The document summarizes the harmonized microbial limit tests established in 2006 by the USP, EP, and JP pharmacopeias. The tests include microbial enumeration tests to determine total aerobic microbial count and total yeast and mold count, as well as tests for specified microorganisms like E. coli, Salmonella species, and Candida albicans. The tests involve preparing samples, incubating them in various growth media, and observing colonies to quantify microbes and identify pathogens based on standardized methods, limits, and interpretations. The harmonization aligned the structure, methods, and acceptance criteria used across different pharmacopeias to ensure microbial safety of non-sterile pharmaceutical products.

1. June 2014
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MicrobialMicrobial
Limit TestLimit Test

2. The Microbial Limit Tests are designed to
perform the qualitative and
quantitative estimations of viable
microorganisms present in samples..

3. Prior to 2006,
The USP,EP and JP
used test methods to ensure microbial safety of
non-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.

4. 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 historically
referred to as “Microbial Limit Tests”
and appeared in
USP chapter <61> and JP 4.05.

5. 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.

6. 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

7.
Enumeration Methods
Membrane Filtration Method,
Plate Count Method
MPN method

8.
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)

9.
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)

10.
Limits: For FP/RM
For TAMC- NMT 1000 CFU/g
For TYMC- NMT 100 CFU/g
For FP Liquid prod.
For TAMC- NMT 100 CFU/mL
For 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 2
If there is No Growth observed in the plates
Report it as <10 CFU/g or mL

11.
Tests for Specified Microorganisms
(Pathogens)
Bile-Tolerant Gram-Negative Bacteria
E .Coli
Salmonella sp
Pseudomonas aeruginosa
Staphylococcus aureus
Clostridia
Candida albicans

12.
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

13.
Tests for Specified Microorganisms (Pathogens)
Candida albicans
From Sample prep. (SCDM)
Transfer 10mL to 100mL SDB
(Incubate at 20-25ºC/ 3 - 5 days )
Subculture
on SDA
(20-25ºC/ 24-48 Hrs )

14.
Tests for Specified Microorganisms (Pathogens)
E. coli
After 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 )

15.
Tests for Specified Microorganisms (Pathogens)
Salmonella. sps
After 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

16.
Tests for Specified Microorganisms (Pathogens)
Pseudomonas. aeruginosa
After 18-24 Hrs, From Sample prep. (SCDM)
Subculture on CA
(Incubate at 30- 35ºC/ 18-72 Hrs)

17.
Tests for Specified Microorganisms (Pathogens)
Staphylococcus. aureus
After 18-24 Hrs, From Sample prep. (SCDM)
Subculture on MSA
(Incubate at 30- 35ºC/ 18-72 Hrs)

18.
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)

19.
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

20.
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21.
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