1

GUIDELINES FOR LABORATORY SAFETY

The microbiology laboratory or most laboratories can be a safe place to work and provide a
rewarding laboratory experience, when the guidelines for personal and environmental safety are
adhered to. The former reflects concern for personal safety in terms of avoiding accidents, while
the latter ensures maintaining a scrupulously clean laboratory that prevents contamination from
exogenous sources.

Since the procedures require the use of viable microorganisms (Biosafety level I) the use
of aseptic techniques is an integral part of all sessions. All microbes should be treated as
potential pathogens and hence handled carefully. Besides, special care must be exercised when
working with human body fluids, corrosive chemicals and fragile glassware.

Standard microbiological practices:

1. Laboratory coats, or uniforms should be worn to prevent contamination or staining of

clothes
2. Wash hands after handling viable materials, after removing gloves, and before leaving the
laboratory. Wash hands with ethanol before performing experiments.
3. Eating, drinking, handling contact lenses and storing food for human use are not permitted
in the work areas.
4. Mouth pipetting is prohibited; Use mechanical pipetting devices
5. All cultures, stocks, and other regulated wastes should be decontaminated before disposal by
using any of decontamination methods such as autoclaving.
6. In case of accidental spilling of cultures clean the place with disinfectant
7. Clean the place before leaving the laboratory
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BASIC APPARATUS 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 cotton plug or by using caps made of
metal such as stainless steel. 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. 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 1µl. 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.
 3

Fig: a) Inoculation needle b) Inoculation loop

3. 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.

4. Refrigerator:
Used for maintenance and storage of stock cultures, samples and chemicals at a
temperature between 0°C to 4°C. In low temperature bacteria shows no metabolic activity and
there will be no growth of microorganisms. Thus refrigeration is bacteriostatic. Deep freezer (-
20°C and -80°C) 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.

5. Equipments for sterilization:

Sterilization is the process of destroying all forms of microbial life. Common methods
used for sterilization is outlined below
 4

A. 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/in2 at 121°C. 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

B. 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 160°C is sufficient for sterilization.
C. 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
 5

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.
 6

EXP 1: PREPARATION OF MEDIA

DATE:

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 100°C and solidifies at 40°C. Thus microorganisms can be grown at 37°C 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.
 7

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 Simlar 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
 8

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:
 9

EXP: 2 PREPARATION OF AGAR PLATES, SLANTS AND STABS

DATE:

AIM:
To prepare nutrient agar plates, slants and stabs for culturing microorganisms

MATERIALS REQUIRED:
Sterile media, test tubes and Petri dishes

PROCEDURE:
1. Clean the laminar flow chamber and light the burner
2. Place the required sterile culture tubes and plate inside the laminar flow chamber.
3. Melt the agar in conical flask and let it cool. Make sure it does not solidify
4. Unplug the cotton from conical flask and show the mouth of the flask in flame
5. Stab culture: Transfer 3ml of nutrient agar medium aseptically into the culture tube and
keep the culture tube in an upright position for the medium to solidify
6. Slant culture: Transfer 3ml of nutrient agar into a culture tube and keep the tube in
slanting position.
7. Petri plate: Pour 10ml of melted agar to a Petri plate to 1/3rd of its volume. Take care to
spread the media evenly without any bubble formation.
8. Incubate the pour plates in incubator overnight to check contamination. If there is any
contamination during handling there will be formation of colonies after overnight
incubation
Note: Name, type of media, date should be labeled properly in all culture tubes and Petri plates.
Culture tubes should not be taken outside of the hood without plug it with cotton
If media solidifies before transfer, it can be reheated in an oven
RESULT:
 10

EXP:3 ISOLATION OF PURE CULTURES-

DATE: 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. 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. Don’t let the loop touch any previously streaked
areas. Cover the agar plate and keep in incubator at inverted position
 11

Fig : Quadrant streaking

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.

Fig : Continuous streaking

RESULT:
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EXP: 4 INOCULATION OF NUTRIENT BROTH, NUTRIENT

DATE: 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 37°C in the incubator for overnight for the growth of pure culture
 13

Fig: a-c- Stab technique for transferring bacteria

d- Streaking of surface of the slant with the loop
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 37°C 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 37°C in the incubator

RESULT:
 14

EXP:5 ENUMERATION OF MICROORGANISMS – PLATE COUNT

DATE: (VIABLE COUNT) METHOD

AIM

To estimate the total bacterial count in samples of soil, pond water 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
 15

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.

Fig :Plate Count Dilution Procedure.

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.
 16

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 9 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.
6. Transfer 0.1 ml of the appropriate dilution on the sterile NA plates and spread them
uniformly using alcohol sterilized, cooled glass spreader.
7. Incubate the plates at 37°C 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.
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RESULTS:

A. Plate Count

1. Choose a plate that appears to have between 30 and 300 colonies.

2. Count the exact number of colonies on that plate.

3. Calculate the number of CFUs per ml of original sample as follows:

The number of CFUs per ml of sample = The number of colonies (30-300 plate) X

The dilution factor of the plate counted

____________ = Number of colonies

____________ = Dilution factor of plate counted

____________ = Number of CFUs per ml

4. Record your results.

 18

EXP: 6 WORKING OF MICROSCOPE

DATE:

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.
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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 µm. 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 µm), 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:
 20

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 have the capacity to reconstitute the various points of the specimen
into the various corresponding points in the image, sometimes called the “anti-points”.
 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”
 21

 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
 Correction for lens errors is incorporated in the finest condensers, an important feature
for research and photography.
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
condenser’s 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
 22

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.

PROCEDURE:

1. Set microscope on a tabletop or other flat, sturdy surface. Plug the microscope's power
cord into an outlet. Compound microscopes have a mirror to focus don’t need electrical
light it uses natural light instead.
2. Switch on your microscope's light source and then adjust the diaphragm to the largest
whole diameter, allowing the greatest amount of light through. If you have an iris
diaphragm, slide the lever till the most light comes through.
 23

3. Rotate the nosepiece to the lowest-power objective usually 4x for 40x magnification). It
is easiest to scan a slide at a low setting, since you have a wider field of view at low
power.
4. Place a microscope slide on the stage, either under the stage clips or clipped onto the
mechanical stage if your microscope has one. A prepared slide works best when you do
this for the first time. (If you do not have a prepared slide, place a strand of colored yarn
or thread on a blank slide place a coverslip over it. Move the slide until the specimen is
under the objective lens.
5. Adjust the large coarse focus knob until the specimen is in focus. Slowly move the slide
to center the specimen under the lens, if necessary. Do this by nudging it gently with your
fingers or by turning the slide control knobs if you have a mechanical stage.
6. Adjust the small fine focus knob until the specimen is clearly in focus. Then adjust the
diaphragm to get the best lighting. Start with the most light and gradually lessen it until
the specimen image has clear, sharp contrast.
7. Scan the slide (right to left and top to bottom) at low power to get an overview of the
specimen. Then center the part of the specimen you want to view at higher power.
8. Rotate the nosepiece to the 10x objective for 100x magnification. Refocus and view your
specimen carefully. Adjust the lighting again until the image is most clear (you will need
more light for higher power). Repeat with the 40x objective for 400x magnification,
which will enable you to see all of the specimen detail that's necessary for high school
biology lab work.
9. Optional: If your microscope has a 100x oil-immersion lens, you'll need to put 1-2 drops
of immersion over the slide coverslip (the piece of glass over the middle of the slide)
before viewing it at highest power. Move the 100x objective lens into position, and then
slowly move the stage up until the lens makes contact with the oil. Continue focusing
with the coarse focus knob until the color or blurred outline of the specimen appears.
Finish focusing with the fine focus knob. With the 100x lens, you will be able to see
additional cell detail, but you will need to take extra care with focus and contrast for a
clear image. When you are done using the slide, clean the oil off of the slide and the lens
with lens cleaning paper and solution.
 24

EXP: 7 IDENTIFICATION OF MICRO ORGANISMS: STAINING TECHNIQUES –

DATE: SIMPLE STAINING

AIM:
 25

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.
 26

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.
 27

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 re-
labeling is important.

Bacterial Morphology:

Bacteria are very small unicellular microorganisms ubiquitous in nature. They are
micrometers (1µm = 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 (diplo-
bacilli) 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:
 28

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.

Preparing Smears from Solid Media

1. Obtain and prepare a slide by drawing a circle on the slide. Once again, turn the slide
upside-down to keep the wax out of the smear.
2. Obtain a slant inoculated with the micro organism
3. Place a drop of water in the circle.
4. Without gauging the agar, obtain a small amount of bacteria and mix withthe water
on the first circle. Flame the needle.
5. Place the slide off to the side to dry
6. After the slide from this prep has dried, heat fix the organisms as was done in Prep 1.
7. Then proceed to Procedure 1.
 29

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% Loeffler’s 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 AND DISCUSSION:

EXP 8: IDENTIFICATION OF MICRO ORGANISMS: STAINING TECHNIQUES –

DATE: GRAM STAINING

AIM:
 30

 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
 31

Fig: Gram positive bacteria

2. Gram negative bacteria: Stain red or pink due to retaining the counter staining dye called
Safranin. Example: Escherichia coli

Fig: Gram negative bacteria

Gram Stain Mechanism:

Gram Positive Cell Wall:

Gram-positive bacteria have a thick mesh-like cell wall which is made up of

peptidoglycan (50-90% of cell wall), which stains purple. Peptidoglycan is mainly a
polysaccharide composed of two subunits called N-acetyl glucosamine and N-acetyl muramic
acid. As adjacent layers of peptidoglycan are formed, they are cross linked by short chains of
 32

peptides by means of a transpeptidase enzyme, resulting in the shape and rigidity of the cell wall.
The thick peptidoglycan layer of Gram-positive organisms allows these organisms to retain the
crystal violet-iodine complex and stains the cells as purple.

Lipoteichoic acid (LTA) is another major constituent of the cell wall of Gram-positive
bacteria which is embedded in the peptidoglycan layer. It consists of teichoic acids which are
long chains of ribitol phosphate anchored to the lipid bilayer via a glyceride. It acts as regulator
of autolytic wall enzymes (muramidases: Bacterial enzymes located in the cell wall that causes
disintegration of the cell following injury or death.

Medical Relevance of Gram Positive Cell Wall:

LTA also has antigenic properties that stimulate specific immune responses when it is
released from the cell wall after cell death. Cell death is mailnly due to lysis induced by
lysozymal activities, cationic peptides from leucocytes, or beta-lactam antibiotics.

Gram Negative Cell Wall:

Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall) and
lose the crystal violet-iodine complex during decolorization with the alcohol rinse, but retain the
counter stain Safranin, thus appearing reddish or pink. They also have an additional outer
 33

membrane which contains lipids, which is separated from the cell wall by means of periplasmic
space.

Medical Relevance of Gram Negative Cell Wall:

The cell wall of Gram-negative bacteria is often a virulence factor that enables
pathogenic bacteria to cause disease. The virulence of Gram-negative bacteria is often associated
with certain components of the cell wall, in particular, the lipopolysaccharide (otherwise known
as LPS or endotoxin). In humans, LPS elicits an innate immune response characterized by
cytokine production and activation of immune system. Inflammation occurs as a result of
cytokine production, which can also produce host toxicity.

Stain Reaction:

The four basic steps of the Gram Stain are:

1) Application of the primary stain Crystal Violet (CV) to a heat-fixed smear of bacterial culture.

CV dissociates in aqueous solutions into CV+ and Cl – ions. These two ions then penetrate
through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The
CV+ ions later interacts with negatively charged bacterial components and stains the bacterial
cells purple.
 34

2) Addition of Gram’s Iodine.

Iodine (I – or I3 –) acts as a mordant and as a trapping agent. A mordant is a substance that

increases the affinity of the cell wall for a stain by binding to the primary stain, thus forming an
insoluble complex which gets trapped in the cell wall. In the Gram stain reaction, the crystal
violet and iodine form an insoluble complex (CV-I) which serves to turn the smear a dark purple
color. At this stage, all cells will turn purple.

3) Decolorization with 95% ethyl alcohol.

Alcohol or acetone dissolves the lipid outer membrane of Gram negative bacteria, thus leaving
the peptidoglycan layer exposed and increases the porosity of the cell wall. The CV-I complex is
then washed away from the thin peptidoglycan layer, leaving Gram negative bacteria colorless.

On the other hand, alcohol has a dehydrating effect on the cell walls of Gram positive bacteria
which causes the pores of the cell wall to shrink. The CV-I complex gets tightly bound into the
multi-layered, highly cross-linked Gram positive cell wall thus staining the cells purple.

The decolorization step must be performed carefully, otherwise over-decolorization may occur.
This step is critical and must be timed correctly otherwise the crystal violet stain will be removed
from the Gram-positive cells. If the decolorizing agent is applied on the cell for too long time ,
the Gram-positive organisms to appear Gram-negative. Under-decolorization occurs when the
alcohol is not left on long enough to wash out the CV-I complex from the Gram-negative cells,
resulting in Gram-negative bacteria to appear Gram-positive.

4) Counterstain with Safranin

The decolorized Gram negative cells can be rendered visible with a suitable counterstain, which
is usually positively charged safranin, which stains them pink. Pink colour which adheres to the
Gram positive bacteria is masked by the purple of the crystal violet (Basic fuschin is sometimes
used instead of safranin in rare situations).
 35

Note that the success of the Gram stain relies upon the integrity of the cell wall. Gram positive
bacteria that have been overly heat fixed resulting in destruction of all or parts of their cell wall
can appear to be pink (Gram negative) or have pink areas. This is an artifact! Further, old
moribund cultures of Gram positive cells can appear pink. This is because the cell wall has
allowed the challenge rinse to enter the cell. Successful Gram stains should be done on young,
growing cells.

Fig: Colour changes that occur at each step in the staining process

Typical Gram-negative bacteria:

1. Bordetellapertusis, the causative agent of whooping cough

2. Salmonella typhi, the causative agent of typhoid

3. Vibrio cholera, the causative agent of cholera

4. Escherichia coli, the normally benign, ubiquitous, gut-dwelling bacteria

Typical Gram-positive bacteria:

1. Staphylococci such as Staphylococcus epidermidis and Staphylococcus aureus which is

a common cause of boils.
 36

2. Streptococci such as the many species of oral streptococci, Streptococcus pyogenes

which causes many a sore throat and scarlet fever and Streptococcus pneumoniae which
causes lobar pneumonia.

3. Clostridia such as Clostridium tetani, the causative agent of tetanus (lockjaw).

4. Actinomyces such as Actinomycesodontolyticus which is found in mouth.

5. Species of the genus Bacillus such as Bacillus subtilis which are common microbes
living in soil.

Generally cocci are Gram-positive but there are exceptions. The most significant from a clinical
point of view is the gonococcus, Neisseria gonorrhoea which typically appears as a Gram-
negative diplococcus looking very much like a pair of kidney bean.

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
 37

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.

Please note: It is very important to prevent preparing thick, dense smears which contain an
excess of the bacterial sample. A very thick smear diminishes the amount of light that can pass
through, thus making it difficult to visualize the morphology of single cells. Smears typically
require only a small amount of bacterial culture. An effective smear appears as a thin whitish
layer or film after heat-fixing.

Part 4: Heat Fixing

 38

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 Gram’s 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 drythe slide with bibulous paper.
10. View the smear using a light-microscope under oil-immersion.

RESULT:
 39

EXP: 9 MICROBIAL GROWTH KINECTICS

DATE:

AIM:

1. To study the different phases of bacterial growth.

2. To plot standard growth curve of Staphylococcusaureus.

3. To determine the generation time of given bacteria.

PRINCIPLE:

The increase in the cell size and cell mass during the development of an organism is
termed as growth. It is the unique characteristics of all organisms. The organism must require
certain basic parameters for their energy generation and cellular biosynthesis. The growth of the
organism is affected by both physical and Nutritional factors. The physical factors include the
pH, temperature, Osmotic pressure, Hydrostatic pressure, and Moisture content of the medium in
which the organism is growing. The nutritional factors include the amount of Carbon, nitrogen,
Sulphur, phosphorous, and other trace elements provided in the growth medium. Bacteria are
unicellular (single cell) organisms.

When the bacteria reach a certain size, they divide by binary fission, in which the one cell
divides into two, two into four and continue the process in a geometric fashion. The bacterium is
then known to be in an actively growing phase.

To study the bacterial growth population, the viable cells of the bacterium should be
inoculated on to the sterile broth and incubated under optimal growth conditions. The bacterium
starts utilising the components of the media and it will increase in its size and cellular mass. The
dynamics of the bacterial growth can be studied by plotting the cell growth (absorbance) versus
the incubation time or log of cell number versus time. The curve thus obtained is a sigmoid curve
and is known as a standard growth curve.
 40

The increase in the cell mass of the organism is measured by using the
Spectrophotometer. The Spectrophotometer measures the turbidity or Optical density which is
the measure of the amount of light absorbed by a bacterial suspension. The degree of turbidity in
the broth culture is directly related to the number of microorganism present, either viable or dead
cells, and is a convenient and rapid method of measuring cell growth rate of an organism. Thus
the increasing the turbidity of the broth medium indicates increase of the microbial cell mass
(Fig 1) .The amount of transmitted light through turbid broth decreases with subsequent increase
in the absorbance value.

Fig 1: Absorbance reading of bacterial suspension

The growth curve has four distinct phases

1. Lag phase

When a microorganism is introduced into the fresh medium, it takes some time to adjust
with the new environment. This phase is termed as Lag phase, in which cellular metabolism is
accelerated, cells are increasing in size, but the bacteria are not able to replicate and therefore no
increase in cell mass. The length of the lag phase depends directly on the previous growth
condition of the organism. When the microorganism growing in a rich medium is inoculated into
nutritionally poor medium, the organism will take more time to adapt with the new environment.
The organism will start synthesising the necessary proteins, co-enzymes and vitamins needed for
their growth and hence there will be a subsequent increase in the lag phase. Similarly when an
organism from a nutritionally poor medium is added to a nutritionally rich medium, the organism
can easily adapt to the environment, it can start the cell division without any delay, and therefore
will have less lag phase it may be absent.
 41

2. Exponential or Logarithmic (log) phase

During this phase, the microorganisms are in a rapidly growing and dividing state. Their
metabolic activity increases and the organism begin the DNA replication by binary fission at a
constant rate. The growth medium is exploited at the maximal rate, the culture reaches the
maximum growth rate and the number of bacteria increases logarithmically (exponentially) and
finally the single cell divide into two, which replicate into four, eight, sixteen, thirty two and so
on (That is 20, 21, 22, 23.........2n, n is the number of generations) This will result in a balanced
growth. The time taken by the bacteria to double in number during a specified time period is
known as the generation time. The generation time tends to vary with different organisms.
E.colidivides in every 20 minutes, hence its generation time is 20 minutes, and for
Staphylococcus aureus it is 30 minutes.

3. Stationary phase

As the bacterial population continues to grow, all the nutrients in the growth medium are
used up by the microorganism for their rapid multiplication. This result in the accumulation of
waste materials, toxic metabolites and inhibitory compounds such as antibiotics in the medium.
This shifts the conditions of the medium such as pH and temperature, thereby creating an
unfavourable environment for the bacterial growth. The reproduction rate will slow down, the
cells undergoing division is equal to the number of cell death, and finally bacterium stops its
division completely. The cell number is not increased and thus the growth rate is stabilised. If a
cell taken from the stationary phase is introduced into a fresh medium, the cell can easily move
on the exponential phase and is able to perform its metabolic activities as usual.

4. Decline or Death phase

The depletion of nutrients and the subsequent accumulation of metabolic waste products
and other toxic materials in the media will facilitates the bacterium to move on to the Death
phase. During this, the bacterium completely loses its ability to reproduce. Individual bacteria
begin to die due to the unfavourable conditions and the death is rapid and at uniform rate. The
 42

number of dead cells exceeds the number of live cells. Some organisms which can resist this
condition can survive in the environment by producing endospores.

Fig 2: Different phases of growth of bacteria

CALCULATION:

The generation time can be calculated from the growth curve(Fig 3).

Fig 3: Calculation of generation time

 43

The exactly doubled points from the absorbance readings were taken and, the points were
extrapolated to meet the respective time axis.

Generation Time = (Time in minutes to obtain the absorbance 0.4) – (Time in minutes to obtain
the absorbance 0.2)

= 90-60 = 30 minutes

Let No = the initial population number

Nt = population at time t

N = the number of generations in time t

Therefore,

Therefore,

The growth rate can be expressed in terms of mean growth rate constant (k), the number of
generations per unit time.

Mean generation time or mean doubling time (g), is the time taken to double its size.
 44

Therefore,

Substituting equation 4 in equation 3

(Since the population doubles t= g)

Therefore,

Mean growth rate constant

Mean generation time,

MATERIALS REQUIRED:

Nutrient broth, Sterile petriplates, Micropipettes, Cuvette, Conical flask, Sterile tips, Culture –
Overnight culture of Staphylococcus aureus, Colorimeter
 45

PROCEDURE:

1. An isolated colony of the organism (Staphylococcus aureus) was inoculated into 15 ml

nutrient broth and kept for overnight incubation

2. Following day, the OD of this culture was measured and confirmed.

3. In order to adjust the OD of the inoculum to the standard value (0.05) the following
dilution formula was used

OD1V1 = OD2V2
Where,
OD1 = OD of the broth culture, inoculated the previous day.
V1 = volume of this broth culture to be added to the inoculums
OD2 = OD of the inoculum (as a standard, this value was adjusted to 0.05)
V2 = volume of the inoculums (in this experiment, 50 ml)

4. Substitute the values in the equation and V1 was calculated.

5. That much amount (V1) of the inoculums was pipetted out before adding an equivalent
amount of the broth to it, so that the net volume remains constant.

6. The OD was checked at every 30 minutes interval and recorded.

7. Using this OD value, a standardized growth curve of the organism was plotted.
(Absorbance verses time).

8. Generation time was calculated.

RESULT:
 46

EXP 10: PHYSICAL FACTORS AFFECTING GROWTH: EFFECT OTEMPERATURE

DATE: ON MICROBIAL GROWTH

AIM:
To study the effect of temperature on microbial growth

PRINCIPLE:
There are many factors controlling microbial growth, including temperature, oxygen, pH,
and water availability. Microbes do not have homeostatic (maintenance of equilibrium)
mechanisms and are therefore directly affected by any changes in the environment. All bacteria
have a minimum, maximum, and optimum temperature for growth. At low temperatures,
enzymatic activity is slowed and growth is minimal. At high temperatures, enzymatic activity is
high, and growth is rapid. Some bacteria are strict aerobes, and will not grow well under
conditions where O2 concentration is low. Others are facultative anaerobes, which grow best with
lots of O2, but can switch to anaerobic respiration if O2 levels are reduced. Some microbes are
halophiles (have an absolute requirement for moderate to high concentrations of salt) or
halotolerant (can tolerate some salt in their environment, but do not require it for growth).
Microbes that cannot tolerate high salt become dehydrated and cannot grow.

In this lab, we will investigate the effects of either temperature or oxygen on the growth of three
different species of bacteria.

There are 3 temperature groupings observed by microbiologists.

Psychrophiles - This group multiplies best at a range of 0°C to around 20°C.Members of this
group are often found causing spoilage of food in the refrigerator.

Mesophiles - Members of this group live in a range of temperatures from20°C to about 40°C.
Most human pathogens are members of this temperature group as well as normal animal flora.
They are also found in abundance in topsoil.
 47

Thermophiles - These microbes thrive at high temperatures. Normally found in hot springs, spas,
desert soils, and other areas that are hot environments, they multiply well in temperatures in
excess of40°C.

MATERIALS REQUIRED:

Sterile nutrient agar plates, nutrient broth tubes, cultures of E.coli and S.cerevisiae,
micropipette, sterile tips for pipetteman, inoculating loop and alcohol, refrigerator set at 4°C, 3
incubators set at 20°C, 30°C and 60°C

PROCEDURE:

1. Score the underside of the NA plates into 2 halves and label with the names of test
bacteria and temperature.
2. Aseptically inoculate the plates with E.coli and B.thuringiensis by means of a single line
of inoculation in the appropriately labelled section.
3. Gently shake the S.cerevisiae culture to suspend the microbes.
4. Using the micropipette, transfer aseptically 20 µl of the suspension into the sterile NB.
5. Incubate the plates in an inverted position at appropriate temperatures of 4°C, 20°C, 30°C
and 60°C for 24-48 hours.
6. Observe all cultures for the presence of growth.
7. Record the growth as 1+ for scant growth, 2+ for moderate growth, 3+ for abundant
growth and – for absence of growth.
8. Tabulate the results for all microbes

RESULT:
 48

EXP 11: PHYSICAL FACTORS AFFECTING GROWTH: EFFECT OF pH ON

DATE: MICROBIAL GROWTH

AIM:
To study the effect of pH on microbial growth

PRINCIPLE:

Each organism has a pH range within which growth is possible, and most have well
defined pH optima. Most natural environments have pH values between 5 –9, and most
organisms have pH optima in this range. Very few species can grow at pH values below 2 or
above 10.

Acidophiles have their growth optimum between pH 0 and 5.5; neutrophiles, between
pH 5.5 and 8.0; and alkalophile sprefer the pH range of 8.5 to 11.5. Extreme alkalophiles have
growth optima at pH 10 or higher. In general, different microbial groups have characteristic pH
preferences. Most bacteria and protozoa are neutrophiles. Most fungi prefer slightly acid
surroundings, about pH 4 to 6; algae also seem to favor slight acidity.

Although microorganisms will often grow over wide ranges of pH and far from their
optima, there are limits to their tolerance. Drastic variations in cytoplasmic pH can harm
microorganisms by disrupting the plasma membrane or inhibiting the activity of enzymes and
membrane transport proteins. Prokaryotes die if the internal pH drops much below 5.0 to 5.5.
Changes in the external pH also might alter the ionization of nutrient molecules and thus reduce
their availability to the organism.

MATERIALS REQUIRED:

Flasks containing Sterile Nutrient Broth, cultures of E.coli, B.thuringiensis and S.cerevisiae,
micropipette, sterile tips for pipetteman, incubators set at 37°C
 49

PROCEDURE:

1. Adjust the pH of 4 sets of sterile NB flasks to 3, 4, 5 and 9 with 1N HCl and 1N

NaOH.
2. Prepare saline suspensions of 24 hour cultures and adjust to an OD of 0.05 at 600 nm.
3. Aseptically inoculate the flasks with E.coli and by transferring 0.1 ml of the saline
suspension to each flask.
4. Label each flask appropriately.
5. Repeat steps 3 and 4 with B.thuringiensis and S.cerevisiae .
6. Incubate the flasks with bacteria at 37°C for 24-48 hours and yeast for 48-72 hours.
7. Using a spectrophotometer record OD of all cultures at 600 nm
8. Tabulate the results for all microbes

RESULT:
 50

EXP 12: PHYSICAL FACTORS AFFECTING GROWTH: EFFECT OF UV

DATE: RADIATION ON MICROBIAL GROWTH

AIM:
To study the effect of UV radiation on microbial growth

PRINCIPLE:

Radiation is the process of emitting radiant energy in the form of waves or parti-cles.
Whether radiant energy is useful or destructive to microorganisms depends on its wavelength
Radiation of wavelengths of 400 nm and below (ultraviolet, X, and γ radiation) is damaging to
the structure of DNA and is thus both mutagenic and carcinogenic.

Ultraviolet (UV) radiation (10 to 400 nm wavelength) is of special interest because it is

used in certain environments to kill microorganisms. For example, hospital operating rooms are
often lined with UV lights, called germicidal lamps. When people are not using these areas, the
lamps are turned on to kill microbes on the surfaces of walls, floors, ceilings, and bench tops,
which helps to keep the work environment relatively sterile. Ultraviolet light is considered to be
a germicide rather than a sterilant because it only has the potential for killing dividing cells and
does not kill bacterial spores.

The most germicidal region of the UV spectrum occurs from about 240 nm to 300 nm.
DNA absorbs light of 260 nm strongly, which is where the greatest lethal effects occur. Several
effects of UV on DNA are known, but the most thoroughly studied is the formation of thymine
dimers.

Although very little UV radiation below 290 to 300 nm reaches the earth’s surface, near-
UV radiation between 325 and 400 nm can harm microorganisms. Exposure to near-UV
radiation induces tryptophan breakdown to toxic photoproducts. It appears that these toxic
tryptophan photoproducts plus the near-UV radiation itself produce breaks in DNA strands. The
precise mechanism is not known, although it is different from that seen with 260 nm UV.
 51

Low doses of radiation may not produce any adverse affects on cells. If one lengthens the
exposure time, or increases the intensity of the UV light, an increase in the number of unrepaired
dimers and an increase in mutations probably occur. If a mutation occurs in an essential gene, the
cell may die and is said to contain a lethal mutation.

MATERIALS REQUIRED:

Sterile nutrient agar plates, cultures of E.coli and S.cerevisiae, UV radiation source,
micropipette, sterile tips for pipetteman, forceps, glass spreader and alcohol

PROCEDURE:

1. Divide the NA plates into 2 sections by scoring the underside of each plate with a
glass marker.
2. Label each section with the name of the organism.
3. Using sterile technique, inoculate the plates by streaking in each section.
4. Label the cover of each plate with exposure time to UV radiation as 0 (control), 1
minute, 3 minutes, 5 minutes. Label two plates as 7 minutes, one which will serve as
the covered irradiated control.
5. Irradiate all inoculated plates for the designated time period by placing them 12
inches from the UV light source. Ensure that all plates have the covers removed
except the 7 minute control.
6. Incubate all plates in an inverted position for48 hours at 37°C.
7. Observe all cultures for the presence of growth.
8. Record the growth as 1+ for scant growth, 2+ for moderate growth, 3+ for abundant
growth and – for absence of growth.
9. Tabulate the results for all microbes

Precaution: Do not expose your eyes to the UV light.

RESULT:
 52

EXP 12: THE EFFECTS OF CHEMICAL AGENTS OF BACTERIA –

DATE: PHENOL COEFFICIENT

AIM:
To determine the effectiveness of some chemical disinfectants used as antimicrobial
agents and calculate a 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. In

general, the former method is called sterilization while the later is termed disinfection.
Sterilization is always aimed at both pathogenic and nonpathogenic bacteria, while disinfection,
in its true sense, applies only to the pathogenic ones. A disinfectant is any agent, such as heat or
a chemical (like iodine) that kills pathogenic microorganisms. The term generally applies to
preparations, usually liquids, intended for use on inanimate objects, as distinguished from living
tissues (antiseptics). Sterilization is achieved by any process that completely removes or
destroys all living organisms in or on an object.

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.
Disinfectants, however, are considered germicidal or bacteriocidal. Germicides are chemicals
that are usually lethal to bacteria and are meant to be used on non-living areas such as floors,
work benches, etc.

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
 53

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. The first part of this exercise will examine
this effect of several chemicals. 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 of a compound is the ratio of the minimal sterilizing concentration of phenol
(under standard conditions) to that of the compound.

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. The second part of this experiment will enable you to calculate a phenol
coefficient for a select chemical.

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.
 54

Agents Used to Control Microbial Growth

A. Antiseptics and Disinfectants

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 oligo-
dynamic action such as combining with the sulfhydryl (-SH) groups and denaturing proteins.
Examples include silver nitrate, mercurochrome, and copper sulfate.
 55

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.
 56

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 are diluted with tap water to obtain 1:400, 1:450, and 1:500 dilutions.

PROCEDURE:

1. Label a set of 9 nutrient broth tubes for the 3 disinfectants, with the name and dilution of
the disinfectant and time interval of sub-culturing, as given in the table.

2. Place them on test tube racks.

 57

3. Place the test tubes with disinfectant dilutions in separate racks.

4. Using a pipette rapidly introduce 0.05 ml (one drop) of the E.coli culture into each of the
test tubes with disinfectant. Note the time of inoculation.

5. Mix the tubes well, to ensure contact of the disinfectant with the microbe.

6. At intervals of 5, 10, and 15 minutes, using sterile technique transfer one loopful from
each test into the appropriate sterile tube of nutrient broth.
7. Incubate all nutrient broth cultures for 48 hours at 37°C.
8. Observe all cultures for the presence of growth.
9. Record + for the presence of growth and 0 for absence of growth.
10. Tabulate the results for the 3 disinfectants.

Tabulation:

Chemical agent and Dilution 5 min 10 min 15 min

Phenol

1:80

1:90

1:100

Dettol

1:400

1:450

1:500

Lyzol

1:400

1:450
 58

1:500
 59

EXP: 13 ANTIBIOTIC SENSITIVITY ASSAY

DATE:

AIM:
To test the sensitivity of E.coli to antibiotics like tetracycline, streptomycin.

PRINCIPLE

Antibiotic sensitivity testing aims to determine the susceptibility of an isolate to a range

of potential therapeutic agents. This can be with a view to individualizing the antibiotic to be
administered or to monitor resistance patterns developing in that environment, gathering this
information is important for revising and updating the standard antibiotic prescribing policy for a
particular population or institution.

Resistance to antibiotics can either be naturally occurring for a particular organism/drug

combination or acquired resistance, where mis-use of anti-microbials results in a population
being exposed to an environment in which organisms that have genes conferring resistance
(either spontaneously mutated or through DNA transfer from other resistant cells) have been able
to flourish and spread.

One method that is used to determine antibiotic susceptibility is the sensitivity disk method of
Kirby- Bauer (named after W. Kirby and A. W. Bauer in 1966). In this method, antibiotics are
impregnated onto paper disks and then placed on a seeded Mueller-Hinton agar plate using a
mechanical dispenser or sterile forceps. The plate is then incubated for 16 to 18 hours, and the
diameter of the zone of inhibition around the disk is measured to the nearest millimeter. The
inhibition zone diameter that is produced will indicate the susceptibility or resistance of a
bacterium to the antibiotic. Antibiotic susceptibility patterns are called antibiograms.
Antibiograms can be determined by comparing the zone diameter obtained with the known zone
diameter size for susceptibility. For example, a zone of a certain size indicates susceptibility,
zones of a smaller diameter or no zone at all shows that the bacterium is resistant to the
antibiotic.
 60

Frequently one will see colonies within the zone of inhibition when the strain is antibiotic
resistant. Many factors are involved in sensitivity disk testing and must be carefully controlled.
These include size of the inoculum, distribution of the inoculum, incubation period, depth of the
agar, diffusion rate of the antibiotic, concentration of antibiotic in the disk, and growth rate of the
bacterium. If all of these factors are carefully controlled, this type of testing is highly satisfactory
for determining the degree of susceptibility of a bacterium to a certain antibiotic.

The Kirby-Bauer method is not restricted to antibiotics. It may also be used to measure the
sensitivity of any microorganism to a variety of antimicrobial agents such as sulfonamides and
synthetic chemotherapeutics.

Fig : Diameter of the various zones of inhibition of agar plate inoculated with S. aureus and various
antibiotics.

REQUIREMENTS:

Sterile nutrient agar plates, culture of E.coli, Bunsen burner, antibiotic discs, micropipette, sterile
tips for pipetteman, forceps, glass spreader and alcohol.

PROCEDURE

1. Flame the glass spreader and allow cooling.

 61

2. Flame the mouth of the test tube containing the bacterial suspension.
3. Using the micropipette, transfer aseptically 500 µl of the suspension on to the surface of
the agar plate.
4. Spread the suspension uniformly with the sterile glass spreader.
5. Close the petri plate and label.
6. Distribute 3 individual antibiotic discs in each plate at equal distances with forceps
previously sterilized by flaming with alcohol.
7. Gently press each disc down to ensure that the discs adhere to the agar surface.
8. Indicate the position of each disc on the underside of the plate.
9. Incubate the plates at 37°C. for 24-48 hours.
10. Observe the plates for zones of inhibition.
11. Tabulate the diameters of the inhibition zones for the different antibiotics used.

RESULT: