Exp.

No:

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 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

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 160°C 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 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.

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 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.

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

b) ISOLATION OF PURE CULTURES- POUR PLATE METHOD

AIM:
To perform pour plate procedure for isolation of colony from a mixed culture

PROCEDURE:
1. Dilute specimen to yield approximately 30 to 300 CFU per aliquot to be plated
2. Inoculate labeled empty petri dish with the aliquot of diluted specimen
3. Pour 15 mL of melted Plate Count Agar (45o C) into the inoculated petri dish.
4. Cover and mix thoroughly by gentle tilting and swirling the dish. Do not slop the agar over
the edge of the petri dish.
5. Place on a flat surface undisturbed for about 10 minutes to allow the agar to completely gel. In
this illustration, the agar is completely gelled and the surface is "smooth as glass."
6. Invert and incubate at 37o C for 24-48 hours.
7. Count, record, calculate:

RESULTS:

c) 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 37°C in the incubator for overnight for the growth of pure culture

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:

EXP:
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 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:

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
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
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.

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.
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, non-
spore forming rod-shaped species which ferment lactose with the production of acid and gas
within 48 h at 37°C. 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 ºC.

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 McCready’s 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 37ºC for 24 to 48 h.
4. Any amount of gas in the inverted Durham’s 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 10ºC.
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

Lauryl tryptose broth with inverted Durham’s tube, EMB or Endo agar plates, Brilliant green
lactose bile broth (BGLB) with inverted Durham’s tube NA Plates and water sample.

Procedure
(A) Presumptive Test
(1) Inoculate 10ml of water sample into each of 3 LT broth tubes (double strength)
Inoculate 1ml and 0.1 ml of water into each of 3 lauryl tryptose broth tubes (single
strength).

(2) Incubate all tubes at 37ºC for 24-48 h. Any amount of gas in the inverted Durham’s
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 37ºC 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 37ºC for 24 h and observe for gas in the LTB
tube
4. Perform the IMViC tests.

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 37ºC 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.

Differentiation of Fecal from Non-Fecal Coliform Organisms using IMViC Tests

The general practice is to classify the members of coliform group into fecal E-coil and non-fecal
E. freundii and Aerobacter aerogenes. The classification is based on the results of four tests
namely (I) indole (M)methyl red(V) Vogus Proskauer and (C) Sodium citrate. (IMViC) The letter
i between V and C is added solely for euphony.
Indole Test

Medium: Tryptone water

Reagent: Kovac’s reagent

Indole is a putrefactive compound produced from tryptophan by some bacteria. Some bacteria
can produce indole others cannot. The test is therefore of value in the identification and
classification of bacteria. Tryptophan is not present in all the proteins. Casein is rich in
tryptophan and is frequently used for the preparation of peptone. Thus tryptone is the peptone
which is rich in amino acid tryptophan.

Inoculate the medium (tryptone water) with appropriate culture and incubate at the optimum
growth temperature for 48 h. Add 0.5 ml of Kovac’s reagent shake tube gently and allow to
stand. A red or pink upper layer is recorded as an indole positive test. In the presence of indole a
deep red colour develops which separates out in the alcohol layer.

Composition

Kovac’s reagent:

Amyl or isoamyl alcohol - 150 ml

P-dimethyl amino benzalde-hyde - 10 g

C0onc. HCl - 50 ml

Dissolve aldehyde in alcohol and slowly add acid.

Methyl Red Test

Medium: Glucose phosphate broth

Reagent: Methyl red indicator

Culture of Escherichia rapidly ferments lactose with the formation of acids until the pH drops to
about 5.0. This acidity is sufficient to prevent it’s further growth. The amount of buffer present
influences the final pH attained by an organism when grown in the presence of a fermentable
substance. As the buffer content is increased the final acidity is decreased. The medium used for
the test contains 0.5% glucose and is buffered with di-potassium phosphate and peptone to give a
limiting pH of about 5.0 when inoculated with typical Escherichia. The methyl red indicator is
added (few drops) into the medium after incubation. If it turns red then the test is positive
indicating sufficient acid production. In case of Aerobacter it turns yellow indicating negative
MR test.

Voges - Proskauer test

Medium - Glucose phosphate broth

Reagent - (α-naphthol + 40% KOH) Omeara’s reagent.

Distinct differences exist in the carbohydrate metabolism of fecal (Escherichia) and non-fecal
(Aerobacter) coliforms. Aerobacter produces Acetylmethyl carbinol during carbohydrate
metabolism but not by E coli. This is a test for the production of acetyl methyl carbinol from
glucose. To the inoculated medium after incubation alkali is added in the presence of which any
acetyl methyl carbinol present becomes oxidized to diacetyl. The diacetyl will combine with
argenine present in peptone to give a rose colouration.
To inoculated and incubated tube add 0.6 ml of α-naphthol (5 g naphthol in 100ml 95% alcohol)
followed by 0.2 ml of 40% KOH. Shake well. A positive test is indicated by the appearance of a
red colour.

Simmon’s citrate test

Medium: Simmon’s citrate agar

Reagent: Medium contains bromo thymol blue as a pH indicator.
Coliforms could be separated into two groups on the basis of their action on sodium citrate Fecal
coliforms are unable to utilize citrate as the only source of carbon and non-fecal coliforms utilize
citrate readily.
Utilization of citrate and growth on citrate agar results in an alkaline reaction so that
bromothymol blue indicator in the medium changes from green to bright blue. When no growth
occurs and citrate is not utilized the colour of the medium remains unchanged.

Standard biochemical results of E coli, Klebsiella pneumoniae

Organism Glucose Lactose Indole MR VP Citrate

+ + + + - -
E. coli.
K. p. + + - - + +