The Growth Promotion Test - bdcint.com.do · 2018-10-29 · 8 hour stability* 10 tests per vial of...
Transcript of The Growth Promotion Test - bdcint.com.do · 2018-10-29 · 8 hour stability* 10 tests per vial of...
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!