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Presentation Focus
1. Microbiology
2. Gram Staining
3. Microbiology in Sterile Operations
4. Sterility & Statistical Capability
Disclaimer
The views expressed in this
presentation are not necessarily
of the Drug Regulatory Authority
of Pakistan
It is an informal communication
that represents the best current
judgment and does not constitute
an advisory opinion or binding or
otherwise obligate or commit the
DRAP, to the views expressed.
This presentation is designed by
the speaker after consulting
international reference
documents
Karachi University
National Seminars,
Conferences
Experience of Hospital Pharmacy
Experience of
Regulatory Services
International Meetings &
Trainings
Microbiology
Tiny living things
Microorganisms that exist as single cells capable of growth, respiration, reproduction independent of other cells of the same or different kind.
Gram Staining
“I have therefore published
the method, although I am
aware that as yet it is very
defective and imperfect; but
it is hoped that also in the
hands of other investigators
it will turn out to be useful”
Hans Christian Gram, 1884
Environmental Requirements
Nutrients Respiration Temperature
Humidity pH
Water also carry the microorganisms and supports growth too
Respiration
Obligate Aerobes
• Must have oxygen to grow
Facultative Aerobes
• Grow with or without oxygen
Obligate Anaerobes
• Cannot grow if oxygen is present
Temperature
Psychrophiles
• Low temperature 0-20°C
Mesophiles
• Mid temperature 15-45°C
Thermophiles
• High temperature 40-70°C
Extremes
Volcanic Vents
• High temperature
Antarctica
• Low temperature
• Low nutrients
Mount Everest
• O2, low temperature, low nutrients
Sahara Desert
• low moisture, high temperature, low nutrients
WFI Systems
• Low nutrients
Microorganisms
Fungi
Eukaryotes
More animal than plant
No Photosynthesis Use carbon fixed by other organisms
Microscopic Fungi
Yeast
• Single cell
Mold
• Multi-cellular filaments
Microorganisms
Bacteria
• Prokaryotes
• Bacteria
• Most abundant of all microorganisms
Virus
• Small
• Genetic material with a protective coat
• Must infect host cell to reproduce
Endospores
Dormant, tough, non-reproductive structure
Forms in response to environmental stress
Can survive for extended periods of time
Produced by Gram Positive (+) Bacillus (rod)
Microbe Movement
Microbes do not …
• Crawl
• Swim
• Fly
Microbes must be carried by …
• Air flow
• Water flow
• Contact and transfer
Putting it all to use
Human Operation/ skin/ hands & hair
1Small/
medium colony
White, beige to yellow
Circular and
convexButtery
Staphylococcus species
Cocci, Gram +ve
Putting it all to use
Environment such as soil (seasonal factor)Through shoe, air etc.
2 Medium/ large colony
Off white to beige
Circular/ irregular
and convex/ raised
Mucoid, dry to grainy
Clostridium species
Rods, Gram +ve
Putting it all to use
Generally found in water (equipment piping )
3Small to medium colony
White, off white to beige
Circular/ irregular
and convex
Mucoidto
buttery
Pseudomonades, E. coli species
Rods, Gram -ve
Compendial Organisms (USP … ICH)
Staphylococcus aureus
• Aerobic
• Gram +ve
• Coccus
Pseudomonas aeruginosa
• Aerobic
• Gram -ve
• Rod
Bacillus subtilis
• Aerobic
• Gram +ve
• Rod
• Spore forming
Clostridium sporogenese
• Anaerobic
• Gram +ve
• Rod
• Spore forming
Candida albicans
• Yeast
Aspergillusbrasiliensis
• Mold
Sterility Test-Growth Promotion (support the growth of wide range of organisms)
Sterility
A strict definition of sterility is the complete absence of life or inability to reproduce
• Cont’d
However, sterility as defined in the pharmaceutical industry has a more complex technical definition, and is expressed in quantitative terms
Sterility
The probability definition for sterility derives from the fact that microbial death follows a geometric progression .i.e. the cells do not all die at once when exposed to lethal conditions
• Cont’d
The US FDA defines sterilization as the
“reduction of a microbial population to 10° (or one cell) plus an additional 6 log-cycles”
Sterility (Statistical Capability-1 in a million)
Sterility is defined as the “probability of a non-sterile unit (PNSU) in a lot or batch of product”
PNSU is also referred to as the “Sterility Assurance Level” or SAL
A product is considered sterile when the probability of a non-sterile unit is < 1 in 1, 000, 000 units; or a SAL of 10-6
Exponential Death
Microorganisms die at a constant
rate over a period of time
For e.g. one million bacteria
with a 90% death rate/ min
1st min
900, 000 dead/ 100, 000 survivors
2nd min
90, 000 dead/ 10, 000 survivors
3rd min
9, 000 dead/ 1, 000 survivors
4th min
900 dead/ 100 survivors
5th min
90 dead/ 10 survivors
6th min
9 dead/ 1 survivor
7th min
• ?
Constant set of lethal conditions develop resistance