Experiment : 1

No.
Aim : RULES AND SAFETY IN MICROBIOLOGY LABORATORY.

INTRODUCTION :

All hazards that are common to all laboratory work can be encountered in the
microbiology laboratory. However “ biohazards” are biggest and most constant
problem. A biohazard is potentially dangerous living microbes. Hence it is essential
that students of microbiology learn and consistently practice the fundamental rules of
laboratory safety.

LABORATORY SAFETY RULES :

1) Note carefully all instruction given by your instructor.

2) Every student must wear Lab coat. Do not use coat for wiping infectious
material, stains & apparatus outside the laboratory.
3) Do not put paper, pencils etc in your mouth. Do not moisten your fingers with
saliva for turning pages of books.
4) Do not bring foodstuff into Laboratory.
5) Care should be taken in handling microscope, culture tubes, etc.
6) Microscope and other items should be returned to their proper places after
cleaning.
7) Electricity and gas should be turned off when not in use.
8) Disinfect your hands with spirit after working with infectious material and before
using towels.
9) All glassware containing infectious material should be disinfected before
washing. Hollow slides and cover slips must be sterilized by placing them in a
disinfectant solution.
10) Instruments like scalpels, syringes should be thoroughly boiled before being put
away.
11) Cultures are not to be taken out of the laboratory.
12) All cultures must be grown in the incubator.
13) Colures and chemicals are not to be taken out of the laboratory.
14) Report all accidents and breakages of culture tubes to in charge.

Page 1 of 44
15) Use every precaution to avoid accidents.

Table for Laboratory Requirements :-

Sr. Laboratory Laboratory Laboratory Other Miscellaneous

No. Instruments Glassware Pastiweares Accessories Things
1 Laminar air flow Test tubes Beakers Bunsen Cuvette
chamber Burner
2 Microscope Pipettes Funnel Chemicals Scissors

3 Autoclave Pasture Measuring Inoculating Stop watch

pipette cylinder Wire loop
4 Hot air oven Measuring Loops Thermometer
cylinder Straight wire
5 PH meter Volumetric Culture Wire gauze
flask Media

6 Orbital Shaker Conical Test-tube

flask holder

7 Colony Counter Beakers Pipette holder

8 Refrigerator Durham’s Centrifuge

tube tube

9 Centrifuge Microscopic Desiccator

slide
10 Spectrophotometer Cover slip Reagent
holder
11 Water bath Petri dishes Test-tube
stand
12 Incubators Spreader Reagent bottle

13 Metal distillery Cavity slide Sterile cotton

14 Glass distillery Funnel Blotting paper

15 Vortex Sugar tubes Aluminum foil

Page 2 of 44
Experiment : 2
No.
Aim : OBSERVATION OF MICROSCOPE

INTRODUCTION :

The word microscope is derived from two Greek words “micro” meaning small and
“scope” meaning to view. In other words a microscope is an instrument used for
visual examination of small objects, which cannot be properly examined by the
unaided eye.

The microscope is the instrument most characteristic of the microbiology laboratory.

The magnification it provides enables us to see microorganisms and their structures
otherwise invisible to the naked eye. The magnification attainable by microscopes
range from 100x to 400,000 x.

In addition, several different kinds of microscopy are available, and many techniques
have been developed by which specimens of microorganisms can be prepared for
examination. Each type of microscopy and each method of preparing specimens for
examination offer advantages for demonstration of specific morphological features.

FUNCTIONS OF VARIOUS PARTS :

The component parts of Microscope, their locations and functions are follows.

1) Base or Stand : the “U” shaped foundation, which gives stability to the
instrument.

2) Arm or Handle : by means of which it may be moved or carried to which are

attached the magnifying and adjustment systems.

3) Stage : a plate form, which provides a surface for the placement of slide with its
specimen over the opening. In addition to the fixed stage, most microscopes
have a mechanical stage that can be moved vertically or horizontally by means
of adjustment knobs.

4) Clips : the se of removable spring clips which hold in place the slide with object.

5) Mirror : the light reflecting device placed under stage and employed to
illuminate the object on the stage.

6) Condenser : a system of lenses placed under the stage; its function is to focus
a strong beam of light upon the object being examined.

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7) Iris diaphragm : a device placed beneath the condenser and capable of
manipulation so as to adjust the quantity of light.

8) Body tube : a hollow cylindrical tube of the magnification system through which
light passes from the objective lenses as its bottom to the eyepiece lenses at
the top.

9) Nosepiece : a revolving disk at the bottom of the body tube to which the
objectives are to be attached.

10) Objectives : a system of small lenses, which constitute primary magnifying

mechanism and are attached to the nosepiece. There are usually three
objectives, low power, high power & oil immersion objectives.

11) Eyepiece or Ocular : a combination of lenses, placed in the upper portion of

the body tube, which magnifies the image formed by the objective lens system.
Two eyepieces ---5X and ---10X are generally provided.

12) Coarse adjustment : the screw mechanisms by which the body tube and its
magnification system may be raised or lowered quickly over wide range to bring
the objective approximately into focus.

13) Fine adjustment : the screw mechanisms which moves the body tube slowly,
and over very limited range, by means of which the object is brought into sharp
or exact focus:

Page 4 of 44
Page 5 of 44
PRINCIPLE :

Microscopes are of two categories light (or optical) and electron, depending upon the
principle on which magnification is based.

Light microscopy, in which magnification is obtained by a system of optical lenses

using light waves, includes :

1) Bright field microscopy

2) Dark-field microscopy
3) Fluorescence microscopy
4) Phase contrast microscopy.

The electron microscope, as the name suggests, uses a beam of electrons in place
of light waves to produce the image. Specimens can be examined by either
transmission or scanning electron microscopy.

1) BRIGHT FIELD MICROSCOPY :

In bright field microscopy, the microscopic field (the area observed) is brightly
lighted and the microorganisms appear dark because they absorb some of the
light. Ordinarily, microorganisms do not absorb much light, but staining them with
a dye greatly increases their light-absorbing ability resulting in greater contrast
and color differentiation. The optical parts of a typical bright field microscope and
the path the light rays follow to produce enlargement or magnification, of the
object is shown in fig. A. Generally microscopes of this type produce a useful
magnification of about 100x to 200x. At magnifications greater than 200x the
image becomes fuzzy.

2) DARK-FIELD MICROSCOPY :

The effect produced by the dark-field technique is that of a dark background

against which object are brilliantly illuminated. This is accomplished by equipping
the light microscope with a special kind of condenser that transmits a hollow cone
of the light from the source of illumination. Most of the light directed through the
condenser does not enter the objective; the field is essentially dark. However,
some of the light rays will be scattered (diffracted) if the transparent medium
contains objects such as microbial cultures. This diffracted light will enter the
objective and reach the eye; thus the object or microbial cell, in this case, will
appear bright in an dark microscopic field. Dark-field microscopy is particularly
valuable for the examination of unstained microorganisms suspended in fluid.

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3) FLUORESCENCE MICROSCOPY :
Many chemical substances absorb light. After absorbing light of a particular
wavelength and energy, some substances will then emit light of a longer
wavelength and a lesser energy content, such substances are called fluorescent
and the phenomenon is formed fluorescence. Application of this phenomenon is
the basis of fluorescence microscopy. In practice, microorganisms are stained
with a fluorescent dye and then illuminated with blue light; the blue light is
absorbed and green light emitted by the substances.

4) PHASE CONTRAST MICROSCOPY :

Phase Contrast-microscopy is extremely valuable for studying living unstained
cells and is widely used in applied and theoretical biological studies. It uses a
conventional light microscope fitted with a phase-contrast objective and a phase I
contrast condenser. This special optical system makes it possible to distinguish
unstained structures within a cell, which differ only slightly in their refractive
indices or thick nesses.
In principle, this technique is based on the fact that light passing through one
material and into mother material of and into another material of a slightly different
refractive index and / or thickness will undergo a change in phase. These different
in phase, or wave front irregularities, are translated in to variations in brightness of
the structures and hence are detectable by the eye.

5) TRANSMISSION ELECTRON MICROSCOPY :

Electron microscopy differs markedly and in many respects from the optical
microscopic techniques. The electron microscope provides tremendous useful
magnification; because of the much higher resolution obtainable will the extremely
short wavelength of the electron beam used to magnify the specimen. The
electron microscope uses electron beams and magnetic fields to produce the
image, whereas the light microscope uses light waves and glass lenses.

6) SCANNING ELECTRON MICROSCOPY :

In scanning electron microscopy the specimen is subjected to a narrow electron
beam, which rapidly moves over (Scans) the surface of the specimen. This
causes the release of a shower of secondary electrons and other types of
radiation from.
The specimen surface. The intensity of these secondary electrons depends on the
shape and the chemical composition of the irradiated object. The secondary
electrons are collected by a detector, which generates which generates an
electronics signal. These signals are then scanned in the manner of television
systems to produce an image on a cathode ray tube. The image system for the
scanning electron microscope.

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A) LIGHT MICROSCOPE:

(Up side down)

Light Source

Condenser Lens

Specimen

Objective Lens

Projection lens
Or eye piece

Final image

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B) TRANSMISSION ELECTRON MICROSCOPE:

V Electron Gun

Condenser Lens

First Aperture

Specimen

Objective Lens

Second Aperture

Intermediate Lens

Projection Lens

Final Aperture

Final Image

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C) SCANNING ELECTRON MICROSCOPE:

V Electron Gun

First Aperture

First Condenser Lens

Second Aperture

Second Condenser Lens

Third Aperture

Scanning Coils

Final Condenser Lens

Final Aperture

Electron Collector
Signal

Signal For Electron

Specimen Image Formation

Page 10 of 44
Experiment : 3
No.
Aim : SIMPLE STAINING OF BACTERIA

INTRODUCTION :

In simple staining the smear is stained with single stain in a single step process;
hence it is known as monochrome stain. In such staining all cells are stained in a
similar way, however, structural differences in size, shape and the arrangement can
be easily visualized.

Many theories have been advanced to explain the phenomenon of staining. All
attempts to explain the process are on a purely physical or chemical basis.

PHYSICAL :

A physical process may be defined a reaction between two substances without the
formation of a new compound. When bacteria are stained, there is no evidence that
the stain has been changed chemically to form a new compound. It is usually
possible to extract all or nearly all the stain from the cells by sufficiently long
immersion in water, alcohol or other solvent. The bacterial protoplasm never
completely removes all the stain from solution. This is generally believed to be
contrary to a chemical reaction, in which a new compound is formed having
properties different from either component entering into its formation.

CHEMICAL :

Some parts of cell are acidic in reaction whereas other parts are basic. The synthetic
stains are either anionic (acidic) or cationic (basic) i.e. the color portion of the
molecule is either the negative or positive ion.

The proponents of the theory stated that the acidic constituents of the cell (nucleus)
reacted with basic stains and the basic constituents (cytoplasm) reacted with acidic
stains.

Summarizing, it may be stated that; available evidence seems to point to the fact that
staining is neither entirely physical nor entirely chemical but probably a combination
of both.

REQUIRMENTS :

1) Methylene Blue staining solution

2) Young cultures of Escharichi coli, Bacillus subtilis and Staphylococcus aureus.

Page 11 of 44
PROCEDURE :

1) Prepare a suspension of bacterial colony in a sterile 0.85% NaCl (saline) to

disperse the bacterial cells.
2) Take a slide and mark by pencil.
3) Prepare heat fixed smear of bacterial suspension.
4) Cover the smear with methylene blue.
5) After 5 minute wash with tap water
6) Air-dry the slide.
7) Observe under an oil immersion lens.

OBSERVATION :

RESULT :

The monochrome staining shows the organisms stained blue in colour.

NOTE :

Staining time differs with the type, the concentration and the composition of staining
solution. It also depends on the type of the organism to be stained. For example,
crystal violet requires 30 to 60 seconds for staining; while carbol fuchsin takes
approximately 15 to 30 seconds for the similar staining.

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Experiment : 4
No.
Aim : GRAM’S STAINING (DIFFERENTIAL STAINING) OF BACTERIA.

INTRODUCTION FOR STAINING :

It is extremely difficult to visualize microorganisms in living state, because they are
minute, transparent and colorless when suspended in aqueous medium. Hence, it is
necessary to stain them in order to visualize and to study their properties. Staining
also helps to differentiate microorganisms into specific groups as well as aids in
diagnosis of disease.

DYES (STAIN) :
A dye (stain) is “ any colored compound that reacts with or is adsorbed by or
dissolves in another phase and renders that phase colored. “ The phase so stained
may not always be solid.
Chemically a stain may be defined as ”an organic unsaturated cyclic compound,
usually a benzene ring] with chromophore and auxochrome group”. The color is
usually due to chromophores and dyeing property of salt formation is due to
anxochrone.
In a chemical possessing only chromophore group may be a good chromogen
[colored compound] but may not be a good stain / dye unless and until it has an
auxochrome group. Without an auxochrome group the chromogen is not able to bind
to cells or tissues or fibers.
The ability of a stain to bind to macromolecular cellular components depends on the
electrical charge found on the chromogen portion as well as on the cellular
components to be stained.

INTRODUCTION FOR GRAM’S STAINING :

Christain Gram discovered gram’s staining in the year 1884. It is a very important
differential staining because it separates bacteria into two board categories namely
gram positive and gram negative.
This differential staining requires the use of at least four reagents that are applied
sequentially. The first reagent is called the primary stain – crystal violet (CV – I)
[because it is applied first] that will impart its color to all cells in conjunction with the
second reagent iodine, the mordant. In order to establish color contrast the third
reagent used is the decolorizing agent - alcohol. It may or may not remove the
primary stain from the cell, depending upon its cellular composition. Finally, to stain
the decolorized cells, another secondary stain - safranin of different color is applied.

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The cells which are not decolorized [i.e. those which have retained primary stain], will
not be affected by the counter stain.

PRINCIPLE :

Several theories have been proposed to explain the mechanism of Gram’s staining,
however, the one based on the physicochemical nature of cell wall of bacteria is
widely accepted. The cell walls of gram-negative bacteria are generally thinner than
those of gram-positive bacteria. Gram-negative bacteria possess higher percentage
of lipids in their cell wall as compared to gram-positive. During staining the primary
stain crystal violet forms complex with mordant iodine (CV-I) in the cell wall. When
the smear is decolorized with alcohol the lipids from gram-negative cells are
extracted thereby increasing the porosity or permeability of the cell. Hence the CV-I
complex is extracted from the cell an gram-negative organisms are decolorized.
These cells subsequent take on the color of the safranin counter stain. In contrast to
this gram-positive bacteria, because of their different composition [lower lipid
content], are dehydrate during treatment with alcohol. As a result the pore size
decrease permeability is reduced and CV-I complex cannot be extracted. Therefore
those cells remain purple violet.

REQUIREMENTS:

1) Young bacterial culture (i.e. E.coli, Bacillus subtilis, Staphylococcus, aureus)

2) Crystal violet, gram’s iodine, 95% ethanol (decolorizer) and safrainin

PROCEDURE :

1) Prepared a heat fixed smear of the bacterial culture

2) Cover the smear with crystal violet stain for 1 minute
3) Wash with water
4) Flood the smear with the gram’s iodine for 1 minute
5) Wash with water
6) Decolorize with 95% ethanol. Add alcohol drop by drop till the crystal violet fails to
come out from the smear.
5) Wash with water
6) Counter stain with safranin for 2 minutes
7) Wash with water, drain, plot, air dry and examine in oil immersion lence of
microscope

OBSRVATION :

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RESULTES :

Gram’s positive bacteria are stain purple violet while gram negative bacteria are
stained pink

FACTOR AFFECTING GRAM REACTION :

1. Age of bacterial cell: as the culture ages the gram-positive cells tend to lose
their ability to retain the primary stain and may appear to be gram variable [i.e.
some of the cells may appear pink].

2. Decolorization: it is the most critical step of the procedure, hence the nature,
concentration and the time of decolorizer used should be carefully controlled.
Over decolorization results in loss of primary stain, causing gram-positive
organisms to appear gram negative. Similarly under decolorization will not
completely remove CV-I complex, causing gram-negative organism to appear
gram-positive.

3. Excessive fixation of smear leads to loss in gram positiveness.

4. pH of the culture medium also influence the gram reaction.

5. Overcrowding of cells in the smear affect the result due to improper

decolorization

EXAMPLE :

Gram – Positive Bacteria Gram -Negative bacteria

Bacillus subtilis Escherichia coli

Streptococcus Pneumoniae Proteus vulgaris
Bacillus megaterium Enterobacter aerogenes,
Sporosrcina ureae Desulphococcus Spp.
Bacillus cereus Pseudomonas aeruginosa
Cellulomonas SP. Neisseria gonorrhoeae
Corynebacterium diphtheriae Methanococcus spp

Page 15 of 44
Experiment : 5
No.
Aim : NEGATIVE STAINING OF BACTERIA

INTRODUCTION:
Negative staining is so called because the dye does not stain the cell instead it stains
the background dark, thus creating the contrast, which makes the organisms visible.

PRINCIPLE:
Negative staining uses acidic dyes like nigrosine, eosin or Congo red. Acidic dyes
have negatively charged chromogen, which will not bind (react) with the bacterial
cells because of the similar (negative) charge on the surface of the bacterial cells.
Therefore the cells are unstained and can be easily visualized against the dark
background, practical applications of negative staining are

1) Natural shape and size of the cell is not distorted because the heat fixation is not
required during the staining.
2) It is possible to observe those bacteria, which are difficult to stain, such a
spirochetes.

REQUIREMENT:
1) Young cultures of E.coli, B. Subtilis, and S. aureus.
2) Nigrosine staining solution (10%)

PROCEDURES :
1) Place a drop of nigrosine and a loopful of culture at one end of the slide, and mix
them well.
2) Using the edge of a second slide held at a 30ºC angle push the mixture to form a
thin smear.
3) Allow the slide to air dry, and examine in oil immersion lense of microscope (do
not heat fix the slide)

OBSERVATION :

RESULT :
Organisms are observed as transparent objects against the dark background.

Page 16 of 44
Experiment : 6
No.
Aim : CELL WALL STAINING OF BACTERIA.

INTRODUCTION :

Most species of bacteria (except Mycoplasma, Spiroplasma, and certain

Archaeobacteria) have the outermost rigid layer known as cell wall.

FUNCTIONS :

1) It gives shape to the cell

2) Protects the cell from osmotic shock and toxic substances.
3) The cell walls of many pathogens have components that contribute to their
pathogenicity, (e.g. M Protein)
4) It is also the site for the attachment of bacteriophages.
5) It also imparts the antigenicity to the cell

COMPOSITION :

Bacteria can be divided into two groups on the basis of their differences in cell walls.
The gram-positive cell wall consists of a single 20-80 nm thick homogenous layer
made up of peptidoglycan (murein) surrounded by teichoic acids. In contrast, the
gram-negative cell wall has 1-3 nm peptidoglycan as inner layer, surrounded by 7-8
nm thick outer membrane, which is made up of lipopolysaccharides.

BACTERIA WITHOUT CELL WALL :

1) Mycoplasma / Spiroplasma / Archaeobacteria)

These are independent genera of naturally occurring bacteria, which lack cell wall.
They are stable and don’t require hypertonic environment for maintenance.

2) L-Forms
Cell wall deficient forms of bacteria can be produced in laboratory or may be
naturally formed by mutation. They are more stable than protoplast and
spheroplast. They can replicate on ordinary media.

3) Spheroplast
They are usually obtained from gram-negative bacteria, by growing in presence of
penicillin. They retain some residual but nonfunctional cell wall material, which
can regenerate under optimum conditions to form complete cell wall. They are
osmotically fragile and requires hypertonic medium for growth.

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4) Protoplast
They are usually obtained from gram-negative bacteria and totally lack cell wall.
They are unstable and osmotically fragile, requires hypertonic conditions for
maintenance. They can be produced in laboratory by lysozyme.

STAINING METHOD :
Cell wall has low affinity for stain and therefore, it is not stained with the routine stains
which are used for the cytoplasm staining. Most techniques use mordants, which
increase the affinity of cell wall for stain. It also increases the apparent thickness of
cell wall due to deposition of fine precipitates.

PRINCIPLE :
Since the cell wall has low affinity for the stain, tannic acid is used as the mordant
which increases the thickness of the wall by depositing on it as well as increases the
affinity between stain and the basic dye (crystal violet). Finally when the acidic dye
(Congo red) is applied the red background gives the clear contrast of the structure.

REQUIREMENTS :
1) Young culture of Bacillus megaterium
2) 10% tannic acid, 0.5% crystal violet and 0.5% Congo red

PROCEDURE :
1) Prepare a heat fixed smear.
2) Flood it with 10% tannic acid for 1 minutes
3) Wash the slide with water.
4) Stain the smear with 0.5% crystal violet for 2 minutes.
5) Wash the slide with water
6) Counter stain with 0.5% Congo red and spread it evenly over the smear with
another slide. Air dry and examine.

OBSERVATION:

RESULT :
Cell wall is violet in color with colorless cytoplasm with the red background.

Page 18 of 44
Experiment : 7
No.
Aim : CULTIVATION OF BACTERIA.

INTRODUCTION :

Cultivation is the process of growing microbial populations in artificial environment

{culture media} under laboratory conditions. Such a growth of particular type of
microorganisms on or with in a solid medium, or in liquid medium, obtained as result
of prior inoculation followed by incubation is called culture.

In order to cultivate microorganisms following conditions should be fulfilled.

1) They should be provided with utilizable source of nutrition for their cellular build up
and energy.
2) The environmental condition like pH, temperature, osmotic pressure, aeration etc
should be optimal.
3) Competition by other organisms must be minimal.

For majority of experimental works in microbiology, microorganisms are cultivated in

laboratory glassware, such a condition is known as in vitro. However, certain
bacteria and viruses require living host for growth, such a condition is called in vivo.

A culture produced by incubation of inoculated medium in presence of air is known as

aerobic culture; while the one produced in absence of oxygen is called anaerobic
culture.

A culture that contains only one kind {species} of microorganism is called pure
culture. The culture, which is genetically pure, is known as axenic culture {i.e. the
population derived from the multiplication of the single parent cell}. In contrast to this,
a culture containing two or more species or strains of organisms is known as mixed
culture.

When a sterile medium is exposed to non- sterile conditions, the extraneous

organisms may enter the medium and grow to gather with the inoculated organisms
this forms the contaminated culture.

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BROTH CULTIVATION :

Since the liquid medium does not provide any supportive base, organisms when
multiply and divide, they remain suspended and dispersed in the medium; hence
they do not form the colony. Due to this reason liquid find limited use for isolation
of organisms in pure culture.

REQUIREMENTS :

1) 5 tubes of nutrient broth

2) Sterile distilled water tube
3) Fertile garden soil [1 gm].
4) Sterile 1 ml pipettes, nicrome wire loop
5) Young cultures of E. coli, B. subtilis.

PROCEDURE :

1) Suspend 1 gm of fertile soil in 10 ml sterile distilled water.

2) Mix contents well and set aside the tube undistributed for 15 minutes, so as to
allow the soil particles to be settled down.
3) Withdraw about 0.2 ml of supernatant with the help of sterile pipette and inoculate
it into nutrient broth tube.
4) Inoculate one loopful or 0.2 ml of pure culture of E. coli, B. subtilis.
5) Label broth tubes with the name of organisms inoculated.
6) Leave one nutrient broth tube uninoculated as control.
7) Incubate all tubes at 370 C for 24 hour.
8) After incubation examine the broth tubes for the growth of microorganism, and
compare it with control tube.
9) Record and interprete the results.

RESULT:

Microorganisms grow in nutrient broth and growth can be described as follows.

ORGANISM GROWTH INTERPRETATION

E.coli Uniform turbidity facultative anaerobic
B.subtilis Pellicle {surface} strict aerobic
Garden soil turbidity/pellicle/ sediment Mixed flora present
Control no growth uninoculated

Page 20 of 44
CULTIVATION ON SOLID MEDIA :
Solid and semi-solid media are also used for the cultivation of bacteria. Solidifying
agent most commonly used for the cultivation is agar, which at a concentration of 1.5-
2.5 % forms a firm, transparent and colorless gel. At times other solidifying agents
like gelatin and silica gel are also used to make the medium solid. Solid media are
highly used for isolation of organisms and studying their growth {colony}
characteristics.
Semisolid media can be prepared with agar concentration of 0.5%. These gives a
soft, curd like consistency and are used for the cultivation of microaerphilic bacteria,
and also for the study of bacterial motility.
Solid and semi- solid media are used for cultivation of microorganisms with different
aims and hence can be prepared in different forms like plate slant medium and stab
medium.

PLATE CULTURE :

Plate culture is the growth of organisms usually on a medium poured in a petri dish.
Growth may be present as continuous layer or lawn or film [confluent growth] or as
discrete [isolated] colonies depending on the method of inoculation as well as size of
inoculums.

SLANT CULTURE :

Slant culture is also known as slope culture, is the growth on a solid medium, which
has been allowed to set in the test tube or bottle placed at horizontal angle. Such
slant cultures are used for maintenance and preservation of microorganisms.

STAB CULTURE :

Stab culture is the growth by deep inoculation of a semi-solid or solid in a tube with a
straight wire. The wire is plunged {stabbed or punctured} vertically into the medium
so that the inoculum is distributed along the length of the stab and growth is
observed along the line of inoculation.

Page 21 of 44
Experiment : 8
No.
Aim : ISOLATION OF MICROORGANISMS.

INTRODUCTION :
Isolation is a procedure in which a given species of present in a sample or specimen
is obtained in a pure culture. In nature, microorganisms normally grow in complex,
mixed populations containing several species. Hence in order to characterize and
study a particular organism it is essential to isolate them in pure forms.
There are several methods to obtain pure cultures.
A) Isolation by plating methods:
1. Streak plate method
2. Pour plate method
3. Spread plate method

B) Isolation in liquid media: Serial dilution technique.

C) Single cell isolation {micromanipulation}
D) Selective methods

 With the use of chemical agents

1) Enrichment culture: by selective favoring the growth of particular species
2) Selective technique: by selective inhibition of certain species

 With the use of physical agents:

1) Heat treatment: e.g. for selective isolation of spore formers
2) pH of the medium: e.g. for selective isolation of acidophiles or alkalophiles.
3) Incubation temperature: e.g. for selective isolation of thermophiles or
psychrophiles

Each technique has certain advantages and disadvantages, and no single method
can be used for isolation of all bacteria.
Some of the methods most commonly used are plating methods and serial dilution
techniques, which will be discussed in detail

ISOLATION BY PLATING METHODS :

When a mixture of cells is spreaded or streaked or immobilized on the surface of the
appropriate agar medium, it is assumed that the every viable unit {cell} grows into

Page 22 of 44
distinct colony {provided all other conditions are permissive for the optimum growth}.
“Colony is a microscopic visible growth of microorganisms on a solid medium.”

STREAK PLATE TECHNIQUE :-

PRINCIPLE :
A sterilized wire loop or swab is charged with appropriately diluted suspension of
organisms and is then placed on the agar- solidified surface of the nutrient medium at
one end of the petri dish. The wire is streaked across the surface of agar plate. The
inoculum is progressively diluted with each successive stroke. As a result, a
dilution gradient is established across the face of the plate. Hence, overlapping
colonies occurs on a part of the plate where the bacterial cells are not sufficiently
separated; while isolated colonies develop in another region of the plate.
Theoretically, each well-isolated colony arises from a single bacterium and
represents a clone of pure culture. The isolated colonies can then be picked, using a
sterile inoculating loop and restreaked onto a fresh medium to ensure purity.

There are several modification of streak plate method.

1. DIRECT STREAKING : In this method the wire loop is charged and direct stokes
are made on the plate from top to bottom. Such method is preferred when the
load of inoculum is low.
2. SECTOR METHOD : several sectors are drawn on the periphery of the plates in a
such a manner that tail of one sector overlaps the beginning of the next sector.
[See figure]. The wire loop charged with the inoculum is streaked parallel to the
line of first sector. For al other coming sectors the uncharged wire loop is streaked
in the similar manner. In this way only the organisms laying in the common
[overlapping] region are carried to the next sector and hence the dilution of the
organism is achieved rapidly. This is a widely accepted technique for the isolation
of the isolation of organisms when the load of organism is high.
3. FOUR -FLAME METHOD : It is a modification of sector method in which the wire
loop is flamed every time before the next sector is streaked. This helps in
immediate reduction of the load of organisms when the wire loop is flamed,
resulting into excellent isolation.

Fig. A) Direct streaking B) Four flame method

Page 23 of 44
REQUIREMENTS :

1) Nutrient agar plate, nutrient agar slant

2) Nicrome wire loop
3) Mixed cultures of Staphylococcus aureus, E. coli, Bacillus subtilis.

PROCEDURE :

1) Sterilize a nicrome wire loop by flaming it in Bunsen- burner’s flame.

2) Allow the wire loop to cool, and withdraw a loopful of suspension from the mixed
culture.
3) Streak the nutrient agar plate in a manner; so as to get well isolated colonies.
4) Incubate the plate at 370 C in an inverted position for 24 hrs.
5) After incubation well isolated colonies should appear on the plate.
6) Select a well-isolated colony of each species and note down their colony
characteristics.
7) Pickup different types of colonies with the help of sterile wire loop and emulsify
them in little amount of sterile distilled water.
8) Prepare a smear from each of the suspension, and perform Gram’s staining.
9) Examined the stained smear to check the purity of the suspension. The smear
should show only one type of bacteria.
10) If the slide does not show a pure culture, continue to examine well-isolated
colonies until a pure culture s confirmed.
11) If the Gram’s staining confirms the purity of the suspension, streak the loopful of
suspension on the nutrient agar slant.
12) Incubate the slants at 370 C for 24 hrs.
13) Next day examine slants for the growth of organisms. Slant should show the
colonies of single type.
14) If necessary the purity can be confirmed by Gram’s staining once again.
15) If the purity of slant is verified, it indicates that the organism has been isolated in
pure form from the mixture; and the slant is preserved in refrigerator for future
use.

Page 24 of 44
POUR PLATE TECHNIQUE :-

PRINCIPLE :
In this technique the mixed suspension is diluted several times in order to reduce the
microbial population sufficiently enough to obtain separate colonies upon plating.
The small volumes of several diluted samples re added to sterile melted agar that
have been cooled to 42-45 0 C. The bacteria and agar medium are mixed well and
the mixture is immediately poured into sterile petri dishes using aseptic technique.
The agar is allowed to solidify, trapping the bacteria at separate discrete positions
with in the matrix of the medium. Although the medium holds bacteria in place, it is
soft enough to permit the growth of bacteria and allow the formation of discrete
isolated colonies both with in the medium and on the surface of the agar.
As the dilution increases the microbial load decreases and there are more chances of
getting well-isolated colonies. This method can also be used to estimate the
population of microorganisms in the sample [quantitative analysis].

REQUIREMENTS :
1) Nutrient agar plate, nutrient agar slant
2) Sterile petri dishes, water bath, pipettes
3) Sterile 9 distilled water tubes.
4) Mixed cultures of Staphylococcus aureus, E. coli, Bacillus subtilis.

PROCEDURE :

1) Dilute the mixed culture several times by transferring 1 ml culture to 9 ml of

sterile distilled water tubes.
2) Melt 2-3 tubes of nutrient agar in boiling water bath.
3) Allow the melted medium to cool, approximately to 500 C
4) Inoculate 1 ml of highest dilution into cooled melted agar.
5) Mix the contents of the tube well by rotating the tube between palms of the
hand.
6) Pour the inoculated agar into the sterile petri dish.
7) Similarly second dilution is also poured into sterile petri dish.
8) Allow the agar to solidify at room temperature.
9) Mark the plate, invert and the incubate at 370 C for 24 hrs.
10) Proceed further by following the similar procedure as described under the
streak plate method..

Page 25 of 44
SPREAD PLATE TECHNIQUE :-

PRINCIPLE :

In the spread plate method a small volume [0.1 – 0.25 ml] of suspension of
microorganisms is placed on the center of an agar plate and spread over the surface
of the agar using a sterile glass rod. The glass rod is normally sterilized by dipping in
alcohol and flaming to burn off the alcohol.

By spreading the suspension over the plate, an even layer of cells is established so
that individual microorganism are separated from the other organisms in the
suspension and deposited at discrete location.

In order to achieve the desired development of isolated colonies, it is often necessary

to dilute the suspension before inoculating it to the agar plate so as to prevent
overcrowding and the formation of confluent growth.

This method often used for the isolation of fungi and bacteria from soil or similar
sample. The following method describes the isolation of bacteria from the soil.

REQUIREMENTS:

1) Fertile soil sample

2) Sterile distilled water tubes
3) Glass spreader
4) Alcohol
5) Nutrient agar plate

PROCEDURE:

1) Weigh approximately 1 gm of soil sample and transfer it to 10 ml of sterile

distilled water under aseptic conditions.

2) Mix the contents of the tube well and set the tube aside to allow the soil particles
to settle down.

3) Using the sterile pipette, withdraw 0.2 ml of supernatant from the soil
suspension and inoculate it to the center of agar plate.

4) Sterilize the glass spreader by dipping it in alcohol and flaming it. Allow the
glass spreader to cool.

5) Spread the suspension placed in the center of the plate with the help of sterile
glass spreader.

6) Mark the plate, and incubate it at 370 C for 24 hrs.

Page 26 of 44
7) After incubation well-isolated colonies should appear on the plate.

8) Select a well-isolated colony of each species and note down their colony
characteristics.

9) Pickup different types of colonies with the help of sterile wire loop and emulsify
them in little amount of sterile distilled water.

10) Prepare a smear from each of the suspension, and perform Gram’s staining.

11) Examined the stained smear to check the purity of the suspension. The smear
should show only one type of bacteria.

12) If the slide does not show a pure culture, continue to examine well-isolated
colonies until a pure culture s confirmed.

13) If the Gram’s staining confirms the purity of the suspension, streak the loopful of
suspension on the nutrient agar slant.

14) Incubate the slants at 370 C for 24 hrs.

15) Next day examine slants for the growth of organisms. Slant should show the
colonies of single type.

16) If necessary the purity can be confirmed by Gram’s staining once again.

17) If the purity of slant is verified, it indicates that the organism has been isolated in
pure form from the mixture; and the slant is preserved in refrigerator for future
use.

Page 27 of 44
Experiment No. : 9
Aim : CULTIVATION OF ANAEROBIC BACTERIA [Clostridium Spp.]

INTRODUCTION :
Lavoisier in 1775 described the role of oxygen for combustion and respiration; this
led to the conclusion that free oxygen [air] is necessary to all life. However, in 1861
Pasteur proved that certain yeast and bacteria could multiply in absence of air. He
devised the term anaerobiosis to describe life without air.

MICROORGANISMS AND OXYGEN :

Microorganisms are divided into several groups with respect to their relation to free
oxygen.
1) Strict or obligate aerobes grow in presence of atmospheric oxygen, because
oxygen serve as the final electron acceptor in biological oxidation or aerobic
respiration [e.g. Bacillus, Azotobacter, Thiobacillus etc.]
2) Strict or obligate anaerobes cannot tolerate oxygen at all and die or are
inhibited in its presence. They generate their energy through fermentative or
anaerobic respiration. [e.g. Clostridium. Fusobacterium, Methanogens etc.]
3) Facultative anaerobes do no require oxygen for growth, but do grow better in its
presence [e.g. Esherichia, Enterobacter etc.]
4) Aerotolerant anaerobes simply ignore oxygen and grow equally well whether it
is present or not. [e.g. Streptococcus, Lactobacillus]
5) Microaerophiles are damaged by normal atmospheric concentration of oxygen
[20 %] and require oxygen concentration below 2 % for growth [e.g. Leptospira]

CULTIVATION OF CLOSTRIDIA FROM SOIL :

Clostridia are obligately anaerobic, gram- positive; spore bearing rods. Unlike spores
of genus Bacillus, the species of the genus Clostridium produce spores with
prominent and elaborate bulging, moreover the spore are round or oval and occurs at
the terminal end; hence they have drum stick or racket shaped appearances.
Clostridia are commonly found in soil, manure, sewage-polluted lands and intestinal
tract of man and animals.

PRINCIPLE:
Selective isolation of clostridia from soil can be achieved by;

1) Heat treatment of the soil suspension: heat shock destroys the sensitive
vegetative non- spore forming bacteria. However, Clostridia can survive heat
shock because it forms endospores.
2) Providing anaerobic environments: among the surviving spore formers, only
anaerobic organisms [clostridia] will be able to grow.

Page 28 of 44
REQUIREMENTS :
1) Thioglycolate broth.
2) Robertson’s bullock’s heart cooked meat medium [RCM]
3) Sterile distilled water tube
4) Sterile pipettes
5) Sterile paraffin oils
6) Soil sample from sewage polluted lands or horse manure.
PROCEDURE :
1) Suspend 1 gm of soil or manure in 10 ml sterile distilled water.
2) Mix contents of the tube well and hold the tube in water bath at 90 0C for 10
minutes, so as to destroy vegetative cells.
3) After heat shock, place the tube aside and leave it undistributed to allow
particulate matter to settle down.
4) Place tubes of thioglycollate broth and RCM medium in boiling water bath for 10
minutes to expel out oxygen present in tubes.
5) Remove media tubes from water bath and immediately cool to 50 0C.
6) With the help of 1 ml sterile pipettes inoculate 0.5 –1.0 ml of soil supernatant
deep into thioglycollate and RCM medium.
7) With the help of 10 ml sterile pipette overlay sterile paraffin oil up to about 2- 3
cm above the medium.
8) Incubate both media at room temperature for 5- 7 days.
9) After the incubation observe the changes occurred in the medium.
10) Withdraw a loopful of suspension from bottom of the tubes and prepare smear.
Stain the smear by gram’s staining.
11) Examine the slide under microscope and search for gram- positive rods having
bulging terminal or subterminal spores; i.e., Racket shaped or drum stick
shaped cells.
12) Presence of such cells indicates the growth of clostridium in an anaerobic
medium.
INTERPRETATIONS :
 Thiglycollate broth:
Change in colour of litmus or methylene blue to colorless state indicates the
anaerobic conditions o the medium, and the development of turbidity in the
medium indicates the growth of organisms. Gram’s staining showing the presence
of gram-positive racket shaped cells indicates the growth of clostridia.
 Robertson’s cooked meat medium : Following changes are observed in the
medium.
1) The color of the meat pieces change from pink to black.
2) Breaking of meat pieces.
3) Putrefaction of the meat pieces, which generates foul smell.
Gram’s staining showing the presence of gram- positive racket shaped cells indicates
the growth of clostridia.

Page 29 of 44
Experiment No. : 10
Aim : BACTERIOLOGICAL ANALYSIS OF WATER.

Routine bacteriological procedures consist of

1) Standard plate count

2) Test for Coliforms
3) Enumeration of Coliforms

1) STANDARD PLATE COUNT (SPC) OR TOTAL VIABLE COUNT (TVC) :-

Standard plate count is also known as Hetrotopic Plate Count. It is a quantities
bacteriological analysis which enumerate total viable populations of sample (soil,
water, sewage and fresh water) capable of growing under given set of conditions.
The results are expressed in term of colony forming units (CFU) rather than the
number of microorganisms.

PRINCIPLE :
TVC / SPC is based on assumption that each viable bacterium develops into a
distinct colony. Hence, original number of microorganisms in the sample can be
calculated from number of colonies and then multiplying it with dilution factor.

REQUIREMENTS :
1) Water Sample (expect water also utilized soil, sewage and fresh water depending
on our own purpose.)
2) Sterile distilled water in dilution tubes (4.5 ml or 9.0 ml.)
3) Sterile melted nutrient agar tubes
4) Sterile Petri dishes and sterile pipettes.

PROCEDURE :
1) Take a tube containing 9 ml sterile distilled water and add 1 ml of sample. This
makes a 10-1 dilution
2) Take a 1 ml of solution from a first tube add and mix it properly this makes 10-2
dilution
3) By this method prepared 10-3,10-4…..…dilutions of water sample (if necessary)
4) From each of the dilution transferred 0.2 ml into sterile malted nutrient agar tubes
(previously cooled to 50 0C ) mix it 12 and pour immediately sterile Petri dishes.
5) Label plates (Petri dishes) clearly indicating the dilutions and volume plated
respectively
6) Incubate all plates at 37 °C for 24 hours.
7) Count total number of colonies that has developed on each of the plate by using
colony counter
8) Calculate the final number of organism present in the waster sample as follows.

Page 30 of 44
CALCULATION & INTERPRETATION OF RESULT:

For a accuracy of results, countable plates are those which have colonies in between
30-300. Fewer than 30 colonies are not acceptable for statistical reasons, and more
than 300 colonies on a plate is likely to produce colonies too close to each other to
be distinguished as individual CFUs

CFUs / ml = Average number of colonies / Dilution x Volume Plated

Example : Average number of colonies = 170

Dilution = 10-3
Volume Plated = 0.1 ml

CFUs / ml = 170 x 1000 x 0.1

= 17000

CALCULATION :

RESULT :

The total viable count for water sample is ____________ CFUs / ml.

Page 31 of 44
2) TEST FOR COLIFORMS :-

To detect Coliforms, a three-stage procedure (the presumptive test, confirmed test,

and completed test) is carried out in systematic order according to the results of each
step.

 PRESUMPTIVE TEST :

PRINCIPLE :

It is based on the principle that coliforms if present in water will ferment lactose to
produce acid and gas within 24-48 hours. Production of acid is indicated by pH
indicator and gas is collected in Durham’s vial, both of which are present in the
medium.
Media used are highly selective for coliforms, which inhibit growth of gram-positive
organisms. MacConekey’s lactose bile broth (MLBB) of formate lactose glutamate
medium or laurel tryptose broth or brilliant green lactose bile broth (BGLB) can be
used for presumptive test.
Different volumes of water ware inoculated in MLBB or BGLB which permits growth
of coliforms only. Coliforms will ferment lactose to produce acid and gas within 24-48
hours and test is considered as positive. Hence, it may be presumed that coliforms
may be present in water hence the name presumptive test.

REQUIREMENTS :
1) Water Sample
2) Sterile 1 ml and 10 ml pipettes.
3) 5 MLBB tubes, each with 10 ml double strength medium (2X)
4) 10 MLBB tubes, each with 5 ml single strength medium (X)

PROCEDURES :
1) Shake the water sample vigorously to ensure uniform distribution of organisms.
2) With sterile graduated pipettes inoculate the water sample as follows.
 5 MLBB double strength tubes with 10 ml water sample
 5 MLBB single strength tubes with 1.0 ml water sample
 5 MLBB single strength tubes with 0.1 ml water sample
3) One tube of MLBB single strength is not inoculated and hence, serve as control.
4) Incubate all tubes at 37 °C for 24 hours.
5) Examine tubes for the presence of acid and gas after 24 hours.
6) If non of the tubes show acid and gas, re-incubate all the tubes for another 24
hours (total 48 hours)
7) Record the presence or absence of acid & gas at each examination, & interpret as
follows.

Page 32 of 44
INTERPRETATIONS :

1. Absence of gas even after 48 hours indicate a negative test and the presumptive
test is terminated. The water sample is assumed to be potable.
2. Presumptive test is considered positive if any one or more of tubes show acid and
gas, positive tubes are retained for confirmed test.

NOTE :

1. Double strength Broth: (Contains double the concentration of ingredients except

water) is used when large volumes of water are to be inoculated, because the
medium would otherwise be too diluted and may not support the growth of
bacteria.
2. False Positive presumptive test may be produced due to:
a. Presence of lactose fermenting organisms other than coliforms.
b. A synergistic association where the joint action of two organisms on a
carbohydrate results into production of gas which will not be formed by either
species when grown separately. Synergism is frequently caused by a joint
action of gram-positive and gram-negative organisms growing together e.g. ;
Staphylococcus aureus + Proteus vulgaris
Streptococcus faecalis + Salmonella paratyphi A
Staphylococcus aureus + Salmonella schottmulleri
False positive presumptive test can be overcome by adding bile salts and
triphenylmethane dyes which inhibit growth of gram-positive bacteria and
thereby eliminate synergistic effect.
3. Over fermentation: these kinds of results are obtained when ratio of number of
organisms to the amount of sugar is comparatively very high. Due to less
amount of sugar, acid produced is not sufficient enough to lower the pH at which
growth of organism is inhibited. Thus, growth continues and organisms switch
over to peptones when sugars are depleted. Since end products of protein
metabolism are alkaline in nature the basicity of medium increases. Hence,
MLBB appears yellow even though the test is positive

 CONFIRMED TEST :

This test is named so because, positive presumptive tubes having acid and gas are
subjected to further confirmation that positive results were due to coliforms only. Test
involves, streaking of eosin methylene blue (EMB) agar or Endo’s agar plate and
looking for the growth of typical &/or atypical colonies of coliforms.

Page 33 of 44
REQUIREMENTS :

1) Positive presumptive tubes

2) EMB Agar plates

PROCEDURE :

1) Streak a loopful of suspension from a positive presumptive tube (which shows the
highest amount of gas production), so as to get well-isolated colonies.
2) Incubate the plate at 37 °C for 24 hours.
3) Record results and interprete them as follow.

INTERPRETATIONS :

EMB agar plate permits three types of colonies to develop:

1. Typical: Small nucleated with or without greenish metallic sheen

2. Atypical: large, opaque, pink, non-nucleated, mucoid, which tends to merge with
each other.
3. Negative: All other types of colonies developing on the plate. Growth of typical
colonies indicate confirmed test positive and has to proceed for completed test.

If only atypical colonies develop, the test can’t be considered negative since at times,
coliforms fail to form typical colonies, or colonies develop slowly. Hence the test
should be completed.
However, if only negative (others) colonies develop on the plate; the confirmed test is
recorded, as negative and further tests are not necessary.

 COMPLETED TEST :

In this test the typical &/or atypical colonies growing on EMB agar plate are subjected
to morphological and biochemical verification so as to prove that they are coliforms.
This is determined by checking whether the organisms isolated from positive
presumptive test (i.e. typical / atypical colonies) are

1) Gram-negative non spore forming short rods, and

2) Able to ferment lactose with production of acid and gas.

Since this test completes and finishes the “ Presumptive test for coliforms” it is
referred to as completed test.

Page 34 of 44
REQUIREMENTS :

1) EMB/Endo’s agar plate having typical / atypical colonies isolated from positive
presumptive tube.
2) Lactose broth tube (other than the one used in presumptive test eg, Brilliant green
lactose bile broth (BGLB), or nutrient lactose broth with Andrade’s indicator and
Durham’s vial.
3) Nutrient agar slant

PROCEDURE :

1) Select and mark a well isolated typical / atypical colony on EMB or Endo’s agar
plate.
2) With the help of nicrome wire loop, pick up half of the previously marked typical /
atypical colony and transfer it to BGLB or nutrient lactose broth tube.
3) From the remaining half of the same colony streak over the surface of a nutrient
agar slant.
4) Incubate slant and broth at 37 °C for 24 hours.
5) Check lactose broth for presence of acid an gas
6) Prepare Gram’s stain of the growth from the surface of agar slant and observe the
slide. Look for the presence of gram-negative non-spore forming short rods.
7) Record results and interpret as follows.

INTERPRETATION :

1) Presence of gram-negative, non-spore forming short rods capable of producing

acid and gas from lactose indicates the completed test positive.
2) Absence of gram-positive non-spore forming short rods and the absence of acid
and gas from the lactose constitutes a negative completed test.

RESULT / CONCLUSION OF PRESUMPTIVE TEST :

Positive presumptive test indicates presence of coliforms in water sample, which

points out the faecal contamination of water; hence the water is non-potable, as it
may carry potentially pathogenic microorganisms. However, as started earlier
coliforms include wide range of bacteria whose primary source may not be the
intestinal tract of human being; so further differentiation of coliforms in recommended.

Page 35 of 44
3) ENUMERATION OF COLIFORMS BY MPN (MOST PROBABLE NUMBER)
TECHNIQUE :

PRINCIPLE :
It is a statistical method based on the probability theory. In this technique, the sample
is serially diluted till the number of organisms reaches the point of extinsion. From
each of these dilutions several multiple tubes of a specific medium are inoculated.
The presence of organism is indicated by acid or gas in the medium. The pattern of
positive and negative test results are then used to estimate the number of bacteria in
original sample. Since the test gives the most probable number of organisms present
in the sample, it is also known as MPN test.

REQUIREMENTS :
1) Water Sample
2) Sterile 1 ml and 10 ml pipettes.
3) MLBB tubes, each with 10 ml double strength medium (2X)
4) 10 MLBB tubes, each with 5 ml single strength medium (X)

PROCEDURES :
1) Shake the water sample vigorously to ensure uniform distribution of organisms.
2) With sterile graduated pipettes inoculate the water sample as follows.
5 MLBB double strength tubes with 10 ml water sample
5 MLBB single strength tubes with 1.0 ml water sample
5 MLBB single strength tubes with 0.1 ml water sample
3) One tube of MLBB single strength is not inoculated and hence, serve as control.
4) Incubate all tubes at 37 °C for 24 hours.
5) Examine tubes for the presence of acid and gas after 24 hours.
6) If non of the tubes show acid and gas has formed, re-incubate all the tubes for
another 24 hours (total 48 hours)
7) Record the presence or absence of acid & gas at each examination, & interpret
as follows.

INTERPRETATIONS :

McCardy in 1918 computed the tables, regarding the most probable number of
organisms present in 100 ml of water, on the basis of various combinations of
positive and the negative results in the amounts used for the test. The number of
organisms / 100 ml is read from the McCardy’s table, and the number is multiplied
by the dilution factor (if any), to come to the final number.

Page 36 of 44
RESULT :

The MPN number of water sample is _____________.

McCardy’s MPN Table:

MPN INDEX FOR VARIOUS COMBINATIONS OF POSITIVE RESULTS WHEN

FIVE TUBES USED FOR DILUTION
[AMOUNT INOCULATED 10 ml, 1.0 ml, and 0.1 ml]

COMBINATION OF MPN index / COMBINATION OF MPN index /

POSITIVES 100 ml POSITIVES 100 ml
10 ml 1.0 ml 0.1 ml 10 ml 1.0 ml 0.1 ml
0 0 0 < 1.8 0 4 3 13.0
0 0 1 1.8 0 4 4 15.0
0 0 2 3.6 0 4 5 17.0
0 0 3 5.4 0 5 0 9.4
0 0 4 7.2 0 5 1 11.0
0 0 5 9.0 0 5 2 13.0
0 1 0 1.8 0 5 3 15.0
0 1 1 3.6 0 5 4 17.0
0 1 2 5.5 0 5 5 19.0
0 1 3 7.3 1 0 0 2
0 1 4 9.1 1 0 1 4
0 1 5 11.0 1 0 2 6
0 2 0 3.7 1 0 3 8
0 2 1 5.5 1 0 4 10
0 2 2 7.4 1 0 5 12
0 2 3 9.2 1 1 0 4
0 2 4 11.0 1 1 1 6.1
0 2 5 13.0 1 1 2 8.1
0 3 0 5.6 1 1 3 10
0 3 1 7.4 1 1 4 12
0 3 2 9.3 1 1 5 14
0 3 3 11.0 1 2 0 6.1
0 3 4 13.0 1 2 1 8.2
0 3 5 15.0 1 2 2 10
0 4 0 7.5 1 2 3 12
0 4 1 9.4 1 2 4 15
0 4 2 11.0 1 2 5 17
1 3 0 8.3 2 3 0 12
1 3 1 10 2 3 1 14

Page 37 of 44
 COMBINATION OF MPN index / COMBINATION OF MPN index /
POSITIVES 100 ml POSITIVES 100 ml
10 ml 1.0 ml 0.1 ml 10 ml 1.0 ml 0.1 ml
1 3 2 13 2 3 2 17
1 3 3 15 2 3 3 20
1 3 4 17 2 3 4 22
1 3 5 19 2 3 5 25
1 4 0 11 2 4 0 15
1 4 1 13 2 4 1 17
1 4 2 15 2 4 2 20
1 4 3 17 2 4 3 23
1 4 4 19 2 4 4 25
1 4 5 22 2 4 5 28
1 5 0 13 2 5 0 17
1 5 1 15 2 5 1 22
1 5 2 17 2 5 2 23
1 5 3 19 2 5 3 26
1 5 4 22 2 5 4 29
1 5 5 24 2 5 5 32
2 0 0 4.5 3 0 0 7.8
2 0 1 6.8 3 0 1 11
2 0 2 9.1 3 0 2 13
2 0 3 12 3 0 3 16
2 0 4 14 3 0 4 20
2 0 5 16 3 0 5 23
2 1 0 6.8 3 1 0 11
2 1 1 9.2 3 1 1 14
2 1 2 12 3 1 2 17
2 1 3 14 3 1 3 20
2 1 4 17 3 1 4 23
2 1 5 19 3 1 5 27
2 2 0 9.3 3 2 0 14
2 2 1 12 3 2 1 17
2 2 2 14 3 2 2 20
2 2 3 17 3 2 3 24
2 2 4 19 3 2 4 27
2 2 5 22 3 2 5 31
3 3 0 17 4 3 0 27
3 3 1 21 4 3 1 33
3 3 2 24 4 3 2 39
3 3 3 28 4 3 3 45
3 3 4 31 4 3 4 52
3 3 5 35 4 3 5 59
3 4 0 21 4 4 0 34

Page 38 of 44
 COMBINATION OF MPN index / COMBINATION OF MPN index /
POSITIVES 100 ml POSITIVES 100 ml
10 ml 1.0 ml 0.1 ml 10 ml 1.0 ml 0.1 ml
3 4 1 24 4 4 1 40
3 4 2 28 4 4 2 47
3 4 3 32 4 4 3 54
3 4 4 36 4 4 4 62
3 4 5 40 4 4 5 69
3 5 0 25 4 5 0 41
3 5 1 29 4 5 1 48
3 5 2 32 4 5 2 56
3 5 3 37 4 5 3 64
3 5 4 41 4 5 4 72
3 5 5 45 4 5 5 81
4 0 0 13 5 0 0 23
4 0 1 17 5 0 1 31
4 0 2 21 5 0 2 43
4 0 3 25 5 0 3 58
4 0 4 30 5 0 4 76
4 0 5 36 5 0 5 95
4 1 0 17 5 1 0 33
4 1 1 21 5 1 1 46
4 1 2 26 5 1 2 64
4 1 3 31 5 1 3 84
4 1 4 36 5 1 4 110
4 1 5 42 5 1 5 130
4 2 0 22 5 2 0 49
4 2 1 26 5 2 1 70
4 2 2 32 5 2 2 95
4 2 3 38 5 2 3 120
4 2 4 44 5 2 4 150
4 2 5 50 5 2 5 180
5 3 0 79 5 4 3 280
5 3 1 110 5 4 4 350
5 3 2 140 5 4 5 430
5 3 3 180 5 5 0 240
5 3 4 210 5 5 1 350
5 3 5 250 5 5 2 540
5 4 0 130 5 5 3 920
5 4 1 170 5 5 4 1600
5 4 2 220 5 5 5

Page 39 of 44
Experiment : 11
No.
Aim : ISOLATION OF FUNGI FROM SOIL

INTRODUCTION :
Fungi are placed in the kingdom Fungi. Microbiologists use the term fungus to
include eukaryotic spore bearing organisms without absorptive nutrition and no
chlorophyll that reproduce sexually and asexually. Fungi usually have filamentous
structures whose cell walls generally contain chitin and cellulose.
CULTIVATION AND ISOLATION :
Fungi grow more slowly than bacteria, hence bacteria overgrow on fungi when mixed
inoculum used. The inhibition of bacterial contamination is one of the important factor
during cultivation of fungi. This can be prevented by making the medium selective for
fungal growth. I.e. conditions which favors fungal growth but are not optimal for
bacterial growth, For example;
1) Addition of antibiotic [Streptomycin, Chloromphenicol], which acts only on 70 s
ribosome, there by inhibiting bacteria not fungi.
2) Addition of dye Rose Bengal [makes the medium acidic].
3) Adjusting the pH of the medium slight acidic.
4) Making the medium rich in carbohydrates.
5) Incubation of medium at low temperature.
REQUIREMENTS :
1) Fertile soil sample
2) Sterile distilled water tubes
3) Sterile pipettes, Spatula
4) Sabouraud’s dextrose agar, Rose Bengal agar
PROCEDURE :
1. With the help of sterile spatula add 1 gm of fertile soil in sterile distilled water
under aseptic condition.
2. Mix well and allow the soil particles to settle down.
3. With the help of sterile pipette inoculate 0.5 ml of soil supernatant in sterile melted
medium [previously cooled to 50 0C]. MIX the inoculum well and pour in sterile
petri plate.
OR
4. With sterile pipette inoculate 0.2 ml of soil supernatant on a medium. Spread it
uniformly on the surface of the medium with the help of the glass spreader.
5. Inoculate plates at room temperature for 3-7 days.
6. After incubation observe the plates for the growth and study their colony
characteristics.

RESULT : After incubation the fungal growth is found on the plates.

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Experiment : 12
No.
Aim : WASTEWATER TREATMENT TECHNOLOGY

The processes involved in municipal wastewater treatment plants are usually

classified as being part of primary, secondary, or tertiary treatment.

PRIMARY TREATMENT :

The wastewater that enters a treatment plant contains debris that might clog or
damage the pumps and machinery. Such materials are removed by screens or
vertical bars, and the debris is burned or buried after manual or mechanical removal.
The wastewater then passes through a comminutor (grinder), where leaves and other
organic materials are reduced in size for efficient treatment and removal later.

1) GRIT CHAMBER:

In the past, long and narrow channel-shaped settling tanks, known as grit
chambers, were used to remove inorganic or mineral matter such as sand, silt,
gravel, and cinders. These chambers were designed to permit inorganic particles
0.2 mm (0.008 in) or larger to settle at the bottom while the smaller particles and
most of the organic solids that remain in suspension pass through. Today, spiral-
flow aerated grit chambers with hopper bottoms, or clarifiers with mechanical
scrapper arms, are most commonly used. The grit is removed and disposed of as
sanitary landfill. Grit accumulation can range from 0.08 to 0.23 cu m (3 to 8 cu ft)
per 3.8 million liters (about 1 million gallons) of wastewater.

2) SEDIMENTATION:

With grit removed, the wastewater passes into a sedimentation tank, in which
organic materials settle out and are drawn off for disposal. The process of
sedimentation can remove about 20 to 40 percent of the BOD5 and 40 to 60
percent of the suspended solids.

The rate of sedimentation is increased in some industrial waste-treatment stations

by incorporating processes called chemical coagulation and flocculation in the
sedimentation tank. Coagulation is the process of adding chemicals such as
aluminum sulfate, ferric chloride, or polyelectrolytes to the wastewater; this
causes the surface characteristics of the suspended solids to be altered so that
they attach to one another and precipitate. Flocculation causes the suspended
solids to coalesce. Coagulation and flocculation can remove more than 80 percent
of suspended solids.

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3) FLOTATION:

An alternative to sedimentation that is used in the treatment of some wastewaters

is flotation, in which air is forced into the wastewater under pressures of 1.75 to
3.5 kg per sq cm (25 to 50 lb per sq in). The wastewater, supersaturated with air,
is then discharged into an open tank; there the rising air bubbles cause the
suspended solids to rise to the surface, where they are removed. Flotation can
remove more than 75 percent of the suspended solids.

4) DIGESTION:

Digestion is a microbiological process that converts the chemically complex

organic sludge to methane, carbon dioxide, and inoffensive humus like material.
The reactions occur in a closed tank or digester that is anaerobic—that is, devoid
of oxygen. The conversion takes place through a series of reactions. First the
solid matter is made soluble by enzymes, and then the substance is fermented by
a group of acid-producing bacteria, reducing it to simple organic acids such as
acetic acid. The organic acids are then converted to methane and carbon dioxide
by bacteria. Thickened sludge is heated and added as continuously as possible to
the digester, where it remains for 10 to 30 days and is decomposed. Digestion
reduces organic matter by 45 to 60 percent.

5) DRYING:

Digested sludge is placed on sand beds for air-drying. Percolation into the sand
and evaporation are the chief processes involved in the dewatering process. Air-
drying requires dry, relatively warm weather for greatest efficiency, and some
plants have a greenhouse like structure to shelter the sand beds. Dried sludge in
most cases is used as a soil conditioner; sometimes it is used as a fertilizer
because of its 2 percent nitrogen and 1 percent phosphorus content.

SECONDARY TREATMENT :

Having removed 40 to 60 percent of the suspended solids and 20 to 40 percent of

the BOD5 in primary treatment by physical means, the secondary treatment
biologically reduces the organic material that remains in the liquid stream. Usually the
microbial processes employed are aerobic—that is, the organisms function in the
presence of dissolved oxygen. Secondary treatment actually involves harnessing and
accelerating nature's process of waste disposal. Aerobic bacteria in the presence of
oxygen convert organic matter to stable forms such as carbon dioxide, water,
nitrates, and phosphates, as well as other organic materials. The production of new
organic matter is an indirect result of biological treatment processes, and this matter
must be removed before the wastewater is discharged into the receiving stream.

Several alternative processes are also available in secondary treatment, including a

trickling filter, activated sludge, and lagoons.

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1) TRICKLING FILTER :

In this process, a waste stream is distributed intermittently over a bed or column

of some type of porous medium. A gelatinous film of microorganisms coats the
medium and functions as the removal agent. The organic matter in the waste
stream is absorbed by the microbial film and converted to carbon dioxide and
water. The trickling-filter process, when preceded by sedimentation, can remove
about 85 percent of the BOD5 entering the plant.

2) ACTIVATED SLUDGE:

This is an aerobic process in which gelatinous sludge particles are suspended in

an aeration tank and supplied with oxygen. The activated-sludge particles, known
as floc, are composed of millions of actively growing bacteria bound together by a
gelatinous slime. Organic matter is absorbed by the floc and converted to aerobic
products. The reduction of BOD5 fluctuates between 60 and 85 percent.

An important companion unit in any plant using activated sludge or a trickling filter
is the secondary clarifier, which separates bacteria from the liquid stream before
discharge.

3) STABILIZATION POND OR LAGOON:

Another form of biological treatment is the stabilization pond or lagoon, which

requires a large land area and thus is usually located in rural areas. Facultative
lagoons, or those that function in mixed conditions, are the most common, being
0.6 to 1.5 m (2 to 5 ft) in depth, with a surface area of several acres. Anaerobic
conditions prevail in the bottom region, where the solids are decomposed; the
region near the surface is aerobic, allowing the oxidation of dissolved and
colloidal organic matter (see Colloid). A reduction in BOD5 of 75 to 85 percent can
be attained.

TERTIARY TREATMENT :

If the receiving body of water requires a higher degree of treatment than the
secondary process can provide, or if the final effluent is intended for reuse, advanced
wastewater treatment is necessary. The term tertiary treatment is often used as a
synonym for advanced treatment, but the two methods are not exactly the same.
Tertiary, or third-stage, treatment is generally used to remove phosphorus, while
advanced treatment might include additional steps to improve effluent quality by
removing refractory pollutants. Processes are available to remove more than 99
percent of the suspended solids and BOD5. Dissolved solids are reduced by
processes such as reverse osmosis and electro dialysis. Ammonia stripping,
denitrification, and phosphate precipitation can remove nutrients. If the wastewater is
to be reused, disinfection by ozone treatment is considered the most reliable method
other than breakpoint chlorination. Application of these and other advanced waste-
treatment methods is likely to become widespread in the future in view of new efforts
to conserve water through reuse. See Absorption; Osmosis; Precipitation.

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LIQUID DISPOSAL:

The ultimate disposal of the treated liquid stream is accomplished in several ways.
Direct discharge into a receiving stream or lake is the most commonly practiced
means of disposal. In areas of the United States that are faced with worsening
shortages of water for both domestic and industrial use, municipalities and state and
federal agencies are turning to reuse of appropriately treated wastewater for
groundwater recharge, irrigation of nonedible crops, industrial processing, recreation,
and other uses. Many reuse projects are located in California, Arizona, and Texas.

The first large-scale wastewater-reclamation plant in the United States is the Denver
Water Department's Potable Reuse Demonstration Plant. The one-million-gallon-per-
day plant was built to demonstrate the quality, reliability, and economic potential of
reuse on a large scale. The quality and health-effects testing program, ended in
1993, after successfully meeting its goal of producing drinkable water from reclaimed
water. The reused water was tested against the regular drinking water received by
Denver residents and found to be equally drinkable. The treatment process involves
conventional primary and secondary treatment followed by lime clarification to
remove suspended organic compounds. During this process, an alkaline (high-pH)
condition is created to improve the process. In the next step, recarbonation is used to
bring the pH level to neutral. Then the water is filtered through multiple layers of sand
and charcoal, and ammonia is removed by ionization. Pesticides and any other
dissolved organic materials still present are absorbed by a granular, activated-carbon
filter. Viruses and bacteria are then killed by ozonization. At this stage the water
should be cleansed of all contaminants, but, for added reliability, second-stage
carbon adsorption and reverse osmosis are used, and chlorine dioxide is added to
attain the highest possible water standard. Similar reuse programs are underway in
the southwestern United States, Saudi Arabia, and the Netherlands.

SEPTIC TANK:

A sewage treatment process commonly used to treat domestic wastes is the septic
tank: a concrete, cinder block or metal tank where the solids settle and the floatable
materials rise. The partly clarified liquid stream flows from a submerged outlet into
subsurface rock-filled trenches through which the wastewater can flow and percolate
into the soil where it is oxidized aerobically. The floating matter and settled solids can
be held from six months to several years, during which they are decomposed
anaerobically.

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