Top 10 Tips for Growth Promotion Testing

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The Growth Promotion Test can be a hassle, but it is necessary for determining if a new batch of media is acceptable. Use our top 10 tips for achieving Growth Promotion success with Microbiologics products to make your testing process as smooth as possible.

1. Test in parallel.
According to the USP, growth on new batches of Tryptic Soy Agar, Sabouraud Dextrose Agar and Potato Dextrose Agar must not differ by a factor greater than two from the calculated value for a standardized inoculum. The CFU value of the standardized inoculum can be determined by counting the number of colonies on the previously approved batch of agar.

Test a new batch of agar in parallel with the previously approved batch. Test in duplicate or triplicate. The average number of colonies on the new batch should be within a factor of two of the average number on the previously approved batch. By testing side-by-side, a laboratory eliminates all variables except the media. The microorganism suspension, environmental conditions and technician are the same.

2. Double the inoculum for selective medium, when necessary.
When using EZ-Accu Shot™, EZ-Accu Shot™ Select, EZ-CFU™ and EZ-CFU™ One Step, you may need to inoculate selective media, such as MacConkey Agar, with 0.2 ml instead of 0.1 ml of the microorganism suspension. This is because selective medium can be slightly inhibitory towards the organism it is supposed to grow. To determine if the inoculum needs be doubled, test the selective medium in parallel with a non-selective medium such as Tryptic Soy Agar. If no colonies grow on the selective medium but less than 50 colonies grow on the non-selective medium, the inoculum may be doubled.

Remember, you do not need to worry about the factor of two when testing the growth-promoting properties of selective media because the USP states growth on the new batch of media should be “comparable” to growth on the previously approved batch. The new batch and the previously approved batch should be tested side-by-side. Cetrimide Agar is very inhibitory. Test the agar with fresh colonies grown from a KWIK-STIK™ Plus device. KWIK-STIK Plus is only two passages from reference culture.

BDI_2015_9_30-5353. Use non-selective agar as a control when testing liquid media.

To determine if a new batch of liquid media is acceptable, test in parallel the new batch of media, the previously approved batch of media, and non-selective agar. The non-selective agar is necessary to determine the CFU concentration of the inoculum. The new batch of liquid media is acceptable if:

  • It is comparable in turbidity to the previously approved batch of media.
  • The number of colonies on the non-selective agar meets specifications (100 or less colonies when testing for growth promoting properties).

4. Routinely calibrate pipettes.

5. Warm lyophilized pellets before beginning test.
After taking lyophilized pellets out of the refrigerator, let them warm to room temperature before removing them from their vials. This takes approximately 30 minutes. In the case of EZ-Accu Shot, do not open the packet during this time because there is a desiccant inside. Moisture, caused by cool air condensing in the vial, is detrimental to lyophilized organisms.

6. Use correct vial of hydrating fluid when reconstituting pellets.
Pseudomonas speciesuse a different type of hydrating fluid from the other strains of organisms. The fluid contains Magnesium Chloride.

BDI_2015_9_25-3597. Mix, mix, mix.
In order to obtain a homogeneous suspension:

  • Warm EZ-CFU and EZ-CFU One Step pellets in pre-warmed hydration fluid at 34°C to 38°C for 30 minutes. This step allows the gelatin to melt
  • After adding EZ-CFU, EZ-CFU One Step or EZ-Accu Shot pellet(s) to the hydrating fluid, mix the fluid until the pellet(s) can no longer be seen and the microorganism suspension is homogeneous.
  • Spread the suspension across an agar plate with a spreader. The agar plate should be dry before use.
  • Remix the suspension periodically if performing multiple tests.

8. Use environmental conditions required by species.

  • Follow pharmacopeia guidelines.
  • Aerobes such as Pseudomonas aeruginosa and Bacillus subtilis need oxygen. When testing them in broth, provide
    head space in the tube or the container and leave cap slightly loose.
  • Use an anaerobic indicator when growing anaerobes such as Clostridium sporogenes.
  • Check and record incubator temperatures twice a day.
  • Validate incubators and calibrate thermometers on a routine basis to ensure incubators stay in correct temperature
    range.

9. Keep organisms warm…but not TOO warm.

  • Using the pour plate method? The molten agar should be maintained in a water bath at a temperature not more than
    45°C. Higher temperatures may harm the microorganisms.
  • The molten agar should not be kept in the bath for more than three hours.
  • Sterile, solidified agar can be re-melted only once.

10. Make certain you can find all objectionable organisms in your product.
Organisms other than the ones listed in the pharmacopeia can be deemed objectionable resulting in product damage or hurt a consumer. To be detected, some objectionable organisms may require special media or growth conditions other than the ones described in the USP. To ensure your laboratory can detect these organisms on media, you may want to preserve them for use as quality control organisms. Microbiologics offers custom lyophilization.

QC Microorganisms
Microbiologics offers four unique QC microorganism products designed specifically for the Growth Promotion Test that provide 10-100 CFU per 0.1 ml of hydrated suspension as required by the USP guidelines. Each product offers different features and benefits:

  EZ-Accu Shot™ Select EZ-Accu Shot™  EZ-CFU™ One Step EZ-CFU™
Available Strains 5 compendial strains in
1 convenient kit
19 licensed, traceable
strains
35 licensed, traceable
strains
29 licensed, traceable
strains
Number of Tests • 10 tests per vial
• 10 tests for each
organism per kit
• 10 tests per vial
• 50 tests per kit
• 19 tests per vial
• 190 tests per kit
• 90+ tests per vial
• 900+ tests per kit
Hydration Fluid (Included in Kit)  Equilibrate to room temp  Equilibrate to room temp  Pre-warm hydrating fluid 30 minutes Pre-warm hydrating fluid 30 minutes
Lyophilized Organism Pellet Use 1 pellet per
suspension to obtain
10-100 CFU / 0.1 ml
Use 1 pellet per
suspension to obtain
10-100 CFU / 0.1 ml
Use 2 pellets per
suspension to obtain 10-
100 CFU / 0.1 ml
Use 2 pellets per
suspension to obtain 10-
100 CFU / 0.1 ml
Resuscitation of Organism Quick-dissolve pellet, no
pre-incubation
Quick-dissolve pellet, no
pre-incubation
Incubate suspension for
30 minutes before
inoculation
Incubate suspension for
30 minutes before
inoculation
Dilutions No dilution step No dilution step No dilution step One log dilution required
Stability of Hydrated Suspension  8 hour stability* 8 hour stability*   8 hour stability*   30 minute stability

*Exceptions to the 8 hour stability are catalog numbers 0484A (EZ-Accu Shot and EZ-Accu Shot Select), 0318Z and 0320Z (EZ-CFU One Step) which must be used within 30 minutes of hydration.

11 Comments

  1. Raja

    Hello if I use Accu shot triplicat in the same day to approve media can I use this media as comparable approved media for another newly prepared media by using home prepared standardise ATCC?

    Reply
    1. Microbiologics

      Yes, to perform GPT testing on previously approved batches of media versus newly prepared batches, you can prepare your own standardized suspensions. However, preparing your own suspensions (and the subsequent dilutions that are required) can be subjective and therefore lead to unforeseen CFU variability in your inoculum. Because of this, you may have to plate several dilutions in order to achieve the correct inoculum level (of 10-100 CFU).

      Routinely using our EZ-Accu Shot products (on each and every lot of media) will eliminate the variability and subjectivity, and ultimately ensure that the correct inoculum level is consistently being used.

      Reply
  2. Tim Sandle

    Good advice, well presented.

    Reply
  3. N

    Hi. Im working in a new lab and we have just bought in our first lots of selective media to begin verification work. We have no previously qualified lots or historical data for comparison during growth promotion. What can I do in this situation? Can I use TSA as the control lot?

    Reply
    1. Microbiologics

      We would recommend that you plate 10 plates to create a mean to use and to set the standard for that media. Once you have established a mean, you will then move forward with comparison of recovery to the new lot of media compared to the previously approved lot. Also keep in mind that selective media is going to have less recovery than non-selective media (TSA), so we would recommend plating in parallel to TSA so that you know the percent recovery of the selective media compared to TSA.

      Reply
  4. Dani Soria

    Hello, I work in a pharmaceutical laboratory where they bought:

    P. AERUGINOSA ATCC 9027 KWIK-STIK DUOPACK

    S. TYPHIMARIUM ATCC 14028 KWIK-STIK DUOPACK

    S. AUREUS ATCC 6538P KWIK-STIK DUOPACK

    C. ALBICANS ATCC 10231 KWIK-STIK DUOPACK

    E. COLI ATCC 8739 KWIK-STIK DUOPACK

    I could use this product for testing to promote growth in new media culture? How can I know the number of CFU / 0.1 ml in KWIK-STICK DUOPACK?

    Reply
    1. Microbiologics

      Hello Dani,

      Our KWIK-STIKs are a non-enumerated product line; therefore, we do not guarantee a certain CFU beyond > 1000 CFU/pellet. If you would like to get a rough estimate of the CFU/pellet for a specific lot number, please reach out to our Technical Support team at 320-229-7045 or techsupport@microbiologics.com.

      If you are looking for products for Growth Promotion Testing, we would suggest using EZ-Accu Shot. Follow this link to learn more: http://microbiologics.com/Products/EZ-Accu-Shot

      Thank you

      Reply
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  6. Koos

    Hello,

    We perform Grotwh Promotion Testing on the media we receive.

    I want to introduce Growth Promotion testing on (TS)Agarstrips for Environmental Control from Millipore we receive. How do I perform Growth Promotion Testing on this product?

    Just spread 10 – 100 cfu over the surface of the strip?

    Reply
    1. Microbiologics

      Unfortunately we do not perform Growth Promotion Testing on (TS)Agarstrips. Although, I would suspect that you could distribute the 0.1 mL inoculum of our Growth Promotion products over the surface of the (TS)Agarstrips.

      Please let us know if you have any additional questions or concerns. Thank you and have a wonderful day!

      Reply
  7. Hassan

    Do you have any link for growth promotion technique of phosphate and peptone buffers ?

    If not can you suggest the optimal method ?

    Reply

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