Date:

Page No.
Experiment No. 1

Aim: Isolation and Purification (Pure Culture Techniques)

Principle: Inoculating needles and loops are used to aseptically transfer microorganisms from
broth, slant, or agar cultures to other media. Both may consist of handles, a shaft, and a turret,
which holds a nickel chromium or platinum wire. If the wire is straight, it is an inoculating
needle; if a loop is present, it is an inoculating loop. Before using either, the end of the wire must
be sterilized by passing it slowly through the tip of the flame. Microorganisms are transferred
from one culture medium to another by subculturing, using specific procedures and aseptic
technique. Since microorganisms are always present in the laboratory, if aseptic technique is not
followed, there is a good possibility that external contamination will result and will interfere with
the results. Proper aseptic technique also protects the laboratory worker from contamination with
the culture.

Usually the sample from which the microbes are to be isolated is first collected aseptically. The
sample may be from soil, water, food or medical or biological sources. Once the samples are
taken they are diluted through serial dilution and then are added to the media plates through
pipette in a definite amount. Then it is spread plate and incubated for overnight. After incubation
as per the choice a single colony is selected and streaked into a media plate for further isolation of
a particular type of colony forming bacteria.

Once discrete, well-separated colonies develop on the surface of the streak plate, selected ones
may be picked up with an inoculating needle and transferred to separate culture tubes. Where
possible, bacteria from the center of a colony are transferred, because the center is less likely to be
contaminated than the edges. Each tube now represents the growth of a single species of
microorganism and is called a pure or stock culture. One of the more important problems in a
microbiology laboratory is the maintenance of pure stock cultures over a long period. The best
way to preserve many stock cultures for long periods is through lyophilization (freeze-drying).
This eliminates the need for periodic transfers and reduces the chance of mutations occurring in
the stock culture.

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 Date:
Page No.
Experiment No. 1

Requirements:
a) Sample Source (Soil, Air, Water, Food etc.)
b) Nutrient Agar1
c) Inoculating loop
d) Spirit Lamp
e) Culture tube / Test tube with cotton plug
f) 1 mL pipettes
g) Distilled water
h) Test tube rack
i) Water bath
j) Incubator
k) 100 mL Beakers
l) 100 mL Conical flasks
m) Spreader
n) Petri plates
o) Saline (0.85 % NaCl)
Comments/Remarks:
1) It is a very routine work in microbiology.
2) The principle and the techniques involved were thoroughly discussed.
3) The experiment was demonstrated and the compound microscopy was understood.
.

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