Lecture Notes (Theory and Practice of Sterilization)

Lecture Notes (M.Sc.
Biotechnology, IVth Semester)
Theory and Practice of Sterilization
1. Introduction:
It is an important technique required to get rid of contaminants present in the

sample.
The process can also be employed to eliminate the germs from the
laboratory space as well.
The practice of sterilization of the sample and media is a very crucial step in
the development of a good laboratory practice.
2. Sample Processing:
The sample after being collected from the site was rinsed thoroughly under running tap
water to remove the excess dirt and other suspended debris from its surface.
The rinsed sample was then dried with the help of filter paper to remove excess moisture
from the surface of sample.
The sample was then subjected to surface sterilization and finally, the above sample was
rinsed thoroughly three to four times with sterile water for further use.
3. Surface Sterilization:
The surface sterilization of the sample was done with 70% ethanol. Ethanol is a known to
be the powerful sterilizing agent.
The sample material was sterilized by subjecting it to ethanol solution only for few
seconds. Finally, the sterilized sample was then rinsed with sterilized distilled water.
The rinsed sample was then dried with the help of filter paper to remove excess moisture
from the surface of sample.
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4. Media Preparation
(a) Potato Dextrose Agar

Peel off potato and weight 250 g. cut it into small pieces and put in a beaker containing
500 ml distilled water.
Boil it for about 30 min and collect the extract. Add the required amount of glucose and
agar and mixed thoroughly.
Raise the volume to 1000 ml. The pH was adjusted to 5.6 ± 0.2 with dilute acid or alkali.
The media was transferred in 250 ml conical flasks, corked and sterilized by autoclaving
at 15 lb pressure (121˚C) for 15 min.
(b) Potato Dextrose Broth

Peel off potato and weight 250 g. cut it into small pieces and put in a beaker containing
500 ml distilled water.
Boil it for about 30 min and collect the extract. Add the required amount of glucose and
mixed thoroughly.
Raise the volume to 1000 ml. The pH was adjusted to 5.6 ± 0.2 with dilute acid or alkali.
The media was transferred in 250 ml conical flasks, corked and sterilized by autoclaving
at 15 lb pressure (121˚C) for 15 min.
5. Serial Dilution Method:
Collect soil samples at random, minimum five, from a field, mix thoroughly to make a
composite sample for microbiological analysis.
Label 9 ml sterile water blanks as 1,2,3,4,5,6 and 7 & sterile petri dishes as 10-2, 10-3,10-
4
,10-5,10-6,10-7,10-8,10-9,10-10 with a marker.
Add 1 g sample of finely pulverized, air dried soil into numbered 1 water blank to make
10-1.
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Vigorously shake the dilution on a magnetic shaker for 20-30 min to obtain uniform
suspension of microorganisms.
Transfer 1 ml of suspension from flask number 1 into water blank number 2 with sterile
pipette under aseptic condition to make 10-2 and shake it well for about 5 min.
Prepare another dilution 10-3 by pipetting 1 ml of the suspension into water blank number
3, using a fresh sterile pipette and shake it.
Make further dilution 10-4 to 10-7 by pipetting 1 ml suspension into additional water
blanks (4, 5, 6, 7) as prepared above.
Transfer 1 ml aliquots each from 10-2 dilution blank into 3 sterile Petri dishes,from 10-3
dilution blank to 6 sterile Petri dishes, from 10-4 to 9 sterile petri dishes, from 10-5 to 9
Petri dishes, from 10-6 to 6 petri dishes and form 10-7 to 3 petri dishes.
Add approximately 15 ml of potato dextrose agar to each petri dish. Upon solidification
of the media on the plates, the above prepared serial dilutions were homogenized
thoroughly and 0.1 ml from the same was dispensed onto the respective petri dish and
further all the plates were incubated in an inverted position at 28-300C for 48 hours.
Serial dilution method
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6. Warcup Method:
Collect the soil sample at random in sterilized polythene bags.
Add approximately 15 ml of potato dextrose agar to each petri dish. Finally, allow the
petri plates to solidify.
Add 0.005 to 0.15 g (approximately) air dried soil each in sterile petri dishes with the
help of a sterilized cooled loop or transfer needle.
Dispense soil particles throughout the medium by gentle rotation of the petri dishes.
Incubate all the inoculated plates in incubator at 280C for 48 hours.
7. Pure Culture and Preservation
The potato dextrose agar slants were prepared and labeled with a wax pencil.
Sterilize the inoculating needle by holding it in the hottest portion of the bunsen burner
flame. Flame until the entire wire becomes red hot and allow the needle to cool for a few
seconds.
Touch the tip of the needle to the surface of selected discrete fungal colonies to be
preserved by lifting the lid of the agar mother plate at 450C.
Now, inoculate the potato dextrose agar slant towards the center over the hardened agar
surface with the help of inoculating needle and recap the cotton plug of the slant.
Inoculate fungi (BKS-W1, BKS-W2, BKS-W3, BKS-W4, BKS-B1, BKS-B2, BKS -S1,
BKS-S2, BKS-S3 and BKS-S4) and incubate all the fungal cultures in the incubator at
28-300C for 48 h.
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8. References:
K. Wilson and J. Walker: Principle and Techniques of Biotechnology and Molecular
Biotechnology.
Upadhya and Upadhya: Biophysical Chemistry.
David, L. Nelson and Michael, M. Cox: Lehniger: Principal of Biochemistry. 4th Edition.
W.H. Freeman and Company, New York.
Anthony J.F. Griffiths, William M. Gelbart, Richard C. Lewontin and Jeffrey. H. Miller;
Modern Genetic Analysis; Publisher: W. H. Freeman
Ralf Portner; Animal cell biotechnology: methods and protocols; Humana Press

Lecture Notes (Theory and Practice of Sterilization)

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Lecture Notes (Theory and Practice of Sterilization)

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Lecture Notes (M.Sc.

Biotechnology, IVth Semester)

Theory and Practice of Sterilization

It is an important technique required to get rid of contaminants present in the

(a) Potato Dextrose Agar

(b) Potato Dextrose Broth

5. Serial Dilution Method:

Serial dilution method

Collect the soil sample at random in sterilized polythene bags.

Incubate all the inoculated plates in incubator at 280C for 48 hours.

7. Pure Culture and Preservation

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