SOP for Preparation of Culture Media

OBJECTIVE

The objective of this SOP is to define a preparation procedure for cultural media for microbiological evaluation of raw materials, bulks, purified and drinking water, intermediates and finished products.

SCOPE

This SOP describes the execution of preparation of cultural media in microbiology laboratory.

RESPONSIBILITIES

Microbiologist:

• To interface this SOP to the actual practices of flow patterns.
• To ensure that the SOP is updated & reflects the actual execution.
• To review the SOP in case of any change or whenever it is due for revision.

AM Microbiology:

• To follow the instructions laid down by this SOP.
• To keep the relevant SOPs valid and updated.

Manager Quality Control:

To provide adequate resources for the implementation of this SOP as define.

MEDIA PREPARATION PROCEDURE

General Requirements

• Before preparation check the expiry date and label the bottle with name and date of preparation.
• Complete instructions for the preparation of culture media are given on the label of each bottle, according to it,
• Open the culture medium bottle away from direct air flow and moisture. Weigh the desired quantity of required medium quickly, accurately and without creating 'clouds of dust'.

• Reclose the medium bottle as soon as possible.
• Always use freshly prepared purified water.
• Pour half of the required volume of purified water in a clean bottle which is twice the final volume of the medium to allow adequate mixing.
• Then transfer the weighed quantity of medium slowly along with agitation as the medium can not accumulate to make a clump.
• Pour the rest of the purified water down the sides of the vessel to wash any adherent medium back into the solution. This is an important step because dry culture media powder above the level of the water may not be sterilized in the autoclave and may be a source of contamination.
• Sterilize using a validated process of autoclave, then check its pH value, if the adjustment is required then adjust it by using NaOH/HCl.

NOTE: The pH of a dehydrated medium should not require adjustment provided it has been prepared correctly using pure water and clean equipment, and it has not been over-autoclaved. The manufacturer’s instructions must be followed strictly.

• After sterilization, pre-incubate the sterilized medium at 20 – 25°C for 3 – 5 days, if the medium shows any sign of turbidity then DO NOT it for testing purpose.
• Broth medium is kept in the same medium bottle whereas agar medium is poured in pre-sterilized Petri dishes when the medium temperature drops at 45 – 50°C.
• Mix the medium gently by rotating the flask or bottle. Avoid forming air bubbles. Dispense aseptically about 15 – 20 mL medium into Petri dishes (90 – 100 mm diameter) under the laminar airflow cabinet.
• Agar plates should be of an even depth and of a firm gel.
• When the medium has gelled and cooled, stack the plates and preincubate them for 3 – 5 days, if plates show any sign of colony then DO NOT use it for testing purpose.
• Record the medium preparation activity in its specified log book.

Media and their Typical Preparation Concentration

Media	Required % & pH	* Required Quantity per 100 mL	** Sterilization °C, Time & Pressure
Tryptic Soy Agar	4.0%, 7.3	4 g	121°C, 15 minutes,  15 psi
Tryptic Soy Broth	3.0%, 7.3	3 g	121°C, 15 minutes,  15 psi
Sabouraud Dextrose Agar	6.5%, 5.6	6.5 g	121°C, 15 minutes,  15 psi
Sabouraud Dextrose Broth	3.0%, 5.6	3 g	121°C, 15 minutes,  15 psi
Enterobacteria Enrichment Mossel Broth	4.5%, 7.2	4.5 g	121°C, 5 minutes,  15 psi
Mannitol Salt Agar	10.8%, 7.4	10.8 g	121°C, 15 minutes,  15 psi
Cetrimide Agar Selective & Pseudomonas Cetrimide agar	4.53%, 7.2	4.53 g	121°C, 15 minutes,  15 psi
Reinforced Medium for Clostridia	3.8%, 6.8	3.8 g	121°C, 15 minutes,  15 psi
MacConkey’s Broth	3.5%, 7.3	3.5 g	121°C, 15 minutes,  15 psi
MacConkey’s Agar	5.0%, 7.1	5 g	121°C, 15 minutes,  15 psi
Rappaport Vassiliadis Salmonella Enrichment Broth	4.18%, 5.2	4.18 g	115°C, 15 minutes,  15 psi
Violet Red Bile Glucose Agar	3.95%, 7.4	3.95 g	Heat in Water bath, boil for 2 minutes, don’t autoclave
Xylose Lysine Deoxycholate Agar	4.0%, 7.1	4 g	Don’t autoclave, sterile on boiling
Columbia Agar	4.2%, 7.3	4.2 g	121°C, 15 minutes,  15 psi
Buffered Peptone Water	2.55%, 7.0	2.55 g	121°C, 15 minutes,  15 psi
Antibiotic medium 11	3.05%, 8.3	3.05 g	121°C, 15 minutes,  15 psi

* Weigh medium as your volume required.                                  ** Sterilization time can increase up to 20 min.

Avoid inhaling the powder and prolonged skin contact.

PRECAUTIONS

1. During weighing, care must be taken and weigh accurately according to the manufacturer’s specifications.
2. Before weighing, properly label the bottles with name, percentage and date.
3. Weigh the desired quantity of required medium quickly, accurately and without creating 'clouds of dust'.
4. After weighing, reclose the bottle as soon as possible because the dehydrated medium is hygroscopic.
5. Always wear a mask and gloves for avoiding inhalation and skin contact.
6. After sterilization, Do Not make heavy agitation for mixing as air bubbles do not form, mix in a gentle clockwise motion, especially in agar medium.
7. If shrinkage OR dryness of agar surface is found, then it should be discarded without its usage for test.
8. Contaminated medium either broth OR agar medium should be disposed-off according to SOP.
9. Do Not use agar plates that contain heavy moisture.

TRAINING

Training about the contents of this SOP must be imparted to all the concerned personnel once in a year and also to new employees before starting with the job and the records documented according to the training SOP.

REVISION HISTORY

Nil

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