STANDARD OPERATING PROCEDURE Growth Promotion & Inhibition Test Procedure of Microbial Media

	STANDARD OPERATING PROCEDURE
	Department: Quality Control	SOP No.: QC/MB/xxx
Growth Promotion & Inhibition Test Procedure of Microbial Media		Effective Date:
		Review Date:
		Page No.:

1.0            OBJECTIVE:

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

2.0            SCOPE:

2.1                 This procedure is applicable to all media which is used for microbiological testing in Microbiology laboratory at  Pharma Private Limited.

3.0            RESPONSIBILITY:

3.1                 Microbiologist – Responsible for execution of activity.

3.2                 Section In-Charge – Responsible for review the procedure.

3.3                 QC Head – Responsible for implementing the Procedure.

3.4                 QA Head – Overall compliance.

4.0            PROCEDURE:

4.1                 Each lot of media received to be tested for its growth promotion and inhibition qualities.

4.2                 Growth Promotion Test shall be performed for one container of each lot.

4.3                   Prepare required quantity of media for growth promotion test as per as per current version of SOP QC/MB/021.

4.4                   For daily prepared media GPT shall be performed as positive control test with the respective microorganisms mentioned in table – I.

4.5                   Prepare culture suspension as per current version of SOP QC/MB/xxx and select the dilution which gives 10-100 cfu/mL for growth promotion test.

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

4.6.1             Prepare required quantity of media to be tested and distributed in flasks or tubes and sterilized as per current version of SOP QC/MB/xxx or as per manufacturer’s instructions.

4.6.2             Transfer the sterile tubes/flasks containing media to LAF bench and allow it to cool to room temperature.

4.6.3             Record the following details on the conical flasks /tubes.

                 Name of the Media:               

                 Lot No. Of Medium:           

                 Organism being used for growth promotion:

                 Tested by / date:

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

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

4.6.6             Negative control should be kept along with the test samples without adding culture suspension.

4.6.7             After specified incubation period, observe the tube/flask and record the observations in annexure I.

4.6.8             Acceptance criteria:

4.6.8.1       Growth/ turbidity shall be observed in test sample.

4.6.8.2       Growth/turbidity shall not be observed in negative control.

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

4.7.1             Transfer the sterile tubes/flasks containing media to micro LAF bench and allow it to cool to room temperature.

4.7.2             Record the following details on the conical flasks /tubes.

Name of the Media:                   

  Lot No. Of Medium:              

  Organism being used for growth inhibition:     

  Tested by / date:

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

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

4.7.5             Negative control should be kept along with the test samples without adding culture suspension.

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

4.7.7             Acceptance criteria: Growth/turbidity should not be observed in test sample and in negative control.

4.8                 Test for Growth Promotion and Indicative Properties of solid media:

4.8.1             Transfer the sterile media, sterile petriplates to micro LAF bench.

4.8.2             Record the following details on the bottom of the Petri plate.

  Name of the Media:                                                                        

  Lot No. of Media:                                                     

  Organism being used for growth promotion:   

  Tested by / date:      

4.8.3             Transfer 1.0 mL of culture suspension having 10-100 cfu/mL into two sterile petriplates and aseptically pour the 15-20 mL of test sample at 40 to 45 °C into the plates.

4.8.4             Incubate the plates as per the conditions mentioned in Table – I.

4.8.5             Negative control should be kept along with the test samples without culture suspension.

4.8.6             After specified incubation period, observe the plates for growth and count the colonies obtained in each plate and record the results in Annexure-II.

4.8.7             The average of the colonies obtained in duplicate plates is used in the calculation of microbial recovery.

4.8.8             Acceptance Criteria:

4.8.9             The recovery of microbial cells must not differ by a factor greater than 2 from the calculated value for a standardized inoculum.

4.8.10         The colonies are comparable in appearance and indication reactions to those previously obtained with a previously tested and approved batch of medium.

4.8.11         No growth should be observed in negative control.

4.9                 Test for Growth Inhibitory properties of solid media:

4.9.1             Transfer the sterile media, Petriplates to micro LAF bench.

4.9.2             Record the following on the bottom plate.

  Name of the Media:

  Lot No. of Media:

  Organism being used for inhibition:

  Tested by / date:

4.9.3             Transfer 1.0 mL of culture suspension having 10-100 cfu/mL into two sterile petriplates and aseptically pour the 15-20 mL of test sample at 40°C to 45°C into the petriplates.

4.9.4             Incubate the plates as per the conditions mentioned in Table – I.

4.9.5             Negative control should be kept along with the test samples without adding culture suspension.

4.9.6             After 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.

4.9.7             Acceptance criteria:

4.9.7.1       Growth should not be observed in test sample and in negative control.

4.10               Growth indicative properties shall be performed for the selective media as mentioned in      Table –I and record the observations in Annexure II.

4.11             GPT passed media shall be used for routine microbial analysis.

4.12             In case the media fails in the growth promotion test then reject the media.

4.13             Precautions:

4.13.1         Media should be stored at 10°C to 30°C or as per instructions given by manufacturer.

4.13.2         Check the physical condition of dehydrated media powder, if lumps are formed in the media discard that media.

4.13.3         Prepare & sterilize all the media as per manufacturer’s instructions.

4.13.4         Care should be taken while handling the cultures.

Table-1

Name of The Media	Property	Micro-organism to be tested	Incubation Temperature	Incubation Period
Soyabean casein digest media	Growth promoting	Bacillus subtilis, Staphylococcus aureus & Pseudomonas aeruginosa	30°C to 35ºC	NMT 3 days
Tryptone Soya Agar/ Plate Count Agar/ Soyabean casein digest  Agar/ Nutrient agar/Nutrient Broth	Growth promoting	Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans & Aspergillus brasiliensis	30°C to 35ºC	NMT 3 days
R2 A Agar	Growth promoting	Bacillus subtilis or Pseudomonas aeruginosa	30°C to 35º	NMT 3 days
Sabouraud Dextrose Agar/Potato Dextrose Agar/Sabouraud Dextrose broth/ Sabouraud chloramphenicol agar	Growth promoting	Candida albicans & Aspergillus brasiliensis	20°C to 25ºC	NMT 5 days
Mac Conkey Broth*/ Brillient Green Bile Broth/ Lactose Lauryl Tryptose Broth	Growth Promotion	Escherichia coli	42°C to 44°C	24 hours
	Inhibitory	Staphylococcus aureus	42°C to 44°C	48 hours
Mac Conkey agar# / Eosin Methylene Blue agar/ Levine Eosin – Methylene blue agar	Growth Promotion+ indicative	Escherichia coli	30°C to 35ºC	24 hours
Rappaport Vassiliadis salmonella enrichment broth*	Growth promoting	Salmonella Species	30°C to 35ºC	24 hours
	Inhibitory	Staphylococcus aureus	30°C to 35ºC	24 hours
Xylose lysine Deoxycholate agar#  /Bismuth Sulphite Agar/Brilliant green Agar/ Triple sugar iron agar/ Deoxycholate citrate Agar	Growth promoting+ indicative	Salmonella Species	30°C to 35ºC	24 hours
Cetrimide Agar*#	Growth promoting + Indicative	Pseudomonas aeruginosa	30°C to 35ºC	24 hours
	Inhibitory	Escherichia coli	30°C to 35ºC	72 hours
Mannitol salt agar*#/ Baired Parker agar/Vogel Johnson Agar	Growth promoting+ Indicative	Staphylococcus aureus	30°C to 35ºC	24 hours
	Inhibitory	Escherichia coli	30°C to 35ºC	72 hours
Reinforced medium for clostridia / Columbia agar	Growth promoting	Clostridium sporogenes	30°C to 35ºC	48 hours
Enterobacteria enrichment broth-Mossel*	Growth promoting	Escherichia coli Pseudomonas aeruginosa	30°C to 35ºC	24 hours
	Inhibitory	Staphylococcus aureus	30°C to 35ºC	48 hours
Violet red bile glucose agar#	Growth promoting + Indicative	Escherichia coli Pseudomonas aeruginosa	30°C to 35ºC	24 hours

                

* Growth Inhibition Tests shall be performed.

# Growth indicative properties shall be formed.

5.0           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

7.0           REFERENCES:

7.1                 United States Pharmacopeia Chapter <61> &<62> (Microbiological Examination of non-sterile Products: Microbial Enumeration test and Test for specified microorganisms).

7.2                 European Pharmacopeia chapter 2.6.12 & 2.6.13 (Microbiological Examination of non-sterile Products: Microbial Enumeration test and Test for specified microorganisms).

7.3                 USP <1117> Best Microbiology Lab Practices.