SOP for Growth Promotion & Inhibition Test Procedure of Microbial Media

OBJECTIVE

To lay down a procedure for evaluating the growth promotion and inhibition properties of media used for Microbiological testing.

SCOPE

This procedure applies to all media which is used for microbiological testing in the Microbiology laboratory at PharmaInfo Limited.

RESPONSIBILITY

Microbiologist – Responsible for the execution of an activity.

Section In-Charge – Responsible for reviewing the procedure.

QC Head – Responsible for implementing the Procedure.

QA Head – Overall compliance.

PROCEDURE

• Each lot of media is received to be tested for its growth promotion and inhibition qualities.
• A growth Promotion Test shall be performed for one container of each lot.
• Prepare the required quantity of media for the growth promotion test as per SOP for Growth Promotion Test.
• For daily prepared media GPT shall be performed as a positive control test with the respective microorganisms mentioned in Table – 1.
• Prepare culture suspension as per the current version of SOP for Preparation of Culture Media and select the dilution that gives 10-100 cfu/mL for the growth promotion test.

Test for Growth Promotion properties of liquid media (Broth):

• Prepare the required quantity of media to be tested and distributed in flasks or tubes and sterilized as per the current version of SOP for Preparation and Sterilization of Microbial Media or as per manufacturer’s instructions.
• Transfer the sterile tubes/flasks containing media to the LAF bench and allow it to cool to room temperature.
• Record the following details on the conical flasks /tubes.

1. Name of the Media:
2. Lot No. of Medium:
3. Organisms being used for growth promotion:
4. Tested by / date:

• Inoculate 1.0 mL of culture suspension having 10-100 cfu/mL into a tube/flask containing sterile liquid media or broth (test sample).
• Incubate the tubes/flasks as per the conditions mentioned in Table – 1.
• A negative control should be kept along with the test samples without adding culture suspension.
• After a specified incubation period, observe the tube/flask and record the observations in Annexure I.

Acceptance criteria:

• Growth/ turbidity shall be observed in the test sample.
• Growth/turbidity shall not be observed in the negative control.

Test for Growth Inhibition properties of liquid media (Broth):

• Transfer the sterile tubes/flasks containing media to a micro LAF bench and allow it to cool to room temperature.
• Record the following details on the conical flasks /tubes.

1. Name of the Media:
2. Lot No. Of Medium:
3. Organisms being used for growth inhibition:
4. Tested by / date:

Inoculate 1.0 mL of culture suspension having 10-100 cfu/mL into a tube/flask containing sterile liquid media or broth (test sample).

Incubate the tubes/flasks as per the conditions mentioned in Table – 1.

A negative control should be kept along with the test samples without adding culture suspension.

After the specified incubation period, observe the tubes/flasks and record the observations in Annexure - I.

Acceptance criteria:

• Growth/turbidity should not be observed in the test sample and in the negative control.

Test for Growth Promotion and Indicative Properties of solid media:

• Transfer the sterile media, and sterile Petri plates to the micro LAF bench.
• Record the following details on the bottom of the Petri plate.

1. Name of the Media:
2. Lot No. of Media:
3. Organisms being used for growth promotion:
4. Tested by / date:

• Transfer 1.0 mL of culture suspension having 10-100 cfu/mL into two sterile Petri plates and aseptically pour the 15-20 mL of the test sample at 40 to 45 °C into the plates.
• Incubate the plates as per the conditions mentioned in Table – I.
• A negative control should be kept along with the test samples without culture suspension.
• After a specified incubation period, observe the plates for growth and count the colonies obtained in each plate and record the results in Annexure-II.
• The average of the colonies obtained in duplicate plates is used in the calculation of microbial recovery.

Acceptance Criteria:

• The recovery of microbial cells must not differ by a factor greater than 2 from the calculated value for a standardized inoculum.
• The colonies are comparable in appearance and indication reactions to those previously obtained with a previously tested and approved batch of medium.
• No growth should be observed in the negative control.

Test for Growth Inhibitory properties of solid media:

• Transfer the sterile media, and Petri plates to the micro LAF bench.
• Record the following on the bottom plate.

1. Name of the Media:
2. Lot No. of Media:
3. Organisms being used for inhibition:
4. Tested by / date:

• Transfer 1.0 mL of culture suspension having 10-100 cfu/mL into two sterile Petri plates and aseptically pour the 15-20 mL of the test sample at 40°C to 45°C into the Petri plates.
• Incubate the plates as per the conditions mentioned in Table – I.
• A negative control should be kept along with the test samples without adding culture suspension.
• After the specified incubation period, observe the plates for growth and count the colonies, if any, obtained in each plate and record the results in Annexure-II.

Acceptance criteria:

• Growth should not be observed in a test sample and in the negative control.

• Growth indicative properties shall be performed for the selective media as mentioned in Table –I and record the observations in Annexure II.
• GPT-passed media shall be used for routine microbial analysis.
• In case the media fails in the growth promotion test then reject the media.

Precautions:

• Media should be stored at 10°C to 30°C or as per instructions given by the manufacturer.
• Check the physical condition of dehydrated media powder, if lumps are formed in the media discard that media.
• Prepare & sterilize all the media as per the manufacturer’s instructions.
• Care should be taken while handling the cultures.

* Growth Inhibition Tests shall be performed.

# Growth indicative properties shall be formed.

ABBREVIATIONS

QC: Quality Control

QA: Quality Assurance

SOP: Standard Operating Procedure

LAF: Laminar Air Flow

GPT: Growth Promotion Test

cfu: Colony Forming Units

NMT: Not more than

NLT: Not less than

REVISION HISTORY

Nil

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