Pure Culture Techniques

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Group 3 Submitted to:

2nd year 3rd Omer Ali Hassen
semester. MSc Medical
Microbiology
Introduction
 Pure culture is a laboratory technique in
which technique grow a particular or single
micro-organism.
 A pure culture may be isolated by use of
special media with a specific chemical or

physical agent that allow the selection of one
organism over another.
 Both procedures (spread plating and streak
plating) require understanding of the aseptic

technique.
Cont…
 However, the term usually applied to any
technique designed to keep unwanted
microorganisms from contaminating sterile
material.
 The best way to obtain a pure culture is to
start with a single bacterial cell.

 This cell divides quickly, and may produce
millions of cells within 24 hours.

Cont…
 Contaminants = other microorganisms present in the sample 
 Isolated colois= a population of millions of cells that are identical
and are descendent from a single founder cell

 Stock Culture = a culture that already contains cells.
 It is used a source of cells from which to inoculate new cultures.
 culture medium: rich/selective
 growth inhibitors
 liquid/solid
 temperature
 source of energy
 sources of carbon, nitrogen, ...

 Aseptic technique:
 sterilization of medium and equipment

 proper handling
Procedure for aseptic transfer.
 1. Flame the loop.
 2.Without setting the loop down, open the
first culture tube and flame the mouth. Do

not set the cap on the bench. The cap should
be held in the same hand as the loop.
 3. Insert the loop into the culture medium,
then withdraw it.

 4. Flame the mouth of the first culture tube
again, and replace the cap.

Remember
 About the Bacteria
 1. Are everywhere!
 2. On every surface of the body
 3. Including digestive tract
 4. Harmless
 5. Beneficial
 6. Pathogenic
 7. Absorb nutrients and release toxins that damage cells
and tissues.
 8. Bacterial toxins can cause disease even when bacteria
are destroyed
 9. Bacteria are Prokaryotes
Bacteria needs other things to grow :-
 Nutrition
 Temperature
 pH
 Osmolarity
Necessary equipments
why we used sterile equipment or
materials:-
Cont…
 Basically to prevent the contamination of
bacterial sample from environmental
microorganisms.
 1) Autoclave the media (heat to 121 °C and 20
psi)
 2) Use sterile petri dishes, pipette tips, plastic
tubes in a laminar hood sprayed with ethanol.
3) Use common sense!
 Use gloves at all times
 Don’t eat/touch other things/attend to your
cell phones while handling bacterial cultures

Methods of culture
 Indications for culture:-
 Isolate bacteria in pure cultures.
 Demonstrate their properties.
 Obtain sufficient growth for preparation of
antigens & for other tests.

 Typing bacterial isolates.
 Antibiotic sensitivity.
 Estimate viable counts.
 Maintain stock cultures.
Methods are used for pure culture of
micro-organisms
 Pour plate method
 Streak plate method
 Spread plate method or picking method
 Serial dilution method
 Single cell isolation method
Streak plate
 “Streaking” bacteria on media plates
Pour plate
 Autoclaved media (nutrients + agar) is poured on petri
dishes. Agar is a matrix that solidifies on cooling. Freshly
poured After coolingPouring
Picking method
 This method used to isolate colony can be picked and
restreaked on another nutrient agar plate to get a pure
culture.

Picking a colony and inoculating liquid culture.
Put the picked

colony in liquid
media to grow
bacteria
Serial dilution method
 This method commonly used to obtain pure
culture of those microorganisms that not yet
been successfully cultivated on solid media and
grow only in liquid media.
 A microorganism that predominates in a mixed
cultures can be isolated in pure form by a series
of dilutions. The inoculum is subjected to serial
dilution in a sterile liquid media and a large
number of tubes of sterile liquid medium are
inoculated with aliquots of each successive
dilution.
Cont…
 If we take out 1 ml of this media and mix it
with 9 ml of fresh sterile liquid media , we
would then have 100 micro-organisms in 10
ml or 10 micro-organisms/ml. If we add 1 ml
of this suspension to another 9 ml. of fresh
sterile liquid media each ml would now
contain a single micro-organism.
Cont…
Single cell isolation method
 A single cell of the required kind is picked out
by this method from the mixed culture and is
permitted to grow.
 Capillary pipette method:- In this method
several small drops of a suitably diluted culture

medium are put on a sterile glass coverslip by a
sterile pipette drawn to a capillary. Then each
drop under the microscope until one finds such
a drop which contains only one micro-
organism. This drop is removed with a sterile
capillary pipette to fresh medium.
Cont…

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special media with a specific chemical or

plating) require understanding of the aseptic

start with a single bacterial cell.

millions of cells within 24 hours.

and are descendent from a single founder cell

 sources of carbon, nitrogen, ...

 sterilization of medium and equipment

first culture tube and flame the mouth. Do

then withdraw it.

again, and replace the cap.

cell phones while handling bacterial cultures

antigens & for other tests.

Put the picked

several small drops of a suitably diluted culture

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