1. Microbial Limit Testing Technology Overview Advisory Committee for Pharmaceutical Science July 19, 2001

2. ACPS2 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

3. July 19, 2001ACPS3 Compendial Methods ADVANTAGES Simple Counts only viable organisms DISADVANTAGES Incubation time (48-72 hours) Not all organisms will grow on a single medium

4. July 19, 2001ACPS4 Microbial Viability Only interested in viable microorganisms “New” methods must only count viable organisms Viability indicators needed

5. July 19, 2001ACPS5 Esterase Detection ChemScan ®, Chemunex Enzyme ubiquitous in microorganisms Chem-Chrome, a non-fluorescent fluorescein derivative cleaved by esterase to fluorescein

6. July 19, 2001ACPS6 ChemScan ® Procedure Filter Sample Expose membrane to reagent Membrane analyzed by laser-scanning

7. July 19, 2001ACPS7 ATP Bioluminescence MicroStar (TM) & MicroCount (TM), Millipore ATP- Primary energy source for all organisms Luciferin/luciferase (reagent) reacts with ATP to produce light

8. July 19, 2001ACPS8 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