Preparation
of agar media
To describe the method
of preparing media plates for carrying out different
microbiological tests.
2. Scope:-
This procedure applies to
preparation of Microbiological media in aseptic conditions in Microbiological
Lab.
3. HSE Statement:-
Face mask and gloves should be used.
4.
Responsibilities:-
i)
Manager Quality Control is responsible
to ensure that procedure & formats are followed entirely as approved.
ii)
Microbiologist is responsible
to perform the test.
5.
Materials:-
5.1 Tryptic
Soya agar (prepared and sterilized) in 500 ml conical flask with cotton plug
covered with butter paper kept in water
bath at 48-50°C.
OR
5.2 Other media such as Sabouraud Dextrose Agar, Eosin Methylene
Blue, MacConkey Agar, Antibiotic Media etc. prepared and sterilized, kept in
water bath at 48-50°C.
5.3 Petri-dishes sterilized at 200°C for at least two hours.
5.4 LFC kept ‘on’ for 30 minutes prior to prepare plates.
5.5 Disinfectant.
5.6 Sterilized cotton duster.
5.7 Incubator for pre-incubation.
6.0 Definitions:
6.1 Media:
Media is the nutritive source for the
cultivation of microorganisms. The media usually prepare from protein by acid
or enzymatic digestion. Medias are usually available in dehydrated form. We
have only to mix required measured quantity in distilled water. Follow
instructions for preparation as mentioned on the label of media container.
6.2 Sterilization:
It is the process of killing or
removing microorganisms. Media is sterilized by autoclaving at
1210C for 15 minutes at a pressure
of 15 PSI.
6.3
Agar:-
Complex sulfated polysaccharide derived from certain marine algae(normally
red algae) that is a
gelling agent for solid or semisolid
microbiological media. Agar consists of about 70% agarose and 30% agaropectin.
Agars can be melted at temperature above 100ºC; gelling temperature is 40-50ºC.
Agar is used for performing viable counts
7. Flow Chart:-
8.0
Description:-
8.1 Procedure:-
8.1.1
Prepare and sterilize agar media as per SOP.
8.1.2 Transfer all the required materials
to LFC bench taking all necessary microbiological precautions.
8.1.3 Remove plates from SS box on to the
bench and let them cool.
8.1.4 After cooling of the plates obtain
Media from water bath and mop outer surface with duster wet
with disinfectant.
8.1.5
Carefully and slowly remove the
cotton plug first uncovering the butter paper on it.
8.1.6
Gently heat the mouth of the flask
or bottle containing media for decontamination of outer
surface.
8.1.7
Now carefully take one plate and
half open it by one hand and pour approximately 20 ml of agar media and close
the plate. Care should be taken to not to lean over the open plate or media nor
take your hand or arm over opened plate.
8.1.8
Similarly pour all the plates as
soon as possible as agar media starts solidifying.
8.1.9
Pour media in such a way to
inhibit the bubble formation in the agar media.
8.1.10 Let plates as such for at least 20 minutes
for solidification.
8.1.11 After solidification label each plate
with abbreviation of media name, date of preparation.
8.1.12 Gather all the plates and transfer in
a container into the appropriate incubator for pre-incubation
in inverted position for at
least 24-48 hours.
8.1.13 Before conducting any test observe the plates carefully for the
evidence of any growth
8.2 Precautions:-
8.2.1 Store the plate in refrigerator if
testing is delayed.
8.2.2 Plates should be used as early as
possible as media starts drying.
8.2.3 See that media is not overheated.
8.2.4 Check that the sterilization cycles
are satisfactory.
8.2.5 Check the pH of the media after
sterilization of the media.
8.2.6 Check the expiry date of dehydrated
media.
8.2.7 Check the washing process of the
glassware.
8.3 Growth Promotion Test of
Medias (Positive Control):
Growth promotion test of each lot of media
prepared should be performed as per SOP.
9. Forms and Records:-
9.1 Sterilization and Preincubation record of
Medias.
9.2 Log of Sterilization
Cycle.
9.3 Growth Promotion test.
10. References:-
USP 35
11. Distributions:-
This SOP has to be distributed in below mentioned
Departments:-
|
Sr. NO
|
Distributed to
|
Received
(Current)
|
Returned
(Obsolete)
|
|
1
|
Quality Control
Department
|
|
|
|
2
|
Quality Management
Department
|
|
|
12.
Revision History:-
N/A