EXPERIMENT No.

10
ISOLATION OF PURE CULTURE BY “THE POUR PLATE TECHNIQUE”.

APPARATUS/ MATERIALS REQUIRED:

 Loop wire
 Test tube
 Petri dishes
 Water bath
 Autoclave
 Bunsen burner
 Laminar flow cabinet
 Culture medium
 Bacterial culture as test sample

THEORATICAL BACKGROUND:
Inoculum
A small amount of substance containing bacteria from a pure culture which is used to start a
new culture or to infect an experimental animal.

PRINCIPLE:
Pour Plate Technique
Pour plate technique is usually the method of choice for counting the number of colony-
forming bacteria present in a liquid specimen. In this method, fixed amount of inoculum
(generally 1 ml) from a broth/ sample is placed at the center of sterile petri dish using a sterile
pipette.
In pour plate method, either 0.1 ml or 1.0 ml of dilution of bacterial suspension is introduced
into a petridish. The nutrient medium in which agar is kept liquid by holding it in a water bath at
about 50°C, is poured over the sample, which is then mixed into the medium by gentle agitation
of the plate.
or
Pour plate technique is used for the isolation of bacteria so the original sample is sufficiently
diluted in such a way that a small amount of microbial (bacterial) suspension is added to the
tubes containing molten agar. Then cool to about 45°C. The bacterial medium is then mixed
well pour into the sterilized petri-dish and then subject to incubation.
PROCEDURE:
1. First I prepared a bacterial suspension by taking a bacterial colony using a loop wire into
9 ml of water and mixed well until homogenous suspension was formed.
2. Then I prepared a series of dilutions by taking 1 ml of bacterial suspension into 9 ml of
distilled water to make dilution 10 ml marked as test tube no. 01 and similarly prepared
dilution no. 02 taken 1 ml from diluted test tube no. 01 and 9 ml of H 2O, and no. 03 from
test tube no. 02 and so on.
3. The pour-plate technique requires a serial dilution of the mixed culture by means of a
loop or pipette.
4. Molten agar cooled to 45°C, is poured into a petri dish containing a specified amount of
the diluted sample (inoculum).
5. Following the addition of the molten-then cooled agar, the cover is replaced, and the
plates gently rotated in a circular motion to achieve uniform distribution of
microorganisms.
6. This procedure is repeated for all dilutions to be plated.
7. Dilutions should be plated in duplicate for greater accuracy, incubated overnight (24 –
48 hrs.) and observed under microscope.
8. If the sample to be tested is an aqueous liquid, a known volume is simply in the base of
petridish and 10 – 15 ml of molten culture medium is poured onto it and quickly mixed by
gentle swirling.
9. If the sample is a solid that is soluble in water that solid would normally be dissolved,
but if it were insoluble then a suspension (uniformly dispersed) would be used.
RESULT/ INFERENCE:
I performed the pour plate method in the laboratory and isolated colonies. Then observed
these isolated colonies under microscope.
PRECAUTIONS:
1. Apparatus should be sterilized.
2. Distilled water should be used.
3. Agar in test tubes should remain in molten state at 45°C.
4. Experiment should be done in sterile environment especially inoculation in laminar flow
cabinet.