Isolation &Preservation of

industrial important
Microorganism

NAME : HARSHAD S. ROHIT

ROLL NO : 12
SUBJECT : MICROBIOLOGY
INSTITUTION :GUJARAT ARTS AND
SCIENCE COLLEGE
SOME BASIC TERMS-
WHAT IS A CULTURE?
Population of microorganisms grown under well defined conditions.
WHAT IS MIXED CULTURE?
When a particular species of microbe is present in a very small
number in comparison to the total number of microorganisms , such
culture is called as mixed culture.
WHAT IS PURE CULTURE?
A culture containing only one species of microbe is called pure
culture.
SPECIES- a collection of bacterial cells which share an overall similar
pattern of traits in contrast to other bacteria whose pattern differs
significantly.
STRAIN- A strain is a subset of a bacterial species differing from
other bacteria of the same species by some minor but identifiable
difference.
 ISOLATION OF MICROBIAL PURE
CULTURE -
 Microorganisms are generally
found in nature (air, soil and water)
as mixed populations.

 Even the diseased parts of plants

and animals contain a great number
of microorganisms, which differ
markedly from the microorganisms
of other environments.

 To study the specific role played by

a specific microorganism in its
environment, one must isolate the
same in pure culture..
 COMMON METHODS OF
ISOLATION OF PURE CULTURE

The process of screening a pure culture by separating one type

of microbes from a mixture is called Isolation. Some common
isolation methods are-

I. ISOLATION BY II. MICRO

STREAK PLATE MANIPULATOR
TECHNIQUE ISOLATION METHOD
METHODS
III.ENRICHMENT IV .SERIAL
CULTURE
DILUTION
METHOD
METHOD
 I. ISOLATION BY STREAKING OR
STREAK PLATE TECHNIQUE
 This method is used most commonly to isolate pure cultures
of bacteria.
 In This method the tip of a fine structure wire loop called
Inoculation needle consist of a wooden or glass handle with a
nichrome wire the end of which is bend to form a loop is used
to transfer microbes from culture. .
 The straight wires are similar to wire loop except they do not
have loop.These are used to transfer culture in colony formed
on solid culture medium.
 In such cases,the colony from solid medium is streaked on the
surface of nutrient agar medium in a sterile petridish.
 This technique consist of the following steps-
A. Hold the broth culture containing tube in left hand and shake it.
B. Sterilize the wire loop of the inoculation needle on burner flame .
C.Remove the cotton plug of the broth culture tube by little
finger of right hand.
D.Flame the mouth of the test tube immediately.
E.Insert the wire loop to form a thin film and replace the cotton
plug.
F.The thin film in the loop is streaked in either a zig-zag manner
by removing the loop backwards and forwards firmly.Care should
be taken that loop should not be firmly pressed against the agar
surface.
G.Incubate the petri dish in incubator at a required temperature.
H.Growth of the bacteria will be visible (afteran
overnight incubation)on the streaked marks.
II-MICROMANIPULATOR METHOD-
 Micromanipulators have been built, which permit one to pick
out a single cell from a mixed culture. This instrument is used
in conjunction with a microscope to pick a single cell
(particularly bacterial cell) from a hanging drop preparation.

ADVANTAGES OF MICROMANIPULATOR METHOD-

 The advantages of this method are that one can be reasonably
sure that the cultures come from a single cell and one can
obtain strains with in the species.

DISADVANTAGES-
 The disadvantages are that the equipment is expensive,its
manipulation is very tedious, and it requires a skilled operator.
 III-ISOLATION BY USING SELECTIVE
OF ENRICHMENT
MEDIA/ENRICHMENT METHOD-
A.Generally, it is used to isolate those microorganisms, which
are present in relatively small numbers or that have slow growth
rates compared to the other species present in the mixed culture.

B.The enrichment culture strategy provides a specially designed

cultural environment by incorporating a specific nutrient in the
medium and by modifying the physical conditions of the
incubation.

C.The medium of known composition and specific condition of

incubation favors the growth of desired microorganisms but, is
unsuitable for the growth of other types of microorganisms.
D. Chemical dyes, such as malachite green and
crystal violet, are used to inhibit the growth of
bacteria and yeast. Sodium azide is a metal-binding
agent that inhibits the growth of anaerobic
bacteria,but does not affect the anaerobic lactic
acid bacteria.
IV- SERIAL DILUTION METHOD-
This method is commonly used to obtain pure cultures of
those microorganisms that have not yet been successfully
cultivated on solid media and grow only in liquid media. A
microorganism that predominates in a mixed culture can be
isolated in pure form by a series of dilutions.

The inoculum is subjected to serial dilution in a sterile liquid

medium, and a large number of tubes of sterile liquid medium
are inoculated with aliquots of each successive dilution.
The aim of this dilution is to inoculate a series of tubes with a
microbial suspension so dilute that there are some tubes showing
growth of only one individual microbe. For convenience, suppose we
have a culture containing 10 ml of liquid medium, containing 1,000
microorganisms i.e., 100 microorganisms/ml of the liquid medium.
 Continued….
.
If we take out 1 ml of this medium and mix it with 9 ml of
fresh sterile liquid medium, we would then have 100
microorganisms in 10 ml or 10 microorganisms/ ml.
If we add 1 ml of this suspension to another 9 ml. of fresh
sterile liquid medium, each ml would now contain a single
microorganism.
If this tube shows any microbial growth, there is a very high
probability that this growth has resulted from the introduction of
a single microorganism in the medium and represents the pure
culture of that microorganism
.A. Yeasts-
Yeast, a type of fungi (plural for fungus), is found in many
places from nature, to research labs and even everyday kitchens
for baking. Yeast colonies generally look similar to bacterial
colonies. Some species, such as candida, can grow as white
patches with a glossy surface.
For example:

ROUND YEASTCOLONIES PINK YEASTCOLONIES

B. BACTERIA-

1.Bacillus subtilis 2.Proteus vulgaris 3.Staphyloccus

aures

Each distinct circular colony should represent an individual

bacterial cell or group that has divided repeatedly. Being kept
in one place, the resulting cells have accumulated to form a
visible patch. Most bacterial colonies appear white, cream, or
yellow in color, and fairly circular in shape.
2.COLONY FORMS-
The colony shape may be circular, filamentous, rhizidoidal,
punctiform(dot like),irregular, or spindle shape.
3.COLONY ELEVATION-This form is used to describe the
depth of the colony developed by microbes.A colony may be
flat(thin film over the agar surface), raised, convex or umbonate
or with papillae surface.
4.COLONY MARGINS-The margins may be entire,
undulate(wavy), crenate, dentate , lobate, rhizoidal or
filamentous.
5.OPTICAL DENSITY-The colony may be transparent or
transluscent (foggy in appearance) or opaque(not permitting light
to pass through it ) or irrediscent (rainbow colour).
6.COLOUR-Many microbes develop colonies which are
pigmented.Such coloured substances are either water soluble or
insoluble.The soluble pigments diffuse out in the medium.
7.COLONY ODOUR--Some microbe produce a characteristic
smell which sometimes helps in identifying the microbe.
PRESERVATION OF INDUSTRIALLY
IMPORTANT MICROORGANISMS
 The isolation of a suitable organism for a commercial
process may be a long and very expensive process.

 So it is essential that organism retain the desirable

characteristics that led to its selection.

 Thus the preservation techniques have been

developed to maintain cultures in a state of
‘suspended animation’ by storing.

 Storing either at reduced temperature or in

dehydrated form.
 Objectives:
 Retain the viability.
 Avoid contamination.
 Eliminate genetic change.

Repeated sub-culture may results in:

 Small probability of mutations and
degeneration.
 Risk of contamination may occur.
TRADITIONAL METHOD

 Culturing isolates on agar slants on

suitable media.
 Then subculture onto fresh slants at
regular intervals.
 After incubation at suitable temperature,
store the subculture in a refrigerator until
required, or until the next sub-culturing is
due.
DISADVANTAGES OF
TRADITIONAL METHOD
 Risk of contamination – in time, it is possible
for important isolates to be completely
overgrown by contaminants.
 Loss of viability – if subculturing is not carried
out at the required intervals and the cultures
are inadequately stored, sensitive isolates
may lose viability and be irrecoverable.
 Genetic drift and mutation – every subculture
carries a potential for genotypic and
phenotypic changes such as loss of virulence
and resistance factors, or reduced motility.
TYPE OF STORAGE / PRESERVATION

Storage at • Storage on agar slope

reduced • Storage under liquid
temperature nitrogen

• Dried Cultures
Storage in
dehydrated form • Use freeze-drying
technique
STORAGE AT REDUCED
TEMPERATURE
 Storage on agar slope

 Culture grown on agar

slope may be stored in
refrigerator (5º C) or a
freezer (-20º C, -80º C)

 Sub-culture at
approximately 6 month
interval or can be
extended to one year if
the slopes are covered
with sterile medicinal
grade mineral oil.
 Storage under liquid nitrogen.

 Cryogenic storage.
 The metabolic activities of
microorganisms may be reduced
by storage at very low temperature
(-150º C to – 196º C) using liquid
nitrogen storage containers.

 May use cryoprotectant beads for Liquid nitrogen

storage containers
long term microbial stock culture
storage; porous beads is designed
to allow spores and cell to attach
to their surface, providing some
degree of protection against
damage during freezing.
.
Cryoprotectant
beads
 Disadvantages :

 expensive

 Liquid Nitrogen evaporates and must be

replenished regularly

 If it is not done, or apparatus fail then the

consequences are the loss of the
collection
PROCEDURE FOR STORAGE UNDER
LIQUID NITROGEN
Growth culture to max stationery
phase in liquid medium

Separate cell from liquid medium

Resuspend the cell in cryoprotective

agent (10-15 % glycerol) to minimise
damage.

The suspension is dispensed into suitable

containers such as small screw capped
vials, which are then immersed in or
suspended in liquid nitrogen

Storage under liquid nitrogen

STORAGE IN DEHYDRATED FORM

 Dried Cultures technique :

 Dried soil cultures have been used widely for

culture preservation, particularly for sporulating
mycelial organism.
 Moist sterile soil may be inoculated with a culture
and incubated for several days for some growth
to occur & then allowed to dry at room
temperature for approx. 2 weeks.
 The dry soil preferably store in refrigerator.
Dried culture technique

 The technique has been used extensively for

storage of fungi & Actinomycetes

 Pridham et al. (1973) observed that of 1800

actinomycetes dried on soil about 50% were
viable after 20 years of storage.
Freeze drying

 It involves the freezing of a culture followed by its

drying under vacuum, which result in the
sublimation of cell water.
 The technique involves growing the culture to the
maximum stationary phase & resuspending the
cells in a protective medium such as milk, serum
or sodium glutamate.
 A few drop of a suspension are transferred to
ampules which is subjected to high vacuum until
sublimation complete, then ampoule is stored in
refrigerator.
 This cell remain viable for 10yrs or more.
PROCEDURE FOR FREEZE-DRYING
TECHNIQUE
A thick suspension of bacterial cells or fungal
spores prepared in a suitable suspending
medium, such as 10% skim milk, or a specific
lyophilisation buffer.

This suspension is then dispensed into small glass

vials or a specific container and frozen.

Once frozen, the plugged or loosely capped

vials are placed in the drying chamber of a
freeze dryer and dried under vacuum for 2 – 24
hours to remove water in the frozen state.
A secondary drying step may also be applied by
attaching the vials to the manifold of the freeze
dryer for a further 2 – 12 hours.

When drying is complete, the vial is sealed

and then stored in the dark at 8º C or less.
 Freeze-drying technique
 lyophilisation.

 method that can be used to suspend the

metabolism of bacterial and fungal
cultures and to stabilise the for long term
storage.

 standard practice for large commercial

and national culture collections.

 not suitable for smaller laboratories that

only need to maintain a small stock
culture collection, need for costly freeze-
drying equipment, the process is time
consuming and requires skilled staff to
ensure that no contamination occurs.
 Quality control & preservation of
stock cultures
 Whichever technique is used for the preservation it is
essential to be certain the quality of stock.

 Each batch of newly preserved cultures should be

routinely checked to ensure their quality.

 Procedure as per Lincoin (1960) a single colony to be

preserved is inoculated into shake flask & the typical
growth pattern is observed.

 In ampules at least 3% of the ampoules are

reconstituted and the culture assessed for the purity,
viability & Productivity.
Reference

 Perlman, D. and Kikuchi, M. (1977) culture

maintenance, Annual report of fermentation
process vol 1, Academic press, London

 P.F. STAMBURY, A.WHITAKER &S.J. HALL(1995)

SECOND EDITION, Culture Preservation
techniques, Elsevier Publication