Tuesday, 17 August 2021

Growth Promotion Test for Media as per USP


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

 

Rahul Kashyap is the executive in a Leading Pharma Company, he author in-depth guide that teach pharma industry owner and workers way to follow, manage and grow the quality work in this Field.

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