The learner will understand various methods like streak plate technique, pour plate technique, spread plate technique and micromanupulator technique

1. Pure culture technique
Mrs. V. A. Warad
Assistant Professor
Pharmaceutical Microbiology
PES, Modern College of Pharmacy, (for Ladies), Moshi, Pune
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2. 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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3. 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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4. Selective methods
1. Chemical methods of selection.
2. Physical methods of selection.
3. Biological methods of selection.
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5. 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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6. 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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7. 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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8. 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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9. 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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10. 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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11. 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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12. Advantages of streak plate technique:
1. Less material required
2. Less laborious
3. Surface colonies
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13. Streak plate technique - Four quadrant
method
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14. 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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15. 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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16. Pour plate technique
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17. 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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18. 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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19. Spread plate technique
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20. 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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