger of the right hand.
3. Flame the neck of the bottle.
4. Lift the lid of the Petri dish slightly with the right hand and pour the mixture into the Petri
dish and replace the lid.
5. Flame the neck of the bottle and replace the lid.
6. Gently rotate the dish to ensure that the medium covers the plate evenly.
7. Allow the plate to solidify.
8. Seal and incubate the plate in an inverted position.
Figure 5: Pouring the inoculated medium.
46
INCUBATION
The lid and base of an agar plate should be taped together with 2-4 short strips of adhesive
tape as a protection from accidental (or unauthorized!) opening during incubation. (Although tape
is the preferred method Parafilm could be used as an alternative for sealing the plates.)
Agar plates must be incubated with the medium-containing half (base) of the Petri dish
uppermost otherwise condensation will occur on the lid and drip onto the culture. This might cause
colonies to spread into each other and risk the spillage of the contaminated liquid.
The advantages of incubators are that they may be set at a range of temperatures and reduce
the possibility of cultures being interfered with or accidentally discarded. However, many cultures
suitable for use in schools will grow at room temperature in the interval between lessons and can
beincubated satisfactorily in a cupboard. The temperature of an incubator varies from the set
temperature, oscillating by several degrees in the course of use.
Water baths are used when accurately controlled temperatures are required, e.g. for enzyme
reactions and growth-temperature relationships, when temperature control of incubators is not
sufficiently precise. They should be used with distilled or deionised water to prevent corrosion and
emptied and dried for storage.
Maintaining stock cultures
It may be convenient to maintain a stock of a pure culture instead of re-purchasing it when
needed. Most of those considered suitable for use are also relatively easy to maintain by subculturing on the medium appropriate for growth but maintenance of stock cultures needs to be well
organized with attention to detail. Be prepared to transfer cultures four times a year to maintain
viability. Cultures on streak plates are not suitable as stock cultures.
Slope/ Slant cultures in screw cap bottles are preferred because the screw cap reduces
evaporation and drying out and cannot be accidentally knocked off (cf. a streak plate culture).
Slope cultures are preferred to broth (i.e. liquid medium) cultures because the first sign of
contamination is much more readily noticed on an agar surface. Two stock cultures should be
prepared; one is the working stock for taking sub-cultures for classes, the other is the
permanent stock which is opened only once for preparing the next two stock cultures. Incubate at
an appropriate temperature until there is good growth.
For growing strict aerobes it may be necessary to slightly loosen the cap for incubation (but
close securely before storage) if there is insufficient air in the headspace.
As soon as there is adequate growth, store the cultures at room temperature in either a
47
cupboard or drawer. Keep on the lookout for contamination.

Checking cultures for contamination
Evidence for a culture being pure or otherwise is given by the appearance of colonies on a
streak plates and of cells in a stained microscopical preparation. There should be uniformity of
colony form and cell form (and consistency with the appearance of the original culture!). It is
sensible to check purity on suspicion of contamination of the working stock culture from time to
time and of the permanent stock when preparing new stock cultures.
If a culture becomes contaminated, it is not advisable to try to remedy the situation by
taking an inoculum from a single colony from a streak plate of the mixed culture because of the
possibility of(1) not being able to distinguish between the colony forms of the contaminant and the
original culture, and (2) culturing a variant of the original culture that does not behave as the
original culture did. Instead, go back to the working (or permanent) stock cultures; that are what
they are for! Preventing contamination of cultures and the environment.
RESULT:
48
VIVA QUESTIONS:
1. How will you avoid contamination during culturing the microorganisms?
2. What is streak plate method and pour plate method?
3. What is Swabbing?
4. What are the different types of streaking methods available?
5. How will you prevent the contamination of culture?
49
EXPERIMENT NO: 5
STAINING TECHNIQUES & MOTILITY
TEST
50
EXP. NO: 5a
STAINING TECHNIQUES GRAM STAINING
DATE:
AIM:
To perform Gram stain to differentiate two principal groups of bacteria: Gram +ve and
Gram -ve
PRINCIPLE:
There are several types of stains which are commonly used in microbiology. The first is a
simple stain, which uses only one reagent which provides contrast between the background and
the heat-fixed bacterium itself. The bacterium takes up stain and becomes colored, while the
background remains unstained. Simple stains are typically used on bacterial smears which have
been heat-fixed and thus contain non-living microbes.
A second type of stain is a negative stain, which uses a single reagent to provide contrast
between the background and the living bacterium. Thus, the background is stained, while
bacterium does not take up any stain. Negative stains are typically used when observing live
bacteria is desired.
A differential stain is a type of staining that allows you to distinguish between types of
bacteria or between specific structures in a bacterium for example, Gram Positive and Gram Negative
bacteria. A differential stain typically uses two or more reagents a primary stain (Crystal Violet)
and a counter stain (Safranin). Bacteria stain differentially because of chemical and physical
differences in their cell wall. G +ve cell wall consists of many layers of Peptidoglycan. The
Crystal Violet iodine complex is larger than than the crystal violet or iodine molecules that
initially enter the cell and hence it cannot pass through the thick peptidoglycan layer. So the
counter stain cannot be absorbed by the cells and it retains the primary stain colour. In Gram ve
cells, alcohol dissolves the outer lipopolysaccharide layer and the Crystal violet iodine complex
will be washed out through the thin layer of peptidoglycan. So, G ve cells will absorb the counter
stain.
Chemically, there are two main types of stains: basic stains, which have a positivecharge
(cationic) and acidic stains, which have a negative charge (anionic). Basic stains have an affinity
for negative components of cells, and include dyes such as methylene blue, crystal violet, and
carbolfuchsin. Acidic stains have an affinity for positive components of cells, and include dyes
such as nigrosin, India ink, and picric acid. Since cell walls are negatively charged, a positive dye
will be attracted to and stain the cell wall, whereas a negative dye will be repulsed by the cell wall
51
and not directly stain the cell.

PROCEDURE FOR PREPARING A BACTERIAL SMEAR
1. Obtain a glass slide and clean if necessary.
2. Using a broth culture:
a. Gently agitate your culture broth tube to disperse the bacteria.
b. Sterilize your inoculating loop using an incinerator or a Bunsen burner, and
let cool for 20-30 seconds.
c. Place loop in the bacterial broth and put the loopful of the broth onto the glass
slide. Rub the drop into a nickel-sized smear. Sterilize the loop again to kill any
remaining bacteria. Let the smear air dry completely. Do not use heat to dry
your smear!
3. Using an agar plate:
a. Place a small drop of water in the center of the slide.
b. Sterilize your inoculating loop using an incinerator or a Bunsen burner, and let cool
for 20-30 seconds.
c. Use the sterile loop to pick up a small amount of bacterial growth from the surface
of the plate. Do not dig into the agar. Put the loopful of bacteria into the drop of
water on the glass slide, and rub the drop into a nickel-sized smear. Sterilize the
loop again to kill any remaining bacteria Let the smear air dry completely. Do not
use heat to dry your smear!
4. Heat fixthe slide.
a. Incinerator method: Hold the slide with a wooden clothes pin approximately
1cm over the barrel of a hot incinerator for 2030 seconds. Let the slide cool.
b. Bunsen burner method: Hold the slide with a wooden clothes pin, and pass 1012 times through the flame. Let the slide cool.
5. Optional: After the slide has cooled us a marker or wax pencil to outline the area of
thesmear on the underside of the slide. This will help you locate your sample later.
GRAM STAIN
The Gram stain is a differential stain which distinguishes bacteria based on cell wall
properties. Bacterial cell walls are composed primarily of peptidoglycan and bacteria can be
classified into two main groups dependent on the amount of peptidoglycan present in their cell
52
wall. Gram-positive organisms have a thick layer of peptidoglycan, whereas Gram-negative

organisms have a thin layer of peptidoglycan, plus an additional outer membrane that is absent
in Gram-positive organisms.
In the gram staining procedure, the primary stain is crystal violet, and all cells take up
the purple crystal violet stain. Following the primary stain, Grams Iodine is applied to
thebacterial smears. The iodine acts as a mordant, enhancing the ability of the stain to enter
and bind to the bacteria. Specifically, the iodine binds with crystal violet and locks it into
peptidoglycan of bacteria. It also intensifies the purple color. The decolorizing agent used in
the gram staining procedure is 95% ethanol, which is a lipid solvent that melts the Gramnegative outer membrane and leads to decolorization of Gram-negative cells. It also dehydrates
proteins, helping the primary stain to remain in Gram-positive cell walls. The counter stain
then used is Safranin, which stains the decolorized Gram-negative cells pink. Thus, at the endof
the staining procedure, Gram-positive cells are purple and Gram-negative cells are pink.
Note: It is preferable to use fresh cultures for the Gram stain. Old cultures may stain Gramvariable (a mix of purple and pink) because they decolorize easily.
CULTURES NEEDED:
Nutrient broth tubes or plates of the following:
Escherichia coli
Staphylococcus xylosus
Bacillus megaterium
PROCEDURE:
1. Prepare a bacterial smear with a mixture of all 3 organisms (i.e. 1-2 loop full of each) listed
above and heat fix it.
2. Place the slide on a staining tray, and cover the smear with crystal violet. Allow to stain for
60 seconds.
3. Tilt the slide and gently rinse with distilled water until the stain is removed.
53
4. Cover the smear with Grams Iodine, and allow to sit for 60 seconds.
5. Tilt the slide and gently rinse with distilled water.
6. IMPORTANT STEP: Tilt the slide and let 2-3 drops of Decolorizer run over the slide.
If the last drop is still purple, continue decolorizing, 2-3 drops at a time, until the
decolorizer runs clear. Rinse with distilled water.
7. Cover the smear with Safranin, and stain for 45 seconds.
8. Tilt the slide and rinse with distilled water.
9. Place the slide in a book of Bibulous paper and blot to dry. You do not need a cover slip!
Observe the slide under oil immersion, and draw what you see in the results section below.
You should see: Small purple cocci (spheres) which are the gram-positive S.xylosus, large
purple rods, which are the gram-positive B. megaterium, and small pinkrods, which are the
gram-negative E. coli. Label these in your drawing.
10. Clean your microscope with lens cleaner, paying extra attention to the 40X and 100X
objectives.
OBSERVATION:
54
RESULT:
55
VIVA QUESTIONS:
1. What are the several advantages of differential staining procedures compared with simple
staining techniques.
2. Give the purpose of each of the following reagents in a differential staining procedure:
a. Primary stain
b. Counter stain
c. Decolorizing agent
d. Mordant.
56
3. Why is it important for the counter stain to be a lighter color than the primary stain?
4. What is the purpose of staining bacteria?
5. What is the most common differential staining procedure used in microbiology?
57
EXP. NO: 5b
MOTILITY TEST
DATE:
AIM:
To learn to make hanging drop slides and then use this technique to observe the motility of
the live bacteria
PRINCIPLE:
Motility is an important characteristic used to identify microorganisms. Three bacteria will
be used which vary in size, shape and arrangement of flagella, and types of motion. Specifically
Pseudomonas aeruginosa, a monotrichous bacterium exhibits high motility using a polar
flagellum, Bacillus cereus that moves by peritrichous flagella and Spirilliumvolutans, a helical cell
that moves using large bipolar tufts of flagella (possible mixture of amphitrichous and
lophotrichous organisms) (Refer to Figure 1).
Flagellae are, in effect, rotary motors comprised of a number of protein rings embedded in
the cell wall. In action the filament rotates at speeds from 200 to more than 1,000 revolutions per
second, driving the rotation of the flagellum. The direction of rotation determines the movement
of the cell. Counterclockwise rotation of polar flagella thrusts the cell forward with the flagellum
trailing behind. Periodically, the direction of rotation is briefly reversed, causing what is known as
a "tumble" resulting in reorientation of the cell.
58
Investigation of the movement of live bacteria by microscope is possible e. g. with hangingdrop preparation (Figure 2). A suspension of microorganisms is placed in the centre of a cover slip
and turned over with a special glass slide with a hollow depression in the centre. When observing live
bacteria, be careful not to confuse motility with Brownian motion resulting from bombardment by
water molecules.
In Brownian motion, organisms all vibrate at about the same rate and maintain a relatively
constant spatial relationship with one another, whereas bacteria that are definitely motile progress
continuously in a given direction. Motility can be observed most satisfactorily in young cultures (24
or 48 hours), because older cultures tend to become non-motile. An old culture may become so
crowded with inert living and dead bacteria that it is difficult to find a motile cell. In addition, the
production of acid or other toxic products may result in the loss of bacterial motility.
Figure 2:Hanging drop preparation: (a) From the examined bacterial culture, (b) Prepare a
weak suspension in a drop of water in the center of cover slip. (c) Put the glass slide with the
hollow depression upside down over the cover slip preparation so that the drop of the culture is
in the center of the depression, and then quickly turn it over.
MATERIALS AND EQUIPMENTS:
Glass slide with hollow depression
Cover slip
Inoculating loop
Bunsen burner
Pipette, sterile pipette tips
Light microscopy
Immersion oil
Benzene
59
PROCEDURE:
1. Clean the cover slip such that it is free from grease and wipe it with a dry cotton tissue.
2. Place a thin film of mountant around the edge of the cover slip.
3. Place a loop full of culturein the centre of the cover slip.
4. Turn the slide carefully upside down to make the drop hang in the cavity.
5. Observe the edge of the dropusing a high power objective under a microscope to visualize the type
of motility of the bacteria.
OSERVATION:
60
RESULT:
61
VIVA QUESTIONS:
1. Name some organisms that are motile in nature.
2. What is the importance of performing motility test?
3. Explain the term monotrichous
4. What is chemotaxis?
5. What is gliding motility?
62
EXPERIMENT NO: 6
QUANTITATION OFMICROORGANISMS
63
EXP. NO: 6
QUANTITATION OFMICROORGANISMS
DATE:
AIM:
To learn the technique that will help in quantifying the number of microorganisms present in
the given sample.
PRINCIPLE:
The counting of bacteria is important if you want to know the number of bacteria in a sample.
The common methods are: Plate count, Direct count, and Turbidometric.
Plate count- is used on the premise that each viable bacterium will produce a colony when
growing on a agar plate. A sample of the material to be counted is suspended in liquid and placed in
a empty petri plate. Next, melted agar is poured into the plate. After incubation, each organism
produces a colony in the agar that can be counted.
There are two main advantages of the plate count over other methods. Only viable organisms
are counted, which are the ones considered to be important. Samples with small methods can also be
counted. The disadvantages are related to size and frequency. Bacteria are usually present in large
numbers. E.coli could easily contain over one billion cells/ml. Some bacteria will stick together,
giving rise that two different organisms may produce the appearance of only one colony.
Direct count - Organisms in a suspension of bacteria are placed on a slide that has been ruled
into squares and can hold a specific amount of volume. By counting the bacteria that appear on the
grid areas, the number of organisms in a sample can be calculated.
The direct count is much faster than the plate count but has its disadvantages. There must
be a certain multiple number of organisms before there are enough to be seen, and both viable
and nonviable organisms appear the same under the microscope.
Turbidometric - turbid simply means cloudy. In this method, a spectrophotometer measures
the turbidity on bacteria in a broth. The more bacteria present, the cloudier the broth.
PLATE COUNT: PROCEDURE
The purpose of this exercise is to quantify the number of bacteria in a broth culture of E.
coli. In microbiological research, it is often necessary to be able to quantify the number of
living bacteria in a particular sample. One of the major ways to do this is using viable plate
counts, in which bacterial cells from a liquid culture are spread onto an agar plate. The plate is
64
incubated, the number of colonies that grow on the plate are counted, and the number of original
bacterial cells in the culture is determined. In most cases, however, the liquid culture being
quantified contains too many cells to be directly plated onto agar plates there would be so
much growth that it would be impossible to count individual colonies! Therefore, the liquid
culture needs to be diluted, often 1-million-fold, before it can be plated.
When such a large dilution is required, an accurate dilution cannot be made in a single
dilution step and it is necessary to make serial dilutions. Serial dilutions are a step-wise set of
dilutions which sequentially dilute the bacterial culture. One or more of the dilutions are then plated
on the agar plates to determine the number of colonies present in the original culture.
Figure 1: Serial Dilution.

Only plates containing between 30 and 300 colonies are counted to ensure statistically
significant data. To estimate the number of bacterial in the original culture, the # of colonies on the
plate is multiplied by the total dilution plated. For example, suppose 0.1 ml of a 10-6dilution was
plated, and 123 colonies were counted following incubation. The total dilution plated would be 107
(since only 0.1 ml was plated), and the number of bacteria/ml of the original culture would be: (123) x
1/10-7= 1.23 x 109CFU/ml. Note that the results are expressed as colony forming units(CFU) per ml.
MEDIA NEEDED: (per group of four)

3 Nutrient agar plates
7 Dilution blanks containing 9 ml water
65
CULTURES NEEDED:
Overnight broth culture of Escherichia coli
PROCEDURE:
1. Label the dilution blanks as follows: 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7.
2. Label the agar plates as follows: 10-6, 10-7and 10-8.
3. Using a sterile pipette, transfer 1 ml of the E. coli broth culture into the tube labeled 10-1. Mix
thoroughly.
4. Using a new sterile pipette, transfer 1 ml of the 10-1 tube into the tube labeled 10-2. Mix
thoroughly.
5. Using a new sterile pipette, transfer 1 ml of the 10 -2 tube into the tube labeled 10-3. Mix
thoroughly.
6. Using a new sterile pipette, transfer 1 ml of the 10 -3tube into the tube labeled 10-4. Mix
thoroughly.
thoroughly.
8. Using a new sterile pipette, transfer 1 ml of the 10 -5tube into the tube labeled 10-6. Mix
thoroughly.
thoroughly.
10. Using a new sterile pipette, transfer 0.1 ml of the 10-5 tube to the nutrient agar plate labeled 10-6,
and spread the liquid thoroughly and evenly over the surface of the plate using a sterile
disposable spreader. Be careful not to let the spreader dig into the agar!
[Note: Since were only plating 0.1 ml of the 10-5dilution, the total dilution plated is 10-6.]
and spread the liquid thoroughly and evenly over the surface of the plate using a sterile disposable
spreader.
and spread the liquid thoroughly and evenly over the surface of the plate using a sterile disposable
spreader.
13. After the liquid has absorbed into the plates, tape them closed on both sides. Make sure your
66
plates are labeled with your name, date, and the organism, and incubate upside down at 30 C.
OBSERVATION:
Examine the nutrient agar plates for growth, and count the number of colonies on each
plate.Remember, the number has to be between 30 and 300 in order to be statistically accurate. If
your plate has fewer than 30 colonies, record the number as TFTC for too few to count. Ifyour
plate has more than 300 colonies, record the number as TNTC for too numerous to count. Then,
use the formula on the previous page to determine the number of CFU/ml of the
original broth culture.
Dilution
Number of colonies
Number of CFU/ml of original broth culture

(Remember to use scientific notation!)
10-6
10-7
10-8
67
RESULT:
68
VIVA QUESTIONS:
1. Why do you think it is important to be able to quantify the number of viable bacteria in a
sample?
2. Give an example of an industrial setting where quantifying viable bacteria would be a useful
tool.
3. Name the methods available for quantifying the bacteria
4. What is the principle behind in the turbidometric method?
5. What is CFU?
69
EXPERIMENT NO:7
CHEMICAL CONTROL OF
MICROORGANISMS & ANTIBIOTIC
SENSITIVITY ASSAY
70
CHEMICAL CONTROL OF MICROORGANISMS
EXP. NO:7a
DATE:
AIM:
To study the effect of chemicals on the growth of microorganisms.

PRINCIPLE:
Antimicrobial chemicals, disinfectants, and antiseptic solutions are routinely used to
decontaminate surfaces, objects, and even skin and tissues. Examples include household cleaners
such as Lysol and Clorox, first aid treatments such as isopropanol and hydrogen peroxide, and
hospital cleaners such as Amphyll and Vesphene. Chemical agents work through two main
mechanisms: (1) Alteration of cell walls or cytoplasmic membranes and (2)Interference with
protein and nucleic acid structure. Like antibiotics, chemical agents may be
static or cidal in action, although most of the newer chemical agents are microbiocidal.
There are many methods to test the effectiveness of antimicrobial chemicals, including the
disk diffusion assay. In this assay, small filter disks are impregnated with the chemical to be tested,
and are placed on a plate inoculated to form a bacterial lawn (even, confluent bacterial growth). The
plates are incubated to allow growth of the bacteria and time for the chemicals to diffuse into the
agar. As a chemical diffuses into the agar, it becomes less concentrated. If an organism is
susceptible to a chemical, a clear zone of inhibition will appear around the disk where the growth
has been inhibited. The size of this zone of inhibition depends on the sensitivity of the bacteria to
the specific chemical and the chemical's ability to diffuse through the agar.
We will be testing four different chemicals today using the disk diffusion assay. Students
may test the chemicals provided in the laboratory, or they are welcome to bring in their own
disinfectants for testing.
MATERIALS NEEDED: (per group of 4)
1 Mueller-Hinton plate
CULTURES NEEDED:
Escherichia coli
Staphylococcus aureus
71
PROCEDURE:
1. With a marker, divide the bottom of the plate into quadrants. Label A, B, C, and D. (Key:
A = isopropanol; B= hydrogen peroxide; C= Lysol antibacterial disinfectant;
D= Micro-90 dish detergent) Note: If you brought your own disinfectants to test,
record those here: A =
C=
B=
D=
2. Select a broth culture of either E. coli or S. aureus. Coordinate with another lab group in your
class so that each group chooses a different one. Write the name of the bacteria on the plate, as
well as your initials and the date.
3. Gently agitate your broth tube to resuspend any bacteria that have settled to the bottom of the
tube. With a sterile swab, dip into the broth tube and completely swab the surface of the plate.
Rotate the plate 90 and repeat, dipping a fresh sterile swab into the broth tube and completely
swabbing the surface of the plate. There should be a blanket of uniform growth following
incubation.
4. Using forceps dip a sterile filter disk in Solution A, allow the excess solution to drip off, and
then place the disk in the center of Quadrant A. Repeat with Solutions B, C, and D.
5. GENTLY press each disk onto the agar to make sure it stays, but do not puncture the agar.
6. Tape the plate, and incubate at 37 C for 48 hours.
OBSERVATION:
Using a plastic ruler, measure the diameter in millimeters (mm) of the zone of inhibition for
each chemical tested. Record your results in the chart below.
Chemical agent
E. coli Zone of Inhibition
72
S. aureusZone of Inhibition
RESULT:
73
VIVA QUESTIONS:
1. What is bacteriostatic?
2. What is bacteriocidal?
3. What is zone of inhibition?
4. What is disc diffusion method?
5. Using your textbook as a reference, describe the mechanisms of action of isopropanol,

hydrogen peroxide, and Clorox bleach against bacteria.
74
EXP. NO: 8b
ANTIBIOTIC SENSITIVITY ASSAY
DATE:
AIM:
To study the effect of antibiotics in the growth of microorganisms.
PRINCIPLE:
Antimicrobial chemotherapy is the use of chemicals to inhibit or kill microorganisms in or
on the host. Antimicrobial therapy is based on selective toxicity. This means that the agent used
must inhibit or kill the microorganism in question without seriously harming the host. In order to
be selectively toxic, a chemotherapeutic agent must interact with some microbial function or
microbial structure that is either not presents in the host, or is substantially different from that of
the host.
For example, in treating infections caused by prokaryotic bacteria, the agent may prevent
peptidoglycan synthesis or inhibit bacterial enzymes such as RNA polymerase or DNA gyrase.
Human cells do not contain peptidoglycan or DNA gyrase, and have structurally different RNA
polymerases. Therefore, drugs with these targets will have little, if any, effect on the host.
Based on their origin, there are 2 general classes of antimicrobial chemotherapeutic
agents:
(1) Antibiotics, which are substances produced as metabolic products of one microorganism
which inhibit or kill other microorganisms.
(2) Antimicrobial chemotherapeutic chemicals, which are chemicals synthesized in the
laboratory which can be used therapeutically on microorganisms.
Today the distinction between the two classes is not as clear, since many antibiotics are
extensively modified in the laboratory (semisynthetic) or even synthesized without the help
ofmicroorganisms.
Some antimicrobial agents are cidal in action: they kill microorganisms (e.g.,
penicillins, cephalosporins, and neomycin). Others are static in action: they inhibit microbial
growth long enough for the bodys own defenses to remove the organisms (e.g., tetracyclines,
erythromycin, and sulfonamides).
Antimicrobial agents also vary in their spectrum. Drugs that are effective against a variety
of both gram-positive and gram-negative bacteria are said to be broad spectrum (e.g.,
tetracycline, streptomycin, cephalosporins, ampicillin, and sulfonamides). Those effective
againstjust gram-positive bacteria, just gram negative bacteria, or only a few species are termed
75
narrowspectrum(e.g., penicillin G, erythromycin, clindamycin, and gentamicin). The major

classesofantibiotics and their mechanisms of action have been covered in the text used in lecture.
Antibiotic sensitivity testing is used to determine the susceptibility of bacteria to various
antibiotics. This standardized test is used to measure the effectiveness of a variety of antibiotics on
a specific organism in order to prescribe the most suitable antibiotic therapy. A series of antibioticimpregnated paper disks are placed on a plate inoculated to form a bacterial lawn (even, confluent
growth). The plates are incubated to allow growth of the bacteria and time for the antibiotics to
diffuse into the agar. As the drug diffuses into the agar, its strength decreases.
If an organism is susceptible to an antibiotic, a clear zone of inhibition will appear around
the disk where the growth has been inhibited. The size of this zone of inhibition depends on the
sensitivity of the bacteria to the specific antibiotic and the antibiotic's ability to diffuse through the
agar. After incubation, the zones of inhibition are measured and compared with tables giving the
interpretation of measurement for each antibiotic.
MEDIA NEEDED: (per pair)
1 Mueller Hinton Agar plate
CULTURES NEEDED:
Escherichia coli, Staphylococcus aureus
PROCEDURE:
1. Each pair will choose one of the five cultures listed above. Make sure to label your plate
with your names or initials and the name of the organism.
2. Gently agitate your broth tube to resuspend any bacteria that have settled to the bottom of
the tube. With a sterile swab, dip into the broth tube and completely swab the surface of
the M-H plate. Rotate the plate 90 and repeat, dipping a fresh sterile swab into the broth
tube and completely swabbing the surface of the plate. There should be a blanket of
uniform growth following incubation.
3. Carefully place the provided antibiotic discs onto the plate using the disc dispenser
provided. (Your lab instructor will demonstrate how to properly use the disc dispenser.)
You will need to take sterile forceps and lightly touch each disc to make sure it will stay
in place.
4. Incubate plates inverted at 37 C for 48 hours.
76
OBSERVATION:
Using a plastic ruler, measure the diameter in millimeters (mm) of the zone of inhibition for
each antibiotic tested. Record your results in the chart below.
Inhibition Zone Diameter
(mm)
Antibiotic
RESULT:
77
VIVA QUESTIONS:
1. What are the classes of anti-microbial chemotherapeutic agents?
2. What is broad spectrum antibiotic?
3. What is narrow spectrum antibiotic?
78
4. Name some examples of antibiotics
5. What is antibiotic sensitivity test?
79
EXPERIMENT NO: 9
BACTERIAL GROWTH CURVE
80
EXP. NO: 9
BACTERIAL GROWTH CURVE
DATE:
AIM:
To study the growth curve of bacteria.
PRINCIPLE:
Bacterial population growth studies require inoculation of viable cells into a sterile broth
medium and incubation of the culture under optimum temperature, pH, and gaseous conditions.
Under these conditions, the cells will reproduce rapidly and the dynamics of the microbial growth can
be charted by means of a populationgrowth curve, which is constructed by plotting the increase in
cell numbers versus time of incubation and can be used to delineate stages of the growth cycle. It also
facilitates measurement of cell numbers and the rate of growth of a particular organism under
standardized conditions as expressed by its generation time, the time required for a microbial
population to double.
The stages of a typical growth curve (figure below) are:
1. Lag phase: When the cells are adjusting to their new environment. During this phase, cellular
metabolism is accelerated, resulting in rapid biosynthesis of cellular macromolecules,
primarily enzymes, in preparation for the next phase of the cycle.Although the cells are
increasing in size, there is no cell division and therefore no increase in numbers.
2. Logarithmic
(log)/Exponential
phase:
Under optimum nutritional and physical
conditions, the physiologically robust cells reproduce at a uniform and rapid rate by binary
fission.
Thus there is rapid exponential increase in population, which doubles regularly until a
maxim um number of cells is reached. The length of the log phase varies, depending on the
organism s and the composition of the medium, although the average may be estimated to last
6 to 12 ho urs.
3. Stationary phase: During this stage, the number of cells undergoing division is equal to the
number of cells that are dying. There is no further increase in cell number and the population
is maintained at its maximum level for a period of time. The primary factors responsible for
this phase are the depletion of some essential metabolites and the accumulation of toxic acidic
or alkaline en d products in the medium.
4. Decline or death phase: Because of the continuing depletion of nutrients and buildup of
metabolic wastes, the microorganisms die at a rapid and uniform rate. This decrease in
81
population closely parallels its increase during the log phase. Theoretically, the entire
population should die during a time interval equal to that of the log phase. This does not
occur, however, since a small number of highly resistant organisms persist for an
indeterminate length of time.
Figure 1: Bacterial growth curve.

Hypothetical growth curve for an hypothetical population. Note the four phases of growth:
lag; exponential; stationary and death.
Construction of a complete bacterial growth curve requires that aliquots of a 24-hour shakeflask culture be measured for population size at intervals during the incubation period; however,
such a procedure does not lend itself to a regular laboratory
session. This experiment is
designed to include only the lag, log and possibly stationary phases of population growth.
INDIRECT METHOD
You will determine generation time with indirect and direct
collect,
once
it
has
been plotted onto a
methods by using data you
graph like
the
one shown below.
Indirect determination is made by simple extrapolation from the log phase as illustrated in the
figure b elow. Select two points on the optical density (OD) scale, such as 0.2 and 0.4, that represent a
doublin g of turbidity. Using a ruler, extrapolate by drawing a line between each of the selected
optical densities on the ordinate and the plotted line of the growth curve. Then draw perpendicular
lines from these end points on the plotted line of the growth curve to their respective time intervals on
the abscissa.
With this information, determine the generation time as follows:
Generation Time GT = t (O.D. 0.4 t (O.D. 0.2)
82
Figure 2: To Calculate generation time.

Generation time is determined using two points within the exponential portion of the
growth curve w here the population doubles in size.
In this example:GT= 263 minutes -222 minutes =43 minutes, so the generation time of
this hypotheticalPopulation is 43 minutes.
PROCEDURE
Starting at time 0, you will do the following every 30 minutes for a total of 4 time points:
1. Set and calibrate the spectrophotometer. To do this, set the wavelength knob (top of
instrument) to 600 nm.
Table supplies
Spectrophotometer (Spec 20)
Side arm flask with E. coli in TSB (each table Media blankswill have a different start
time for inoculation) Then, adjust the meter needle to zero by rotating the zero control knob (left
side, front of instrument, see figure below).
2. Blank the spectrophotometer. To do this, insert a test tube containing the medium
you are using (called a blank) into the sample holder. Adjust the meter needle to read
100 % transmittance by rotating the light control knob.
3. Remove the blank from the instrument.
4. Swirl culture flask to resuspend organisms and carefully pour medium into side arm.
Wipe down the side arm if it is at all dirty on the outside. Insert side arm into sample
83
holder.Try to maintain the same orientation of arm to flask for each time point reading.
5. Read and record the % transmittance and the optical density (OD) of the culture.
OBSERVATION:
Record your indirect measurements of OD and % transmittance in the table below.
Plot the OD on the Y axis and incubation times on the X axis of the provided semi-log
graph paper.
Identify the log phase (if present) for the graph and determine the generation time of your
bacterial culture using the indirect method described in the introduction to this lab exercise.
Remember that the straight line portion of the curve will represent the log phase. Find two points
that show a doubling of the O.D. Determine the time between to estimate doubling, generation
time.
84
RESULT:
85
VIVA QUESTIONS:
1. What are the different phases of growth curve?
2. What is doubling time?
3. What is log phase?
4. In which phase of the bacterial growth primary metabolites will be formed?
5. In which phase of the bacterial growth secondary metabolites will be formed?
86
EXPERIMENT NO: 11
EFFECT OF DIFFERENTPARAMETERS
ON BACTERIAL GROWTH
(TEMPERATURE, UV RADIATION)
87
EXP. NO: 11
EFFECT OF DIFFERENT PARAMETERS ON BACTERIAL GROWTH

(TEMPERATURE AND UV IRRADIATION)
DATE:
AIM:
To study the effect of different parameters on bacterial growth (Temperature, UV

irradiation)
Effect of Temperature on growth
OBJECTIVES
1. To determine optimum growth temperature and observe effects of temperature on
such processes as pigment production.
2. Define the different temperature categories.
PRINCIPLE
Microbes grow over a broad temperature range that extends from below 0 C to above
100C. Their range is dependent upon their specific cellular enzymes, which will increase in
activity as the temperature increases until the point at which they denature. Temperature ranges
for individual species consist of the following cardinal temperature points: minimum growth
temperature, optimum growth temperature, and maximum growth temperature. Based on these
ranges, they can be grouped into the following categories:
1. Psychrophiles: Optimum growth between -5 C and 20 C. These bacteria can be found in
supercooled waters of the arctic and Antarctic.
2. Psychrotrophs: Mesophilic bacteria that can grow a low temperatures, such as 4 C, or
refrigeration temperatures.
3. Mesophiles: Optimum growth between 20C and 50 C. Most bacteria fall into this
category, forexample human pathogens that grow at an optimum of 35 C to 40 C.
4. Thermophiles: Optimum growth between 50 C and 80 C. Bacteria in this group occur in
soilswhere temperatures reach greater than 50 C or in compost piles where fermentation
activity can cause temperatures to exceed 60- 65C.
5. Hyperthermophiles: Growth optimum above 80 C. These organisms include many of the
Archaea, including those that live in deep sea hydrothermal vents heated by volcanic hot
springs.
88
PROCEDURE:
1. Inoculate 100 l of your organism into different tubes of nutrient broths that are
labeled with different temperatures.
2. Incubate all broths at the appropriate pH.
RESULTS: (At OD 600nm)

Temp 1 (0C)
Species
Temp 2 (0C)
Temp 3 (0C)
Temp 4 (0C)
Lethal effects of ultraviolet light

To perform experiments to test the varying resistance to UV light of different microbes
PRINCIPLE
UV light is non-ionizing short wavelength radiation that falls between 4 nm and 400 nm
in the visible spectrum.
Most bacteria are killed by the effects of UV light and it is routinely used to sterilize
surfaces, such as work areas of transfer hoods used for the inoculation of cultures. The primary
lethal effects of UV are due to its mutagenic properties. When DNA absorbs UV light, it
causes the formation of pyrimidine dimers. These form when a covalent bond forms between
two adjacent thymine or cytosine molecules in a strand of DNA. These dimers deform the DNA
molecule, so that DNA polymerase is unable to replicate the strand of DNA past the site of
dimer formation and genes can no longer be transcribed.
Cells have evolved various repair mechanisms to deal with the damage (including photoreactivation, nucleotide excision repair and the SOS system). However, if sufficiently large
numbers of dimers form in the DNA, the systems cannot cope and begin making errors by
89
inserting incorrect bases for the damaged bases, eventually resulting in cell death.
Figure: Effect of UV radiation on microorganisms.

PROCEDURE:
1. Label 5 plates for your test organism: control, 5 seconds, 1 min, 2 min, and 5 min.
2. Mix culture tube and transfer 0.1 ml onto each plate. Top spread with a metal spreader.
3. For UV exposure plates, remove Petri dish lid from plate and cover of plate with an
index card. Being careful not to expose your eyes to the UV light, place the plate under
the UV illuminator box and expose to UV for proper amount of time. Keep plates at least
3 from UV illuminator, too close is too powerful and will kill everything.
4. For control plate, keep lid on so not to expose to UV.
5. Once complete, replace Petri dish lids, invert and incubate all plates at appropriate
temperature.
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RESULTS:
Record the results: substantial growth (+++), moderate growth (++), little growth (+), or
no growth (-).
Organisms
Survival
Growth
Exposure time
Control
5 sec
1min
91
2min
5min
VIVA QUESTIONS:
1. What are mesophiles?
2. What are acidophiles?
3. How does UV light cause lethal effects to microbes?
4. What are hyperthermophiles?
5. Name some examples of mesophile.
92

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Laboratory safety and aseptic

Microscopy light microscopy

Culture media Types,

Bacterial growth curve

Effect of different parameters on

LABORATORY SAFETY AND ASEPTIC TECHNIQUES

However, many types of microorganisms are potentially pathogenic.

Staphylococcus aureus, Staphylococcus epidermis, and Streptococcus mutans.

Complete all operations as quickly as possible, but without any hurry.

Vessels must be open for the minimum amount of time possible.

Using a wire loop

Flaming the neck of bottles and test tubes

Horizontal flow type

Vertical flow type

HEAVY METALS AND THEIR COMPOUNDS:

2. How will you avoid the exposure to microorganisms?

3. How will you dispose the microbiological waste?

4. Why the aseptic technique needs to be followed?

5. If any accidents take place what will you do?

MICROSCOPY LIGHT MICROSCOPY

To prepare a wet mount.

The compound light microscope uses visible

Figure 1: Light Microscope.

science of microscopy is based on the following concepts and principles:

Prepared slides of bacteria

PROCEDURE FOR CLEANING A MICROSCOPE:

VIVA QUEST IONS:

e. Mechanical stage control

4. What is refractive index?

5. Name the types of microscopy techniques

CULTURE MEDIA TYPES, PREPARATION OF

Mac conkey agar:

Mannitol salt agar:

COMPOSITION OF NUTRIENT BROTH:

Distilled water 100 ml

COMPOSITION OF NUTRIENT AGAR:

Distilled water 100 ml

1. Dissolve the ingredients in 100 ml of distilled water.

2. What is the role of agar in culture media?

3. How will you prepare nutrient broth?

4. How will you prepare nutrient agar?

5. Why do we need to prepare culture media?

EXP. NO: 4 CULTURING OF MICROORGANISMS - IN BROTH AND IN PLATES

(SPREAD PLATE, POUR PLATE, STREAK PLATE)

Figure 1: Bacterial colony morphology description

culture and aerosol formation.

Figure 2: Flaming a loop

Figure 3: Flaming the neck of the bottles.

Culturing bacteria in plate

every cell grows into a complete separate colony. A

bottle/test tube on bench.

Figure 4: Streaking of Microorganisms.

1. Loosen the top of the bottle containing the inoculum.

Figure 5: Pouring the inoculated medium.

cupboard or drawer. Keep on the lookout for contamination.

2. What is streak plate method and pour plate method?

4. What are the different types of streaking methods available?