The growth promotion test (GPT) can be a hassle, but it’s necessary for determining if a new batch of media is acceptable. Follow our best practices below to make your testing process as smooth as possible when using Microbiologics products to perform GPT.

1. Test in parallel.
According to the United States Pharmacopeia (USP) Chapter <61>, 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 colony forming unit (CFU) value of the standardized inoculum can be determined by counting the number of colonies on the previously approved batch of agar.

We recommend testing 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™, or 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. USP Chapter <62> 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. Read our 9 Tips for Growth Promotion Testing on Selective Media for more on this topic.

3. Use non-selective agar as a control when testing liquid media.
To determine if a new batch of liquid media is acceptable, test the new batch of media in parallel with a previously approved batch of media and non-selective agar. The non-selective agar is necessary to determine the CFU concentration of the inoculum and to show you inoculated with fewer than 100 colonies. 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 fewer colonies when testing for growth promoting properties).

4. Routinely calibrate pipettes.

5. Be sure a homogeneous suspension is achieved.

• EZ-Accu Shot pellets are quick dissolve and can be vortexed immediately after being added to the hydration fluid. Mix the suspension until the pellet is completely dissolved and the suspension is homogenous.
• 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 excipient in the pellets to melt and ensure the pellet will dissolve in the hydration fluid. Following the 30 minute incubation step, vortex the fluid until the pellets can no longer be seen and the microorganism suspension is homogeneous.
• Spread the suspension evenly across an agar plate with a spreader. The agar plate should be dry before use.
• Remix the suspension periodically if performing multiple tests.

6. Use environmental conditions required by species.

• Always 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.

7. 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.
• Read our 11 Pour Plate Method Best Practices post for more tips.

8. 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 consumers. 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.

Learn more about isolate preservation and custom controls from Microbiologics.

QC Microorganisms
Microbiologics offers four unique QC microorganism products designed specifically for GPT that provide 10-100 CFU per 0.1 ml of hydrated suspension as required by USP guidelines.

*Catalog numbers 0353A and 0484A (EZ-Accu Shot and EZ-Accu Shot Select), and 0320Z (EZ-CFU One Step) must be used within 30 minutes of hydration.

Custom Controls for Environmental Monitoring
Microbiologics can simplify GPT for objectionable organisms in your laboratory. Send us your isolates for custom preservation and we’ll create a GPT kit designed for your laboratory with your isolates. Visit our website to learn more.

Read Next – Non-Sterile Pharmaceuticals: Next Steps After Finding an Environmental Isolate