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 the preparation of cultural media in a 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:
- 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 defined.
MEDIA PREPARATION PROCEDURE
General Requirements
- Before preparation check the expiry date and label the bottle with the 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 airflow 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 purposes.
- 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 purposes.
- Record the medium preparation activity in its specified log book.
Media and their Typical Preparation Concentration
Media
|
Required
% & pH
|
* Required Quantity / 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
|
McConkey
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 a Water bath,
boil for 2 minutes, and 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 requires.
** Sterilization time can increase up to 20 minutes.
Avoid inhaling the powder and prolonged skin contact.
PRECAUTIONS
- During weighing, care must be taken and weighed accurately according to the manufacturer’s specifications.
- Before weighing, properly label the bottles with names, percentages, and dates.
- Weigh the desired quantity of required medium quickly, accurately, and without creating 'clouds of dust'.
- After weighing, reclose the bottle as soon as possible because the dehydrated medium is hygroscopic.
- Always wear a mask and gloves to avoid inhalation and skin contact.
- 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.
- If shrinkage OR dryness of agar surface is found, then it should be discarded without its usage for test.
- Contaminated medium either broth OR agar medium should be disposed of according to SOP.
- 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 a year and also to new employees before starting the job and the records documented according to the training SOP.
REVISION HISTORY
Nil
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