Microbiology Flashcards
What are the basic types of micro-organism?
What are the two main types of bacteria?
Bacteria
Fungi
Yeast
Virus
Mycoplasm
Gram positive and gram negative bacteria
What types of culture media are there?
Broad spectrum- TSB
Selective;
Cetrimide agar- Pseudomonas
Mackonkey- Gut organisms (E-Coli)
Baird-Parker- Staphlococcus Aureus
Anaerobic;
Fluid Thyoglycolide media (Antioxidant)
What are specifications for incoming media?
Sterility
Appearance, colour, PH, Fill volume.
Growth promotion
How would you validate a particular culture method?
Using a plate of media…
Selectivity of organism
Repeatability
Range (concentration ranges of CFU)
Linearity (CFU dose/response curve)
What are the sources of microbial organisms?
Man
Materials
Environment
Equipment/Utilities
Facility
What are the different types of organism enumeration methods?
Pour plate -Sample + Media
Spread plate -Sample on media
Membrane filtration - Sample filtered and filter membrane incubated
Miles Mirsa- Dilution 4x and incubate
Most probable number- Serial dilution and incubate
Dip slides- Buy slides, dip into sample.
What factors do you consider important while validating microbial methods?
Product toxicity (Neutralisation requirements)
Recovery efficiency
PhEur
Growth promotion organisms;
- Staph Aureus
-Pseudomonas Arugenosa
-Bacillus Subtilis
- Aspergillus Niger
- Candida Albicans
Incubate NMT 100Cfu, recovery must be 50-200CFU.
Outline a process for identification of micro-organisms
Obtain a pure culture- Spread plate method
Morphology (Size, shape, colour, colonies)
Discriminatory tests- Gram stain with crystal violet and Oxidase test for aerobic/anaerobic.
Specific tests for metabolism, Antibiotic resistance, Phage typing (virus response) and serology (Antibody presence).
Coagulase test (Staph Epidermis vs aureus)
Catalase (Staph vs Strep)
Oxidase (Aerobe vs anaerobe)
What would you expect in each phase of a micro lab OOS Lab investigation?
1a-
Negative control growth
Incubator failure
Obvious sample contamination
Equipment failure
Wrong method
1b-
Trend data
Training, plate dilutions
Review of plate specifications
2-
Raise deviation
Manufacturing investigation
Retest may not provide any useful data (micro!)
Inform stability team if on stability sample
3-
Post rejection
plant impact
Impact to other products
What are raw material micro specifications?
Consider risk for final product (Sterile/non sterile) and nature of sources (Synthetic vs natural)
Raw material standards same as for OSD
Parenteral use;
TAMC 100cfu
TYMC 10 CFU
What limit of water activity means unlikely to support microbial growth?
0.6aW
What are the pharmacopeial limits for micro for the following dosage forms;
1
Oromucosal
Gingival
Nasal
Ear
Cutaneous
Transdermal
- Oral Liquid
- Oral Solid
- Rectal
- Vaginal
- Inhalation
1
100cfu/ml or g TAMC
10cfu/ml or g TYMC
Absence of Staph Aureus and Pseudomonas Aurgenosa.
- 100cfu/ml or g TAMC
10cfu/ml or g TYMC
Absence of E-coli - 1000cfu/ml or g TAMC
100cfu/ml or g TYMC
Absence of Ecoli - 1000cfu/ml or g TAMC
100cfu/ml or g TYMC - 100cfu/ml or g TAMC
10cfu/ml or g TYMC
Absence of Staph Aureus, Pseudomonas Argenosa, Candida Albicans - 100cfu/ml or g TAMC
10cfu/ml or g TYMC
Absence of Staph Aureus, Pseudomonas Argenosa, Bile tollerant organisms (E-coli/salmonella)
What methods are used to test for pyrogens?
How would you validate the main test?
Rabbit test- pyrogenic temperature response.
LAL test- horeshoe crab blood
-Turbidometric
-Chromogenic
Recombinant factor C - synthetic clotting factor to replace crab
Monocyte activation test- Human cells - read by Eliza
LAL test validation;
- Establish Lysate sensitivity (dose/ response curve with reference standard endotoxin)
- Establish recovery efficiency from product/ water (neutralisation)
- Determine maximum valid dilution to allow recovery of endotoxin result
- Determine interfering factors- proteins, product, peptodiglycan.
Outline how you wold expect sterility testing to be preformed for your product.
What are compendial growth promotion organisms for each test?
What would you consider for validation of the sterility tests?
What events would lead you to invalidate a sterility test?
Following PhEUR guidance
Stats mean that 90% chance of pass result with 1/100 contaminated units- test for gross contamination.
TSB- Incubate depot from direct inoculation at 20-25 degrees for 14 days. Detection of aerobes, yeast, fungi.
Fluid thyoglycolate- Incubate depot from direct inoculation at 30-35 degrees for 14 days. Detection of anaerobes and some aerobes.
Compendial organisms;
TSB- Candida Albicans, Aspergilus Niger, Bacillus Subtilis
Fluid Thyoglycollate- Costridium sporonges, Pseudomonas Aurgenosa, Staph aureus.
Validation of sterility test;
Growth promotion of compendial organisms with and without product (toxicity and recovery). 5 day inoculation- look for comparable growth.
Invalidating a sterility test;
With caution- need to have direct link.
- EM of sterility test shows unequivocal fault
- Fault in test procedure
- Growth in negative controls
- Link to ID found outside of production
How would you expect preservative effectiveness testing to be performed for a multi dose product?
What are the compendial organisms used for PET?
PhEur test guidance
Final product container
10 to the 6 organisms innoculated into product and held at 20-25 degrees.
Acceptance criteria varies for dosage forms.
Parenteral/ occular
Ear/nasal/Inhalation
Oral/rectal
Maximum 3 log reduction in 24 hours, no recovery after 28 days.
6hr, 24hr, 7, 14, 28day time points.
Compendial organisms;
Saph Aureus
Pseudomonas Aurgenosa
Candida Albicans
Aspergillus Brazilliens
E-coli
