Anaerobic Culture Suspension preparation and maintenance SOP

Objective:

This procedure is for Maintenance & preparation of culture suspension for anaerobic microorganisms in Microbiology Department.  it includes maintenance of culture & preparation of culture suspension as per Culture Dilution Method.

PROCEDURE:

Maintenance:

Prepare Nutrient Agar as follows 

he composition can be adjusted to obtain optimal performance, for 1 liter of medium:

Composition of nutrient agar
Ingredients	Gram / liter
Tryptone	5,0g
Meat extract	1,0g
Yeast extract	2,0 g
Sodium chloride	5,0 g
bacteriological Agar	12,0 g

Preparation of nutrient agar (Dehydrated medium from himedia) 

Suspend 28.0 grams in 1 litre of purified/distilled or deionized water.

Heat to a boil to completely dissolve the medium.

Sterilize by autoclaving at a pressure of 15 lb (121°C) for 15 minutes.

After autoclaving, cool to 45-50°C.

Pour nutritious agar into Petri dishes (until the agar is solidified).

Keep boxes in the refrigerator at 2-8°C.

Pour 5 mL of Nutrient Agar in clean 18 mm rimless test tube.

Plug the tube with cotton and wrap the cotton plug with butter paper and label the tubes for type of media, autoclave lot no and date of sterilization.

Steam sterilize the media slants, as per the validated autoclave cycle.

Place the media tubes under laminar airflow (LAF) at approximately 30º from the surface.

Allow the media to solidify.

After the solidification of the media slants, 30 to 35ºC for 48 hours for checking of any contamination

Streak the surface of the Nutrient Agar with Culture and incubate under Anaerobic Condition at 30-350C for 72 hours

Observe for the growth and perform gram staining.

Clostridium Sporogenes shall be Maintained as per Annexure

Preparation of Culture Suspension:

Prepare Reinforced Medium and Columbia Agar in a conical flask.

Reconstitute the media with the required volume of purified water.

Boil the media in the water bath to uniformly dissolve the medias.

Dispense 15 ml of the media in a clean 18 mm rimless test tube.

Plug the tubes with cotton plug and wrap the cotton plug of the tube with butter paper and label the tubes for type of media, autoclave lot no and date of sterilization

Similarly prepare normal saline solution (0.9% w/v sodium chloride solution) for harvesting.

Transfer 9 ml of the normal saline solution in test tubes and label the tubes with autoclave lot no and date of sterilization.

Plug the test tube with cotton plug and wrap the plug with Butter paper.

Steam sterilize normal saline solution tubes as per the validated autoclave cycle.

After steam sterilization remove the normal saline solution tubes and peptone Solution tubes from the autoclave.

Transfer the culture slant and the dilution tubes to microbial limit test room.

Prepare culture suspension by washing and scraping the surface of the slant by means of sterile inoculating loop in 10 ml of 0.9% saline Clostridium Sporogenes

Transfer the culture suspension in a sterile test tube.

Collect the suspension in a sterile test tube.

Vortex the culture suspension to obtain a uniform suspension.

Carry out serial dilution so as to obtain a culture suspension of 10-100 cfu/ml by following the steps given below.

• Transfer 1 ml of the suspension to 9 ml sterile normal saline solution – 10¹
• 1 ml of 10¹ Dilution to 9 ml sterile normal saline solution – 10²
• 1 ml of 10² Dilution to 9 ml sterile normal saline solution – 10³
• 1 ml of 10³ Dilution to 9 ml sterile normal saline solution – 10⁴
• 1 ml of 10⁴ Dilution to 9 ml sterile normal saline solution – 10⁵
• 1 ml of 10⁵ Dilution to 9 ml sterile normal saline solution – 10⁶
• 1 ml of 10⁵ Dilution to 9 ml sterile normal saline solution – 10⁷

Note : For preparation of Clostridium sporogenes microbial dilution add 1mL of immersion oil on the surface of the normal saline solution.

Pipette out 1 ml of the each inoculum from last three dilution tubes into sterile Petri plate in duplicate and perform Pour Plate Technique.

Incubate the Columbia Agar plates of Clostridium sporegenes anaerobically at 30 to 35ºC for 72 hours.

Till the observation of the microbial counts preserve all the dilution tubes at 2 to 8ºC.

After incubation count the colonies and note the microbial count in the format attached as Annexure

Note the dilution, which is giving a microbial count in between 10 to100 CFU/ml.

Preserve the previous dilution which is giving 10 to100 CFU/ml.

This dilution shall be preserved for microbial inoculum and from this dilution 100μl of the suspension shall be used for testing. Eg., if the 106 dilution is giving microbial count in between

10 to 100 CFU/ml the 105 dilution tubes shall be preserved and 100μl of the suspension shall be used to give microbial count in between 10 to 100 CFU/ml.

Label should be given to each tubes and it should contain SOP No. , Name of Microorganism, strain no, dilution count & due on. ti maintain the documentary records of the same annexes should be added in SOP.

Annexure like – Enumeration anaerobic culture suspension record . culture maintenance record & Inoculum preparation record.

Reference :- British Pharmacopoeia.