Last reviewed: November 2026 by Dr. Farrukh Mehmood, Ph.D., 16+ years in pharmaceutical QC/QA
Key takeaways
- The growth promotion test (GPT) proves that each batch of culture medium can recover low numbers of microorganisms before it is used for testing.
- Inoculate not more than 100 CFU of each specified strain. For inhibitory tests, use at least 100 CFU.
- Solid media: recovery must be within a factor of 2 of the inoculum or reference count (50–200%). Liquid media: clearly visible growth, comparable to an approved batch.
- Selective media in USP <62> are also tested for inhibitory and indicative properties.
- Use cultures not more than 5 passages from the original reference strain.
Every microbiological result is only as reliable as the culture medium it was grown on. A medium that was overheated, prepared at the wrong pH or stored too long can fail to recover organisms, and that gives you false negatives. The growth promotion test is how a QC microbiology lab proves that a batch of medium works before it is released for use. This guide explains the GPT step by step for USP <61>, <62> and <71> media, with organisms, incubation conditions, acceptance criteria, a worked recovery calculation and a free GPT record template.
What is the growth promotion test?
The growth promotion test (also called the media suitability test or the test of fertility) checks that a culture medium supports the growth of a small, known number of specified microorganisms. For selective media, it also checks that the medium suppresses organisms it should inhibit, and that target organisms show the expected colony appearance or reaction.
The pharmacopoeias require the test for each batch of ready-prepared medium and each batch of medium prepared from dehydrated medium or from individual ingredients. In practice this means every lot of purchased ready-to-use plates and every batch you prepare and sterilize in-house.
The three properties tested
| Property | What it shows | Inoculum | Pass criterion |
|---|---|---|---|
| Growth-promoting | The medium recovers low numbers of the specified organisms | NMT 100 CFU | Solid: within a factor of 2. Liquid: clearly visible growth, comparable to an approved batch |
| Inhibitory | A selective medium suppresses non-target organisms | At least 100 CFU | No growth after incubation for not less than the longest time specified in the test |
| Indicative | Target organisms show the expected colony appearance or reaction | NMT 100 CFU | Colonies comparable in appearance and reaction to an approved batch |
Test microorganisms
Use reference strains from a recognized culture collection. Maintain them with a seed-lot technique so that the working culture is not more than 5 passages from the original master seed lot. Equivalent strains from other collections (NCIMB, CIP, NBRC) are acceptable where the pharmacopoeia lists them.
| Organism | Strain | Used for |
|---|---|---|
| Staphylococcus aureus | ATCC 6538 | <61> TAMC media, <62>, <71> FTM |
| Pseudomonas aeruginosa | ATCC 9027 | <61> TAMC media, <62>, <71> FTM |
| Bacillus subtilis | ATCC 6633 | <61> TAMC media, <71> SCDM |
| Candida albicans | ATCC 10231 | <61> TAMC and TYMC media, <62>, <71> SCDM |
| Aspergillus brasiliensis | ATCC 16404 | <61> TAMC and TYMC media, <71> SCDM |
| Escherichia coli | ATCC 8739 | <62> (growth-promoting and inhibitory control) |
| Salmonella enterica serovar Typhimurium | ATCC 14028 (or serovar Abony NCTC 6017) | <62> test for Salmonella |
| Clostridium sporogenes | ATCC 11437 or ATCC 19404 | <62> test for Clostridia, <71> FTM |
For media used in environmental monitoring and water testing, many labs also include representative in-house environmental isolates, because they reflect the organisms the medium must actually recover in your facility.
Preparing an inoculum of not more than 100 CFU
- Grow the bacteria on Soybean-Casein Digest Agar or Broth at 30–35 °C for 18–24 hours. Grow C. albicans on Sabouraud Dextrose Agar or Broth at 20–25 °C for 2–3 days. Grow A. brasiliensis on Sabouraud Dextrose Agar or Potato Dextrose Agar at 20–25 °C for 5–7 days, or until good sporulation.
- Suspend the growth in buffered sodium chloride–peptone solution pH 7.0 or phosphate buffer pH 7.2. For A. brasiliensis, add 0.05% polysorbate 80 to help suspend the spores.
- Dilute the suspension with serial tenfold dilutions to reach about 100–1,000 CFU/mL, so that the volume you inoculate delivers not more than 100 CFU.
- Use the suspension within 2 hours at room temperature, or within 24 hours if stored at 2–8 °C, unless your lab has validated a longer period.
- Confirm the count: plate the same volume of inoculum in parallel on a previously approved batch of medium (or on the standardized inoculum’s counting medium). This is the reference count for the recovery calculation.
Growth promotion test procedure, step by step
| Step | What to do | What to record |
|---|---|---|
| 1. Identify the batch | Note the medium name, manufacturer, dehydrated lot, preparation date, sterilization load and final pH. | Medium details section of the GPT record |
| 2. Select the plates or tubes | Take representative units from the batch: start, middle and end of the pour or fill. | Number of units tested |
| 3. Prepare the inocula | Prepare NMT 100 CFU of each specified organism (at least 100 CFU for inhibitory tests). | Strain, passage number, suspension ID, time of preparation |
| 4. Inoculate | Plates: spread or pour-plate the inoculum (in duplicate). Broths: add the inoculum directly to the tube or bottle. Run the approved reference batch in parallel. | Volume inoculated, time |
| 5. Sterility check | Incubate un-inoculated units from the same batch alongside the test. | Observation |
| 6. Incubate | Use the temperature and the shortest time specified in the test method for growth promotion (see the tables below). | Incubator ID, start and end time |
| 7. Read and calculate | Count colonies on solid media and calculate the ratio to the reference count. Check broths for visible growth. | Counts, ratio, growth +/− |
| 8. Conclude and release | Release the batch only if every organism meets the criterion and the sterility check shows no growth. | Conclusion, signatures, QA approval |
GPT of media for microbial enumeration (USP <61>)
| Medium | Organisms | Incubation |
|---|---|---|
| Soybean-Casein Digest Agar and Broth (TAMC) | S. aureus, P. aeruginosa, B. subtilis | 30–35 °C, NMT 3 days |
| Soybean-Casein Digest Agar and Broth (TAMC) | C. albicans, A. brasiliensis | 30–35 °C, NMT 5 days |
| Sabouraud Dextrose Agar (TYMC) | C. albicans, A. brasiliensis | 20–25 °C, NMT 5 days |
GPT of selective media (USP <62>)
| Test for | Medium | Growth-promoting | Inhibitory | Indicative |
|---|---|---|---|---|
| Bile-tolerant Gram-negative bacteria | Enterobacteria Enrichment Broth Mossel | E. coli, P. aeruginosa | S. aureus | – |
| Violet Red Bile Glucose Agar | E. coli, P. aeruginosa | – | E. coli, P. aeruginosa | |
| Escherichia coli | MacConkey Broth | E. coli | S. aureus | – |
| MacConkey Agar | E. coli | – | E. coli | |
| Salmonella | Rappaport Vassiliadis Salmonella Enrichment Broth | Salmonella Typhimurium or Abony | S. aureus | – |
| Xylose Lysine Deoxycholate (XLD) Agar | Salmonella Typhimurium or Abony | – | Salmonella Typhimurium or Abony | |
| Pseudomonas aeruginosa | Cetrimide Agar | P. aeruginosa | E. coli | – |
| Staphylococcus aureus | Mannitol Salt Agar | S. aureus | E. coli | S. aureus |
| Clostridia | Reinforced Medium for Clostridia; Columbia Agar | C. sporogenes | – | – |
| Candida albicans | Sabouraud Dextrose Broth | C. albicans | – | – |
| Sabouraud Dextrose Agar | C. albicans | – | C. albicans |
For growth-promoting and indicative properties, incubate for not more than the shortest period specified in the test. For inhibitory properties, incubate for not less than the longest period specified.
GPT of sterility test media (USP <71>)
| Medium | Organisms | Incubation |
|---|---|---|
| Fluid Thioglycollate Medium (FTM) | S. aureus, P. aeruginosa, C. sporogenes | 30–35 °C, NMT 3 days |
| Soybean-Casein Digest Medium (SCDM) | B. subtilis, C. albicans, A. brasiliensis | 20–25 °C, NMT 3 days for bacteria and NMT 5 days for fungi |
Sterility test media must show clearly visible growth of every organism within these times. Many labs run this test before or in parallel with the sterility test, so that a failed medium does not invalidate a batch release.
Acceptance criteria and recovery calculation
For solid media, the count on the test medium must not differ by more than a factor of 2 from the calculated count of a standardized inoculum. With a freshly prepared inoculum, it must be comparable to the count on a previously tested and approved batch. In practice, the ratio must fall between 0.5 and 2.0, which means 50–200% recovery.
Ratio = mean count on test medium ÷ mean count on reference medium (or calculated inoculum)
Example 1: test plates 44 and 50 CFU (mean 47); reference plates 58 and 62 CFU (mean 60). Ratio = 47 ÷ 60 = 0.78 (78%) → complies.
Example 2: test mean 24 CFU; reference mean 60 CFU. Ratio = 0.40 (40%) → does not comply.
Some labs use an internal criterion of 70% recovery. That is stricter than the pharmacopoeial factor of 2. If your SOP uses it, apply it consistently, but do not describe it as the USP requirement. For liquid media, the medium complies when clearly visible growth (turbidity) occurs, comparable to an approved batch, within the specified time.
Acceptance criteria at a glance
| Check | Typical media | Inoculum | Incubation | Acceptance criterion |
|---|---|---|---|---|
| Growth-promoting, solid (USP <61>/<62>) | TSA, SDA, selective agars | NMT 100 CFU | Not more than the shortest specified time | Count within a factor of 2 of the inoculum or approved batch (ratio 0.5–2.0, 50–200%) |
| Growth-promoting, liquid (USP <61>/<62>) | TSB, enrichment broths | NMT 100 CFU | Not more than the shortest specified time | Clearly visible growth, comparable to an approved batch |
| Inhibitory (USP <62>) | Selective media, e.g. MacConkey broth, Mossel broth | At least 100 CFU | Not less than the longest specified time | No growth of the test organism |
| Indicative (USP <62>) | Differential agars, e.g. MacConkey, mannitol salt, XLD | NMT 100 CFU | Within the specified time range | Colony appearance and indicative reactions comparable to an approved batch |
| Sterility test media (USP <71>) | FTM, SCDM | NMT 100 CFU | NMT 3 days (bacteria), NMT 5 days (fungi) | Clearly visible growth |
| Sterility check | Uninoculated units of the batch | None | As for the test | No growth |
The organisms for each medium are listed in the USP <61>, <62> and <71> tables above. Want to check your understanding? Try our QC microbiology quiz.
What to do if the growth promotion test fails
- Do not use the batch. Quarantine it and record a deviation according to your SOP.
- Check the test itself first: Was the inoculum count within range? Was the culture within 5 passages and viable? Did the reference batch perform as expected?
- Check the medium preparation: weighing, water quality, pH, over-sterilization or repeated melting, storage temperature, exposure to light, and expiry date.
- Repeat only with a documented, justified cause. If the medium itself is at fault, reject the batch and consider whether test results obtained on it need to be assessed.
- Trend failures by supplier and lot. Repeated failures may require supplier action or a change control.
Common mistakes
- Using an inoculum that is too high (for example 500 CFU). It makes almost any medium “pass” and hides poor performance.
- Not counting the inoculum in parallel, so there is no reference for the recovery calculation.
- Using cultures with too many subcultures, which can change their characteristics.
- Incubating growth-promoting tests for longer than the shortest specified time.
- Testing only one plate from a large batch, instead of representative units.
- Skipping inhibitory and indicative checks for selective media.
Download the free GPT record template
Growth Promotion Test Record (2-page PDF)
A ready-to-print record covering medium details, test organisms and inoculum counts, growth-promoting results for solid media (with a ratio column) and liquid media, inhibitory and indicative properties, the sterility check, the conclusion, sign-offs and a USP <61>/<62>/<71> quick-reference table.
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Frequently asked questions
Is GPT needed for ready-to-use media from a supplier?
Yes. The pharmacopoeias require testing of each batch of ready-prepared medium. Supplier certificates can support your assessment, but the user is responsible for showing that the medium performs after transport and storage. Some labs justify a reduced program for qualified suppliers, but this must be risk-assessed and documented.
Why must the inoculum be not more than 100 CFU?
Real samples usually contain low numbers of stressed organisms. A low inoculum challenges the medium in the same way. A high inoculum can grow on a poor medium and give a false pass.
What is the acceptance criterion for solid media?
Growth must not differ by more than a factor of 2 from the standardized inoculum or the approved reference batch. That means a recovery ratio of 0.5 to 2.0 (50–200%).
Can I use the media while the GPT is still incubating?
Some labs allow concurrent use under a documented procedure, but any results obtained become invalid if the GPT then fails. Releasing media only after a passing GPT is the safer default.
Which Salmonella strain is used for GPT?
Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028, or serovar Abony NCTC 6017. It is not Salmonella Typhi.
Related guides
- Serial dilution method in microbiology (with calculator)
- Total microbial count test (TAMC and TYMC)
- Microbiological testing of primary packaging material
- OOS procedure in pharma
- Microbiology quiz and QC microbiology quiz
References
- USP General Chapter <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests.
- USP General Chapter <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms.
- USP General Chapter <71> Sterility Tests.
- Ph. Eur. 2.6.12, 2.6.13 and 2.6.1 (harmonized equivalents).
Part of our QC microbiology guide, which maps every microbiology test, guide and tool on AskPharmaTutor in the order a lab uses them.
