Pure Culture Techniques and Aseptic Techniques

Vijayanagara Sri Krishnadevaraya
University Bellary.
DEPT : BIOTECHNOLOGY
UNIT-1 STERILIZATION:
PURE CULTURES AND ASEPTIC
TECHNIQUES
Guest faculty : Gulappagouda

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) ROBERT KOCH (1843-1912)

“Father of Microbiology” “Father of Practical
Bacteriology”.
 Agar was dis

Japan
 Agar was first used in microbiology in 1882 by the German
microbiologist Walther Hesse ,an assistant working in Robert
Koch”s 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 or, their progeny , to obtain pure culture
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 in various aspect .
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BIOLOGICAL SAFETY CABINET
Biological safety cabinet i.e. Laminar

hood
HEPA:(High– efficiency particulate
air filters ) filters the exhaust air
 Obtaining Pure Cultures
•Cultures composed of cells arising from a single

progenitor
•Progenitor is termed a CFU
•Aseptic technique prevents contamination of
sterile substances or objects
•Common isolation techniques
 Streak plates method
 Spread plate method
 Serial dilution method
 Pour plates method
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 45 C 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 and Preservation
of Pure Cultures
 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 include preservation of pure cultures are following
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
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 -196°C) helps survival
of pure cultures for long storage times. In this method, the microorganisms
of culture are rapidly frozen in liquid nitrogen at -196°C 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 (-
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.

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Vijayanagara Sri Krishnadevaraya

Guest faculty : Gulappagouda

o Pure culture spontaneous mutation rate is low

LOUIS PASTEUR (1822-1895) ROBERT KOCH (1843-1912)

 Agar was dis

 Sterilization of growth media and glassware

Something either sterile or it is not ; it is probability

 The most common method of

Biological safety cabinet i.e. Laminar

•Cultures composed of cells arising from a single

 Solid agar is prepared by adding agar, a complex

1.The microorganisms look alike microscopically and stain in the

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