LECTURE IV

CULTIVATION AND MAINTAINANCE OF BACTERIAL CULTURE

FACTORS THAT AFFECT BACTERIAL GROWTH:

The growth of microorganisms is greatly affected by the chemical and physical nature of their
surroundings. An understanding of these influences aids in the control of microbial growth and
the study of the ecological distribution of microorganisms. The major physical factors which
affect microbial growth are; water activity, pH, temperature, oxygen level, pressure and
radiation. Nutrient is also a factor affecting growth of microorganisms.

Water Availability: Water activity (aw) is the amount of water available to microorganisms and
this can be reduced by interaction with solute molecules (osmotic effect). Water activity is
inversely related to osmotic pressure; if a solution has high osmotic pressure, its a w is low.
Microorganisms differ greatly in their ability to adapt to habitats with low water activity. In a
low aw habitat, the microorganisms must expend extra effort to grow as it should maintain a
high solute concentration to retain water. Such microorganisms are osmotolerant or can grow
over wide range of water activity or osmotic concentration. Most of the microorganisms grow
at aw =0.98 or higher.

pH: It refers to the acidity or alkalinity of a solution. It is a measure of the hydrogen ion activity
of a solution and is defined as the negative logarithm of the hydrogen ion concentration.

pH = -log [H+] = log (1/H+)

The pH scale ranges from 1.0 to 14.0 and most microorganisms grow vary widely from pH 0 to
2.0 at the acid end to alkaline lakes and soil that may have pH values between 9.0 and 10. The
pH can affect the growth of microorganisms and each species has a definite pH growth range
and pH growth optimum. Acidophiles have their growth optimum between pH 0 and 5.5;
neutrophiles between 5.5 and 8.0 and alkalophiles prefer pH range of 8.5 to 11.5. Most bacteria
and protozoans are neutrophiles, fungi prefer acid surroundings about pH 4 to 6; algae also
seem to favour slight acidity. Drastic changes/variations in cytoplasmic pH can harm
microorganisms by disrupting the plasma membrane or inhibiting the activity of enzymes and
membrane transport proteins. Prokaryotes die if the internal pH drops much below 5.0 to 5.5.
External pH alterations also might alter the ionization of nutrient molecules and thus reduce
their availability to the organism.

Temperature: Temperature profoundly affects microorganisms as the most important factor

influencing the growth of microorganisms, the effect of temperature is observed on sensitivity
of enzyme-catalyzed reactions. Beyond a certain point of higher temperature, slow growth
takes place and damages the microorganisms by denaturing enzymes, transport carriers and
other proteins. The plasma membrane also is disrupted as lipid bilayer simply melts and the
damage is such an extent that it cannot be repaired. At very low temperature, membranes
solidify and enzymes do not work rapidly.

1
Microorganisms are classified into four classes based on their temperature ranges for growth.

1. Psychrophiles: Microorganisms that grow well at 0°C and the optimum growth
temperature of 15°C or lower and maximum at around 20°C. These microorganisms are
isolated from Arctic and Antarctic habitats. They have adapted to their environment in
several ways. Their enzymes, transport systems and protein synthetic mechanisms
function well at low temperatures. The cell membranes have high levels of unsaturated
fatty acids and remain semifluid when cold. At higher than 20°C, the psychrophiles begin
to leak cellular constituents because of cell membrane disruption. Microorganisms such
as Pseudomonas, Alcaligenes, Arthrobacter, Moritella, Photobacterium belong to this
group. The psychrophilic Chlamydomonas nivalis turns a snowfield or glacier pink with
its bright red spores.

2. Mesophiles: Growth optimum around 20°C to 40°C, minimum at 15°C to 20°C and
maximum at 45°C or lower. Most of the organisms fall under or within this category
including human pathogens.

3. Thermophiles: The microorganisms in this group can grow at temperature of 55°C or

higher, minimum is usually around 45°C and growth optima at around 55°C to 65°C.
Mostly prokaryotes and a few algae and fungi belong to this group. The habitats in
which they grow include, composts, self-heating haystacks, hot water lines and hot
springs. Microorganisms have more heat-stable enzymes and proteins synthesis
systems, which function at high temperature. Heat stable proteins have high organized,
hydrophobic interiors, more hydrogen bonds and other non-covalent bonds strengthen
the structure. Amino acids like proline make the polypeptide chain less flexible and
chaperones also aid in folding of proteins to stabilize them. DNA also is stabilized by
specific histone like proteins. The membrane lipids are also stable and tend to be more
saturated, more branched and of higher molecular weight. Archaeal thermophiles have
membrane lipids with ether linkages, which protect the lipids from hydrolysis at high
temperatures. Examples include Bacillus and Stereothermophillus

4. Hyperthermophiles: Few microorganisms can grow at 96°C or above and have

maximum at 100°C; and growth optima between 80°C and about 113°C. Pyrococcus and
Pyrpdictiumoccultum are examples of marine hyperthermophiles found in hot floors of
the sea floor.

Oxygen Concentration: In microorganisms and also bacteria, oxygen is most often uses as
terminal electron accecptor in respiratory pathway. Some organisms will require oxygen for
growth, others do not. An aerobe is an organism able to grow in the presence of atmospheric
O2 and the ones that grow in its absence is an anaerobe. Organisms which completely are
dependent on atmospheric O2 for growth are obligate aerobes, and it serves as the terminal
electron acceptor for the electron transport chain in aerobic respiration and employs it in the
synthesis of sterols and unsaturated fatty acids. Organisms which do not require O 2 for growth
but do grow better in its presence are called facultative anaerobes . Aerotelerant anaerobes

2
such as Enterococcus faecalis simply ignore O2 and grow equally well whether it is present or
not. Obligate anaerobes like Bacteroides, Fusobacterium, Clostridium pasteurianum,
Methanococcus, Neocallimastix, do not tolerate O2 at all and die in its presence. Aerotelerant
and obligate anaerobes cannot generate energy through respiration and must employ
fermentation or anaerobic respiration pathways for the purpose. Microaerophiles are those
organisms that are damaged by the normal atmospheric levels of O 2 (20%) and require O2 levels
between the range of 2% to 16% for growth. Helicobacter pylori is a microaerophilic bacterium.

Nutritional requirements

In addition to a proper physical environment, microorganisms also depend on an available

source of chemical nutrients. Therefore bacteria nutrients should contain;

a. Energy source

1. Phototrophs use radiant energy (light) as their primary energy source.

2. Chemotrophs use the oxidation and reduction of chemical compounds as their primary
energy source.

b. Carbon source

Carbon is use to form the structural backbone of the organic compounds that make up a living
cell.

c. Nitrogen source

Nitrogen is needed for the synthesis of such molecules as amino acids, DNA, RNA and ATP.
Depending on the organism, nitrogen, nitrates, ammonia, or organic nitrogen compounds may
be used as a nitrogen source.

d. Minerals

Trace elements are elements required in very minute amounts, and like potassium, magnesium,
calcium, and iron, they usually function as cofactors in enzyme reactions. They include sodium,
zinc, copper, molybdenum, manganese, and cobalt ions. Cofactors usually function as electron
donors or electron acceptors during enzyme reactions.

e. Growth factors

Growth factors are organic compounds such as amino acids, purines, pyrimidines, and vitamins
that a cell must have for growth but cannot synthesize itself. Organisms having complex
nutritional requirements and needing many growth factors are said to be fastidious.

3
Bacterial growth curve:

This is a curve on a graph that shows the changes in size of a bacterial population over time in a
culture. The bacteria are cultured in sterile nutrient medium and incubated at the optimum
temperature for growth. Samples are removed at intervals and the number of viable bacteria is
counted. A logarithmic growth curve is plotted, which shows various phases;

1. Lag phase
2. Log phase or exponential phase
3. Stationary phase
4. Death phase or decline phase

1. Lag phase:

 When bacteria are inoculated into new fresh media, it does not divide immediately.
Bacteria take some time to adjust to the new environment. The time period in which
bacteria is metabolically active but do not divide is called as lag phase.
 lag phase is characterized by the period during which there is no increase in number of
cell.
 Size of bacteria increase continuously so the bacteria have largest size at the end of lag
phase.
 In this phase, microorganism tries to adopt in new environment. It is the phase of
adjustment necessary for the synthesis of enzymes and co-enzymes for physiological
activities.
 Time is required for adjustment in physical environment around each cell.
 Duration of lag phase varies according to conditions and species of bacteria.
o If the culture organism is taken from old culture, the duration will be longer but
if the culture is fresh, duration is short.
o Similarly if the culture media is different from the previous culture then duration
is long because bacteria takes some more time to adjust in the new media.
 At the end of lag phase, bacteria become fully prepared for cell division.

2. Log phase or exponential phase:

 During this phase bacteria divides continuously at constant rate and the number of
bacteria increase exponentially.
 In this phase all bacteria are in their rapid stage of cell division and show balanced
growth.
 Due to rapid cell division, bacteria have smallest size in this phase.
 Bacterial population is nearly uniform in terms of their metabolic activities, chemical
composition of cell and other physiological characteristics.

4
  Biochemical and physiological characteristics that are commonly used for identification
of bacteria are manifested during log phase of growth.
 Generation time of bacteria is usually determined during log phase. However it is not
same for all bacteria in culture.
 Generation time is shortest during log phase and is strongly dependent upon growth
factors present in the medium.
 This phase lasts for several hours depending on the type of organism, conditions of
growth and density of organism.

3. Stationary phase:

 The bacteria growth reaches a state during which there is no net increase in bacterial
population. This is called as stationary phase.
 In this phase a constant bacterial population is maintained by balance between cell
division and cell death.
 In some bacteria, complete cessation of cell division occurs hence there is no net
increase or decrease in number of bacteria.
 Stationary phase is induced by- increased bacterial cell density, depletion of nutrition in
media and accumulation of toxic secondary metabolic wastes.
 Production of antibiotics such as Penicillin, streptomycin etc. and enzymes by certain
bacteria occur during stationary phase of their growth.
 In endospore forming bacteria, sporulation occurs as the bacteria enter stationary
phase.

4. Death phase or decline phase:

 In this phase, number of bacteria decrease continuously and exponentially.

 During this phase, total count of bacteria may remain constant but the viable count
decreases.
 It is just inverse of log phase. But the death rate is slower than growth rate.
 Death phase is brought about by various reasons, such as depletion of nutrition and
accumulation of toxic wastes.
 Not all bacteria die at same rate, some die faster and some are more resistant and
remain viable for longer time. eg. Spore forming bacteria.

5
 BACTERIAL GROWTH CURVE

Culture media: Medium is a solid or liquid preparation use to grow, transport and store
microorganisms. A culture media is a special medium used in microbiological laboratories to
grow different kinds of microorganisms. A growth or a culture medium is composed of different
nutrients that are essential for microbial growth.

Since there are many types of microorganisms, each having unique properties and requiring
specific nutrients for growth, there are many types based on what nutrients they contain and
what function they play in the growth of microorganisms.

Classification of culture media based on the chemical composition

1. Synthetic or chemically defined medium

a chemically defined medium is one prepared from purified ingredients and therefore
whose exact composition is known.
2. Non synthetic or chemically undefined medium

Non-synthetic medium contains at least one component that is neither purified nor completely
characterized nor even completely consistent from batch to batch. Often these are partially
digested proteins from various organism sources. Nutrient broth, for example, is derived from
cultures of yeasts.

Classification of Bacterial Culture Media based on the basis of purpose/ functional use/
application

Many special purpose media are needed to facilitate recognition, enumeration, and isolation of
certain types of bacteria. To meet these needs, numerous media are available.

1. General purpose media/ Basic media

Basic media are basically simple media that supports most non-fastidious bacteria. Peptone
water, nutrient broth and nutrient agar are considered as basic medium. These media are
generally used for the primary isolation of microorganisms. Example is tryptic soy broth

6
2. Enriched medium (Added growth factors):

Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basic medium makes
them enriched media. Enriched media are used to grow nutritionally exacting (fastidious)
bacteria. Blood agar, chocolate agar, Loeffler’s serum slope etc. are few of the enriched media.
Blood agar is prepared by adding 5-10% (by volume) blood to a blood agar base. Chocolate agar
is also known as heated blood agar or lysed blood agar.

3. Selective media are designed to inhibit unwanted commensal or contaminating bacteria and
help to recover pathogen from a mixture of bacteria. While selective media are agar based,
enrichment media are liquid in consistency. Both these media serve the same purpose. Any
agar media can be made selective by addition of certain inhibitory agents that do not affect the
pathogen of interest. Various approaches to make a medium selective include addition of
antibiotics, dyes, chemicals, alteration of pH or a combination of these.

Examples of selective media include:

1. Thayer Martin Agar used to recover Neisseria gonorrhoeae contains antibiotics;

vancomycin, colistin and nystatin.
2. Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contains 10% NaCl.
3. Potassium tellurite medium used to recover C.diphtheriae contains 0.04% potassium
tellurite.
4. MacConkey’s Agar used for Enterobacteriaceae members contains bile salt that inhibits
most gram positive bacteria.
5. Pseudosel Agar (Cetrimide Agar) used to recover P. aeruginosa contains cetrimide
(antiseptic agent).
6. Crystal Violet Blood Agar used to recover S. pyogenes contains 0.0002% crystal violet.
7. Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by
incorporating malachite green.
8. Wilson and Blair’s Agar for recovering S. typhi is rendered selective by the addition of
dye brilliant green.
9. Selective media such as TCBS Agar used for isolating V. cholerae from fecal specimens
have elevated pH (8.5-8.6), which inhibits most other bacteria.

4. Differential/ indicator medium:

Certain media are designed in such a way that different bacteria can be recognized on the basis
of their colony colour. Various approaches include incorporation of dyes, metabolic substrates
etc, so that those bacteria that utilize them appear as differently coloured colonies. Such media
are called differential media or indicator media. Differential media allow the growth of more
than one microorganism of interest but with morphologically distinguishable colonies.
Examples of differential media include:

7
 1. Mannitol salts agar (mannitol fermentation = yellow)
2. Blood agar (various kinds of hemolysis i.e. α, β and γ hemolysis)
3. Mac Conkey agar (lactose fermenters, pink colonies whereas non- lactose fermenter
produces pale or colorless colonies.
4. TCBS (Vibrio cholerae produces yellow colonies due to fermentation of sucrose)

5. Transport media:
Clinical specimens must be transported to the laboratory immediately after collection to
prevent overgrowth of contaminating organisms or commensals. This can be achieved by using
transport media. Such media prevent drying (desiccation) of specimen, maintain the pathogen
to commensal ratio and inhibit overgrowth of unwanted bacteria. Some of these media
(Stuart’s & Amie’s) are semi-solid in consistency. Addition of charcoal serves to neutralize
inhibitory factors.

 Cary Blair transport medium and Venkatraman Ramakrishnan (VR) medium are used to
transport feces from suspected cholera patients.
 Sach’s buffered glycerol saline is used to transport feces from patients suspected to be
suffering from bacillary dysentery.
 Pike’s medium is used to transport streptococci from throat specimens.

NA (Nutrient agar) and NB (Nutrient broth) Preparation in the laboratory:

Nutrient agar and nutrient broth share almost the same medium composition. The main
difference between them is that nutrient agar contains a solidifying agent, agar powder that
causes the medium to solidify in room temperature, whereas nutrient broth remains in liquid
form.
Nutrient agar contains nutrients that suitable to subculture a wide range of microorganisms
and makes it an excellent agar media to check on the purity before any biochemical or
serological test.

Besides, the addition of agar solidifies nutrient agar, which makes it suitable for the cultivation
of microorganisms. You can also add up to 10% blood or other biological fluids that meet your
experimental purpose.

Preparation of Nutrient Agar

Nutrient agar consist of Peptone, beef extract, salt and agar. The beef extract contains water-
soluble substances (carbohydrates, vitamins and nitrogen compounds). Peptones are the main
source organic Nitrogen while agar is used as a gelling agent. A litre of Nutrient agar is prepared
by dissolving the following in a litre of distilled water:

8
 •3g of beef extract •5g of Peptone • 5g of NaCl •15g of Agar

The beef extract, peptone and NaCl are first dissolved and then heat until the components are
completely dissolved. Then the agar is added with continuous stirring while heating until it is
also dissolved completely. The solution is nutrient agar compounded from the components or
the commercially available nutrient agar is made ready for usage in the following way:

 Suspend 28g of Nutrient Agar powder in 1L of distilled water.

 Mix and dissolve them completely.
 Sterilize by autoclaving at 121°C for 15 minutes.
 Pour the liquid into the petri dish and wait for the medium to solidify. Be sure that you are
preparing the agar in the clean environment to prevent any contamination.
Once the agar solidifies, the agar is ready to use.

Basically, the nutrient broth is the nutrient agar that lack of the solidifying agent, agar powder.
They remain in liquid form at room temperature and are usually used to maintain the stocks of
microorganisms. Therefore when preparing nutrient broth from the components, the
procedure is as for nutrient agar except that agar will not be added or the commercial available
nutrient broth can be made ready for usage as stated below:

Preparation of Nutrient Broth

 Add 13g of nutrient broth powder in 1L of distilled water.

 Mix and dissolve them completely.
 Pour them into the final containers (e.g. conical flask)
 Sterilize by autoclaving at 121°C for 15 minutes.

CULTIVATION OF ANAEROBIC BACTERIA:

Anaerobic bacteria are common causes of infection and will be missed in clinical diagnosis
unless special precautions are taken for their isolation and culture. With anaerobic culture,
microbiologists are not only challenged with obtaining a good specimen, but also with ensuring
that the specimen does not come in contact with air. Anaerobic and micro-aerophilic bacteria
can also be beneficial to humans. These bacteria are used as “waste digesters” by industry to
clean oil spills, methane gas production in waste management, and can actually be a culprit in
the spoilage of beer.

The cultivation of microorganisms in these oxygen-depleted atmospheres involves many

methods which include:

9
 1. Grow in anaerobic jar
2. Grow in gaspak system
3. Usage of Thioglycollate medium
4. Grow in the tissue of dicot plant

Grow in anaerobic jar; Anoxomat uses the widely known McIntosh and Fildes system of
evacuation and replacement to create anaerobic environments. During the anaerobic
evacuation cycle, oxygen within the jar is replaced with hydrogen. After the anaerobic cycle, a
mere 0.16% residual oxygen content is left in the jar, which is then removed by a Palladium
catalyst. The anaerobic recipe leaves the jar with a strict zero oxygen level and 10% carbon
dioxide mix. This method is the most efficient way to create precise, reproducible conditions
because it rapidly removes the oxygenated environment from a jar and replaces it with precise
amounts of anaerobic, or microaerophilic or capnophilic gas mixtures.

Grow in Gaspak system; These are commercially available, disposable sachets containing a dry
powder or pellets, which, when mixed with water and kept in an appropriately sized airtight jar,
produce an atmosphere free of elemental oxygen gas (O 2). They are used to produce an
anaerobic culture in microbiology.

Constituents of gas-pak sachets

1. Sodium borohydride - NaBH4

2. Sodium bicarbonate - NaHCO3
3. Citric acid - C3H5O(COOH)3
4. Cobalt chloride - CoCl2 (catalyst)

Reactions

 NaBH4 + 2 H2O = NaBO2 + 4 H2↑

 C3H5O(COOH)3 + 3 NaHCO3 + [CoCl2] = C3H5O(COONa)3 + 3 CO2 + 3 H2 + [CoCl2]
 2 H2 + O2 + [Catalyst] = 2 H2O + [Catalyst]

Consumption of oxygen

These chemicals react with water to produce hydrogen and carbon dioxide along with sodium
citrate (C3H5O(COONa)3) and water as byproducts. Again, hydrogen and oxygen reacting on a
catalyst like Palladiumised alumina (supplied separately) combine to form water.

Usage of Thioglycollate medium; Thioglycollate medium allows the growth of wide range of
bacteria including the anaerobic ones because it contain wide range of nutrients including
0.075% agar to prevent convection currents from carrying atmospheric oxygen throughout the
medium.

10
Grow bacteria in dicot plant; Bacteria can be culture in the root of dicot plant which achieve its
anaerobic environment as a result of some bacteria inhabitant that convert atmospheric
nitrogen to nitrite. The nitrite inturn react with oxygen in the environment and convert it to
nitrate. This makes the environment oxygen free.

Culturing Microaerophilic Bacteria

A microaerophile is a microorganism that requires oxygen to survive, but requires

environments containing lower levels of oxygen than are present in the atmosphere (~20%
concentration). They require between 2 to 10%. Many microphiles are also capnophiles, as they
require an elevated concentration of carbon dioxide. In the laboratory they can be easily
cultivated in a candle jar. A candle jar is a container into which a lit candle is introduced before
sealing the container’s airtight lid. The candle’s flame burns until extinguished by oxygen
deprivation, which creates a carbon dioxide-rich, oxygen-poor atmosphere in the jar. Many labs
also have access directly to carbon dioxide and can add the desired carbon dioxide levels
directly to incubators where they want to grow microaerophiles.

Stabbing agar slant is another method for culturing microaerophilic bacteria although other
bacteria based on oxygen requirement can also grow on the slant but at different zone. The
slant is stabbed with the suspected bacterium, after incubation at the appropriate temperature,
microaerophiles grow very close to the surface of the agar slant indicating their requirement for
very low concentration of oxygen.

Techniques to Separate Bacteria in a Mixed Culture

There are two main techniques of getting pure culture of bacterium in a mixed culture, these
are; A. Streak plate and B. Pour plate techniques.

A. Streaking: Bacteria streaking require three tools: an agar plate, an alcohol burner and a wire
loop. The agar plate is the growth media to which the bacteria are transferred after growing in
broth. The alcohol burner is a small, alcohol-filled lamp for sterilizing the wire loop — a long,
slender handle with a small loop of fire-resistant wire attached at one end. The loop will hold a
tiny drop of bacteria-filled broth when transferring the bacteria from the broth to the agar
plate.

Streaking bacteria across an agar plate is simple, but you must perform this step correctly or
you will not isolate separate colonies. Heat the loop over the alcohol burner to sterilize the
wire, then dip the tip of the loop into the broth. Streak the loop tip back and forth several times
over a quarter of the agar plate. Sterilize the loop again, and streak across and perpendicular to
the first streaks. Move the loop back and forth a few times over a new section of the plate.
Sterilize the tip. Streak lightly across the last streaked section one time and move the loop back
and forth several times. The streaking is diluting the initial drop of broth to a point where the
last streaks will contain single colonies. Place the plate in an incubator or on a tabletop at room

11
temperature, and wait for the colonies to grow overnight. Single colonies in the last streak will
become isolated colonies of a single bacterium.

B. THE POUR PLATE

Another method of separating bacteria is the pour plate method. With the pour plate method,
serial dilution of the bacteria is first done, each dilution factor is then used to seed a plate with
melted agar until evenly distributed and separated throughout the liquid. The highest number
of dilution will contain colonies well separated. This can be continued (dilution) until pure
culture is obtained after growth. After incubation, discrete bacterial colonies can then be found
growing on the agar plate.

QUALITY CONTROL OF PREPARED MEDIA

The quality of work in a microbiological laboratory depends on the quality of the culture media.
It is to use correct media for the purpose at hand, although the correct media is not always
obvious. The recommendation is provided that the choice of media should be consistent,
appropriate and justified. The following quality control methods should be observed while
preparing the media:

* Accurate weighing of the dehydrated components

* Use of high quality water

* Completely dissolving the dehydrated media or various ingredients

* Control of heating the media to avoid damaging the heat-labile components of the media

* Some recommendations on the labeling and packing of the media are also to be observe

The quality control methods of prepared media are:

* Excess of heat and cold are to be guarded against, as is the potential for dehydration of
poured plate

* The harmonized sterility test and the harmonized microbial limit test have both incorporated
stringent media quality checks.

Maintainance and storage of stock culture:

Once a pure culture has been obtained from the mixture, the microbes may be maintained in
the laboratory over long periods. Several techniques are used to preserve cultures, although
refrigeration is the most common. Cultures are preserved for the following reasons;

12
1. To ensure optimal long-term viability and genetic stability.

2. To keep the properties of the culture until they are deposited at the culture collection
Centre.

3. To have a means of replenishing culture at any point in time etc.

There are several methods of preservation available for microbiologists, the choice of which
depends on the type of culture to be preserved and the period of preservation required. The
methods include;

1. Sub-culturing

2. Soil stock

3. Refrigeration

4. Overlaying culture with mineral oil/ paraffin

5. Lyophilization.

1. Sub-culturing: Normally in laboratories, the pure cultures are transferred periodically onto or
into a fresh medium (subculturing) to allow continuous growth and viability of microorganisms.
The transfer is always subject to aseptic conditions to avoid contamination.

Since repeated subculturing is time consuming, it becomes difficult to maintain a large number
of pure cultures successfully for a long time. In addition, there is a risk of genetic changes as
well as contamination.

2. Soil Stock: This method is widely used for preserving spore forming bacteria and fungi. In this
method, organisms will remain in dormant stage in sterile soil. Soil is sterilized then spore
suspensions are added to it aseptically, this mixture is dried at room temperature and stored in
refrigerator. Viability of organisms found around 70 –80 years.

3. Refrigeration: Pure cultures can be successfully stored at 0-4°C either in refrigerators or in

cold-rooms. This method is applied for short duration (2-3 weeks for bacteria and 3-4 months
for fungi) because the metabolic activities of the microorganisms are greatly slowed down but
not stopped. Thus their growth continues slowly, nutrients are utilized and waste products
released in medium. This results finally in the death of the microbes after sometime. This
method is liable to bring about genetic changes in the microorganism preserved, some
microorganisms such as Blakeslea trispora “a fungus” used in beta carotene production cannot
be stored at refrigerator temperature because they die out relatively quickly at this
temperature.

4. Paraffin Method/ preservation by overlaying cultures with mineral oil

This is a simple and most economical method of maintaining pure cultures of bacteria and
fungi. In this method, sterile liquid paraffin is poured over the slant (slope) of culture and stored

13
upright at room temperature. The layer of paraffin ensures anaerobic conditions and prevents
dehydration of the medium. This condition helps microorganisms or pure culture to remain in a
dormant state and, therefore, the culture can be preserved for months to years (varies with
species).

The advantage of this method is that we can remove some of the growth under the oil with a
transfer needle, inoculate a fresh medium, and still preserve the original culture. The simplicity
of the method makes it attractive, but changes in the characteristics of a strain can still occur.

5. Lyophilization: In this method, the culture is rapidly frozen at a very low temperature (-70°C)
and then dehydrated by vacuum. Under these conditions, the microbial cells are dehydrated
and their metabolic activities are stopped; as a result, the microbes go into dormant state and
retain viability for years. Lyophilized or freeze-dried pure cultures and then sealed and stored
in the dark at 4°C in refrigerators. Freeze- drying method is the most frequently used technique
by culture collection centres.

14