Microbial Limit Test- An Over view

June 2014

Sreenath.SSreenath.SQC-Micro

WelcomeWelcomeMicrobial Microbial Limit TestLimit Test

The Microbial Limit Tests are designed to perform the qualitative and

quantitative estimations of viable microorganisms present in samples.. 

Prior to 2006, The USP,EP and JP

used test methods to ensure microbial safety ofnon-sterile pharmaceutical products that were similar in intent, but widely variable in execution and acceptance

criteria.

HARMONIZED MLT: These three pharmacopoeia collaborated over the

course of years to harmonize their testing methods and specifications for non-sterile pharmaceutical products.

The pharmacopeias released two chapters in 2006 entitled

“Microbiological Examination of Non-Sterile Products: Microbial Enumeration Tests” (TVAC)

and“Microbiological Examination of Non-Sterile Products: Tests

for Specified Microorganisms.” (Pathogens)

These tests were historicallyreferred to as “Microbial Limit Tests”

and appeared in USP chapter <61> and JP 4.05.

With the advent of the harmonization,

the structure of the USP and JP were altered and the two chapters are now USP <61> and <62>,

and JP 4.05 sections I and II.

The EP had a structure similar to the harmonized chapters,

and those tests are still found in EP 2.6.12 and 2.6.13.

The pharmacopeias set the scheduled implementation of the new harmonized methods

to August 1, 2007,

however the implementation was delayed to allow more time for users to comply with the

new requirements. The new harmonized methods become official in 2009

Enumeration Methods

Membrane Filtration Method,Plate Count Method

MPN method

Sample Size Water Soluble products – 1 in 10 dilution

Non fatty products insoluble in water- 1 in 10 dilution with 1g/L of polysorbate 80 (surface active agent)

Fatty products – Dissolve in Isopropyl myristate sterilized by filtration (if req, heat up to 40°C-NMT45°C)

Microbial Enumeration Tests (TVAC- TAMC & TYMC)Sample prep: 10 g /10mL of sample in 100 mL SCDM - & Mix 1 mL each

Transfer to petriplates

in duplicates

Pour 20-25mL of Sterile media at 45ºC

TAMC- SCDA (30-35ºC/5days) TYMC-SDA (20-25ºC/7days)

Incubate the plates

-ve Control with diluent

(30-35ºC/18-24 hrs)

Limits: For FP/RMFor TAMC- NMT 1000 CFU/gFor TYMC- NMT 100 CFU/g

For FP Liquid prod.For TAMC- NMT 100 CFU/mLFor TYMC- NMT 10 CFU/mL

TVAC- Results & Interpretation

Avg No. of Colonies observed on the plate X Dil Factor

For Ex.Plate-1 +Plate-2 X 10 4 + 3 X 10 = 35 CFU/g or mL

2 2If there is No Growth observed in the platesReport it as <10 CFU/g or mL

Tests for Specified Microorganisms (Pathogens)

Bile-Tolerant Gram-Negative Bacteria E .Coli Salmonella spPseudomonas aeruginosa Staphylococcus aureusClostridia Candida albicans

Tests for Specified Microorganisms (Pathogens)BT G-ve Bacteria:

From Sample prep. (after 2 hrs (to NMT 5 hrs) of incubation in RT/20-25°C)

Transfer 10mL to 100mL EEBM (Incubate at 30-35ºC/ 24-48 Hrs )

Subculture on VRBGA(30-35ºC/ 18-24 Hrs )

EEBM & VRBGA- Only Boiling

Tests for Specified Microorganisms (Pathogens)

Candida albicansFrom Sample prep. (SCDM)Transfer 10mL to 100mL SDB (Incubate at 20-25ºC/ 3 - 5 days )

Subculture on SDA(20-25ºC/ 24-48 Hrs )

Tests for Specified Microorganisms (Pathogens)

E. coliAfter 18-24 Hrs, From Sample prep. (SCDM)Transfer 1 mL to 100mL MCB (Incubate at 42- 44ºC/ 24-48 Hrs)

Subculture on MCA(30-35ºC/ 18-72 Hrs )

Tests for Specified Microorganisms (Pathogens)

Salmonella. spsAfter 18-24 Hrs, From Sample prep. (SCDM)Transfer 0.1 mL to 10 mL RVSEM(Incubate at 30- 35ºC/ 18-24 Hrs)

Subculture on XLDA(30-35ºC/ 18-48 Hrs )XLDA- Only Boiling

Tests for Specified Microorganisms (Pathogens)

Pseudomonas. aeruginosaAfter 18-24 Hrs, From Sample prep. (SCDM)

Subculture on CA(Incubate at 30- 35ºC/ 18-72 Hrs)

Tests for Specified Microorganisms (Pathogens)

Staphylococcus. aureusAfter 18-24 Hrs, From Sample prep. (SCDM)

Subculture on MSA(Incubate at 30- 35ºC/ 18-72 Hrs)

Tests for Specified Microorganisms (Pathogens)

Clostridia After 18-24 Hrs, From Sample prep. (SCDM)Transfer 10 mL to 100 mL RIMC in duplicates(Incubate at 30- 35ºC/ 48 Hrs-Anaerobic condition)

Subculture on COA(30-35ºC/ 48-72 Hrs)

Heat one tube at 80ºC for 10min (for spore)Second tube- as such (vegetative)

CAUSIONS

Antimicrobial activity of products-Neutralization

Beta-lactam products- Sterile Penicillinase enzyme

Boiling of media – EEBM/ VRBGA/ XLDA

Incubation of initial sample preparation-SCDM(2- NMT 5 Hrs.)

Aseptic Technique- Analysis in LAF

FUTURE CHALLENGE: Burkholderia cepacia

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