Growth Promotion Test for Media as per USP

Cultures for GPT

·         The growth promotion test is applicable to each batch/Lot of the dehydrated medium received and each lot of autoclaved media to check its suitability.

·         Perform the growth promotion test using one of these cultures for both agar & broth media.

•             Staphylococcus aureus (NCIM 2079, ATCC 6538P)

•             Escherichia coli (NCIM 2065, ATCC 8739)

•             Salmonella abony (NCIM 2257, ATCC 6017)          

•             Pseudomonas aeruginosa (NCIM2200, ATCC 9027) 

•             Shigella Flexneri (NCIM 5265, ATCC 12022)

•             Candida albicans (NCIM3471, ATCC 10231)

•             Aspergillus brasiliensis (NCIM1196, ATCC16404)

•             Bacillus subtilis (NCIM 2921, ATCC9372)

•             Burkholderia cepacia (NCIM 5658, ATCC25416)

The details of media with respect to culture name and incubation temperature are as per format given at the end of this article

·         Prepare the medium as per sop.

·         Prepare the Microbial Culture Suspension as per the Procedure.

·         Prepare 100ml 0.9% w/v Normal saline in volumetric flask.

·         Ensure that solution is clear.

·         Take 7 clean and dry test tubes and mark them as 1,2,3,4,5,6,7 and 8.

·         Using 10 ml calibrated pipette, transfer 10ml normal saline in the tube no.1 and 9ml in tube no. 2 to 8 each.

·         Plug the test tube 1 to 8 and wrap the mouth with foil and autoclave at 121+3oC for 20 minutes.

·         Take out the saline tubes and transfer to the biosafety cabinet allow cool.

·         Take out culture tube for sub culturing, using a sterile inoculating loop, transfer a portion of colony to tube no. 1.

·         Swirl the test tube gently to become uniform distribution of organism in saline. Treat the tube no. 1 as a stock culture suspension.

·         Transfer 1ml of tube no. 1 to tube no. 2 again swirl to homogenize and mark it as 10-1   dilution.

·         Repeat the process of serial dilution to 10-5, 10-6, till tube 8 to get last dilution of 10-8 and pour 1.0 ml of culture suspension from 10-3, 10-4 , 10-5, 10-6 ,10-7 & 10-8  dilution to sterile duplicate plates.

·         Pour 15-20 ml of Soyabean Casein Digest Agar or Sabouraud Dextrose Agar  (Previously qualified) to the plates and incubate the plate at 30-35oC for 24-48 hrs. (for bacteria) or 20-25oC for 48-72 hrs. for yeasts & 96-120 hrs. for molds).

·         After incubation record the observation in Format (Given at the end of article) and select the dilution which contain 10-100cfu/ml.

·         Store the above culture dilution at 2-8oC.

Growth promotion test of Agar/Broth Medium: Growth Promotion Test for Media as per USP

·         Perform growth promotion test of one container from each sterilized lot of media and  on daily basis perform the GPT by pour plate or spread plate method for general purpose medium (SCDA, SDA and R2A) by one organism and for selective/Differential media only perform Growth promotive and indicative test by Streak method.  

·         Consider the Daily GPT as positive control and report in Daily Growth promotion test record. In case of agar media pour 1.0 ml suspension of organisms in sterilize Petri dishes (duplicate). In case of broth media, add 1.0ml culture suspension to the 90-100 ml of specific broth medium for which growth promotion test is being performed.

·         Incubate the agar plates or broth medium at 30-35 C for 48-72 hrs for bacterial growth and at 20-25C for 72-120 hrs for fungal growth.

·         After incubation observe the growth of broth.

·         The growth of the specific organism should be comparable to the initial suspension counts, which were observed after preparation of the suspension.

·         If the media shows good response for GPT culture. Label the media container with the entries

·         Calculate the Microbial Recovery by given formula:

% Recovery = Cfu observed (Mean) X100

                                Inoculated cfu/ml

·         For the initial media Growth promotion test of media, compare the recovery against

                      the previously qualified media.

% Recovery = Cfu observed of Previous Media (Mean) X100

                                Cfu observed of New Media (Mean)

Precautions for Growth Promotion Test for Media as per USP

Biosafety cabinet shall be validated.

All glassware shall be dry and sterilized.

Autoclave shall be validated.   

Use culture carefully.

Acceptance Criteria for Growth Promotion Test for Media as per USP

·         Growth obtained on an Agar (R2A, SCDA and SDA) shall be between 50 -200% of standardized inoculated suspension or not differ by a factor greater than 2 or shall be comparable to that obtained on the same medium previously tested and approved.

·         All Solid and Liquid media except (R2A, SCDA and SDA) shall growth and match their characteristic as per Annexure No. II than approved.

·         For New Agar: Media shall exhibit growth not less than 75% of previously qualified media.

·         For growth inhibition test, there shall not be any growth of the microorganism on the agar and broth medium.

·         For the indicative test, there shall be growth of indicative microorganism on the agar medium.

List of media and their specific cultures for growth promotion test.

S. No	Type of Medium	Microorganism	Properties	Incubation
				Temp.	Period
1.	Enterobacteria enrichment broth mossel	E.coli	Growth Promoting	30-350C	18 to 24 hrs.
		P. aeruginosa	Growth Promoting	30-350C	18 to 24 hrs.
		S. aureus	Inhibitory	30-350C	24 hrs.
2.	Violet Red Bile Glucose agar	E.coli	Growth Promoting & indicative (purplish – red colonies)	30-350C	18-72 hrs.
		P. aeruginosa	Growth Promoting & indicative (colourless)	30-350C	18-24 hrs.
3.	MacConkey Broth	E.coli	Growth Promoting	42-440C	18- 24 hrs.
		S. aureus	Inhibitory	42-440C
4.	MacConkey Agar	E.coli	Growth Promoting & indicative (pink colonies)	30-350C	18-72 hrs.
5.	Burkholderia cepacia Agar/ MacConkey Agar	Burkholderia cepacia	greenish –brown colonies with white halos or white colonies surrounded by a pink red zone	30-350C	18-72 hrs.

 

6.	Rappaport Vassiliadis Salmonella enrichment broth	S. enterica subsp. Enterica serover typhimurium    Or S. enterica subsp. Enterica serovar abony	Growth Promoting	30-350C	18-24 hrs.
		S. aureus	Inhibitory	30-350C	24 hrs.
7.	Xylose Lysine Deoxycholate agar	S. enterica subsp. Enterica serover typhimurium    Or S. enterica subsp. Enterica serovar abony& Shigella	Growth Promoting & indicative (Red colonies with or without black centers)	30-350C	Growth: 24-48 hrs. Indicative: 24-48 hrs.
			Growth Promoting & indicative (Red colonies with or without black centers)	30-350C	Growth: 18 hrs. Indicative: 18-48 hrs.
8.	Cetrimide Agar	P. aeruginosa	Growth Promoting	30-350C	18 to 72 hrs.
		E.coli	Inhibitory	30-350C	18 to 72 hrs.
9.	Mannitol Salt Agar	S.aureus	Growth Promoting & indicative (Yellow or white colonies with yellow zone)	30-350C	Growth: 18 to 72 hrs. Indicative: 18 to 72 hrs.
		E.coli	Inhibitory	30-350C	18 to 72 hrs.
10.	Pseudomonas Agar Medium for Detection of Fluorescein	P. aureuginosa	Indicative	30-350C	18 to 72 hrs.
11.	Pseudomonas Agar medium For Detection of Pyocyanin	P. aureuginosa	Indicative	30-350C	18 to 72 hrs.
12.	Sabouraud Dextrose Agar	C. albicans	Growth Promoting	20-250C	5 days
		Aspergillus brasiliensis	Growth Promoting	20-250C	5 days
13.	Sabouraud Dextrose Broth	C. albicans	Growth Promoting	20-250C	3-5 days
		Aspergillus brasiliensis	Growth Promoting	20-250C	3 Days
14.	Soyabean casein  digest agar/Tryptone Soya Agar	S. aureus	Growth Promoting	30-350C	3 Days
		P. aeruginosa	Growth Promoting	30-350C	3 Days
15.	Soyabean casein  digest medium	P. aeruginosa	Growth Promoting	30-350C	18-24 hrs.
		S. aureus	Growth Promoting	30-350C	18-24 hrs.
16.	R2A Agar	B.subtilis / B.spizezinii	Growth Promoting	30-350C	3 days
		P. aeruginosa	Growth Promoting	30-350C	3 days
16.	GNB	Shigella	Growth Promoting	30-350C	24-48 hrs.
17.	Tryptone Soya broth/Soyabean casein digest broth with 0.5% soya lecithin & 4% Tween 20	P. aeruginosa	Growth Promoting	30-350C	18-24 hrs.
		S. aureus	Growth Promoting	30-350C	18-24 hrs.

 

List of Qualitative or Quantitative test for Liquid and Solid Media

S. No.	Type of Medium	Microorganism	Test Qualitative or Quantitative	Identification
1.	Enter bacteria enrichment broth mossel	E.coli	Qualitative	Colour change from Emerald green colored to Yellow colour
		P. aeruginosa	Qualitative
		S. aureus	Qualitative	No colour change
2.	Violet Red Bile Glucose agar	E.coli	Qualitative	(purplish – red colonies)
		P. aeruginosa	Qualitative	(colorless)
3.	MacConkey Broth	E.coli	Qualitative	Colour change from blue to yellow
		S. aureus	Qualitative	No colour change
4.	MacConkey Agar	E.coli	Qualitative	(pink colonies)
5.	Burkholderia cepacia Agar/ MacConkey Agar	Burkholderia Cepacia	Qualitative	greenish –brown colonies with white halos or white colonies surrounded by a pink red zone
6.	Rappaport Vassiliadis Salmonella enrichment broth	S. enterica subsp. Enterica serover typhimurium    Or S. enterica subsp. Enterica serovar abony	Qualitative	Colour change from green to light white or turbid
		S. aureus	Qualitative	No colour change
7.	Xylose Lysine Deoxycholate agar	S. enterica subsp. Enterica serover typhimurium    Or S. enterica subsp. Enterica serovar abony& Shigella	Qualitative	(Red colonies with or without black centers)
8.	Cetrimide Agar	P. aeruginosa	Qualitative	Greenish colony observed
		E.coli	Qualitative	No growth of colony observed
9.	Mannitol Salt Agar	S.aureus	Qualitative	(Yellow or white colonies with yellow zone)
		E.coli	Qualitative	No growth of colony observed
10.	Pseudomonas Agar Medium for Detection of Fluorescein	P. aeruginosa	Qualitative	Blue green colour colony observed
11.	Pseudomonas Agar Medium For Detection of Pyrocyanin	P. aeruginosa	Qualitative	Greenish colour colony observed
12.	Sabouraud Dextrose Agar	C. albicans	Quantitative	Colony observed
		Aspergillus brasiliensis	Quantitative	Colony observed
13.	Sabouraud Dextrose Broth	C. albicans	Qualitative	Turbidity observed
		Aspergillus brasiliensis	Qualitative	Turbidity observed
14.	Soyabean casein      Digest agar/ Tryptone Soya Agar	S. aureus	Quantitative	Colony observed
		P. aeruginosa	Quantitative	Colony observed
15.	Soyabean casein  digest medium	P. aeruginosa	Qualitative	Turbidity observed
		S. aureus	Qualitative	Turbidity observed
16.	R2A Agar	B.subtilis / B.spizezinii	Quantitative	Colony observed
		P. aeruginosa	Quantitative	Colony observed
16.	GNB	Shigella	Qualitative	Turbidity observed
17.	Tryptone Soya broth with 0.5% soya lecithin & 4% Tween 20	P. aeruginosa	Qualitative	Turbidity observed
		S. aureus	Qualitative	Turbidity observed