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Growth Promotion Test


Learn how to test the microbial culture media for growth promotion using different microbial cultures.

1.0  Equipments 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 SOP.
4.4  After sterilization, transfer the media to microbiology analysis room and allow it to cool at 40 to 450C.
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 450C into both the labeled plates, mix the plates by gently rotating clock wise and anti-clock wise 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 following 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 450C into both the labeled plates, mix the plates by gently rotating clock wise and anti-clock wise 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 manufacturers 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 discard 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 - 450C.
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 350C
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 250C
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 350C
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 350C
48 hrs
Mannitol salt Agar
S.aureus NCIM 2079
E.coli           NCIM 2065
- Yellow colonie  on Mannitol Salt Agar Plates
30 to 350C
48 hrs
Cetrimide agar
Ps.aeruginosa NCIM 2200
E.coli          NCIM 2065
- Greenish Colonies on Cetrimide Agar Plates
30 to 350C
48 hrs
Antibiotic assay Medium No. 11
S. epidermidis NCIM 2493
NA
- Good Luxuriant    colonies on the respective media Plates
30 to 350C
24 hrs
Peptone water
E.coli  NCIM 2065
NA
Luxuriant (Turbid)
growth in the respective media tubes
30 to 350C
48 hrs
Sabourauds Dextrose Agar
C.albicans  NCIM 3471
NA
-white  colonies on Sabourauds  Dextrose Agar Plates
22 to 250C
120 hrs
Selenite F broth
Salmonella abony   NCIM 2257
S.aureus NCIM 2079
-Red ppt. observed in the Selenite F broth
30 to 350C
24 hrs
EMB agar
E.coli   NCIM 2065
S.aureus NCIM 2079
- Blue – Black colonies with metallic sheen on EMB Agar Plates
30 to 350C
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 350C
48 hrs
Nutrient Broth
E.coli  NCIM 2065
NA
-Good Luxuriant (Turbid) growth
 Observed.
30 to 350C
24 hrs
Triple sugar Iron agar
Salmonella abony NCIM 2257
S.aureus        NCIM 2079
-Red and Yellow butt (With or without blackening)
30 to 350C
48 hrs
Vogel Johnson Agar
S.aureus NCIM 2079
E.coli           NCIM 2065
- Black Colonies on Vogel Johnson Agar Plates
30 to 350C
48 hrs
Baired Parker Agar
S.aureus NCIM 2079
E.coli           NCIM 2065
- Black Colonies  surrounded by a clear zone
30 to 350C
48 hrs
Pseudomonas agar(For Fluorescein)
Ps.aeruginosa NCIM 2200
S.aureus NCIM 2079
-  Yellowish Colonies on Pseudomonas Agar Plates
30 to 350C
72 hrs
Pseudomonas agar(For Pyocyanin)
Ps.aeruginosa NCIM 2200
S.aureus NCIM 2079
- Greenish Colonies on Pseudomonas Agar Plates
30 to 350C
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 350C
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 350C
24 hrs
Bismuth sulphite agar
Salmonella abony NCIM 2257
S.aureus NCIM 2079
- Black or green Colonies on Bismuth Sulphite Agar Plates
30 to 350C
48 hrs
Xylose lysine deoxycholate agar
Salmonella abony  NCIM 2257
S.aureus NCIM 2079
- Red Colonies on Xylose lysine deoxycholate Agar
30 to 350C
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



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