sop for Media Growth Promotion Test and various Microbiological test

1.0 OBJECTIVE
1.1 To lay down a procedure for Culture Suspension (Inoculum) preparation to determine 10 to 100 cfu/ml by Serial Dilution Method.

2.0 SCOPE
2.2 This procedure is applicable to the procedure of Culture Suspension (Inoculum) preparation of 10-100 cfu/ml which is used for

Media Growth Promotion Test and various Microbiological test method validation studies in Microbiology lab

3.0 RESPONSIBILITY
3.3 Microbiologist QC

4.0 ACCOUNTABILITY
4.1 Q. C. Manager

5.0 PROCEDURE
5.1 PRECAUTIONS

5.1.1 Always use freshly prepared culture slant for initial suspension which should not be more than 48 hours old.
5.1.2 Always use properly cleaned and sterilized glass containers.
5.1.3 Vortex each dilution before transferring the suspension to next tube of series.
5.1.4 Suspension of culture should be homogenous.
5.1.5 Check calibration status of colony counter before use.
5.1.6 Media should not have the temperature more than 45 oC prior adding to the plate containing dilution.
5.1.7 It is extremely important to make each serial transfer immediately after vortex.

5.2 MATERIALS REQUIRED
5.2.1 Media, Dilution tubes containing sterilized 09 ml normal saline, Micropipette, Sterilized Pipette 10 ml, sterilized

microtips (100-1000µl), Inoculation Loop, Sterilized Petri plates, Sterilized Normal saline, Freshly prepared slant not more than 48hrs old.

5.3 EQUIPMENTS REQUIRED
5.3.1 Vortex Mixer, Colony counter, BOD Incubator, Refrigerator.

5.4 PROCEDURE
5.4.1 Prepare the media.
5.4.2 Sterilize the media at 121oC for 20 minutes in autoclave as per SOP.
5.4.3 Take a freshly prepared culture slant which should not be more than 48 hour old.
5.4.4 Add 10 ml of sterile normal saline to the tube and allow to stand for 10 minutes.
5.4.5 Homogenize the tube by gentle shaking to make a homogenous suspension.
5.4.6 Vortex the tube containing initial suspension for not less than one minute.
5.4.7 Transfer 1ml aliquot from initial suspension to dilution tube containing 9 ml of sterile normal saline

and mark as 10-1 and vortex the dilution tubes for at least 10 seconds (1:10 dilution).
5.4.8 Transfer 1ml from 10-1 to second dilution tube containing 9 ml of sterile normal saline & mark as 10-2.
5.4.9 Repeat the step 5.4.8 eight times to make a dilution series up to 10-5.4

( 1ml from second to third, third to fourth, fourth to fifth so on respectively and mark them as 10-3 , 10-4 , 10-5 …….respectively).
5.4.10Vortex each tube before transferring the dilution to next tube for 10 seconds.
5.4.11Perform the serial dilutions in the same manner ranging from 10-1 to 10-5.4.
5.4.12Pipette 1 ml from 10-4 tube and add to two sterilized Petri plate. Do this for dilutions up to dilution 10-5.4 and mark the Petri plates as per dilution.
5.4.13Pour approximately 20 ml of sterilized melted Soyabean Casein Digest Agar cooled to 45 oC to 50 oC into Petri

plates containing respective dilution for bacterial cultures, Sabouraud Dextrose Agar to all the yeast or molds cultures and

Columbia Agar for Clostridium sporogeneses into the plates.
5.4.14 Swirl to assure adequate mixing and allow the agar to solidify.
5.4.15 Invert the plate and Incubate the plates at 32.5 ± 2.5°C for 48 hrs for bacterial cultures and 22.5± 2.5C for 3 to 5 days

for fungi & for Clostridium sporogeneses in anaerobic jar at 32.5 ± 2.5 ° C for 72 hrs.
5.4.16 Do not discard the dilutions after plating the cultures. Preserve the all dilutions in refrigerator at temperature 2-8°C.
5.4.17 After incubation count the plates and calculate the population by multiplying the count with
dilution factor.
5.4.18 Select the previous dilution of which is having cells between 10 – 100 Cfu/ml.
5.4.19Transfer 1ml from this dilution to the bottle containing 100ml sterile normal saline.
5.4.20 Store the culture suspension for 01 to 10 Days at 2-8°C.
5.4.21 Frequency of culture suspension preparation of bacterial and fungal cultures is once in 10 days.
5.4.22 Label the culture suspension with following details:-

Abbreviation used	Full form of abbreviation used
SOP	Standard Operating Procedure
IPA	Iso Propyl Alcohol
QA	Quality assurance
SCM	Soyabean Casein Digest Medium
IP	Indian Pharmacopeia
HLAF	Horizontal Laminar Air Flow
Cfu	Colony Forming Unit

sop for Media Growth Promotion Test and various Microbiological test

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