The Growth Promotion Test

Growth Promotion Testing (GPT)

What is GPT?How is GPT performed?What are frequent challenges encountered in the lab and how do we overcome them?

GPT: the Global StandardGPT determines the suitability of medium used in pharmaceutical tests

Used by pharmacopeias throughout the world

Suitable Media is Needed for:Type ofProduct

Test Purpose

Non-sterile Microbial EnumerationTests

Counts the total number of bacteria and fungus in a product sample

Non-sterile Tests for SpecifiedOrganisms

Looks for objectionable organisms such as Salmonella in a product sample

Sterile Sterility Tests Checks sterility of a product sample

Four Rules for the GPT1. Test each new batch of medium

2. Inoculate medium with small number of organisms

3. Organisms should be 5 passages or less from Reference Culture

4. Use organisms listed in USP

There are Variations in the GPT test

The Microbial Enumeration Tests, Tests for Specified Organisms, and Sterility Tests vary by:

Microorganisms

Medium

Test method

Acceptance Criteria for AgarCompare number of colonies on previously approved medium to number on new medium The number must be within a factor of 2Example:

• previously approved medium = 40• new batch of medium must =

20 to 80

Previously approved medium

New medium

Acceptance Criteria for BrothCompare turbidity in new medium to turbidity in the previously approved mediumTurbidity should be “comparable”Count colonies on non-selective agar: there should be ≤ 100

Previously approved broth

New broth

Control plates Soybean-Casein Digest Agar

How Microbiologics Can Help You

Four GPT products. Easy to use and guaranteed to deliver 10-100 CFU per 0.1 mlSaves time. No need for spectrophotometer, turbidimeter, or numerous serial dilutions Strains are only 4 passages from reference cultureCertificates of Analysis and Statistical Analysis can be downloaded from the website

New Pharmaceutical PackagingEasy flip-top latchCompact plastic containerRecyclableCondensed IFU cards

EZ-CFU™

Requires a 1:10dilution 30 minute stability> 90 tests per vial ofhydrating fluid

EZ-CFU™ One Step

No dilution step8 hour stability* 19 tests per vial of hydrating fluid

*Exceptions are Bacteroides fragilis, 0320Z, and Clostridium perfringens, 0318Z

EZ-Accu Shot™

Instant dissolve pelletsNo dilution stepNo pre-incubation required8 hour stability*10 tests per vial

*An exception is Pseudomonas aeruginosa, 0484A

EZ-Accu Shot™ Select

Ideal for low volume users10 tests for each organism per kitFive Pharmacopeiacompendial strains for GPTIndividual peel-off label for eachstrain

Microbiologics GPT productsProduct CFU Applications/Specifications

EZ-CFU™ Delivers 10-100 per 0.1 ml when processed as directed

Requires a 1:10 dilution 30 minute stability

> 90 tests per vial of hydrating fluid

EZ-CFU™ One Step Delivers 10-100 per 0.1 ml when processed as directed

No dilution 8 hour stability*

19 tests per vial of hydrating fluid

EZ-Accu Shot™ Delivers 10-100 per 0.1 ml when processed as directed

• Instant dissolve No dilution

8 hour stability* 10 tests per vial of hydrating fluid

EZ-Accu Shot™ Select Delivers 10-100 per 0.1 ml when processed as directed

5 different compendial strains in one package

Instant dissolve No dilution

8 hour stability* 10 tests per vial of hydrating fluid

* Exceptions to the 8 hour stability are catalog numbers 0484A, 0318Z and 0320Z which must be used within 30 minutes.

Method Prep of Reference Culture Strains

Prep of Inoculum

Counting time

Total Hands on time

Total Incubation Time

# of Steps

Spectro-photometric

10 min prep18 hrs or more incubation

85 minutes –Hands on time

20 minutes 115minutes

38 hoursbacteria

21

Turbidi-metric

10 min prep18 hrs or more incubation

85 minutesHands on time

20 minutes 115 minutes

38 hours bacteria

21

EZ-Accu Shot

No prep 10 minutes Hands on time

20 minutes 30 minutes

19 hours bacteria

11

ChallengesFactor of 2Growth on non-selective mediumGrowth on selective medium

Challenge: Factor of 2The pharmacopeia says, “For solid media, growth obtained must not differ by a factor greater than 2 from the calculated value for a standardized inoculum”The value of the standardized inoculum is obtained by counting the number of colonies on the previously approved mediaPerform side-by-side testing for direct comparison of new and previously approved media

Only one variable: the mediumOther variables eliminated,

including:• Temperature• Atmosphere• Technique• Method• Incubation time

Why Side-by-Side Testing?

Variable - Temperature Candida albicans after 48 hours incubation

25°C 20°C

Variable - AtmosphereAspergillus brasiliensis taped and not taped

Taped Not Taped

Variable - AtmosphereBacillus subtilis with much air and with little air

With air Without air

Variable – Incubation TimeBacillus subtilis will form secondary colonies after 24 hours of incubation

Variable -TechniquePipetting angle effects results

998

1000

1002

1004

1006

1008

1010

Sample 1

Sample 2

Sample 3

Sample 4

Pipetting w/angle

Pipetting-no angle

Micro Liters

Variable-MediumVariances in Media Manufacturer

• GPT was performed on various brands of Soybean-Casein Digest Agar using the same suspension of EZ-Accu Shot™, Pseudomonas aeruginosa, 0484A. The test was performed in duplicate.

Tryptic Soy Agar CFU per 0.1 mlBrand A 36Brand B 45Brand C 57Brand D 38Brand E 21Brand F 10

Conclusions• CFU results can vary between media manufacturers• GPT test is important to determine if medium is suitable

Challenge: Achieving Growth on Non-selective Medium

Troubleshoot/consider the variables (for example, were the conditions for the Bacteroides truly anaerobic?)Check broth recovery with agarAn environmental or unusual organism may need a special agar or special growth conditions

Clostridium sporogenesThe same suspension of C. sporogenes was used to inoculate 3 types of non-selective mediaAll the media were incubated anaerobically

Media Recovery

Soybean-CaseinDigest Agar

18 CFU

Anaerobic Blood Agar 44 CFU

Columbia Blood Agar 50 CFU

Best PracticesMix thoroughlyUse dry platesUse correct growth conditionsVerify pipettes deliver correct volumeTest in duplicate or triplicateTest a negative control

Best Practices for Pour PlatesKeep melted medium for pour plates at <50°C (44°C to 46°C is best)Give microorganisms sufficient time to grow. It takes longer for colonies to appear on pour plates than on spread platesA back-lit colony counter can make it easier to see small colonies such as Candida albicans

Effects of OverheatingThe same suspension of E. coli was used to inoculate both pour plates The medium on the left was poured at 45°C The medium on the right was poured at 59°C Both plates were incubated 48 hours

Take-awaysThe purpose of the Growth Promotion Test is to ensure the media used to test products can recover small numbers of organismsTesting in parallel eliminates all variables except for the mediaUse best practices to obtain accurate and consistent results

Thank you!