Microbial Limit Testing

Technology Overview
Advisory Committee
for
Pharmaceutical Science
July 19, 2001

Compendial Methods
USP
Plate Counts- Colony Forming Units
Growth of microorganisms on solid medium
Count visible colonies

MPN- Most Probable Number

Multiple serial dilutions of sample
Based on statistical distribution of organisms in
a sample
July 19,

ACPS

Compendial Methods
ADVANTAGES
Simple
Counts only viable
organisms

July 19,

DISADVANTAGES
Incubation time (48-72
hours)
Not all organisms will
grow on a single
medium

ACPS

Microbial Viability
Only interested in viable microorganisms
New methods must only count viable
organisms
Viability indicators needed

July 19,

ACPS

Esterase Detection
ChemScan, Chemunex

Enzyme ubiquitous in microorganisms

Chem-Chrome, a non-fluorescent
fluorescein derivative cleaved by esterase to
fluorescein

July 19,

ACPS

ChemScan Procedure
Filter Sample
Expose membrane to reagent
Membrane analyzed by laser-scanning

July 19,

ACPS

ATP Bioluminescence
MicroStar(TM) & MicroCount(TM), Millipore

ATP- Primary energy source for all

organisms
Luciferin/luciferase (reagent) reacts with
ATP to produce light

July 19,

ACPS

MicroStar(TM) Procedure
Filter Sample
Place membrane on solid medium for brief
incubation
Disrupt cells to release ATP
Add bioluminescence reagent to membrane
Detect light with CCD and computer
analysis
July 19,

ACPS