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Pure culture technique
Mrs. V. A. Warad
Assistant Professor
Pharmaceutical Microbiology
PES, Modern College of Pharmacy, (for Ladies), Moshi, Pune
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Microbiology/ Pure culture technique/VAW

Introduction
Natural microbial populations are mixed cultures.
Contain large number of organisms – single sneeze
– 104 – 105 bacteria, 1gm feces – 1011 bacteria, 1gm
fertile soil – billions of organisms.
To study properties and to prepare products from
microorganisms, they should be in pure culture.
Definition: Mass of growth of cells containing only
one type of cells is called pure culture.
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Selective methods
Desired microorganism may be less in number in
mixed population or it may be slow growing.
By using selective methods number of desired
microorganism is increased relatively.
This increases chance of isolation of Desired
microorganism.
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Selective methods
1. Chemical methods of selection.
2. Physical methods of selection.
3. Biological methods of selection.
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Chemical methods of selection
1) Use of special carbon or nitrogen source -
enrichment selection -
Use of enrichment medium containing single
source of carbon – α conidendrin – isolation of
bacteria which can degrade α conidendrin
Use of enrichment medium containing single
source of nitrogen- N2 gas - Isolation of nitrogen
fixing bacteria.
2)Use of dilute media – Caulobacter spp. (0.01%
peptone)
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3)Use of inhibitory or toxic chemicals
Isolation of Gram negative bacteria - addition of
bile salts or crystal violate – MacConkys agar
Campylobacter jejuni – vancomycin, polymyxin,
trimethoprim added to medium to inhibit other
interfering intestinal bacteria
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Physical methods of selection
1) Heat treatment – endospore producing bacteria –
80oC – 10 min
2)Incubation temperature –
Thermophiles: 55oC
Psychrophiles isolation: 0-5oC
3)pH of medium –
Isolation of lactobacilli – pH - 5.35 with acetate
buffer to maintain pH
 Isolation of V. cholera – pH – 8.5
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4) Cell size and motility –
Isolation of Treponema spp. from oral cavity
Membrane filter with pore size – 0.15 µm sample from
oral cavity is placed on surface of agar.
Treponema as small in size pass through filter on
underlying agar medium.
They have ability to move swim through solid surface
of agar and grow within the agar.
Other bacteria from oral cavity – neither pass through
filter nor swim trough agar
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Biological methods of selection
Use of susceptible laboratory animals.
Isolation of S. pneumoniae from sputum sample -
by injecting sputum sample of patient in mice. After
mice suffers from pneumonia, S. pneumoniae can
be isolated from blood of mice.
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Methods of isolating pure culture
1) The streak plate technique
2)Pour plate technique
3) Spread plate technique
4) Micromanipulator technique
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1) The streak plate technique
Dilution of bacteria on solid nutrient media.
It is done by streaking the culture over agar surface
Pattern of streaking - Four quadrant method.
Bacteria get separated from each other by sufficient distance.
One organism produces one colony and is considered as pure
culture.
Colonies developed after streak plate method may require repeated
streaking for isolation of bacteria from mixed culture
Roll tube technique – for isolation of anaerobic bacteria - diagram,
description
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Advantages of streak plate technique:
1. Less material required
2. Less laborious
3. Surface colonies
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Streak plate technique - Four quadrant
method
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Roll tube technique
Modification of streak plate technique for isolation of
anaerobic bacteria.
Stoppered anaerobic culture tubes whose inner walls are
coated with pre-reduced media are used. Tubes are filled
with oxygen free nitrogen.
Stopper is removed to keep them, anaerobic are flushed
oxygen free CO2 from gas cannula.
Inoculation done with transfer loop, held against agar
surface as tube being rotated by a motor.
Inoculation is started at bottom and drawing the loop
gradually upward, the inoculum becomes thinned and well
isolated colonies are can be obtained
Tube is re-stoppered and incubated
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2)Pour plate technique
Serial dilutions of mixed culture of sample are made.
Diluted sample is added to tube of sterile sufficiently
cooled agar medium and mixed. --- diagram
Mixture is poured in Petri plate.
After incubation plates are observed for isolated
colonies.
Advantage - Method can be used to determine
number of bacteria in sample - Quantitative method.
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Pour plate technique
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Disadvantage –
1. Laborious
2. Time consuming
3. More material required
4. Some of the colonies are submerged, difficult
to pickup and study.
5. Exposure of organism to 450C - can not be
used to isolate psychrophiles
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3) Spread plate technique
Serial dilutions of mixed culture of sample are made.
Sample of dilution is added onto surface of agar plate.
Sample is evenly spread on surface by sterile glass
spreader.
Advantage –
1. Surface colonies
2. No exposure to higher temperature of agar
3. Quantitative method.
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Spread plate technique
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4) Micromanipulator technique
Uses instrument – micromanipulator.
Uses microscope in conjunction with micromanipulator.
Micromanipulator allows controlled movement of
micropipette
Single cell is picked up with micropipette while observing
cells through microscope.
Single cell is added to nutrient medium. After incubation
pure culture is developed from single cell.
This culture is a clone.
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Pure culture techniques

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Pure culture techniques