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Pure Culture

Techniques

Natural microbial
populations
Microbial population in our environment large &
complex

Many different microbial species normally


inhabit various parts of our body ---such as oral
cavity, skin, intestinal tract

These microbes present extremely large


numbers e.g. single sneeze may disperse 10,000
to 1,00,000 bacteria. 1 g of human/animal
excreta may contain 1011 bacteria

Our environment- air, soil, water consist of


mixed populations of bacteria plus other
microbes

Fertile garden soil microorganism several


billion per gram. It include many species of
bacteria, fungi, algae and protozoa

Pure Culture Technique


Culture : Act of cultivating microorganisms or the
microorganisms that are cultivated

Mixed culture : more than one microorganism


Pure culture : containing a single species of organism.
A pure culture is usually derived from a mixed culture (one
containing many species) by transferring a small sample
into new, sterile growth medium in such a manner as to
disperse the individual cells across the medium surface or
by thinning the sample many times before inoculating the
new medium.

Why important?
Pure cultures are important in microbiology for the following reasons

Once purified, the isolated species can then be cultivated with the
knowledge that only the desired microorganism is being grown.

A pure culture can be correctly identified for accurate studying and


testing, and diagnosis in a clinical environment.

Testing/experimenting with a pure culture ensures that the same


results can be achieved regardless of how many time the test is
repeated.

o Pure culture spontaneous mutation rate is low


o Pure culture clone is 99.999% identical

HISTORY

LOUIS PASTEUR (1822-1895)


Father of Microbiology

ROBERT KOCH (1843-1912)


Father of Practical Bacteriology.

A GREAT DISCOVERY
Agar was discovered around 1658 by Minoya Tarozaemon in
Japan
Agar was first used in microbiology in 1882 by the German
microbiologist Walther Hesse ,an assistant working in Robert
Kochs laboratory

Pure culture technique consist of three


interrelated techniques
Sterilization of growth media and glassware
Introducing desired cells into sterile growth media or
removing samples from pure cultures without
accidentally introducing contaminating microbes ,and
Isolating singles cells to obtain pure culture

STERILIZATION

BUT !

Something either sterile or it is not ; it is probability

Sterilization continue

The most common method of sterilizing is


autoclaving
Heat-sensitive solutions are sterilized by
filtration
Glassware is sterilized by dry heat
Bunsen burner flames help to prevent
contamination during transfer into or out of
containers
Some pathogenic microbes require special
containment facilities .
BSC ( biological safety cabinet) is important

STERLIZATION

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m

BIOLOGICAL SAFETY CABINET

Biological safety cabinet i.e. Laminar


hood
HEPA:(High efficiency particulate
air filters ) filters the exhaust air

Culture techniques
Selective method:
This method favour the growth of desired
species & discourage/kill the other organism
present in the mixed culture

Chemical, physical and biological methods are


used in order to achieve selection of particular
kind of bacterium

(A) Chemical methods of


selection
Use of special carbon or nitrogen source to
enrich the culture medium

(A) Chemical methods of


selection
(a) Use of dilute media
Certain bacteria are capable of growing with low
level of carbon or nitrogen source e.g. broth
containing 0.01% peptone

(A) Chemical methods of


selection
(b)Use of inhibitory or toxic chemicals
Addition of certain chemicals such as dyes, bile
salts, salts of heavy metals or antibiotics to
culture medium for selection of particular kind of
bacteria

(B)Physical methods of
selection
(a) Heat treatment
To select for endospore forming bacteria, mixed
culture can be heated to 80 C for 10 min before
incubate to culture media. Vegetative cells will
be killed, but endosperms will survive and
subsequently germinate and grow

(B)Physical methods of
selection
(b) Incubation temperature
To select psychrophilic or psychrotropic bacteria,
cultures are incubated at low temperature e.g: 0
- 5 C

For selection of thermophiles incubation


temperature may be 55 C

(B)Physical methods of
selection
(c) pH of the medium
To select acid tolerant bacteria low pH medium
can be used

(B)Physical methods of
selection
(d) Cell size and motility
Membrane filter having pore size of 0.15m is
placed on the surface of an agar plate. During
inoculation, mixed culture placed on the surface
of the filter

(C) Biological methods of


selection
Selection of bacteria naturally exist
E.g: high salt concentration water bodies, root
plants, natural infection of humans and animals,
blood samples etc.

Pure Cultures
Particular bacterial species comprises high
proportion in mixed population

It can be isolated in pure culture


The descents of a single isolation in pure culture
comprise a strain

Strain is usually made up of a succession of


cultures and is derived from a single colony

If the strain is derived from single parent cell, it


is termed as clone

Each strain is designated by


Identifying number and its history recorded
Source from which isolation was made
Name of the person who made the isolation
Date of isolation
The culture collection in which the strain is
maintained
From which it can be obtained for study

Methods for isolating


pure culture techniques
Streak-plate technique
By means of transfer loop, portion of the mixed
culture is placed on the surface of an agar
medium & streaked across the surface

This technique thins out the bacteria on the


agar surface, so that individual bacteria are
separated from each other

Methods for isolating


pure culture techniques
Roll tube technique
For stringent anaerobes

Methods for isolating


pure culture techniques
Pour plate and Spread plate
technique

Isolation of pure culture continue.

Solid agar is prepared by adding agar, a complex


polysaccharide derived from marine algae , to
liquid media
The agar is dissolve at high temperature of the
autoclaved and remains liquid as it cool down to
a temperature about 455C and below to this
temperature gets solidify.
Solid media pour in petri plates
To obtain pure cultures microbes are normally
streak onto solid media
Inoculating loop are use for streaking
Then the plate is incubate at desired
temperature . Then after some times ( generally
24 hours) colonies are visible wherever a
microbial cell capable of growth on particular
medium was deposited on the agar surface.
Then colony observation and selection of pure
colony are follow

Different media are required for


different microbes
Different cell have widely varying
requirement for their growth.
Media also called rich media or
complex media
Media is mixture of many different organic
compounds , including all of the amino
acids, purine , pyrimidine , vitamins
(enzyme cofactors) , etc.
Rich media generally contain growth
factors , nutrition , and other supporting
compound for growth of microorganism
Minimal media ( sometimes mineral
media) is another media contains
mineral salts , such as sulfur , nitrogen ,
and phosphorus

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Proof of Purity of Cultures


Assuming that one has isolated a pure culture, how does one
establish that it is pure?
A pure culture is one in which the cells are all of one kind, i.e.,
demonstrate "likeness". Hence, the proof of purity of cultures consists
of demonstrating the "likeness" of microorganisms in the culture. It is
based on certain criteria as follows:
1.The microorganisms look alike microscopically and stain in the
same fashion.
2. When plated, all the colonies formed look alike.
3. Streaks, stabs, etc. are uniform.
4. Several isolated colonies perform identically, i.e., ferment the same
sugars, and so on.

Maintenance & Preservation of


pure cultures
Large collection of strains frequently referred as
stock-culture collection

Once a microorganism has been isolated and grown


in pure culture, it becomes necessary to maintain
the viability and purity of the microorganism by
keeping the pure cultures free from contamination.

Since repeated sub culturing is time consuming, it


becomes difficult to maintain a large number of
pure cultures successfully for a long time.

Methods of maintenance and


preservation
Refrigeration: Strains can be maintained
periodically by preparing fresh stock culture
from previous stock culture. Pure cultures can be
successfully stored at 0-4C 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

Paraffin Method
This is a simple and most economical method of maintaining pure cultures
of bacteria and fungi. In this method, sterile liquid paraffin in poured over the
slant (slope) of culture and stored 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 is preserved for several years.
Cryopreservation
Cryopreservation (i.e., freezing in liquid nitrogen at -196C) helps survival of
pure cultures for long storage times. In this method, the microorganisms of
culture are rapidly frozen in liquid nitrogen at -196C in the presence of
stabilizing agents such as glycerol that prevent the formation of ice crystals
and promote cell survival.
Lyophilization (Freeze-Drying)
In this method, the culture is rapidly frozen at a very low temperature (70C) 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 4C in refrigerators.

Culture collections
In France Institute of Pasteur in Paris
England National Collection of Type Cultures in London
Federal Republic of Germany Deutsche Sammlung Von
Mikroorganismen in Darmstadt

Japan Institute for Fermentation on Osaka


US Americal Type Culture Collection (ATCC) located in
Maryland

India Microbial Type Culture Collection MTCC


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