Exp.

No: 1
Date:
INTRODUCTION, LABORATORY SAFETY, USE OF EQUIPMENT, STERILIZATION
TECHNIQUES; CULTURE MEDIA TYPES AND USE; PREPARATION OF
NUTRIENT BROTH AND AGAR

a) GENERAL LABORATORY SAFETY PROCEDURES

Make sure to read the laboratory exercise before class and plan your work. This creates
awareness of the special safety concerns for the laboratory class and permits efficient use
of class time

Wear laboratory coats and then enter the laboratory.

Wear closed footwear to protect the feet. Long hair should be tied back.

Keep all bags in the racks provided inside the lab

Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food
for human use are not permitted in the work areas.

Do not begin any experimental work without prior orientation by the instructor.
Wash your hands thoroughly with soap and water before starting any experiment.
Mouth pipetting is prohibited. Use mechanical devices for pipetting

Broken glassware must not be handled directly by hand, but must be removed by
mechanical means such as a brush and dustpan, tongs, or forceps.

Spills and accidents should be reported to the instructor

If a piece of equipment fails to work, report it immediately to the lab instructor.

Clean up the work place and replace all reagents in designated place before leaving the
laboratory

b) MICROBIOLOGY SAFETY PROCEDURES

Follow the general guidelines and prepare for experimentation.

Keep your workbench neat and organized for the experiment

Wear disposable latex gloves while handling blood products (e.g. whole blood, plasma,
serum) or cultures

Clean slides carefully and wipe it with alcohol for microscopic work.
Label all cultures and solutions properly with the name of the test organism, the name of
the medium, dilution of the sample, your name or initials, date, course / lab section, prior
to inoculation
Keep culture tubes on test tube racks when not in use and carry them in racks.
Procedures should be performed carefully to avoid splashes or aerosols.
If a bacterial culture splashed in your eye(s) or on your skin, immediately flush with
copious amount of running water
If a culture is spilled, cover the spilled material with paper towels and apply
laboratory disinfectant such as 1% sodium hypochlorite solution or 70% ethanol
over the spill area. Keep the towel on the spill for 20 minutes. Disposable gloves should
be worn while cleaning spills. Inform your instructor of the spill. Place the towel in an
autoclave waste bag provided. Ensure you wash your hands immediately after dealing
with the spill.
Working with hot items, either from the autoclave or heated in the Bunsen burner requires
protection of your hands. Wear protective gloves or handle the hot item with tongs.
Never leave a lighted Bunsen burner unattended.
A fire extinguisher is ready in each laboratory. If your clothes catch on fire "drop and
roll" to smother the flames. Your lab partners should use a fire blanket or their coats to
help smother the flames.

Termination of sessions

Clean up your bench as you work, disposing used items properly.

Place used glass slides and coverslips in glass dishes of disinfectant.

All materials requiring incubation or refrigeration must be appropriately labelled and

placed on the trays provided.

Turn of all equipment after use and reagents and supplies must be returned to their
designated places before leaving the laboratory.

Sterilisation and disposal

Do not throw any bacterial culture in the sink. Do not dispose of any solid material in the
sink.
All cultures, stocks, and other regulated wastes are decontaminated before disposal by an
approved decontamination method such as autoclaving. Dilute the culture with 1 M
sodium hydroxide before autoclaving and disposal.
Place items that require decontamination by autoclaving, including flasks, beakers and
other containers in a cart.
Place glass tube at an angle in baskets to avoid spillage. The caps of all screw-topped
bottles must be loosened before cultures and media are sterilised. It is very important that
instructions for use of the auto clave are followed in order to achieve and maintain
sufficiently high temperatures for a long enough time.

c) USE OF EQUIPMENT AND STERILIZATION TECHNIQUE IN

MICROBIOLOGY LABORATORY
1. Culture tubes and Petri dishes:
Glass test tubes and glass or plastic Petri dishes are used to cultivate microorganisms. A
suitable nutrient medium in the form of broth (liquid medium) or agar (solid medium) may be
added to the culture tubes while only a solid medium is used in Petri dishes. Sterile environment
is maintained in culture tubes by closing the tubes with non absorbent cotton plugs. The
necessary movements of air in and gaseous products out are not prevented by using cotton plugs.
Petri dishes provide a larger surface area for growth and cultivation. It consist of bottom dish
portion contains medium and larger top portion as a loose cover. For routine purposes dishes
approximately 15cm in diameter are used. The sterile agar medium of 15 to 20ml is dispensed to
previously sterilized dishes. After inoculation the Petri dish should be placed in an inverted
position to prevent condensation that forms on during solidification of agar.
2. Equipments for sterilization:
Sterilization is the process of destroying all forms of microbial life. Common methods
used for sterilization is outlined below

A. Bunsen burner
A Bunsen burner, named after Robert Bunsen, is a common piece of laboratory equipment that
produces a single open gas flame, which is used for heating, sterilization, and combustion. The
gas can be natural gas (which is mainly methane) or a liquefied petroleum gas, such as
propane, butane, or a mixture of both.
It is used for sterilization of wire loops and (with alcohol) metal forceps and glass spreaders.
B. Autoclave
It is used for sterilizing media, solutions, discarded cultures and contaminated materials.
Autoclave uses moist heat, steam under pressure for inhibiting or destroying microorganisms.
Steam under pressure provides temperatures above those obtainable by boiling. Autoclave is a
double-jacketed steam chamber equipped with devices which permit the chamber to be filled
with saturated steam and maintained at a designated temperature and pressure for any period of
time. During operation the chamber should be completely replaced by saturated steam. Generally
autoclave is operated at a pressure of approximately 15lb/in 2 at 121C. Time required to achieve
sterility depends on the material to be sterilized, type of the container and the volume. For media
and glass wares 20minutes is required for efficient sterilization
C. Hot air oven
It is recommended when exposure of materials to moist heat is undesirable. It contains
rectangular chamber made up of double walls with insulating material between the wall spaces.
Hot air oven uses electric coils or gases to heat the chamber. For laboratory glass wares 2hr
exposure to a temperature of 160C is sufficient for sterilization.
D. Filters
It is used to remove microorganisms from liquids or gases. High Efficiency Particulate
Air filters (HEPA) is used to deliver to clean air to an enclosure such as cubicle or room.
Together with laminar air flow it is used in biological hoods to produce dust and bacteria-free air.
Laminar air flow chamber also contains germicidal UV-C lamp for sterilizing air in the enclosure
and materials before use. Ultraviolet lamp in the chamber emits radiation in the range of 260 to
270nm which has high bactericidal effect. Disadvantage is that ultraviolet light has very little

ability to penetrate matter. Even a thin layer of glass filters off a large percentage of light. Thus
only the microorganisms on the surface of the object are susceptible for destruction.

3. Materials for transferring microbial cultures:

Microorganisms must be transferred from one vessel to another or from stock cultures to
various media for maintenance. It is called subculturing and must be carried out under sterile
conditions to prevent contamination.

A. Micro Pipettes:
Used for handling small amount of volume from 1ml to 1l. There are two types of
pipettes, Air displacement pipette and positive displacement pipette. Air displacement pipettes
are meant for general use with aqueous solutions. Positive displacement pipettes are used for
high viscosity and volatile liquids.
B. Wire loops and needles:
Made of nichrome or platinum. It is extremely durable and is easily sterilized by
incineration using flame from Bunsen burner. It is used for techniques such as streak plating and
for preparation of stab cultures.

Fig: a) Inoculation needle

b) Inoculation 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. This leaves the little finger free to take hold of the cotton
wool plug/ screw cap of a test tube/bottle.

4. Cultivation chambers:
Microorganism should be grown at their optimum temperature. Incubator is used
to maintain temperature during the necessary growth period. It is an insulated metallic chamber
and is divided into compartments by metallic racks to hold test tubes and Petri dishes. Incubator
uses dry heat and is thermostatically controlled so that temperature can be varied depending on
the requirements of specific microorganisms. Incubator with shaker provides increased
aeration by agitating the vessel. It can be used only for cultivation of organisms in liquid
medium.
5. Refrigerator:
Used for maintenance and storage of stock cultures, samples and chemicals at a
temperature between 0C to 4C. In low temperature bacteria shows no metabolic activity and

there will be no growth of microorganisms. Thus refrigeration is bacteriostatic. Deep freezer (20C and -80C) is used for long term storage of stock cultures, isolated DNA, RNA, Proteins
and enzymes. Stock cultures are stored upon addition of glycerol to maintain the cells in viable
condition.
6. Microwave oven
A microwave oven is used to melt microbiological media, resulting in a substantial
reduction of heat generation and considerable savings in time.
d) CULTURE MEDIA TYPES AND USES; PREPARATION OF MEDIA
AIM:
To prepare nutrient agar and nutrient broth medium for growth of microorganisms
PRINCIPLE:
The survival and growth of microorganisms depends on the adequate supply of
nutrients and a favorable growth environment. A culture medium may be classified by three
ways, based on consistency, nutritional composition and application.
i. Classification based on consistency:
Culture media are solid, liquid or semisolid. A liquid medium which lacks a
solidifying agent is called broth medium. A broth medium supplemented with solidifying agent
like agar results in semisolid or solid medium. Agar is an extract of seaweed; a complex
carbohydrate composed mainly of galactose and it does not contribute any nutritive property as
most of the bacteria cannot hydrolyze agar. Agar is an excellent solidifying agent as it liquefies at
100C and solidifies at 40C. Thus microorganisms can be grown at 37C and slightly above
without liquefaction of medium. Most commonly 1-3% of agar is used for solid medium.
Concentration below this (0.2-0.5%) is used for semi-solid medium.
ii. Classification based on composition:
Chemically defined media: It composed of pure ingredients in carefully measured
concentrations dissolved in double distilled water i.e., the exact chemical composition of the
medium is known. Typically, they contain a simple sugar as the carbon and energy source, an
inorganic nitrogen source, various mineral salts and if necessary growth factors (purified amino
acids, vitamins, purines and pyrimidines).
Complex media: Complex media are rich in nutrients, they contain water soluble extracts of
plant or animal tissue (e.g., enzymatically digested animal proteins such as peptone and
tryptone). Usually a sugar, often glucose is added to serve as the main carbon and energy source.
The combination of extracts and sugar creates a medium which is rich in minerals and organic
nutrients, but since the exact composition is unknown, the medium is called complex.
iii. Classification based on application:

Selective media: It supports the growth of only certain types of bacteria. Media can be made
selective through the addition of substances that enhance or inhibit the growth of particular types
of bacteria. Ex: MacConkey Agar- selective for gram negative bacteria
Differential media: It reveals specific metabolic or metabolic characteristics of bacteria grown
on it. Certain reagents or supplements when incorporated into culture media, allow
differentiation of various kinds of bacteria based on their colony color. Ex: MacConkey agar
contains neutral red (pH indicator) helps to differentiate lactose fermenting bacteria.
Enriched media:

Promotes the growth of a particular organism by providing it with the

essential nutrients, and rarely contains inhibitory substances to prevent the growth of normal
competitors
Media

Purpose

Selective

Suppress unwanted microbes, or encourage desired microbes

Differential Distinguish colonies of specific microbes from others

Enrichment Similar to selective media but designed to increase the numbers of desired
microorganisms to a detectable level without stimulating the rest of the bacterial
population
MATERIALS REQUIRED:
Media components, conical flask, pH meter, Distilled water, Test tubes, Cotton,
Petri plates, Autoclave, Paper
PROCEDURE:
Nutrient broth composition: for 150ml
Peptone- 1.5g
Sodium chloride- 0.7g
Yeast extract- 0.45g
1. Weigh required components and transfer to 250ml conical flask. Make up the volume to
100ml using distilled water.
2. Adjust pH to 7.3 using 0.1M NaOH
3. Make up the volume to 150ml and check pH again
4. Plug the flask with cotton and wrap it with paper.
5. Autoclave at 15Psi for 20min
Nutrient agar composition: for 100ml
Peptone-1g
Sodium chloride-0.5g
Yeast extract-0.3g
Agar-2g

1. Weigh required components and transfer to 250ml conical flask. Make up the volume to
100ml using distilled water.
2. Adjust pH to 7.3 using 0.1M NaOH
3. Make up the volume to 100ml and check pH again
4. Weigh and add 2g of agar.
5. Plug the flask with cotton and wrap it with paper. Autoclave at 15Psi for 20min
RESULT:

EXP: 2
DATE:
CULTURE TECHNIQUES, ISOLATION AND PRESERVATION OF CULTURES
BROTH: FLASK, TEST TUBES; SOLID: POUR PLATES, STREAK PLATES, SLANTS,
STABS

a) ISOLATION OF PURE CULTURES- STREAK PLATE METHOD

AIM:
To perform streak plate procedure for isolation of single colony from a mixed culture
PRINCIPLE:
In nature, microorganisms exist as mixed population in widely differing types.
However, to obtain the knowledge of particular type of microorganisms, it is essential to separate
or isolate these organisms from the mixed population. Various techniques have been employed
for isolation of pure cultures. These techniques initially require that number of organisms in the
inoculums be reduced. It ensures that, following inoculation, individual cells will be sufficiently
far apart on the surface of the agar medium to effect a separation of the different species.
APPARATUS REQUIRED:
Nutrient agar plates, Bunsen burner, Inoculation loop, beaker, 95% ethanol
PROCEDURE:
Quadrant streaking:
1. Clean the laminar hood. Place the nutrient agar plates, loop and inoculum inside the
hood. Flame and cool the loop. Take loopful of mixed culture on the agar surface. Flame
and cool the loop and drag it rapidly several times across the surface of area 1. Flaming is
done to dilute the culture so that fewer organisms are streaked.
2. Reflame and cool the loop and turn the Petri dish 90.Then touch the loop to a corner of a
culture area and drag several times across agar on area 2.
3. Reflame and cool the loop and turn the Petri dish 90. Streak area 3 as above
4. Without reflaming the loop, again turn it to 90 then drag the culture from the corner of
area 3 to area 4 using a wider streak. Dont let the loop touch any previously streaked
areas. Cover the agar plate and keep in incubator at inverted position

Result:

Continuous streaking:
1. Flame and cool the loop. Take loopful of mixed culture on the agar surface.
2. Drag the inoculation loop on the agar surface continuously from left to right as shown in
figure.

RESULT:

b) INOCULATION OF NUTRIENT BROTH, NUTRIENT AGAR SLANTS, STABS

AIM:
To inoculate isolated colony from streak plate in nutrient broth, nutrient agar slants and stabs
PRINCIPLE:
Once discrete colonies develop on the surface of agar plate, each colony may be
picked up from agar plate and grown on nutrient broth, agar or slants. Each of these cultures
represents pure or stock culture and can be used to study cultural characteristics of
microorganisms.
APPARATUS REQUIRED:
Inoculation loop, inoculation needle, Nutrient agar slant, Nutrient agar stab
PROCEDURE:
A. Inoculation of agar slants:
1. Clean the laminar hood and light the burner and place the required materials inside the
laminar hood.

2. Flame the inoculation loop until it becomes red.

3. Cool the flame for 10seconds. A hot loop will damage the bacteria cells. Pick single
colony from streak plate
4. Uncap agar slant culture and show mouth of the tube in flame.
5. Inoculate the culture by drawing the loop over the surface of the agar in zigzag motion.
Care should be taken not to dig the agar slant.
6. Reflame the inoculation loop and mouth of the tube. Plug tube with cotton.
7. Incubate the tube at 37C in the incubator for overnight for the growth of pure culture

B. Inoculation of agar stabs:

1. Flame the inoculation needle and pick single colony from streak plate
2. Uncap the culture tube containing agar and show mouth of the tube in flame.
3. Insert the needle to the bottom of the tube through the agar and withdraw along the line
of insertion
4. Reflame the inoculation needle and mouth of the tube. Plug tube with cotton
5. Incubate the tube at 37C in the incubator for overnight
C. Inoculation into nutrient broth medium
1. Sterilize the inoculation loop and pick single colony from streak-plate
2. Flame the mouth of the culture tube and inoculate into nutrient broth by dislodging the
inoculum from the loop by slight agitation/ rotation in the broth
3. Reflame the inoculation loop and mouth of the tube. Plug tube with cotton
4. Incubate the tube at 37C in the incubator

RESULT:

EXP: 3
DATE:
MICROSCOPY - WORKING AND CARE OF MICROSCOPE
AIM:
1. To identify all the parts of a compound microscope
2. Know how to use the microscope and oil immersion lens

MATERIALS REQUIRED:
Compound microscope, immersion oil, lens cleaner, glass slide, cover slip
THEORY AND PRINCIPLE:
The magnification of small things is a necessary facet of biological research, but the fine
detail in cells and in subcellular components requires that any imaging system be capable of
providing spatial information across small distances. Resolution is defined as the ability to
distinguish two very small and closely-spaced objects as separate entities. Resolution is best
when the distance separating the two tiny objects is small. Resolution is determined by certain
physical parameters that include the wavelength of light, and the light-gathering power of the
objective and condenser lenses. A simple mathematical equation defines the smallest distance
(dmin) separating the two very small objects:
dmin = 1.22 x wavelength / N.A. objective + N.A. condenser
This is the theoretical resolving power of a light microscope. In practice, specimen quality
usually limits dmin to something greater than its theoretical lower limit.
N.A. (Numerical Aperture) is a mathematical calculation of the light-gathering capabilities of a
lens. The N.A. of each objective lens is inscribed in the metal tube, and ranges from 0.25-1.4.
The higher the N.A., the better the light-gathering properties of the lens, and the better the
resolution. Higher N.A. values also mean shorter working distances (you have to get the lens
closer to the object). N.A. values above 1.0 also indicate that the lens is used with some
immersion fluid, such as immersion oil.

From the equation above, you should be aware that the N.A. of the condenser is as important as
the N.A. of the objective lens in determining resolution. It is for this reason that closure of the
condenser diaphragm results in a loss of resolution. In practice, at full aperture and with good oil
immersion lenses (N.A. 1.4 for both the condenser and the objective) it is possible to be able to
resolve slightly better than 0.2 m. From the equation above, it should also be clear that shorter
wavelength light (bluer light) will provide you with better resolution (smaller dmin values).
However, there are practical considerations in how short the wavelength can be. In the early
1950's, a UV microscope was designed, but required quartz objectives and a specialized imaging
device. The quartz lenses provided slightly better resolution (dmin = 0.1 m), but image quality
suffered from an inability on the part of the manufacturers to correct for aberrations caused by
the quartz. The human eye is best adapted for green light and our ability to see detail may be
compromised somewhat with the use of blue or violet. Most manufacturers of microscopes
correct their simplest lenses (achromats) for green light.

- Magnification and Imaging Most microscopes in current use are known as compound microscopes, where a magnified image
of an object is produced by the objective lens, and this image is magnified by a second lens
system (the ocular or eyepiece) for viewing. Thus, final magnification of the microscope is
dependent on the magnifying power of the objective times the magnifying power of the ocular.
Objective magnification powers range from 4X to 100X. Lower magnification is impractical on a
compound microscope stand because of spatial constraints with image correction and
illumination. Higher magnification is impractical because of limitations in light gathering ability
and shortness of working distances required for very strong lenses. Ocular magnification ranges
are typically 8X-12X though 10X oculars are most common. As a result, a standard microscope
will provide you with a final magnification range of ~40X up to ~1000X.

Components of microscope:
1. Objective:
Its basic function is to gather the light passing through the specimen and then to project
an accurate, real, inverted IMAGE of the specimen up into the body of the microscope.
The objective must be constructed so that it will be focused close enough to the specimen
so that it will project a magnified, real image up into the microscope.
The higher power objectives should have a retractable front lens housing to protect the
front lens where the objective requires focusing very close to the specimen.
To the extent possible, corrections for lens errors (aberrations) should be made within the
objective
2. Eyepiece or Oculars:
Its basic function is to look at the focused, magnified real image projected by the
objective and magnify that image a second time as a virtual image seen as if 10inches
from the eye.
The eyepiece houses a fixed diaphragm. It is at the plane of that fixed diaphragm that the
image projected by the objective will be seen
On the shelf of the fixed diaphragm, the eyepiece can be fitted with scales or markers or
pointers or crosshairs that will be in simultaneous focus with the focused image

3. Substage condenser:
Its basic function is to gather the light coming from the light source and to concentrate
that light in a collection of parallel beams onto the specimen.
The light gathered by the condenser comes to a focus at the back focal plane of the
objective
Other components:
The base of the microscope contains a collector lens. This lens is placed in front of the
light source. Its function is to project an image of the light source onto the plane of the
condensers aperture diaphragm. In some instruments a diffusion or frosted filter is
placed just after the collector lens (side closer to the specimen) in order to provide more
even illumination.
Also in the base of the microscope, under the condenser, is a first surface mirror
(silvered on its front surface only). Its function is to reflect the light coming from the
lamp up into the substage condenser.
At the lowest part of the observation tubes (binocular or trinocular) there is incorporated
a tube lens. Its function is to gather the parallel rays of light projected by the objective
(in infinity-corrected systems) and bring those rays to focus at the plane of the fixed
diaphragm of the eyepiece. In the instruments of some manufacturers, the tube lens is
built into the body of the microscope itself.
Mechanical/ Electrical components:

The stand of the microscope houses the mechanical/electrical parts of the microscope. It
provides a sturdy, vibration-resistant base for the various attachments.
The base of the Olympus microscopes is Y-shaped for great stability. It houses the
electrical components for operating and controlling the intensity of the lamp. The lamp
may be placed, depending on the instrument, at the lower rear of the stand or directly
under the condenser fitting. The base also houses the variable field diaphragm. The base
may also have built in filters and a special circuit for illumination intensity for
photomicrography.
Built into the stand is a fitting to receive the microscope stage. The stage has an opening
for passing the light. The specimen is placed on top of the stage and held in place by a
specimen holder.
Attached to the stage are concentric X-Y control knobs which move the specimen
forward /back or left/right.
On the lower right and left side of the stand are the concentric coarse and fine focusing
knobs. These raise or lower the stage in larger / smaller increments to bring the specimen
into focus.
Above the stage, the stand has a nosepiece (may be fixed or removable) for holding the
objectives of various magnifications. The rotation of the nosepiece can bring any one of
the attached objectives into the light path (optical axis). The nosepiece may also have a
slot for special attachments.
Removable observation tubes, either binocular or trinocular, are attached to the stand
above the nosepiece. The binocular is used for viewing and the trinocular is used for
viewing and /or photography. The observation tubes are usually set at approximately a 30
degree angle for comfortable viewing and may be tiltable or telescoping push-pull for
greater flexibility.

EXP: 4
DATE:
IDENTIFICATION OF MICRO ORGANISMS: STAINING TECHNIQUES
SIMPLE STAINING
AIM:
To prepare and stain bacterial smears made from broth and solid media and evaluate cell
morphology.
PRINCIPLE:
The development of staining techniques was of great importance to microbiology. Since
many bacteria do not have pigments, it can be difficult to see individual cells under a light
(bright-field) microscope. Stains enhance the contrast and allow the microscopist to view the cell
more distinctly. Staining not only makes bacteria more easily seen, but it allows their
morphology (e.g. size and shape) to be visualized more easily.
Stains range from simple to complex. Simple stains involve only one reagent, and stain
all bacteria similarly. They are useful solely for increasing contrast so that morphology, size, and
arrangement of organisms can be determined. More complex stains involve multiple reagents,
and are often differential. A differential stain displays the chemical differences in cellular
structures, including the cell wall and cell membrane because the macromolecules within the
structure bind to different components of the stain. This means that they stain different types of
bacteria differently. In some cases, specific stains can be used to visualize certain structures
(flagella, capsules, endospores, etc) of bacterial cells.
Staining is based on the principle that opposite charges attract and that like charges repel.
Most bacteria, when placed in an aqueous environment with the pH at about 7, have a net
electrical charge that is negative. These negatively charged cells will attract positively charged
molecules and repel those molecules that are negative. Stains (dyes) are chemicals containing
chromophores, groups that impart color. Their specificity is determined by their chemical
structure. Stains are generally salts in which one of the ions is colored. (A salt is a compound

composed of a positively charged ion and a negatively charged ion.) In most commonly used
dyes (basic dyes), the cation is the chromophore. Basic dyes include methylene blue, crystal
violet, and safranin. These are used to prepare a simple stain. For example, the dye methylene
blue is actually the salt methylene blue chloride which will dissociate in water into a positively
charged methylene blue ion which is blue in color and a negatively charged chloride ion which is
colorless.
Commonly used microbiological stains generally fall into one of two categories - basic
stains or acidic stains (although there are a few stains such as India Ink) which are neutral). A
basic dye is a stain that is cationic (positively charged) and will therefore react with material that
is negatively charged. The cytoplasm of all bacterial cells have a slight negative charge when
growing in a medium of near neutral pH and will therefore attract and bind with basic dyes.
Some examples of basic dyes are crystal violet, safranin, basic fuchsin and methylene blue.
Acid dyes have negatively charged chromophores and are repelled by the bacterial
surface forming a deposit around the organism. They stain the background and leave the microbe
transparent. Nigrosine and congo red are examples of acid dyes.
Preparing Stains
When preparing a stain, a perfectly clean microscope slide must be used. New slides are
usually the best, however if used slides are used, great care should be taken to clean all greasy
film from the slide. Cleanliness can be tested by dropping a drop of water on the slide. If it
spreads over the entire slide, the slide is clean. Any beading of the water indicates the presence
of a greasy film.
A thin film of bacteria should be spread upon the slide. If the smear is too thick, it is
difficult to see anything because there will be little light passing through. The smear should be
thin and allowed to dry. Once the smear has dried, the slide should be passed over a lit Bunsen
burner several times to affix the organisms. This procedure is known as heat fixing. There is a
slight shrinkage of cells during this process which is normal, but it helps the bacterial cells to
adhere to the slide through several rinses.
If the slide is overheated, the cells will warp and structure will be indistinguishable. If
heat is applied to the cell before the smear is dry, there willbe distortion.
A properly stained bacterial smear should be slightly difficult to see to the naked eye. If
there are dark splotches of color, the bacteria are piled on top of each other.
Finished stained smears will last for months stored in a cool dark place provided no oil is
present on the stain. There are solvents, such as xylol, that can be used to remove excess oil from
slides that are to be saved. Solvents, however, strip any markings made by wax pencils, so relabeling is important.

Bacterial Morphology:
Bacteria are very small unicellular microorganisms ubiquitous in nature. They are
micrometers (1m = 10-6 m) in size. They have cell walls composed of peptidoglycan and
reproduce by binary fission. Bacteria vary in their morphological features.
The most common morphologies are:
Coccus (pleural: Cocci):
Spherical bacteria; may occur in pairs (diplococci), in groups of four (tetracocci), in grape-like
clusters (Staphylococci), in chains (Streptococci) or in cubical arrangements of eight or more
(sarcinae).
For example: Staphylococcus aureus, Streptococcus pyogenes
Bacillus (pleural: Bacilli):
Rod-shaped bacteria; generally occur singly, but may occasionally be found in pairs (diplobacilli) or chains (streptobacilli).
For example: Bacillus cereus, Clostridium tetani

Spirillum (pleural: Spirilla)

Spiral-shaped bacteria
For example: Spirillum, Vibrio, Spirochete species.

Some bacteria have other shapes such as:

Coccobacilli: Elongated spherical or ovoid form.

Filamentous: Bacilli that occur in long chains or threads.
Fusiform: Bacilli with tapered ends.
MATERIALS REQUIRED:
Microscope slides, Cover slips, Inoculating loops, Broth cultures of various bacteria,
Microscopes, Various simple stains
PROCEDURE:
Preparing Smears from Broth Cultures
1. Prepare the slide. A circle made with a grease pencil will provide an area in which to
apply the smear. The slide may be turned over so that the markings of the pencil are on
the bottom of the slide. This keeps any wax from getting into the smear and causing a
viewing problem.
2. Obtain a tube containing E. coli.

3. Resuspend the bacteria in the broth by rolling the tube between the hands. Bacteria must
always be resuspended before removing any inoculum.
4. Using aseptic techniques transfer a loop full of bacteria from the tube to the labelled
circle on the slide. Keep the slide and the tube near the flame. Avoid inhaling any
aerosols. Flame the loop after transfer.
5. Allow the smear to dry.
6. When the smear is completely dry pass the slide through the top of the Bunsen burner
flame several times to heat fix the organisms.
7. Then proceed to Procedure 1.
Procedure 1 - Simple Staining
1. Place the slides on the stain rack over the sink.
2. Cover the slides with one of the stains and allow the stain to stay on the slide for
following intervals.
1. 1% Crystal violet
- 30 seconds to 1 minute
2. 0.1% Basic fuchsin
- 2 to 3 minutes
3. 1% Loefflers Methylene blue - 2 to 3 minutes
4. 0.5% Saffranin
- 1 minute
3. Hold the slide still tilted to the side and begin to rinse with deionizedwater from the
supplied water bottles. Aim around the smear and remove all excess stain. Do not aim
right at the smear as it may result in the removal of the smear.
4. Shake all excess water from the slide.
5. Slides can be air dried, but to avoid any chance of decolorization by water, you may blot
the slides dry in the book of bibulous (absorbent) paper.
6. Examine the stained smears on the microscope. The smears should be examined on every
power including the oil immersion lens.
7. Draw what is seen in the field of view on the oil immersion lens below. Once done,
cleanup work area and dispose of gloves and slides in a biohazard bag.
RESULT :

EXP:5
DATE:
IDENTIFICATION OF MICRO ORGANISMS: STAINING TECHNIQUES
GRAM STAINING
AIM:

To differentiate between the two major categories of bacteria: Gram positive and Gram
negative.
To understand how the Gram stain reaction affects Gram positive and Gram negative
bacteria based on the biochemical and structural differences of their cell walls.

PRINCIPLE:
Staining is an auxiliary technique used in microscopic techniques used to enhance the
clarity of the microscopic image. Stains and dyes are widely used in the scientific field to
highlight the structure of the biological specimens, cells, tissues etc.
The most widely used staining procedure in microbiology is the Gram stain, discovered
by the Danish scientist and physician Hans Christian Joachim Gram in 1884. Gram staining is a
differential staining technique that differentiates bacteria into two groups: gram-positives and
gram-negatives. The procedure is based on the ability of microorganisms to retain color of the
stains used during the gram stain reaction. Gram-negative bacteria are decolorized by the
alcohol, losing the color of the primary stain, purple. Gram-positive bacteria are not decolorized
by alcohol and will remain as purple. After decolorization step, a counter stain is used to impart a
pink color to the decolorized gram-negative organisms.
The Gram stain procedure enables bacteria to retain color of the stains, based on the differences
in the chemical and physical properties of the cell wall.
1. Gram positive bacteria: Stain dark purple due to retaining the primary dye called Crystal
Violet in the cell wall. Example: Staphylococcus aureus
2. Gram negative bacteria: Stain red or pink due to retaining the counter staining dye called
Safranin. Example: Escherichia coli
MATERIALS REQUIRED:

Clean glass slides, Inoculating loop, Bunsen burner, Bibulous paper, Microscope, Lens paper
and lens cleaner, Immersion oil, Distilled water, 18 to 24 hour cultures of organisms
REAGENTS:
1. Primary Stain

Crystal Violet

2. Mordant

Grams Iodine

3. Decolourizer

Ethyl Alcohol

4. Secondary Stain

Safranin

PROCEDURE:
Part 1: Preparation of the glass microscopic slide
Grease or oil free slides are essential for the preparation of microbial smears. Grease or oil from
the fingers on the slides is removed by washing the slides with soap and water. Wipe the slides
with spirit or alcohol. After cleaning, dry the slides and place them on laboratory towels until
ready for use.
Part 2: Labeling of the slides
Drawing a circle on the underside of the slide using a glassware-marking pen may be helpful to
clearly designate the area in which you will prepare the smear. You may also label the slide with
the initials of the name of the organism on the edge of the slide. Care should be taken that the
label should not be in contact with the staining reagents.
Part 3: Preparation of the smear

Bacterial suspensions in broth: With a sterile cooled loop, place a loopful of the broth
culture on the slide. Spread by means of circular motion of the inoculating loop to about
one centimeter in diameter. Excessive spreading may result in disruption of cellular
arrangement. A satisfactory smear will allow examination of the typical cellular
arrangement and isolated cells.

Bacterial plate cultures: With a sterile cooled loop, place a drop of sterile water or
saline solution on the slide. Sterilize and cool the loop again and pick up a very small
sample of a bacterial colony and gently stir into the drop of water/saline on the slide to
create an emulsion.

Swab Samples: Roll the swab over the cleaned surface of a glass slide.

Part 4: Heat Fixing

Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and allows the
sample to more readily take up stains.

Allow the smear to air dry.

After the smear has air-dried, hold the slide at one end and pass the entire slide through
the flame of a Bunsen burner two to three times with the smear-side up.

Now the smear is ready to be stained.

Please Note: Take care to prevent overheating the slide because proteins in the specimen can
coagulate causing cellular morphology to appear distorted.

Part 5: Gram Stain Procedure

1. Place
slide
with
heat
fixed
smear
on
staining
tray.
Gently flood smear with crystal violet and let stand for 1 minute.
2. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle.
3. Gently flood the smear with Grams iodine and let stand for 1 minute.
4. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle. The smear will appear as a purple circle on the slide.
5. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the
alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be
careful not to over-decolorize.
6. Immediately rinse with water.
7. Gently flood with safranin to counter-stain and let stand for 45 seconds.
8. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash
bottle.
9. Blot dry the slide with bibulous paper.
10. View the smear using a light-microscope under oil-immersion.
RESULT:
EXP:
DATE:

QUANTIFICATION OF MICROBES: SAMPLING AND SERIAL DILUTION;

BACTERIAL COUNT IN FOOD PRODUCTS - TVC
AIM:
To estimate the total bacterial count in samples curd, milk shake, fruit juice, sambar , chutney,
soil, tap water , rotten tomato etc using spread plate technique
PRINCIPLE:
As part of daily routine, the laboratory microbiologist often has to determine the number of
bacteria in a given sample as well as having to compare the amount of bacterial growth under
various conditions. Enumeration of microorganisms is especially important in dairy
microbiology, food microbiology, and water microbiology.
There are many techniques for measuring microbial growth or population size, but they can be
divided into two main groups, based on whether the population size is determined directly or
indirectly. Direct counts include counting cells under the microscope (with or without special
stains), using electronic particle counters, or counting colonies on spread plates (also called a
viable plate count). Indirect methods provide an estimate of cell numbers and can be done by
measuring dry weight, the optical density of a culture, or by measurements of total protein.
Indirect methods have the advantage of being more rapid than direct methods, but in order to be
meaningful, an indirect method must first be correlated to a direct method.
A. The plate count (viable count):
The number of bacteria in a given sample is usually too great to be counted directly. However, if
the sample is serially diluted and then plated out on an agar surface in such a manner that single
isolated bacteria form visible isolated colonies , the number of colonies can be used as a measure
of the number of viable (living) cells in that known dilution. However, keep in mind that if the
organism normally forms multiple cell arrangements, such as chains, the colony-forming unit
may consist of a chain of bacteria rather than a single bacterium. In addition, some of the
bacteria may be clumped together. Therefore, when doing the plate count technique, we
generally say we are determining the number of Colony-Forming Units (CFUs) in that known
dilution. By extrapolation, this number can in turn be used to calculate the number of CFUs in
the original sample.

Normally, the bacterial sample is diluted by factors of 10 and plated on agar. After incubation,
the number of colonies on a dilution plate showing between 30 and 300 colonies is determined.
A plate having 30-300 colonies is chosen because this range is considered statistically
significant. If there are less than 30 colonies on the plate, small errors in dilution technique or the

presence of a few contaminants will have a drastic effect on the final count. Likewise, if there are
more than 300 colonies on the plate, there will be poor isolation and colonies will have grown
together.
Generally, one wants to determine the number of CFUs per milliliter (ml) of sample. To find this,
the number of colonies (on a plate having 30-300 colonies) is multiplied by the number of times
the original ml of bacteria was diluted (the dilution factor of the plate counted). For example, if a
plate containing a 1/1,000,000 dilution of the original ml of sample shows 150 colonies, then 150
represents 1/1,000,000 the number of CFUs present in the original ml. Therefore the number of
CFUs per ml in the original sample is found by multiplying 150 x 1,000,000 as shown in the
formula below:
The number of CFUs per ml of sample = The number of colonies (30-300 plate) X
The dilution factor of the plate counted
MATERIALS REQUIRED:
Sterile nutrient agar plates, sterile dilution tubes, sterile 10 ml pipettes, sterile tips for
pipetteman, sterile saline as a diluent, glass spreader, alcohol

PROCEDURE
1. Weigh 10 g of the sample in a sterile beaker and transfer to 90 ml of diluent in a conical
flask. Mix well. This gives a 10-1 dilution.
2. Transfer 0.5 ml of this diluted sample and mix with 4.5 ml of sterile diluent in a test tube.
3. Shake gently to facilitate mixing and dilution.
4. Prepare serial dilution tubes by transferring 4.5 ml of diluent into 5 sterile test tubes.
5. Dilute the supernatant serially to obtain10-3, 10-4, 10-5, 10-6, 10-7, 10-8 ,10-9,10-10
respectively, Transfer 0.1 ml of the appropriate dilution on the sterile NA plates and
spread them uniformly using alcohol sterilized, cooled glass spreader.
6. Incubate the plates at 37C for 24 hours.
7. Count the number of colonies on the agar surface.
8. Calculate the no. of bacteria present as cfu/ml / g of the given sample.
9. Describe the colony characteristics of the major type of organisms seen on the plates used
for counting.

RESULTS:

EXP. No :
Date :
MICROBIOLOGICAL QUALITY OF WATER

The most important bacterial diseases transmitted by water are typhoid, dysentery and cholera.
Since they are intestinal diseases, causative agents are found in sewage. Therefore the presence
of sewage in a water supply means that one or more of these disease-causing organisms may be
present and that the water is potentially dangerous for human consumption.
Coliform organisms in Sewage

The coliform group is defined to include all aerobes, facultative anaerobic, gram-negative, nonspore forming rod-shaped species which ferment lactose with the production of acid and gas
within 48 h at 37C. Probably the most important members found in sewage polluted waters and
relatively easy to isolate are E-coli, E. freundi and Aerobacter aerogenes.

Some coliform species or varieties have been designated fecal because they are commonly found
in feces; others have been called non-fecal because they are believed to be normal inhabitants of
soil. However in the tests which follow, no attempt is made to differentiate between fecal and
non fecal types. Such a differentiation has been shown to be of limited value in determining the
suitability of water for human consumption, as contamination with either type renders the water
potentially dangerous and unsafe from a sanitary standpoint.

Microorganisms as indicators of water quality

In the routine microbiological examination of water to determine its potability, it would not be
satisfactory to base the test upon the presence of (or isolation of) pathogenic microorganisms for
the following reasons:

1. Pathogens are likely to gain entrance into water sporadically, but since they do not
survive for long periods of time, they could be missed in a sample submitted to the
laboratory.
2. If they are present in very small numbers, pathogens are likely to escape detection by
laboratory procedure.
3. It takes 24 h or longer to obtain results from a laboratory examination. If pathogens were
present, humans would be exposed to infection before actions could be taken to correct
the situation.
Indicator microorganisms
The term indicator microorganisms as used in water analysis refers to a kind of microorganism
whose presence in water is evidence that the water is polluted with fecal material from humans
or other warm-blooded animals. This kind of pollution means that the opportunity exists for the
various pathogenic microorganisms, which periodically occur in the intestinal tract, to enter the
water.

Some of the important characteristics of an indicator organism are:

1. It is present in polluted water and absent from unpolluted (potable) water.
2. It is present in water when pathogens are present.
3. The quantity of indicator organism correlates with the amount of pollution.
4. It has greater survival ability than pathogens.
5. It has uniform and stable properties.
6. It is harmless to humans and other animals.
7. It is present in greater numbers than pathogens (making detection relatively easy).
8. It is easily detected by simple laboratory techniques.
Several species, or groups, of bacteria have been evaluated for their suitability as indicator
organisms. Among the organisms studied, Escherichia coli and other coliform group bacteria
most nearly fulfill the requirements of an ideal indicator organism and are regarded as the most
reliable indicators of fecal pollution.

Escherichia coli and other coliform bacteria

Escherichia coli is a normal inhabitant of the intestinal tract of humans and other warm-blooded
animals. Normally, it is not pathogenic. Another member of the coliform group is Klebsiella
pneumoniae, which is widely distributed in nature. It is found in soil, water, and grain, and also
in the intestinal tract of humans and other animals. Enterobacter aerogenes, a coliform bacterium
found in the intestinal tract of humans and other animals, occurs also in soil, water, and dairy
products.

The coliforms as a group are characterized as gram-negative, non-spore forming, aerobic and
facultatively anaerobic, rod-shaped bacteria that ferment lactose with the production of acid and
gas within 48 h at 35 C.

The coliforms have several characteristics in common with members of the genera Salmonella
and Shigella, two genera, which are enteric pathogenic species. However, a major distinctive
biochemical difference is that the coliforms ferment lactose with production of acid and gas;

Salmonella and Shigella do not ferment lactose. The fermentation of lactose is the key reaction
in the laboratory procedure performed to determine potability of water.

Sampling of water
For collection of sample great care is necessary. The water samples collected for bacteriological
analysis should ensure truly representative samples from different sources and prevent
extraneous contamination during collection.

Procedures
Collect the sample in sterilized ground glass stoppered bottle of about 30-50 ml capacity. While
collecting from top allow the water to run for 3-4 m. Sterilize the nozzle of the top by heating it
with a burner or with a piece of cotton wool which is dipped in spirit. Again allow the water to
flow slowly for a minute and then holding the sample bottle in one hand, remove stopper with
other hand. Flame the mouth quickly and allow the bottle to fill. Replace the stopper.

Most Probable Number (MPN) Estimates

These are based on assumption that bacteria are normally distributed in liquid media, that is,
repeated samples of the same size from one source are expected to contain the same number of
organisms on average. Some samples will obviously contain a few more, some a few less. The
average number is the most probable number. This technique is used mainly for estimating
coliforms but it can be used almost for any organism in liquid samples if
growth can be easily observed e.g. by turbidity or acid production. Examples are yeasts and
molds in fruit juices and beverages, Clostridia in food emulsions. For anaerobes back tube MPN
counts can also be done. Double strength broth is used for the larger volumes because the
medium would otherwise be too dilute.

It is possible to calculate the most probable number of organisms per 100ml for any combination
of results from such sample series. Tables have been prepared for samples of 10ml, 1ml and 0.1
ml using five tubes or three tubes of each sample size. Tables indicate the estimated no. of
bacteria of the coliform group present in 100 ml of water corresponding to various combinations
of positive and negative results in the amounts used for the tests. The tables were basically
computed by McCready and therefore are referred to as McCreadys table.

Procedure
1. Inoculate 10 ml of water sample into each of 3 Lauryl Tryptose (LT) broth tubes (double
strength).
2. Inoculate 1ml and 0.1 ml of water sample into each of 3 LT broth tubes (single strength).
3. Incubate all tubes at 37C for 24 to 48 h.
4. Any amount of gas in the inverted Durhams tube constitutes a positive test.
The sample must be collected in a sterile bottle.
The sample must be representative of the supply from which it is taken.
Contamination of the sample must be avoided during and after sampling.
The sample should be tested as promptly as possible after collection.
If there is a delay in examination of the sample, it should be stored at a temperature between 0
and 10C.
The routine bacteriological procedure consists of
(1) A plate count to determine the number of bacteria present and
(2) Tests to reveal the presence of coliform bacteria.
Standard plate count
Colony counts are performed after plating samples of the water. Plate-count standards have not
been suggested for water because water with a few pathogenic bacteria is obviously more
dangerous than water containing many saprophytic bacteria. Nevertheless, water of good quality
is expected to give a low total count, less than 100 per milliliter. Plate counts are useful in
determining the efficiency of the operations removing or destroying organisms-sedimentation,
filtration, and chlorination. A count can be made before and after the specific treatment. The
results indicate the extent to which the microbial population has been reduced.
Tests for the detection of coliform bacteria
Several selective and differential media greatly expedite the examination of water for coliform
organisms. The examination involves three successive steps:
(1) Presumptive test,
(2) Confirmed test and
(3) Completed test

Multiple tube fermentation technique is followed here. The routine standard tests are (A)
Presumptive (B) Confirmed (C) Completed test
EXPERIMENT
Aim
To determine whether the given water sample is potable
Requirements
McConkey broth , EMB or Endo agar plates, Brilliant green lactose bile broth (BGLB) with
inverted Durhams tube NA Plates and water sample.
Procedure:
(A) Presumptive Test
(1) Inoculate 5ml of water sample in 5 test tubes,2.5ml in 5 test tubes,1ml in 5 test tubes
containing 5 ml of Mcconkey broth .
(2) Incubate all tubes at 37C for 24-48 h. Any amount of gas in the inverted Durhams
tube constitutes a positive presumptive test.
The absence of gas formation within that period constitutes negative test and no further tests
need to be performed.
(B) Confirmed Test
From tubes showing positive presumptive test inoculate a loopful into BGLB and streak a
loopful on EMB or Endo agar, incubate the tubes and the plates at 37C for 48h. Gas in the
BGLB tubes or typical colonies on EMB or Endo agar- dark centered pink colonies on these
media constitutes positive confirmed.

(C) Completed Test

1.

Pick up one typical coliform colony from EMB or Endo agar plate and subculture it on a
NA slant.

2.

Prepare a suspension from each colony and inoculate a loopful into LTB

3.

Incubate the slant and broth tube at 37C for 24 h and observe for gas in the LTB
tube

Eosin methylene blue agar (EMB)

This medium is prepared by adding definite quantities of the two stains eosin and methylene blue
to a melted lactose agar base. A loop-full of culture from each positive fermentation tube is
streaked over the surface of EMB agar. The plates are inverted and incubated at 37C for 24 h.
It is used for the isolation, cultivation and differentiation of Gram-negative enteric bacteria based
on lactose fermentation. Bacteria that ferment lactose, especially the coliform bacterium
Escherichia coli, appear as colonies with a green metallic sheen or blue-black to brown color.
Bacteria that do not ferment lactose appear as colourless or transparent light purple colonies.
Colonies of Yersinia pseudotuberculosis are pale pink.

Three types of colonies develop on the medium.

1.

Typical - nucleated with or without metallic sheen

2.

Atypical - Opaque, non-nucleated, pink

3.

Negative - All others.

If typical coliform colonies appear on the plates the confirmed test may be considered positive. If
only atypical colonies appear the confirmed test cannot be considered negative, since some
coliforms fail to produce typical colonies on this medium or the colonies develop slowly. If no
colonies or non-coliforms colonies develop within 24 h, the confirmed test may be considered
negative.

The colour of coliform on this medium depends on 2 factors (1) the reaction of eosin (an acid
stain) with methylene blue (a basic stain) to form a compound of either acidic or neutral in nature
and (2) the formation by lactose-fermenting organisms of sufficient acid to cause this stain
compound to be taken up by individual cells of a colony. The non-lactose-fermenting organisms
are not coloured because the stain compound is not taken up in basic solution.

Endo agar
Metallic gold-like sheen imparted to the surface of the typical colonies. The media is used for the
selective isolation, cultivation and differentiation of coliform and other enteric microorganisms
based on their ability to ferment lactose. Lactose fermenting bacteria appear as dark red colonies
with a gold metallic sheen. Lactose-non-fermenting bacteria appear as colourless or translucent
colonies.

Brilliant green lactose bile broth

A positive test is indicated by the presence of gas in any amount in the inverted vial within
incubation period.

RESULT:

Exp :
Date:
MICROBIOLOGICAL QUALITY OF MILK
AIM:
To evaluate the microbiological quality of milk.
INTRODUCTION:

Milk is one of the most important foods for man but it is also highly susceptible to microbial
contamination and spoilage. A variety of microorganisms, including several pathogenic species
can gain entry into milk during its production and handling. A knowledge of the numbers and
types of bacteria present in raw milk supplies is thus very useful in determining the hygienic
conditions of its production and handling, its keeping quality and its suitability for processing
or manufacture of products.
A number of bacteriological tests are used for checking the quality of milk and these may be
broadly grouped into
(i)

Direct enumeration of total bacterial population in milk (e.g. direct microscopic count)

(ii)

Estimation of the numbers of viable bacteria e.g. standard plate count.

(iii) Testing for the presence of specific types of contaminants (e.g. coliform test)
(iv) Assessing the metabolic activities of bacteria (e.g. methylene blue and resazurin reduction
tests)
(v)

Estimating the chemical changes or products formed in milk by bacterial growth (e.g.
acidity, gas production, pH and proteolysis)

When quality of milk has to be detected on the spot, it is necessary to adopt simple, reliable and
rapid methods of bacteriological examination. Two such methods are usually followed.

Methylene blue reduction test

Introduction
This test is based on the principle that methylene bluet (an oxidation-reduction dye or indicator)
which is blue in its oxidised state, is reduced to a colourless compound (Leuco form) as a result
of the metabolic activities of bacteria in milk. When a solution of methylene blue is added the
organism present in milk consume the dissolved oxygen and lower the O-R potential to a level
when methylene blue and similar indicators are reduced or decolourised. The time taken for the
reduction of the dye (methylene blue reduction time) is influenced by the number and types of
bacteria growing in milk. The greater the number of organisms present in milk and greater their
activity the more rapidly is the dye reduced. The methylene blue reduction time thus gives an
indication of bacterial numbers and activity in milk. The M.B.R. test is therefore, used for (i)
judging the hygienic quality of milk and grading raw milk supplies, (ii) for assessing the
probable quality of milk, and (iii) for detecting post pasteurisation contamination in milk.

Materials

1.

Thermostatically controlled water bath maintained at 37C.

2.

Sterile test tubes without rim (150 x 16 mm) preferably with marking at 10 ml.

3.

Sterilized rubber bungs to fit into the above test tubes. The rubber bungs together with
forceps are held in boiling water for 10 minutes prior to use.

4.

10.0 ml and 1.0 ml pipettes.

5.

Clock, watch or an interval times

6.

Forceps, beakers and flasks.

7.

Standard methylene blue solution.

8.

Four samples of milk in sample bottles ( fresh raw milk, raw mik refrigerated , raw
milk refrigerated after 2 hours , pasteurized milk)
Methylene Blue Solution A standard solution of methylene blue is prepared by dissolving one
tablet of approved methylene blue thiocyanate of chloride in 200 ml of cold sterile glass distilled
water in a sterile flask by gentle heating in water bath or by allowing the mixture to stand for
several hours to facilitate complete solution and then adding 600 ml of sterile, glass distilled
water. One ml of this solution mixed with 10 ml of milk results in obtaining a final concentration
of 1/300,000 for the dye, which has been found to be satisfactory for the test. The stock solution
must be stored in a sterile glass-stoppered amber coloured bottle in a dark place. Fresh solution
must be prepared once in two months.
Procedure
1.

Thoroughly mix the sample of the milk.

2.

Transfer 10 ml of each sample of milk into a test tube.

3. Add 1 ml of the methylene blue solution to the milk in the test tubes and replace the cotton
plugs with sterile rubber bungs using sterile forceps. While transferring methylene blue
solution care should be taken not to contaminate the pipette by touching the milk or
otherwise a fresh pipette will have to be used for transferring methylene blue solution to
another tube.
4. Mix the dye and the milk by inverting the tubes twice.

5.

Place the tubes in the water bath.

6.

Observe the test tubes after every 30 minutes and if there is no sign of reduction
(decolourisation) the tubes are inverted once and returned to the water bath. If the
decolourisation has commenced the tubes should not be inverted or shaken.

7.

9.
10.

Continue the observation until the complete reduction of the dye (complete
decolourisation) occurs or the formation of a persistent blue ring (0.5 mm) at the top.
8.
Two control tubes, one containing 10 ml of milk and 1 ml of the methylene blue
solution, after heating it in boiling water for 30 minutes and another with 10 ml of milk
plus 1 ml of tap water are also kept in the water-bath. These are required for comparing
the colour changes in experimental tubes
Record the times taken for reduction of methylene blue

Tabulate the results

Interpretation
The following standard for methylene blue reduction times are suggested as a guide for grading
of raw milk supplies.
M.B.R. time (Hours)

Quality of milk

5 and above

Very good

3 and 4

Good

1 and 2

Fair

1/2 and below

Poor

RESULT:

EXP:
DATE:

ENUMERATION OF LACTIC ACID BACTERIA FROM FERMENTED FOODS

PLATE COUNT METHOD
AIM
To estimate the total bacterial count (Lactic acid bacteria) in samples of curd, soy sauce, yakult,
batter , butter milk , Old rice, using pour plate technique
PRINCIPLE
As part of daily routine, the laboratory microbiologist often has to determine the number of
bacteria in a given sample as well as having to compare the amount of bacterial growth under
various conditions. Enumeration of microorganisms is especially important in dairy
microbiology, food microbiology, and water microbiology.
There are many techniques for measuring microbial growth or population size, but they can be
divided into two main groups, based on whether the population size is determined directly or
indirectly. Direct counts include counting cells under the microscope (with or without special
stains), using electronic particle counters, or counting colonies on spread plates (also called a
viable plate count). Indirect methods provide an estimate of cell numbers and can be done by
measuring dry weight, the optical density of a culture, or by measurements of total protein.
Indirect methods have the advantage of being more rapid than direct methods, but in order to be
meaningful, an indirect method must first be correlated to a direct method.
A. The plate count (viable count):
The number of bacteria in a given sample is usually too great to be counted directly. However, if
the sample is serially diluted and then plated out on an agar surface in such a manner that single
isolated bacteria form visible isolated colonies , the number of colonies can be used as a measure
of the number of viable (living) cells in that known dilution. However, keep in mind that if the
organism normally forms multiple cell arrangements, such as chains, the colony-forming unit
may consist of a chain of bacteria rather than a single bacterium. In addition, some of the
bacteria may be clumped together. Therefore, when doing the plate count technique, we
generally say we are determining the number of Colony-Forming Units (CFUs) in that known
dilution. By extrapolation, this number can in turn be used to calculate the number of CFUs in
the original sample.
Normally, the bacterial sample is diluted by factors of 10 and plated on agar. After incubation,
the number of colonies on a dilution plate showing between 30 and 300 colonies is determined.
A plate having 30-300 colonies is chosen because this range is considered statistically
significant. If there are less than 30 colonies on the plate, small errors in dilution technique or the
presence of a few contaminants will have a drastic effect on the final count. Likewise, if there are

more than 300 colonies on the plate, there will be poor isolation and colonies will have grown
together.
Generally, one wants to determine the number of CFUs per milliliter (ml) of sample. To find this,
the number of colonies (on a plate having 30-300 colonies) is multiplied by the number of times
the original ml of bacteria was diluted (the dilution factor of the plate counted). For example, if a
plate containing a 1/1,000,000 dilution of the original ml of sample shows 150 colonies, then 150
represents 1/1,000,000 the number of CFUs present in the original ml. Therefore the number of
CFUs per ml in the original sample is found by multiplying 150 x 1,000,000 as shown in the
formula below:
The number of CFUs per ml of sample = The number of colonies (30-300 plate) X
The dilution factor of the plate counted
MATERIALS REQUIRED:
Streile nutrient agar plates, sterile dilution tubes, sterile 10 ml pipettes, sterile tips for
pipetteman, sterile saline as a diluent, glass spreader, alcohol

PROCEDURE
1. Weigh 10 g of the sample in a sterile beaker and transfer to 90 ml of diluent in a conical
flask. Mix well. This gives a 10-1 dilution.
2. Transfer 0.5 ml of this diluted sample and mix with 4.5 ml of sterile diluent in a test tube.
3. Shake gently to facilitate mixing and dilution.
4. Prepare serial dilution tubes by transferring 4.5 ml of diluent into 5 sterile test tubes.
5. Dilute the supernatant serially to obtain10-3, 10-4, 10-5, 10-6, 10-7 ,respectively.
6. Transfer 0.1 ml of the appropriate dilution on the sterile petri plates and spread uniformly
using alcohol sterilized, cooled glass spreader.
7. Incubate the plates at 37C for 24 hours.
8. Count the number of colonies on the agar surface.
9. Calculate the no. of bacteria present as cfu/ml / g of the given sample.
10. Describe the colony characteristics of the major type of organisms seen on the plates used
for counting.
RESULTS:

Exp. No:
Date:
ENUMERATION OF YEAST AND MOLDS
AIM:

To enumerate the yeast and molds from different food.

MATERIALS REQUIRED:
Food samples(tomato, potato , carrot, custard apple juice, guava juice , cocnut chutney) sterile
plates with potato dextrose agar containing 100mg/ml of chloramphenicol, sterile test tube with
4.5ml 0.1% peptone, micropipette, sterile beakers, sterile tips, sterile spatula and spreader rod.

PROCEDURE:

Weigh 0.5g of sample or 0.5 ml of liquid sample using a sterile beaker or sterile pipette.
Take 4.5 ml of peptone water (0.1%) and add the samples to it.
Prepare serial dilution using 7 test tubes containing 0.1% peptone water to obtain 10 -3,
10-4, till 10-7
Using pour plate technique transfer 1 ml of appropriate dilution on sterile petri plates
and pour the potato dextrose agar solution into the petri plates and allow it to solidify
Incubate the plates(22C to 25C ) for 5 days and count the plates containing 15 to 55
colonies and record it chloramphenicol or gentamycin are used because

RESULT:

Exp. No:
Date:
ENUMERATION OF SPORES FROM PEPPER
AIM:
To enumerate the mesophilic bacterial spores from species.
PRINCIPLE:
Bacteria produce spores in response to environmental stress. Spores are dominant forms of cells
which are essential to heat, dehydration, freezing and irradiation compared to vegetative forms.
Spores forming bacteria are Clostridium and Bacillus. Members of the former are strict or

facultative anaerobes whereas the latter are aerobes of facultative anaerobes. They are both
mesophilic spore forming bacteria.
.
PROCEDURE:
1.

Weigh 1 g of sample in a sterile beaker and add it to 99 ml of sterile 0.1% peptone water.

2.

Mix well, put the samples in a water bath at 80 C for 30 m.

3.

This will heat shock the spores and kill the vegetative bacteria.

4.

Remove the flask from water bath, mix well and allow the particles to settle down.

5.

Transfer 0.5 ml of supernatant to a test tube containing 4.5 ml of 0.1% sterile peptone
water. Mix well, this gives 10 3 dilution.

6.

Similarily prepare 10 4, 10 5 dilutions.

7.

Plate each dilution in quadruplicate using the pour plate technique and molten nutrient
agar at 45 C.

8.

Incubate teh plates at 35 C and 50 C.

9.

At the end of 24 h, count the colonies and report mesophilic aerobic spore count and
thermophilic aerobic spore count

RESULT:

Exp. No:
Date:
INHIBITORY EFFECT OF SPICES ON MICROBIAL LOAD
IN RAW POULTRY
AIM:
To evaluate the inhibitory effect of spices on microbial load in raw poultry.
MATERIALS REQUIRED:

Grounded spice sample(Garlic , Ginger , pepper, turmeric), fleshy chicken, sterile plates
with nutrient agar, sterile distilled water , peptone water (0.1%), micropipette, sterile beakers,
sterile tips, sterile spatula and spreader rod.
PROCEDURE:
1. Weigh 1 gm of sample using sterile spatula into a sterile container(test tube).
2. Add 9ml of 0.1% sterile peptone water .
3. Transfer 300l of sample from the test tube on sterile NA plates and spread
them uniformly using alcohol sterilized, cooled glass spreader.
4. Add 2 gm of sample to 8 ml of distilled water in a sterile test tube to prepare
20 % concentration (dilution).
5. Then add 5 ml of the 20 % solution to 5 ml of distilled water to prepare 10%
dilution in another test tube.
6. Then add 5 ml of the 10 % solution to 5 ml of distilled water to prepare 5 %
dilution in another test tube.
7. By well diffusion method punch a small holes using the sterile tips in the agar
plates
8. Transfer 200l of the prepared dilution in three different petri plates exactly
in the small holes made in well diffusion process.
9. Repeat the same step for all the samples .
10.Inoculate the plates at 37C for 48 hrs .
11.Absorb the plates and tabulate the results .

RESULT:

Exp.No
Date
ENUMERATION AND ISOLATION OF E.COLI FROM PROCESSED MEAT /
CHICKEN
AIM:
To enumerate E.Coli which may be present in foods. Homogenize with 90ml of sterile peptone
REQUIREMENTS:
Food samples (Raw meat,chicken, fish),EMB agar, flask containing 90 ml sterile
peptone(0.1%),sterile piptte tips, stirrer.
PROCEDURE:

1. Weight 1g of sample using a sterile spatula into a sterile container(test tube).

2. Add 9 ml of peptone water(this gives 1:10 dilution).
3. Transfer 1 ml of sample from the test tubes on sterile petri plates and pour EMB agar and
allow it to slodify(pour plate technique).
4. Repeat the same procedure for all the 3 samples .
5. Inoculate the plates at 37C for 24 hrs.
6. Observe the plates and count the colonies on the media.
RESULT:

Exp. No:
Date:
THERMAL DESTRUCTION OF MICROORGANISMS : TDT AND TDP

AIM
To study the effect of high temperature, a physical method on the destruction of microbes in
liquid suspension.
PRINCIPLE:
Heat is the most used method for inactivating the microorganisms in food production.
Microbial exposure to heat has two parameters-temperature and exposure time. Heat appears to
kill microorganisms by denaturing their enzymes. Heat resistance varies among different

microbes. These differences can be expressed through the concept of thermal death point.
Thermal death point (TDP) is the lowest temperature at which all the microorganisms in a
liquid suspension will be killed in 10 minutes.
Another factor to be considered in sterilization is the length of time required for the material to
be rendered sterile. This is expressed as Thermal Death Time (TDT), the minimal length of
time in which all bacteria in a liquid culture will be killed at a given temperature. Both TDP and
TDT are useful guidelines that indicate the severity of treatment required to kill a given
population of bacteria.
REQUIREMENT:
Sterile suspension tubes, sterile 1 ml pipettes, sterile recovery tubes containing sterile nutrient
broth, sterile saline, thermometers and water bath.
DETERMINATION OF TDP:
1. To 4.5 ml of 0.1% sterile peptone water add 0.5 of juice sample, this will give 1:10 dilution
2. Similarly prepare 10-2 and 10-3 respectively.
3. To prepare 10-4 dilution, to 45 ml 0.1% sterile peptone water add 5ml of sample from 10 -3
dilution in a sterile conical flask.
4. Transfer 3 ml of sample from 10-4dilution to 3 sterile test tube.
5. Adjust and fix the temperature of water bath to 70c , 80c and 90c respectively for 10
minutes.
6. Cool the test tubes
7. Then Transfer 300 of the sample from the test tubes on the sterile NA plates and spread them
uniformly using alcohol sterilized, cool glass spreader.
8. Incubate the plates at 37C for 24 hrs, count the no. of colonies on the agar surface and
tabulate the results and to determine TDP
DETERMINATION OF TDT:
1. From the 10-4 dilution of sample prepared above transfer 3 ml of sample to 12 sterile test
tubes.
2. Adjust the temperature of the water bath to 70C, 80C, 90C.
3. Incubate 4 test tubes in each water bath kept at three different temperatures.

4. Remove one tube at a regular interval of 5, 10 , 15 and 20 mins respectively from each water
bath.
5. Cool the test tubes.
6. Then transfer 300l of sample from the test tubes on the sterile NA plates and spread them
uniformly using sterilized , cooled glass spreader.
7. Incubate the plates at 37C for 24 hrs
8. Tabulate the results and count the no of colonies and determine the TDT.
RESULT:

EXP .NO:
DATE:
EFFECT OF CLEANING AND DISINFECTION
PHENOL COEFFICIENT TEST
AIM:
To determine the effectiveness of some chemical disinfectants used as antimicrobial
agents and calculate its phenol coefficient.
PRINCIPLE:
Microorganisms are present everywhere and one must be constantly aware of the living
invisible world. There is a strong need to kill bacteria when and where their presence is
undesirable. Therefore, many situations such as preparation of surgical operations,

microbiological studies, and disinfection of infectious materials call forth the need and use of
methods to destroy them.
The destruction of microorganisms may be achieved by physical and chemical means.
Sterilization is defined as the process where all the living microorganisms, including bacterial
spores are killed. Disinfection is the process of elimination of most pathogenic microorganisms
(excluding bacterial spores) on inanimate objects. Sterilization is always aimed at both
pathogenic and nonpathogenic bacteria, while disinfection, in its true sense, applies only to the
pathogenic ones so that there is no or a much reduced threat of disease. A disinfectant is any
agent, such as heat or a chemical (like iodine) that kills pathogenic microorganisms.
Disinfectants are used to reduce the numbers of microbes on non-living surfaces, while
antiseptics are used to reduce the microbial population on living tissue. Antiseptics normally are
more bacteriostatic in that they prevent bacterial multiplication, but do not kill the organism. The
emergence of anti-microbial soaps, lotions and other products has seen a huge increase over the
last few years. The number of choices is excessive and the consumer is often unaware that many
of the anti-microbial agents are no more effective than basic soap and water. The effectiveness of
an anti-microbial is dependent upon many factors such as the concentration of the antimicrobial
agent, the amount of contamination, the sensitivity of the contaminating organisms, temperature,
and length of exposure. This exercise evaluates the influence that specific antimicrobial agents
may or may not have on bacterial growth.
Many factors influence the effectiveness of chemical disinfectants and antiseptics. The
microbicidal (to kill) or microbiostatic (to inhibit) efficiency of a chemical is often determined
with respect to its ability to deter microbial growth. More specifically, the microbicidal
efficiency of a chemical is often determined with respect to phenol and is known as the phenol
coefficient (PC).
The phenol coefficient is calculated by dividing the highest dilution of the antimicrobial
of interest, which kills all organisms after incubation for 10 minutes but not after 5 minutes, by
the highest dilution of phenol that has the same characteristics. Chemicals that have a phenol
coefficient greater than 1 are more effective than phenol, and those that have a phenol coefficient
less than 1 are less effective than phenol. However, this comparison should only be used for
phenol-like compounds that do not exert bacteriostatic effects and are not neutralized by the
subculture media used.
An ideal disinfectant should be highly effective even when diluted, nontoxic, colorless,
odorless, stable in any concentration, harmless to all surfaces, biodegradable, inexpensive and, if
it is a phenolic, a good phenol coefficient.

A list of commonly used antiseptics and disinfectants and their area of application is shown in
table.

Agents Used to Control Microbial Growth

A. Disinfectants and Antiseptics
1. Phenols and phenolics - these compounds inactivate proteins, denature enzymes, and injure
plasma membranes and should only be used on surfaces. Examples include Lysol,
hexachlorophene, and pHisoHex.
2. Halogens may be used on surfaces either alone or as components of organic or inorganic
solutions to inactivate enzymes and other cellular proteins. Tend to be strong oxidizing agents.
Iodine combines with the amino acid tyrosine, chlorine when added to water forms hypochlorous
acid. Betadine is another example often used instead of iodine.
3. Alcohols denature proteins and dissolve lipids. Examples include ethanol and isopropanol.
4. Heavy metals such as silver, mercury, copper, and zinc exert their influence through oligodynamic action such as combining with the sulfhydryl (-SH) groups and denaturing proteins.
Examples include silver nitrate, mercurochrome, and copper sulfate.
5. Surface active agents soaps and detergents decrease the tension between molecules that lie
on the surface of a liquid
6. Quaternary ammonium compounds (quats) cationic detergents attached to NH4+ disrupt
plasma membranes, denature proteins, and inhibit enzymes. Examples include Cepacol and
Zephran.
7. Organic acids used in the food and cosmetic industry to prevent growth of microorganisms.
Examples include sorbic acid, benzoic acid, and propionic acid.
8. Aldehydes formaldehyde and glutaraldehyde attach methyl or ethyl groups to DNA and
proteins making them nonfunctional.
B. Antibiotics
1. Inhibition of cell wall synthesis may inhibit synthesis of petidogylcan. Include penicillins,
cephalosporins, vancomycin, bacitracin, oxacillin, and nafcillin
2. Damage to plasma membrane polymyxin B, nystatin, and amphotericin B
3. Inhibition of protein synthesis streptomycin (causes misreading of codons on mRNA),
chloramphenicol (prevents peptide bond formation between amino acids), tetracyclines (prevents

hydrogen bonding between anticodon on tRNA-aa complex and codon on mRNA), kanamycin,
erythromycin, and gentamicin
4. Inhibition of nucleic acid synthesis rifamycin, actinomycin D, nalidxic acid, ciprofloxacin,
and norflaxacin
5. Structural analogs such as sulfonamides that are structurally similar to cellular metabolites
and compete with these in enzymatic reaction.

MATERIALS REQUIRED:
Sterile nutrient broth tubes, 24 hour culture of E.coli, phenol, commercial disinfectants such as
Lysol, Dettol, test-tube rack, Bunsen burner, inoculating loop, alcohol.
The phenol is diluted with tap water to obtain 1:80, 1:90, and 1:400 dilutions.
The dettol and lyzol, sodium hypochlorite are diluted with tap water to obtain 1:400, 1:450, and
1:500 dilutions.
PROCEDURE:
1. Label a set of 9 nutrient broth test tubes for 1 disinfectants for 3 different dilutions with
name and dilution of disinfectant and time interval of sub-culturing .
2. Place the test tubes with disinfectant dilutions in separate sacs.
3. Using pipette rapidly introduced 0.05 ml (1drop) of the E.coli culture into the test tubes
with disinfectants. note the time of inoculation.
4. Mix the tubes well , to ensure contact of the disinfectant with microbe .
5. At intervals of 5, 10 and 15 mins , using sterile technique transfer one loop full from each
test tube into the appropriate sterile tube of nutrient broth .
6. Inoculate all cultures for the presence of growth .
7. Absorb all cultures for the presence of growth.
8. Record for the presence of growth and for absence of growth .
9. Tabulate the results for three disinfectants .
10. Repeat for all three disinfectants.

Result: