Work  Instruction

Microbiological  Media – Preparation, Sterility and Growth Promotion Tests

Lab Code

                                                              (Microbiology)

Version No.:                                    

Effective Date:                                

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1.            Scope

The document describes the procedure to carry out sterility and growth promotion tests in order to ensure that the microbiological media is acceptable for use. This procedure is applicable for all types of media used in Microbiological Laboratory.

2.            Procedure

2.1.         Criteria for acceptance of dehydrated media

Upon receipt the media container shall be inspected for any visible damages. Upon opening, each container of media shall be inspected for clarity, color and flowability as per the media specification from the manufacturer. Media fulfilling these shall be labeled as below and stored at condition recommended by the manufacturer.

Lot Number
Date Opened
Date of Expiry
Storage Condition
Signature & Date

Media rejected on account of the above shall be sent back to supplier after recording the defects in inventory records. Following information shall be maintained in inventory record for each consignment:

o    Name & lot number

o    Quantity & number of containers

o    Date received

o    Expiry date

o    Storage location

o    Signature & date of person receiving& verifying the lot

o    Remarks (accepted/ rejected)

2.2.         Media Preparation

Media shall be prepared according to manufacturer’s instructions or as provided in the test methods.

Final pH shall be checked taking adequate quantity of autoclaved media aseptically and ensure that the pH is within manufacturer specified tolerance level. Record the final pH, lot number and date of preparation in the LNB.

2.3        Sterility Test

Sterility testing of prepared agar or broth media shall be done by incubating the representative number plates or tubes (5% of total volume / total number of units (plates or tubes), at relevant incubation conditions (30-35°C, 37°C, 20-25°C) for up to 72 hours. Alternate procedures may be employed for broth/ diluent where turbidity cannot be ascertained visually on incubation. 1 ml of the sterile broth/ diluent is plated with tryptic soy agar and observed for microbial growth by pour plate technique.

Acceptance Criteria

Agar Media: Absence of microbial growth

Liquid Media: No growth, no turbidity

If microbial contamination is not detected on the representative plates then the whole lot can be used for testing after growth promotion test. If microbial contamination is detected, identify the root cause and take appropriate measures to avoid the recurrence. Media lot failed in sterility test should be disposed through lab waste. Record all the observations in LNB.

2.4          Growth Promotion Test

Broth Media (general purpose):

Inoculate a loopful from a working culture (18-24 hrs on tryptic soy agar slant) of reference organisms mentioned below:

Pseudomonas aeruginosa ATCC 9027 or equivalent

Staphylococcus aureus ATCC 6538 or equivalent

Broth Media (selective):

Inoculate a loopful from a working culture (18-24 hrs on tryptic soy agar slant) of reference organisms mentioned below:

For example, use Salmonella ATCC 13311 or equivalent in tetrathionate broth

Agar Media (general purpose):

Streak a loopful of inoculum from a working culture (18-24 hrs on tryptic soy agar slant) of reference organisms mentioned below:

Pseudomonas aeruginosa ATCC 9027 or equivalent

Staphylococcus aureus ATCC 6538 or equivalent

Agar Media (Selective):

Streak a loopful of inoculum from a working culture (18-24 hrs on tryptic soy agar slant) of reference organisms relevant to the media in use

For example, Use Escherichia coli ATCC 8739 or equivalent (for promotion) and Staphylococcus aureus ATCC 6538 or equivalent (for inhibition) in Eosin methylene blue agar

Agar / Broth Media for Yeast and Mould:

Streak or inoculate PDA, SDA plates or PDB, SDB from a working culture of reference organisms mentioned below grown in SDA slant.

Candida albicans ATCC10231 or equivalent

Aspergillus niger ATCC16404 or equivalent

Streaked agar media or inoculated broth media are then incubated at appropriate temperature and time (usually 24 hrs for broth and 48 hrs for plates. Yeast & moulds plates should be incubated at least for 72 hrs.).

Examine each inoculated broth or plate for the presence or absence of microbial growth. Record the results in the LNB.

Acceptance Criteria

If the growth promotion test results are positive, the media can be used for testing. In the event of unsatisfactory results identify the root cause and take appropriate measures to avoid the recurrence. Media lot failed in growth promotion test should be disposed through lab waste. Record all the observations in LNB.

2.5        Concurrent Sterility/ Growth Promotion Tests

Whenever a batch of media is prepared and used immediately, sterility and growth promotion tests should be performed as a part of experimental controls. In the event of growth promotion or sterility results fail, the test shall be repeated using fresh set of media checked for sterility and/or growth promotion.

3.         Special Instructions

 Nil

4.         Appendices

5.        Revision History

WI No.	Supersedes No. & Date	Changes made (in brief)