Growth Promotion Test (GPT) for Culture Media

1.0 Equipment Required

LAF
Colony Counter
Incubators
DHS
Autoclave

2.0 Material Required

Dehydrated culture media
Sterile Saline solution
Sterile Petri Plates
Measuring Cylinder
Test organism – E. coli, Staph. aureus, Ps. aeruginosa, C. albicans, A. niger, Salmonella. B. subtilis, Cl. Sporogenes, S. epidermidis.

3.0 Utilities Required

Power

4.0 Procedure

4.1  After receipt of dehydrated culture media, make a necessary entry in receipt register including B. No, Mfg date and use before date.

4.2  Collect minimum 5.0 g sample from each received packs (i.e. batch wise) and mix properly.

4.3  Prepare culture media & sterilize as per SOP for preparation of culture media.

4.4  After sterilization, transfer the media to microbiology analysis room and allow it to cool at 40 to 45°C.

4.5  Start the LAF as per SOP and further proceed works under LAF.

4.6 Test For Growth Promoting Properties, Solid Media

4.6.1  Label the plates with culture name, Media B. No, Date of incubation on the base of petri plates

4.6.2  Add 1.0 ml suspension of specific culture containing 10 to 100 cfu/ml (as specified in Table – I) into two sterile petri plates.

4.6.3  Aseptically pour the cooled media at 40 to 45°C into both the labeled plates, mix the plates by gently rotating clockwise and anti-anticlockwise direction.

4.6.4  Allow the plates to solidify at room temperature under Laminar Air Flow.

4.6.5  Simultaneously run negative control to verify testing conditions, using the same procedure in place of the test organism use diluents i.e. 1.0 ml of saline solution

4.6.6  Incubate the plates at specified temperature and period as listed in Table – 1,

4.6.7  Observe the plates for number of colonies, and count the cfu observed on both plates and express the result in cfu by following formula

                                           P1 + P2 / 2

                                           Where P1 and P2 are plate 1 and plate 2

4.6.8  Calculate the Microbial recovery in percentage by equation –

% Recovery  =  Mean cfu observed X 100

                                Inoculated cfu ml

4.6.9  Recovery should not less than 75%

4.7 Test For Growth Inhibitory Properties, Solid Media

4.7.1  Label the plates with culture name, Media B. No, Date of incubation on the base of petri plates.

4.7.2  Add 1.0 ml suspension of specific culture containing 100 cfu/ml (as specified in Table – I of growth inhibitory property) into two sterile Petri plates.

4.7.3  Aseptically pour the cooled media at 40 to 45°C into both the labeled plates, mix the plates by gently rotating clockwise and anti-anticlockwise direction.

4.7.4  Allow the plates to solidify at room temperature under Laminar Air Flow.

4.7.5  Incubate at specified temperature and period as listed in Table – 1,

4.7.6  Observe the plates for number of colonies, No growth of the test organism occurs.

4.8 Test For Growth Promoting and Inhibitory Properties, liquid Media

4.8.1  Prepare required quantity of liquid culture media, dispense 100 ml in test tubes and sterilize as per manufacturer's instruction.

4.8.2  After sterilization, transfer the media to microbiology analysis room and allow it to cool at room temperature.

4.8.3  Start the LAF as per SOP and proceed further work under LAF

4.8.4  Add 1.0 ml of positive culture of growth promoting properties, containing 100 cells (as per table – 1) into broth tube and label with Media B. No, name of positive culture & date of inoculation.

4.8.5  For Growth Inhibitory Test, add 1.0 ml of positive culture of growth inhibitory properties, containing 100 cells (as per table – 1) into broth tube and label with Media B. No, name of positive culture & date of inoculation.

4.8.6  Simultaneously run negative control to verify testing conditions, using the same procedure in place of the test organism use diluents i.e. 1.0 ml of saline solution

4.8.7  Incubate all the tubes at specific temperature as specified in table –1.

4.8.8  Daily observe the tubes for growth for turbidity.

4.8.9  Satisfactory growth should be observed within 3 days of incubation in the test. There should not be growth in growth inhibitory test & negative control.

4.8.10  In case the media passes the growth promotion test, a approved label shall be affixed on the media container, then the same should be used for analysis.

4.8.11  In case the media fails for the growth promotion test then a rejected label shall be affixed on the container then the same shall be rejected and accordingly the rejection entry should be made in the stock register.

4.8.12  The rejected media should be discarded or returned back to the supplier.

4.8.13  The specimens of the Receipt, Sampled, Approved and Rejected label are attached in Annexure.

5.0 Precaution

5.1  The dehydrated culture media as well as their ingredients are highly hygroscopic and must be stored in a cool dry place away from bright light. These media are meant for laboratory use only and shall never be used for human or animal consumption.

5.2  Use fresh sterile pipette for each transfer.

5.3  The medium to be poured in petri plates should have a temperature of 40 - 45°C.

5.4  The plates should be incubated in an inverted position to prevent collection of condensation on the plate surface.

5.5  If any spillage of cultures, immediately wash with 70% IPA solution.

5.6  Entire operation inside the microbiology room should be carried out under the laminar airflow chamber using gas burner.

5.7  Examine the physical nature of the dehydrated medium. If any unusual colour, odour or physical appearance is noticed, discard the medium.

5.8  Always use a dry spoon or spatula for weighing the dehydrated media. The weighing operation shall be completed as quickly as possible to prevent absorption of moisture by the hygroscopic contents. Wear a face mask while weighing the dehydrated media to avoid inhalation of fine particles of media.

5.9  All dehydrated media must be retest after the release of three months interval and finally media must be discarded after release of one year.

Table - 1

Name of Media	Positive culture to be used for Growth Promotion Test	Positive culture to be used for Growth Inhibitory Test			Expected growth characteristics of the test organism for the respective media	Incubation temperature		Incubation period
Fluid Thioglycollate Medium	B. subtilis  NCIM 2063 Ps.aeruginosa NCIM 2200 S.aureus  NCIM 2079	NA			-Growth (Turbidity)  Observed in the      respective media     tubes	30 to 35°C		72 hrs
Soybean Casein digest Medium	B. subtilis NCIM 2063 C.albicans NCIM 3471 A. niger NCIM 1196	NA			-Growth (Turbidity) Observed in the respective media tubes	20 to 25°C		72 hrs
Soybean Casein digest Agar	E.coli  NCIM 2065 Ps.aeruginosa NCIM 2200 S.aureus  NCIM 2079	NA			-Opaque white  colonies on SCDA Plates -Large grayish colonies on SCDA Plates -Tiny white colonies on SCDA Plates	30 to 35°C		48 hrs
MacConkey Agar	E.coli  NCIM 2065	S.aureus NCIM 2079			- Brick red colonies on MacConkey Agar Plates	30 to 350C		48 hrs
MacConkey broth	E.coli  NCIM 2065	S.aureus NCIM 2079			- Yellow colour change	30 to 35°C		48 hrs
Mannitol salt Agar	S.aureus NCIM 2079		E.coli           NCIM 2065	- Yellow colonies  on Mannitol Salt Agar Plates			30 to 35°C	48 hrs
Cetrimide agar	Ps.aeruginosa NCIM 2200		E.coli          NCIM 2065	- Greenish Colonies on Cetrimide Agar Plates			30 to 35°C	48 hrs
Antibiotic assay Medium No. 11	S. epidermidis NCIM 2493		NA	- Good Luxuriant    colonies on the respective media Plates			30 to 35°C	24 hrs
Peptone water	E.coli  NCIM 2065		NA	Luxuriant (Turbid) growth in the respective media tubes			30 to 35°C	48 hrs
Sabourauds Dextrose Agar	C.albicans  NCIM 3471		NA	-white  colonies on Sabourauds  Dextrose Agar Plates			22 to 25°C	120 hrs
Selenite F broth	Salmonella abony   NCIM 2257		S.aureus NCIM 2079	-Red ppt. observed in the Selenite F broth			30 to 35°C	24 hrs
EMB agar	E.coli   NCIM 2065		S.aureus NCIM 2079	- Blue – Black colonies with metallic sheen on EMB Agar Plates			30 to 35°C	24 hrs
Brilliant green agar	Salmonella abony   NCIM 2257		S.aureus NCIM 2079	- Opaque pinkish to white colonies on Brilliant green agar Plates			30 to 35°C	48 hrs
Nutrient Broth	E.coli  NCIM 2065		NA	-Good Luxuriant (Turbid) growth  Observed.			30 to 35°C	24 hrs
Triple sugar Iron agar	Salmonella abony NCIM 2257		S.aureus        NCIM 2079	-Red and Yellow butt (With or without blackening)			30 to 35°C	48 hrs
Vogel Johnson Agar	S.aureus NCIM 2079		E.coli           NCIM 2065	- Black Colonies on Vogel Johnson Agar Plates			30 to 35°C	48 hrs
Baired Parker Agar	S.aureus NCIM 2079		E.coli           NCIM 2065	- Black Colonies  surrounded by a clear zone			30 to 35°C	48 hrs
Pseudomonas agar(For Fluorescein)	Ps.aeruginosa NCIM 2200		S.aureus NCIM 2079	-  Yellowish Colonies on Pseudomonas Agar Plates			30 to 35°C	72 hrs
Pseudomonas agar(For Pyocyanin)	Ps.aeruginosa NCIM 2200		S.aureus NCIM 2079	- Greenish Colonies on Pseudomonas Agar Plates			30 to 35°C	72 hrs
Urea broth	Salmonella abony  NCIM 2257		S.aureus NCIM 2079	-Luxuriant growth with no color change Observed in the respective media tubes			30 to 35°C	24 hrs
Tetrathionate Brilliant Green Bile Broth	Salmonella abony NCIM 2257		S.aureus NCIM 2079	-White ppt. observed in the TBGB broth tubes			30 to 35°C	24 hrs
Bismuth sulphite agar	Salmonella abony NCIM 2257		S.aureus NCIM 2079	- Black or green Colonies on Bismuth Sulphite Agar Plates			30 to 35°C	48 hrs
Xylose lysine deoxycholate agar	Salmonella abony  NCIM 2257		S.aureus NCIM 2079	- Red Colonies on Xylose lysine deoxycholate Agar			30 to 35°C	48 hrs

6.0  Frequency

6.1  For each batch of consignment

7.0  Abbreviation

SOP  :  Standard Operating Procedure

QA  :  Quality Assurance

QC  :  Quality Control

NA  :  Not Applicable

WFI  :  Water for Injection

LAF  :  Laminar Air Flow