Pure Culture-Technique's of Microorganisms for B.Sc. Biotechnology & Botany Sem-3

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Maintenance, Culture, Preservation & Types of
Agar Medium for Micro-organisms

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UNIT-III PURE CULTURE TECHNIQUES
Single Cell Isolation Methods
❖ Capillary pipette method:
1. Several small drops of a suitably diluted culture medium are put on a sterile
glass-coverslip by a sterile pipette drawn to a capillary.
2. Each drop under the microscope is examined until one finds such a drop,
which contains only one microorganism.
3. This drop is removed with a sterile capillars pipette to fresh medium. The
individual microorganism present in the drop starts multiplying to yield a pure
culture.
❖ Micromanipulator method: -
1. This instrument is used in conjunction with a microscope to pick a single cell
(particularly bacterial cell) from a hanging drop preparation.
2. The micro-manipulator has micrometer adjustments by means of which its
micropipette can be moved right and left, forward, and backward, and up and
down.
3. A series of hanging drops of a diluted culture are placed on a special sterile
coverslip by a micropipette.
4. Now a hanging drop is searched, which contains only a single microorganism
cell.

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5. This cell is drawn into the micropipette by gentle suction and then
transferred to a large drop of sterile medium on another sterile coverslip.
6. When the number of cells increases in that drop as a result of multiplication,
the drop is transferred to a culture tube having suitable medium. This yields
a pure culture of the required microorganism.

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❖ PURE CULTURE TECHNIQUES
Development of pure culture
"A population of organisms cultivated in a medium is called as a culture". While
a culture that contain only one species of microorganism is known as a pure or
axenic culture OR A population of cells arising from a single cell is called as a
pure culture.
While mixed flora is the rule of nature meaning is that the natural
ecosystem like soil, sewage, milk, urine contains mixed population of several
species of micro-organisms.
Historical background
Antony Van Leeuwenhoek, 'Father of microbiology1
in 1863 1SI
time
observed mixed flora in natural samples like faeces, urine, sewage etc. In the
earliest period microbiologists had faced many problems because of
contamination, during their research. Later on Joseph Lister, a 'pioneer of aseptic
surgery' first time developed a method of isolation of single desired bacteria in
pure form by successive dilution of sample using a sterile fluid.
Later on in 1872, a microbiologist 'Schroeter' observed a growth in the form of
compact masses of various colors on the slice of decaying potato. While when he
observed such masses, microscopically he found that each mass contain single type
of organisms. Thus he gave a clue that, it is possible to isolate the organism in pure
form by the use of solid media.
Then in 1880 Robert Koch had succeeded in using gelatin as a solidifying
agent. Nutrient solution with 5 to 10% gelatin can be used as a solid medium for
the growth of organisms. But gelatin has two major drawbacks as -
• In summer days, gelatin melts (at 280
C), thus can't work as a solidifying agent.
• Being proteinic in nature, it can be utilized (or liquefied) by many micro-
organisms as a nutrient.

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By that time many scientists had gathered to Koch's lab from all over the
world. Dr. Walther Hesse was one of them, who was working with air borne
organisms. He was frustrated by gelatin's nature. At that time his wife Fran Fanny
Hesse had suggested her husband to use agar-agar as a solidifying agent instead
of gelatin. Agar is a polysaccharide derived from sea weed. At that time it was
used for stabilizing jellies, thickening of soups. The agar was found to be as a
perfect solidifying agent. Since then up to date agar is successfully used as
solidifying agent.
Therefore isolation of pure culture is nothing but the gain of isolated colony
of desired organism on the surface of agar. Before that one fact should be kept in
mind that a single colony is formed from the population of single organism.
Techniques of pure culture
There are four main techniques by which one can get a pure culture.
1. Streak plate technique.
2. Pour plate technique.
3. Spread plate technique.
4. Single cell isolation

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Significance of Pure Culture
Pure culture is needed for -
• Identification of species
• Large scale production of any desired product.
• The study of molecular structure and biochemical characters of desired
organism.

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Microbial growth to determine growth rates and generation times can be
measured by different methods. Since growth leads to increase both the number
and the mass of the populations, either of the two may be followed. It is necessary
to make it clear that no single technique is always best; the most appropriate
approach depends upon the experimental situation. It is done by—
A) Measurement of Cell Numbers
B) Measurement of Cell Mass
A) Measurement of Cell Numbers
1. Breed Method (Direct Microscopic Count) DMC
2. Counting Chamber Technique
3. Viable Count
4. Coulter Counter
5. Membrane-Filter Technique
1. Membrane-Filter Technique
Microbial cell numbers are frequently determined using special membrane
filters possessing millipores small enough to trap bacteria. In this technique a
water sample containing microbial cells passed through the filter. The filter is
then placed on solid agar medium or on a pad soaked with nutrient broth (liquid
medium) and incubated until each cell develops into a separate colony.
Membranes with different pore sizes are used to trap different microorganisms.
Incubation times for membranes also vary with medium and the microorganism.
A colony count gives the number of microorganisms in the filtered sample, and
specific media can be used to select for specific microorganisms. This technique
is especially useful in analyzing aquatic samples.

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Steps of Membrane Filter Technique
B) Measurement of Cell Mass
1. Dry Weight Technique
2. Measurement of nitrogen content
3. Measurement of Turbidity (Turbidometry)
4. McFarland standards (Barium chloride)
1. Dry Weight Technique
The cell mass of a very dense cell suspension can be determined by this
technique. In this technique, the microorganisms are removed from the medium
by filtration and the microorganisms on filters are washed to remove all
extraneous matter, and dried in desiccator by putting in weighing bottle
(previously weighted). The dried microbial content is then weighted accurately.
This technique is especially useful for measuring the growth of micro fungi. It is
time consuming and not very sensitive. Since bacteria weigh so little, it becomes
necessary to centrifuge several hundred millions of culture to find out a sufficient
quantity to weigh.

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2. Measurement of nitrogen content
As the microbes (bacteria) grow, there is an increase in the protein concentration
(i.e. nitrogen concentration) in the cell. Thus, cell mass can be subjected to
quantitative chemical analysis methods to determine total nitrogen that can be
correlated with growth. This method is useful in determining the effect of
nutrients or antimetabolites upon the protein synthesis of growing culture.
3. Measurement of Turbidity (Turbidometry)
Rapid cell mass determination is possible using turbidometry method.
Turbidometry is based on the fact that microbial cells scatter light striking them.
Since the microbial cells in a population are of roughly constant size, the amount
of scattering is directly proportional to the biomass of cells present and indirectly
related to cell number. One visible characteristic of growing bacterial culture is
the increase in cloudiness of the medium (turbidity). When the concentration of
bacteria reaches about 10 million cells (107
) per ml, the medium appears slightly
cloudy or turbid. Further increase in concentration results in greater turbidity.
When a beam of light is passed through a turbid culture, the amount of light
transmitted is measured, Greater the turbidity, lesser would be the transmission
of light through medium. Thus, light will be transmitted in inverse proportion to
the number of bacteria. Turbidity can be measured using instruments like
spectrophotometer and nephlometer.

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4. McFarland standards
In microbiology, McFarland standards are used as a reference to adjust
the turbidity of bacterial suspensions so that the number of bacteria will be within
a given range.
Original McFarland standards were made by mixing specified amounts of
barium chloride and sulfuric acid together. Mixing the two compounds forms a
barium sulfate precipitate, which causes turbidity in the solution. A 0.5
McFarland standard is prepared by mixing 0.05 ml of 1.175% barium chloride
dihydrate (BaCl2•2H2O), with 9.95 ml of 1% sulfuric acid (H2SO4).
Now there are McFarland standards prepared from suspensions of latex
particles, which lengthens the shelf life and stability of the suspensions.
The standard can be compared visually to a suspension of bacteria in sterile
saline or nutrient broth. If the bacterial suspension is too turbid, it can be diluted
with more diluent. If the suspension is not turbid enough, more bacteria can be
added.
McFarland Nephelometer Standards:
McFarland Standard No. 0.5 1 2 3 4
1.0% Barium chloride (ml) 0.05 0.1 0.2 0.3 0.4
1.0% Sulfuric acid (ml) 9.95 9.9 9.8 9.7 9.6
Approx. cell density (1X108
CFU/ml) 1.5 3.0 6.0 9.0 12.0
% Transmittance* 74.3 55.6 35.6 26.4 21.5
Absorbance* 0.132 0.257 0.451 0.582 0.669
*at wavelength of 600 nm

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❖Cultivation of microorganisms
About 100 years ago, the technique was developed to grow microbes in
pure form in laboratory by removing them from their natural sources. It was a
milestone in the journey of microbiology science. Because of this achievement it
became easy to do close examination of a microbe and its morphological,
physiological and genetical study.
For successful cultivation of organisms in lab, it is necessary to supply all
required nutrients. 'Any nutrient preparation employed to grow micro-organism
in lab is called as culture medium'. In the construction of a culture medium,
primary goal is to provide a balanced mixture of required nutrients for good
growth of desired organism. Culture media have a great significance. Following
are the different purposes for which the culture media are used.
a) For primary isolation of micro-organisms from their natural sources.
b) To determine the biochemical characters of any organism.
c) To determine the growth characters of desired organism.
d) To maintain the culture in laboratory.
e) The transport media are used to preserve the specimen during the time
period between sample collection and clinical analysis.
f) The assay media are used to test the effectiveness of antimicrobial drugs.
g) The enumeration media are used to count number of microbes in milk,
soil, food, water etc. and numerous others.
i) An ideal or satisfactory culture medium should fulfill following
requirements (Properties of good culture medium)
• It should possess all necessary nutrients including growth factors and
vitamins.
• It should have a correct desired pH and should have a provision for
maintenance of pH for ex. use of buffer.
• It should have proper moisture content.
• It should be sterile in microbiological sense.
• It should have desired physical properties like solid or liquid state, clarity
etc.

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There is a great diversity in the nutritional requirements of
microorganisms. It may be as simple as requiring only a few inorganic
compounds or may be as complex as requiring a list of inorganic and organic
compounds. Nowadays in modem era, the culture media are commercially
available in an instant, dehydrated form. But it was a challenging and difficult
task in determining the nutritional requirement of organisms and designing of
medium suitable for organism.
Common components of media and their functions:
For preparation of culture media different components are added in
different combination. Each component performs specific functions in the culture
media. Following are a few commonly used components.
1. Peptone
It is a trypsinised or hydrolysed proteins from animals like meat or casein
or proteins from vegetables like soyabean or cottonseeds. Proteins from above
sources are hydrolysed by proteolytic enzymes like pepsin, trypsin, papain. Due
to hydrolysis, the large complex proteins are broken down to proteoses, peptones,
peptides and amino acids. Along with this proteinic part, peptone also possesses
carbohydrates, many inorganic micronutrients. Therefore it is the most important
ingradient of almost all culture media (synthetic and semisynthetic). Peptone
performs many important functions when it is in the culture media as
i) It supplies most available form of nitrogen to bacterial cell.
ii) It also supplies energy source to micro-organisms,
iii)It supplies assimilable form of phosphorus, sulphur and other essential
elements,
iv) As amino acids are amphoteric in nature, peptone also works as an excellent
buffer prevents drastic changes in pH.
2. Yeast extract:
Brewer's yeasts are the major raw material for manufacture of yeast
extracts, while the Brewer's yeasts are obtained as a byproduct of brewing

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industry. Yeast extracts are produced by autolysis of yeast cells. Autolysis is done
by keeping yeast cells in distilled water where the pH of distilled water is adjusted
to 6.5. Then the distilled water with yeast cells are heated at 45°C for
14 hours, with intermittent stirring. The autolysis is done under controlled
condition to avoid destruction of vitamin of B complex group. Yeast extracts also
performs following functions. It is the best growth factor of bacteria. It is a rich
source of B vitamin. It is used in culture media by replacing meat extracts.
3. Vitamins
Vitamins are most commonly needed growth factor. Vitamins are defined
as an organic compound required in very small amount and cannot be synthesized
by some cells. First discovered growth factor is the vitamin. Vitamins are required
in very minute amount and they show stimulatory effect on the growth of micro-
organisms. They are not an energy source or building blocks of macromolecules.
But vitamins play different functions. Most of them are working as a 'co-enzymes'
of enzymes. Strict autotrophs can synthesize all vitamins but heterotrophs can't
synthesize many of them, thus the growth of heterotrophs depends on the
availability of vitamins. Vitamins which are needed must be incorporated in the
medium. The culture media can be supplemented with vitamins by the use of
yeast extracts, meat extracts, and even peptone.
4. NaCl
NaCl is generally added to culture media, but it is not required for the
growth of microorganisms. It also does not work as buffer in the culture medium.
NaCl is added to a culture medium to maintain the isotonicity of the medium.
Therefore the NaCl is added to medium in the concentration which will be
isotonic with the cytoplasm of cells. Such isotonic condition is not only needed
to prevent plasmolysis or plasmoptysis but also for transport of nutrients into the
cell.
5. Agar agar
It is a dried mucilagenous substance derived from aquatic algae belonging
to the genus Gelidium corneum and related species. These weeds are found

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growing in the water of the coasts of Japan, Srilanka, Malaya, and Southern
California. It is an acidic polysaccharide, polymer of galactose with one sulphate
per ten to fifty residues of galactose. Though it is a polysaccharide, it is fully
indigestable and has no nutritive value. Solid media are found to be more useful
in obtaining a pure culture than liquid media. Agar agar is an ideal solidifying
agent as it possess following important properties.
• It is nontoxic for bacteria.
• Its fibrous structure is fine enough to prevent motility of bacteria within it.
• At the same time it is coarse enough to permit diffusion of even
macromolecules.
• It is transparent and colorless.
• It is insoluble in cold water but it melts at the temperature of boiling and
forms a viscous gel, remains liquid until temperature decreases up to body
temperature, but once it is cooled to body temperature, then it settles in a
solid form, make it suitable for the growth of micro-organisms in the form
of compact masses called as colonies.
• It is attacked by only a few bacterial species like Agrobacterium, Vibrio,
Cytophoga, Pseudomonas or otherwise, it is unutilizable form, therefore
works as a solidifying agent.
• It is easily sterilizable.
• In the culture media 2 to 3% agar-agar powder is satisfactory.
ii) Types of culture media
Depending upon the nature of ingredients, the culture media are of two types.
1. Living media
2. Nonliving media
1 Living media: There are some microorganisms like Viruses, Rickettsia,
Chlamydia etc. which are obligatory intracellular parasites. They cannot be
cultivated in lab on synthetic media. They require live cells for their growth. Thus,
the media which contain living cells are called as living /media. The living media
are mainly of three types -

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a. Bird embryos
b. Tissue culture
c. Live animals.
2. Non-living media: These are the media which totally lack living cells, but
contain non-living material like any pure organic or inorganic chemical or any
natural component like milk, blood etc. Depending upon the nature of ingredient,
the non-living media are classified into three sub-types –
a) Natural media
b) Synthetic media
c) Semi-synthetic media.
a. Natural media:
Natural media are also called as impirical media. Natural media are
prepared using the ingredients which are available in nature like milk, urine,
diluted blood, carrot juice, coconut milk, vegetable juice, etc. Such media were
used in early stages of development of microbiology. These media are prepared
on the basis of previous experience and not on the basis of knowledge about their
exact composition. These media are inexpensive and convenient to use, however,
as the exact composition of chemical nature of ingredient is not known, they are
not reproducible. These media may not be suitable for cultivation of many
important organisms.
b. Synthetic media:
These are the media which contain all ingredients of known chemical
composition (nature). Each component of the media is highly pure and the exact
amount incorporated in the medium is known. Thus such media are reproducible.
Such media are designed according to the knowledge about the exact nutritional
requirement of the desired organism. Such standardized and reproducible media
are most useful in research and in industries and for cell culture, where exact
nutritional need of test organism is known. Synthetic media are of two types –
(i) Inorganic synthetic media,
(ii) Organic synthetic media.

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(i) Inorganic synthetic media:
Inorganic synthetic media are the media where all components are in inorganic
form. They are used generally for autotrophic organisms. For example - The
medium used for the isolation of Thiobacillus thioxidans. The chemical
composition of the medium is given below -
(NH4)2S04 0.2gm.
MgSO4, 7H2O 0.5 gm.
KH2PO4 3.0gm.
CaCl, 0.25 gm.
powdered sulphur 10.00 gm.
Distilled water 1000 ml.
It is a typical inorganic synthetic medium. Chemical nature and exact
amount of each component of the medium is exactly known. The medium
provides all necessary nutrients and energy source for the organism, where CO2
is the carbon source, (NH4)2SO4 is the nitrogen source, powdered sulphur is the
energy source, KH2PO4 is not only the source of, potassium and phosphate but
also works as a buffer. MgSO4, 7H2O, CaCl2 supplies necessary inorganic
elements needed for growth. Thiobacillus synthesizes all components of living
cells using these ingradients.
Winogradsky's medium is another example of inorganic synthetic media.
It is used for the cultivation of Nitrosomonas and Nitrobacter.
(ii) Organic synthetic media: These are the media which contain all ingredients
in organic form. These media are useful for the cultivation of heterotrophic
organisms. Example of this medium is the medium used for the growth of
fastidious pathogenic organism - Corynebacterium diphtherias. The medium
contains 21 different chemically pure organic ingredients and they are in
accurately weighed amounts as shown below -
Amino acids - eight
Vitamines - three
Carbohydrates - several
Esters - several
salts of Ca, Mg, Cu, K, P, S, etc. where carbohydrates and esters serve as carbon
source.

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c. Semi-synthetic media:
The media which contain both natural ingredients and pure chemicals
(organic or inorganic) are called as Semi-synthetic media. Natural ingredients
include meat extracts, peptone, yeast extracts, blood, casein hydrolysate etc. Such
media are also called as complex media, non-synthetic media or undefined media.
Examples of semi-synthetic media are nutrient agar, peptone water, blood agar,
MacConkey's agar, etc.
Nutrient agar is the most common medium used for the cultivation and
enumeration of wide variety of organisms. Peptone water is the most suitable and
economical and used for the cultivation of many non-fastidious organisms. It is
also used to study the ability of organism to ferment different sugars.
The exact chemical composition of all ingredients of semi-synthetic media
is not clearly known. Thus, the media are not exactly reproducible, but they
provide rich mixture of nutrients. Therefore, these media are widely used. They
allow excellent growth as they provide complex nutritional need of different
organisms.
General purpose media are designed to grow a broad spectrum of micro-
organisms. They contain mixture of nutrients and support the growth of pathogens
and non-pathogens alike. But there are some media which are designed especially
for special purpose.
I) Enriched media:
If any general purpose medium or basic medium is added with complex
nutritionally rich organic substance like blood, serum, haemoglobin, extracts of
plants or growth factors (vitamins, amino acids) etc., then such media are called
as enriched media. Such media now become suitable for many fastidious
organisms. Fastidious organisms are those which require growth factors and
complex nutrients for their growth. Fastidious organisms can't grow on basic
medium. Because of addition of nutritionally rich component in the basic
medium, it is called as an enriched medium.For example
1) Blood-agar - It is prepared by addition of sterile rabbit, sheep or horse blood
to a sterile nutrient base. It is suitable for the growth of fastidious organisms like
Streptococcuspyogenes.

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2) Chocholate agar- It is best for the growth of pathogenic fastidious organism
Neisseria gonorrhea. (Red blood cells that have been lysed by slowly heating to
80°C)
Chocolate agar is a non-selective, enriched culture medium used in microbiology
laboratories to isolate and cultivate fastidious bacteria, particularly species like
Haemophilus influenzae and Neisseria gonorrhoeae, by providing essential nutrients
released from lysed red blood cells within the agar, giving it a characteristic
chocolate brown colo
II) Enrichment media:
When anyone wants to isolate a single desired organism from a natural
sample with a large microbial population, where number of desired organism is
very less, and then the sample is added to a special medium which stimulates or
favors the growth of only desired organism and preventing the growth of other
unwanted organisms. Then such medium is called as enrichment medium. The
technique was first time used by Beijerinck and Winogradsky. They used a salt
solution with NaNO2 at pH 8.5 as a nutrient medium; it is then added with soil
sample and incubated at room temperature. It resulted in the enrichment of a
Nitrobacter species from soil.
Enrichment media are generally liquid media. Example –
(1) Tetrathionate broth - It is enrichment medium for Salmonella group of
organisms. The faeces from the patient suffering from typhoid, if inoculated in
tetrathionate broth, then it results in the enrichment of Salmonella group of
organism, simultaneously preventing the growth of other intestinal organisms.
(2) Medium lacking organic or inorganic nitrogen that is nitrogen free mannitol
salt agar is best for nitrogen fixing Azotobacter organism from soil.
III) Selective media:
Selective media contains one or more agents (chemicals), that inhibit the
growth of different micro-organisms (say A, B, C) and do not inhibit the growth
of anyone desired organism (say 'D'). Thus, this medium favours or selects or
encourages the growth of only organism 'D'. Selective media are very important
in primary isolation of specific organism from natural samples (faeces, urine,
saliva, skin, water and soil) containing a dozens of different species.
The isolation is done by suppressing the growth of unwanted background

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organisms and favoring the growth of only desired organism. Examples –
1) Mannitol salt agar - It contains 7.5% NaCl, which is inhibitory for the most
human pathogens except Staphylococcus aureus. Therefore it is a selective
medium for Staphylococcus aureus.
2) MacConkey's agar - It contains bile salt, which is a constituent of human
intestine and faeces. It inhibits the growth of most of the Gram positive and non-
intestinal organism, simultaneously allowing the growth of only intestinal
organisms. Therefore MacConkey's agar is a selective medium for intestinal
organisms like Escherichia coli, Salmonella typhi, Shigella dysentrac-etc.
Table shows some examples of selevtive media.
Name of medium Inhibitory
agent
Inhibiting
the
growth of
Selective for
l. Azide blood agar Azide Gram
negative
Staphylococcus
Streptococcus
2. MacConkey's agar
3. Wilson and Blair’s
medium
bile salt (Sodium
taurocholate)
Bismuth
Sulphite,
Brilliant green
non
intestinal
Coliforms
Intestinal
Salmonella species

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IV) Differential media:
The media which allow the growth of different groups of organisms with
visible differences in the growth patterns, with the help of which organisms can
be differentiated into different groups are called as differential media.
Differentiation is observed as differences or variations in the size and colour of
colony, in the pattern of colour changes, in the formation of precipitate etc. The
variation is achieved because of incorporation of certain chemicals to the medium
and also due to the ways by which microbes react to that chemical.
The medium sometime may contain certain particular nutrient and two different
types of organisms produce two different types of colonies depending upon the
ability of organism to use that specific nutrient. Sometimes dyes or pH indicator
dye is added to the medium as a differentiating agent. It results in the production
of different coloured colonies depending upon the product produced for ex. acid
or base.

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Examples –
1. MacConkey's agar: The medium helps to differentiate the organisms into two
groups as lactose fermenting or lactose non-fermenting. The medium contain
lactose as a sugar and neutral red as a pH indicator dye. Neutral red gives pink or
red colour in acidic condition and yellow when neutral. If the organism in lactose
fermenting, then when ii will ferment lactose, to form organic acids, then pH of
medium changes to acidic, which results in production of pink or red coloured
colony. On the other hand, if organism unable to ferment lactose, then there is
no acid production, no pH change and colonies will remain colourless. E. coli
can ferment lactose to produce acid, thus colour of colony will be pink. But
Salmonella can't ferment lactose no colour change, colonies will be off white.
2. Blood agar: It helps to differentiate the organisms into two groups as
haemolytic and nonhaemolytic. Because of RBCs in the blood, the medium is red
and opaque. If the organisms are haemolytic, there will be lysis of RBCs around
the colony; there will be clear zone around the colony. But if organisms are
nonhaemolytic, then there will be no lysis of RBCs and no zone of clearance
around the colony. Streptococcus haemolyticus, which is haemolytic, forms a
clear zone around the colony while Staphylococcus epidermidis which is non-
haemolytic don't form clear zone around the colony.

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2. EMB agar (Eosin methylene blue agar)
It helps to differentiates coliforms from noncoliforms. Coliform organisms form
a typical that is nucleated black centered colonies with metalic sheen while
noncoliforms form atypical that is pink non nucleated, nucoid colonies without
metallic sheen. E.coli forms typical colonies and Enterobacteraemgenes forms
atypical colonies.
Role of buffers in culture media
A buffer is an aqueous solution consisting of a mixture of a weak acid and
its salt (acidic buffer) or a weak base and its salt (basic buffer). Its pH changes
very little when a small amount of strong acid or base is added to it and

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thus it is used to prevent changes in the pH of a solution. One example of a
buffer solution found in nature is blood. The normal pH of human blood is 7.4.
Media used for cultivation of Bacteria, Fungi, Actinomycetes, Yeasts, Algae
and photosynthetic bacteria
Media used for cultivation of Bacteria
1. Nutrient agar
Distilled Water 100 ml
Peptone 2 gm
Yeast extracts 1 gm
OR
Meat extracts 0.3 gm
NaCl 0.5 gm
Agar agar powder 2.5 gm
pH 7
2. MacConkey’s agar
Distilled water 100 ml
Peptone 2 gm
Sodium taurocholate 0.5 gm
Lactose 1 gm
pH 7
Neutral red (2 % in 50 % Ethanol) 0.3 ml
Agar agar powder 2.5 gm

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Media used for cultivation of Fungi
1. Sabraud’s agar
Glucose 4 gm
Peptone 1 gm
Agar agar powder 2 gm
pH 5.4
2. Potato Dextrose agar
Potato (peeled) 20 gm
Dextrose 2 gm
Agar agar powder 1.5
pH 5.4
Media used for cultivation of Actinomycetes
1. Bennet’s agar medium
Glucose 1 gm
Casein 0.2 gm
Yeast extracts 0.1 gm
Beef extracts 0.1 gm
pH 7.3

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2. Dextrose tryptone agar
Glucose 1 gm
Tryptone 0.5 gm
K2HPO4 0.05 gm
NaCl 0.05 gm
FeSO4. 7H2O 0.01 gm
Agar Agar powder 2 gm
pH 7.2
Media used for cultivation of yeasts
1. Malt extract agar
Malt 1.5 gm
K2HPO4 0.1 gm
NH4Cl 0.1 gm
Citric acid (0.1 N) 1.5 ml
2. Penicillin Streptomycin Blood Agar
Nutrient agar 90 ml
Blood 10 ml
Penicillin 300 units
Streptomycin 300 microgram

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Media used for cultivation of algae
1.Chu’
s medium
Distilled water 1000ml
Calcium nitrate 0.04gm
K2HPO4 0.01gm
Na2CO3 0.02gm
MgSO4.7H2O 0.025gm
Sodium Silicate 0.025gm
Ferric citrate 0.003gm
A 5 trace element stock 1.0 ml
Solution (optional)
A 5 trace element stock solution
Distilled water 1000ml
Boric acid 2.86gm
MnCl2 1.81gm
ZnSO4 0.222gm
Molibdenum trioxide(85%) 0.177gm
Cupric sulphate 0.079gm
pH 8.5 to 9
1. Modified Bristol’s medium
KH2PO4 0.50gm
NaNO3 0.50gm
MgSO4.7H2O 0.15gm
CaCl2.6H2O 0.05gm
NaCl 0.05gm
FeCl3.6H2O 0.01gm

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Media used for cultivation of photosynthetic bacteria
1.For Thiorhodaceae family
Water 100 ml
NH4Cl 0.1 gm
KH2PO4 0.1 gm
MgCl2 0.1 gm
NaHCO3 0.1 gm
Na2S. 9H2O 0.1 gm
pH 8
1.For Chlorobeacae family
Water 100 ml
NH4Cl 0.1 gm
NaCl 0.03 gm
KH2PO4 0.1 gm
MgCl2 0.05 gm
NaHCO3 0.2 gm
Na2S. 9H2O 0.1 gm
Fe 50 µ g
pH 7.3
Cultivation of anaerobes
The isolation of oxygen was successfully done by the scientist Priestley in
1774. Immediately in 1775 Lavoisier observed the role of oxygen in combustion
and respiration. His observation led to conclusion that free air or oxygen is
necessary for all life.
However in 1861, Pasteur proved that certain yeasts and bacteria could
multiply in absence of air. He devised the term 'anaerobiosis', to describe the life
without air. It was one of the epoch making discovery! When further study of
physiology of these micro-organisms was done, it was startling to old ideas of
cell physiology and biochemistry.
Many micro-organisms living without air were then discovered.

31
Table Types of micro-organisms with respect to their relationship to
oxygen along with examples.
Types of organisms Examples
(a) Strict aerobes (i) Most species of genus
Bacillus (ii) Genus Brucella
(iii) Genus Micrococcus (iv)
Genus Pseudomonas
(b) Facultative (i) Genus Staphylococcus
(ii) Genus Streptococcus
(iii)All Coliforms (iv)
Genus Lactobacillus
(c) Strict anaerobes (i) Genus Clostridium (ii)
Genus Actinomyces (iii)
Genus Desulfovibrio (iv)
Genus Neisseria
(d) Microaerophilic (i) Genus Leptospira (ii)
Genus Compylobacter.

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Anaerobic Chamber
1. Anaerobic chamber is an ideal anaerobic incubation system, which provides
oxygen- free environment for inoculating media and incubating cultures.
2. It refers to a plastic anaerobic glove box that contains an atmosphere of H2,
CO2, and N2. Glove ports and rubber gloves are used by the operator to
perform manipulations within the chamber.
3. There is an air-lock with inner and outer doors.
4. Culture media are placed within the air-lock with the inner door. Air of the
chamber is removed by a vacuum pump connection and replaced with
N2 through outer doors.
5. The culture media are now transferred from air-lock to the main chamber,
which contains an atmosphere of H2, CO2, and N2. A circulator fitted in the
main chamber circulates the gas atmosphere through pellets of palladium
catalyst causing any residual O2 present in the culture media to be used up by
reaction with H2.
6. When the culture media become completely anaerobic they are inoculated
with bacterial culture and placed in an incubator fitted within the chamber.
7. The function of CO2 present in the chamber is that it is required by many
anaerobic bacteria for their best growth. A schematic representation of an
anaerobic chamber showing its various parts is given in Fig

33
3) Anaerobic Bags or Pouches:
Anaerobic bags or pouches make convenient containers when only a few
samples are to be incubated anaerobically. They are available commercially. Bags
or pouches have an oxygen removal system consisting of a catalyst and calcium
carbonate to produce an anaerobic, CO2-rich atmosphere.
One or two inoculated plates are placed into the bag and the oxygen
removal system is activated and the bag is sealed and incubated. Plates can be
examined for growth without removing the plates from bag, thus without
exposing the colonies to oxygen.
But as with anaerobic jar, plates must be removed from the bags in order
to work with the colonies at the bench. These bags are also useful in transport of
biopsy specimen for anaerobic cultures.

34
4. Anaerobic Jars (or GasPak Anaerobic System)
i. When an oxygen-free or anaerobic atmosphere is required for obtaining
surface growth of anaerobic bacteria, anaerobic jars are the best suited. The
most reliable and widely used anaerobic jar is the Melntosh-Fildes’ anaerobic
jar.
ii. It is a cylindrical vessel made of glass or metal with a metal lid, which is held
firmly in place by a clamp.

35
iii) The lid possesses two tubes with taps, one acting as gas inlet and the other
as the outlet.
iv) On it’s under surface it carries a gauze sachet carrying palladium pellets,
which act as a room temperature catalyst for the conversion of hydrogen
and oxygen into water. Palladium pellets act as catalyst, as long as the
sachet is kept dry.
v) Inoculated culture plates are placed inside the jar and the lid clamped tight.
vi) The outlet tube is connected to a vacuum pump and the air inside is
evacuated.
vii) The outlet tap is then closed and the gas inlet tube connected to a hydrogen
supply. Hydrogen is drawn in rapidly. As soon as this inrush of hydrogen
gas has ceased the inlet tube is also closed.
viii) After about 5 minutes inlet tube is further opened. There occurs again an
immediate inrush of hydrogen since the catalyst creates a reduced pressure
within the jar due to the conversion of hydrogen and leftover oxygen into
water.

36
ix) If there is no inrush of hydrogen, it means the catalyst is inactive and must
be replaced.
x) The jar is left connected to the hydrogen supply for about 5 minutes, then
the inlet tube is closed and the jar is placed in the incubator. Catalysis will
continue until all the oxygen in the jar has been used up.
xi) The gasPak is now the method of choice for preparing anaerobic jar. The
gasPak is commercially available as a disposable envelope containing
chemicals, which generate hydrogen and carbon dioxide when water is
added. After the inoculated plates are kept in the jar, the gasPak envelope
with water added, is placed inside and the lid screwed tight.
xii) Hydrogen and carbon dioxide are liberated and the presence of a cold
catalyst in the envelope permits the combination of hydrogen and oxygen
to produce an anaerobic environment.
❖ Method for detecting microscopic organisms by using bacteriophages
Kent J. Voorhees apparatus:
Methods and apparatus are provided for detection of microorganisms in a
sample. Methods and apparatus of the invention are based on the specificity that
phage for example bacteriophage, have for target microorganisms, for example
bacterium. Phage adsorption to target microorganisms act as signal, for the
presence of the target microorganism. Typically, the phages are labeled with a
detectable signal. Apparatus of the invention are directed toward concentrating
the phage adsorbed microorganisms at a predetermined site for flag dependent
observation.
A method of determining the presence of a target microorganism in a
sample, the method comprising: combining with the sample an amount of flagged
phage capable of binding to an outside surface of the target microorganism to
create a flag labeled target microorganism; providing conditions and an amount
of time sufficient to allow the flagged phage to bind to the outside surface of the
target microorganism; and assaying the phage exposed sample to detect the
presence of the flag labeled microorganism, without detecting phage amplified
within the target microorganism and, wherein presence of the flag bound to the
outside surface of the target microorganism indicates presence of the
microorganism, wherein the detecting the presence of

37
the flag labeled microorganism is by concentrating the flag labeled
microorganism to enhance the capacity to detect the flag labeled microorganisms,
and wherein the concentrating of flag labeled microorganisms is on a flow strip
device having one or more immobilization zones, wherein the flag labeled
microorganisms are captured and concentrated within the one or more
immobilization zones. The method of claim 1 wherein the flagged phage is a
phage labeled with colorimetric particles. The method of claim 2 wherein the
colorimetric particles are colloidal gold particles. The method of claim 1 wherein
the flagged phage is a phage labeled with a fluorescent tag.

Pure Culture-Technique's of Microorganisms for B.Sc. Biotechnology & Botany Sem-3

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Pure Culture-Technique's of Microorganisms for B.Sc. Biotechnology & Botany Sem-3