Lesson 6. Microbial Enumeration Test of Paracetamol Oral Suspension

Microbial Limits Testing (MLT)

done to quantify viable microorganisms and verifying the absence of specific microorgansisms in finished pharmaceutical products and raw materials

non-sterile products

Microbial Limits Testing (MLT) are used for ____________________________

Microbial Enumeration Tests

used to accurately count mesophilic bacteria and fungi that thrive in aerobic conditions

mesophilic bacteria

bacteria that grows at room temperature (20 - 40 C) which includes ambient and body temperature

safety

Quantifying Viable Microorganisms are used for:

1. _________ reasons

2. _________ which includes

a. ________ - no change in its physical appearance

b. ________ - no change in composition

c. ________ - contains the appropriate amount of microbes as approved by the USP

1 = ?

stability

Quantifying Viable Microorganisms are used for:

1. _________ reasons

2. _________ which includes

a. ________ - no change in its physical appearance

b. ________ - no change in composition

c. ________ - contains the appropriate amount of microbes as approved by the USP

2 = ?

physical

Quantifying Viable Microorganisms are used for:

1. _________ reasons

2. _________ which includes

a. ________ - no change in its physical appearance

b. ________ - no change in composition

c. ________ - contains the appropriate amount of microbes as approved by the USP

2.a. = ?

chemical

Quantifying Viable Microorganisms are used for:

1. _________ reasons

2. _________ which includes

a. ________ - no change in its physical appearance

b. ________ - no change in composition

c. ________ - contains the appropriate amount of microbes as approved by the USP

2.b. = ?

microbiological

Quantifying Viable Microorganisms are used for:

1. _________ reasons

2. _________ which includes

a. ________ - no change in its physical appearance

b. ________ - no change in composition

c. ________ - contains the appropriate amount of microbes as approved by the USP

2.c. = ?

active

Microbiological Enumeration Tests are not used in drugs with microorganisms as ________ ingredient.

Membrane Filter Method

MICROBIAL ENUMERATION TESTS

• its filter has a pore size equivalent to 0.22-0.3 microns provides maximum filtration because its pore size is smaller than typical microorganisms with 0.45 microns as its size

• after filtration, the microorganisms will be left on the membrane filter

• membrane is placed on the culture medium, then incubated

Spread-Plate Method

MICROBIAL ENUMERATION TESTS

Conventional Method

• 0.1 sample is pipetted and spread evenly over the surface of the agar, it is incubated

• results in surface colonies

Pour Plate Method

MICROBIAL ENUMERATION TESTS

Conventional Method

• sample is pipetted into sterile plate before a sterile medium is added, then incubated

• results in surface and subsurface colonies

• technique used when we want to count colonies

Most Probable Number Method

MICROBIAL ENUMERATION TESTS

• used when sample contains particulate matter (ex: soil matter) which contains a smaller number of microbes

• uses a 10-fold dilution (undiluted, 1:10, 1:100) as well as Phenol red (pH color indicator)

• no of growth in medium: undiluted > 1:10 > 1:100

METHOD SUITABILITY TEST (METHOD VALIDATION)

Conducted to demonstrate the applicability of the method for the detection of microbial contamination in the test product

Growth promotion test

METHOD SUITABILITY TEST:

• To demonstrate that the medium supports growth and has the ability to detect organisms in the presence of the test sample

Suitability of counting method

METHOD SUITABILITY TEST:

• Based on sample characteristics and required microbial limits

Staphylococcus aureus

gram-positive coccus

Pseudomonas aeruginosa

non-fermentative gram-negative bacteria

Bacillus subtilis

Spore-forming gram-positive bacillus

Candida albicans

yeast

Aspergillus brasiliensis

Mold

Bacteria

METHOD SUITABILITY TEST (METHOD VALIDATION)

Growth Promotion Test

Test strains are prepared from stock cultures of the organisms

(a) ____________ – incubated in SCD Agar at 300 -350C for 18-24 hours

(b) ____________ – incubated in SDA at 200 -250C for 2-3 days (or 5-7 days for A. brasiliensis)

(c)Fresh cultures of each organism are harvested and standardized to an ________________________________ in phosphate buffer solution (pH 7.2)

a = ?

Fungi

METHOD SUITABILITY TEST (METHOD VALIDATION)

Growth Promotion Test

Test strains are prepared from stock cultures of the organisms

(a) ____________ – incubated in SCD Agar at 300 -350C for 18-24 hours

(b) ____________ – incubated in SDA at 200 -250C for 2-3 days (or 5-7 days for A. brasiliensis)

(c)Fresh cultures of each organism are harvested and standardized to an ________________________________ in phosphate buffer solution (pH 7.2)

b = ?

optical density of 0.1-0.3 at 550 nm

METHOD SUITABILITY TEST (METHOD VALIDATION)

Growth Promotion Test

Test strains are prepared from stock cultures of the organisms

(a) ____________ – incubated in SCD Agar at 300 -350C for 18-24 hours

(b) ____________ – incubated in SDA at 200 -250C for 2-3 days (or 5-7 days for A. brasiliensis)

(c)Fresh cultures of each organism are harvested and standardized to an ________________________________ in phosphate buffer solution (pH 7.2)

c = ?

Seed-lot systems

Growth Promotion Test:

a. _________________ are recommended for long-term storage, and stock culture of each organism is limited to not more than five passages removed from the master seed-stock

b. ________________________ should be used within 2 hours or stored at 2-8 C for not more than 24 hours

a = ?

Standardized cell suspensions

Growth Promotion Test:

a. _________________ are recommended for long-term storage, and stock culture of each organism is limited to not more than five passages removed from the master seed-stock

b. ________________________ should be used within 2 hours or stored at 2-8 C for not more than 24 hours

a = ?

Total Aerobic Microbial Count (TAMC)

counts both bacteria and fungal microorganisms

Total Combined Yeasts and Molds Count (TYMC)

counts all fungal microorganisms

streak plate method

In the Growth Promotion Test, what kind of method did we use to grow the colonies?

SCDA

used in growing both bacteria and fungi

SDA

used in growing fungi

SDA and SCDA

what kind of medium was used in Growth Promotion Test

SCDB (control), SCDA (test product), and SDA (test product)

what kind of medium was used in the Suitability of Counting Method

30-35 C for 3 days

incubation period of bacteria in SCDA and SCDB

30-35 C for 5 days

incubation period of fungi in SCDA and SCDB

20-25 C for 5 days

incubation period of fungi in SDA

20

1. 10^1 cfu – maximum acceptable count is

2. 10^2 cfu – maximum acceptable count is

3. 10^3 cfu – maximum acceptable count is

1 = ?

200

1. 10^1 cfu – maximum acceptable count is

2. 10^2 cfu – maximum acceptable count is

3. 10^3 cfu – maximum acceptable count is

2 = ?

2000

1. 10^1 cfu – maximum acceptable count is

2. 10^2 cfu – maximum acceptable count is

3. 10^3 cfu – maximum acceptable count is

3 = ?

microbiological count

The TAMC and TYMC determines if the ________________ of the product passed the acceptance criterion.

not be less than 50% and not be more than 200%

The mean count of the test organism (TAMCorg and TYMC org) must ___________________________________ from the value of the control.

Growth Promotion Test

demonstrates suitability of SCDA and SDA to detect the microorganisms used in the Microbial Enumeration Test.

Suitability of Counting Method

confirms the ability and suitability of the microbial enumeration test (based on TAMC and TYMC) to detect microorganisms in the presence of a product or raw material.