Micro Flashcards
Typical Gram positive
Typical Gram-positive bacteria
1.
staphylococci such as Staphylococcus epidermidis and Staphylococcus aureus which is a common cause of boils
2.
streptococci such as the many species of oral streptococci, Streptococcus pyogenes which causes many a sore throat and scarlet fever and Streptococcus pneumoniae which causes lobar pneumonia
3.
clostridia such as Clostridium tetani which cause tetanus (lockjaw)
4.
actinomyces such as Actinomyces odontolyticus which is found in mouths
5.
species of the genus Bacillus such as Bacillus subtilis which are common microbes living in soil
Typical gram negative bacteria
Typical Gram-negative bacteria
the bacilli that cause
1.
whooping cough, Bordetella pertussis
2.
typhoid, Salmonella typhi
3.
cholera, Vibrio cholerae
4.
the normally benign, ubiquitous, gut-dwelling Escherichia coli
Generally cocci are Gram-positive but there are exceptions. The most significant from a clinical point of view is the gonococcus, Neisseria gonorrhoea which typically appears as a Gram-negative diplococcus looking very much like a pair of kidney bean.
What are the major groups of microorganisms?
Viruses Mycoplasma Rickettsia Bacteria Fungi Protozoa
What are the differences between prokaryotic and eukaryotic microorganisms?
Prokaryotic: lack defined nucleus or membrane bound organelle
Eukaryotic:nuclear envelop + microtubules in cell devision
What is the difference between streptococcus and staphylococcus?
Staph: coccus in clusters
Strep: coccus in chain
What are the characterisitcs of a gram positive cell wall?
Thickness: 20-80nm thick
No outer membrane/capsule (which is resistant to Ax) - source of endotoxin
Provides structural integrity + transportation of nutrients via protein channels
Attach mechanisms to surfaces
What are the main structural components of a bacterial cell?
Cell wall
Outer membrane
Nucelus + ribosome + mitochondria + pilli + flagelle + cell membrane + cytoplasm
What is a bacterial spore?
From gram positive bacilli
complicated morphological and biochemical process (7 stages)
Result in thick walled, highly resistant endospore with low water content and metabolic activity
Improve Env factors - germinate and return to vegetative form
What are the main phases of microbial growth curve?
Multiply by binary fission lag phase Log phase Stationary phase Death phase
Describe the process of carrying out Gram stain.
Glass slide + culture - dried Crystal violet (1min) - stain gram +ive purple wash with DIW Iodine (mordant) (1min) Wash with DIW Alcohol treatment (5-10sec) wash with DIW neutral red (1min) - stain gram -ive pink wash with DIW Dry
What are the key characteristics of common bacteria found in the pharmaceutical env?
Staphylococcus (staph. Epidermidis)
Human skin
Staph aureus less commonly found - nasal passage + wound infection + boils
Gram positive cocci
Bacillus
Widespread in the env: soil + water + cardboard, paper and wood
Spore forming
Gram positive bacili
Pseudomonas (Ps aeruginosa)
Water: drains and surface water + U bends of sinks + water sys deadlegs + bore hole water supply + Mop heads and disinfectant buckets + equip stored wet
Large number - musty smell
Sensitive to lack of moisture + temp over 45-50 dec
Other water source - acinetobacter, Achromobacter and enterobacter
Gram negative bacilli
What are the key characteristics of common fungi found in the pharmaceutical env?
Eukaryotic microorganisms
Cell wall composed of chitin - not peptidoglycan
Degradation of organic matter
Moulds vs yeasts
Moulds
Spore forming - thoudsands at a time - less resistant than bac spore
requires longer period of incubation + specialised method of staining
Cardboard shippers, exposed plaster and damaged pipework lagging
Yeasts
Large gram positive spheres/ovoids
moderately resistant spores - less resistant buds
require higher level of nutrition and moisture
Spoilage of product
What is a typical formula of a broth? What are limitations?
Tryptone soya broth: Tryptone Soy peptone NaCl Dipotassium phosphate Dextrose Cannot give any indication of the range of organisms present Does not give any indication of the number of organisms at the start of the growth
What are the different types of solid growth media?
General purpose: Tryptone Soy Agar
Enhanced media: Chocolate Blood Agar - Factor X+Y - Neisseria sp
Selective media: Cetrimide agar (contains Ax)
Differential media:Baird Parker (staph aureus and other) - colour change + MacConkey Agar (Bile salts)
Enrichment media: enhance nutrition - sub-lethally damaged
What are the different method of microbiological enumeration?
Miles misra Technique
Serial dilution until 20 -80 bacterial per 0.5mL
4 dilutions are plated and 4 drops per dilution
Useful when accurate count required
starting innoculum level unkown
Use a lot of materials
Pour plate technique
1mL sample pipetted into empty plate then pour media
1 to 500 bacteria per mL
dilution needed if more concentrated
high temp may kill bacteria
colonies may obscure
The Spread Plate Technique
0.1mL sample spread onto the surface of an agar plate
homogenate would obscure the count
10-100 cfu
higher count need furhter dilution
Most Probrable Number Technique
Serial dilution 1 in 10
sample innoculated into tubes containing nutrient broth
Turbitity is then used to calculate and compared with statistical tables
MPN cacluated
The Membrane Filtration Technique
Most widely used in Pharm industry
disolve sample in a neutral carrier liquid
pass the solution through a bacteria retentive membrane filter
Place membrane onto a nutrient agar
Bac retained forms visible cfu
Inhibitory aspects removed by filtration
What are the considerations when validating microbiological methods?
Product toxicity:
antimicrobial / bacteristatic qualties must be neutralised
neutralising agents dependant on nature of the inhibition
membrane filtration adv - common to include a neutralising rinse as part of the test
Contamination recovery:
Innoculate with known numbers of a range of bacteria
Compare results with innoculant level
Assess neutralisation efficacy as well as contamination recovery
Operator validation
replicate sample testings to evaluate operator consistency
Paired operator sample testing
External QA systems
What are the automated methods for identification?
Substrate ultilisation: Reduce medium volume Enhancing sensitivity of result detection Need pure culture to give good results i.e. Biolog Gas chromatography of fatty acids discrimination between organisms on the basis of their fatty acid content as measured by GC i.e. MIDI MIS
What are the types of rapid methods for detection?
detect specific organisms in mixed cultures
PCR based methods
Applies to DNA and RNA
Amplified nucleic acid by PCR - detected using labelled cpmlementary DNA probes
Target: chromosomal DNA and Ribosomal RNA
Adv: Specific + sensitive + quick
Weak: not robust + expensive
What are the main types of rapid enumeration methods?
ATP Bioluminescence Use enzyme luciferase - uses energy of ATP to produce light Sesitivity: 10^3 /mL seconds to get results DEFT Direct epifluorescence filtration testing filtrate through a black filter stain organisms with fluorescent stain Can't tell if it was dead or alive Chemiluminescence Absorption of fluorochrome by organsims detect by laser scanning analysis Impedence Measure impedence in medium due to increase in microorganism numbers
What are the common microorganisms in the atmospheric habitat?
Commonly organisms which can withstand extended periods of desiccation
Spore forming bacteria: Bacillus spp, Clostridium spp
Non spore forming G+ive: Staphylococcus spp + corynebacterium spp
Sporing mould: Penicillum spp + Aspergillus spp, Mucor spp
Yeasts: Rodotorula spp
Enter via vehicle i.e. particles
What are the common microorganisms resides on human bodies?
Skin: Staph + Strep + Corynebacteria + Coliforms (E coli, Enterobacter species + mycobacteria + fungi including yeasts
Eye: Staph + strep + Neisseria + Corynebacteria + branhamella catarrhalis
URT: Acinetobacter + Staph + strep + Branhamella catarrhalis + neisseria + Corynebacteria + candida alb + Spirochetes + Actinomyces
Mouth: Staph + Strep + Lactobacillus + Fusobacterium + Bacteriodes + spirochetes + Veillonella
Intestine: G-ive anaerobic rods (Bacteroides, fusobacterium) Pseudomonas aeruginosa + Lactobacilli +
What are the main reasons for identifying isolates?
Assess risk
ID source
Prepare strategy for control
Recognition of repeated isolation
What are the controls in place to prevent contamination of water sampling?
IMS sample point -contact time
Flush sample point for 2 min - splash back
Sampling procedure must reflect in use procedure - if hose is used, use hose
Container: sterile? Pyrogen free? Label for traceability
transfer to lab 4-6hrs
Staff training
How does LAL test work?
LAL is an aq extract obtained after lysis of blood cells of the horseshoe crab limulus polyphemus
Endotoxin: G-ive bacteria outer memberane lipopolysaccharide + cell death
Unit of measurement: EU/mL
No direct link between endotoxin level and number of bacteria present
What is the structure of LPS?
Polysacchride side chains
Core polysaccharide
Lipid A: di-glucosamine backbone - ling chain fatty acid linked
What are the methods available for the deactivation of endotoxin?
Acid base hydrolysis
Strong acid break linkages between lipd A and KDO
Strong alkalis saponisation of the fatty acids
Oxidation:
H2O2: 0.1% 2 hrs at 100 deg
Dry Heat:
By incineration
purified endotoxin - twice more resistant hence worst case
250 deg
What are the methods of removal of endotoxin?
Rinsing
Rinse with pyrogen free water
i.e. components for filling and eqiupment leaving the cleanroom
Distillation
dobule phase change: liquid - vapour - liquid
Ultrafiltration
Molecular wt cut off: 100,000 delton
Reverse osmosis
micorbiological vulnerability of the sys - not 100% fool proof
Activated carbon
Efficient means
produce fines - need filtration
Electrostatic attraction to charged media
Endotoxin: negatively charged - attract to positively charged matrices
Hydrophobic attraction
Aliphatic polymers: PVDF, PTFE
Describe a LAL test
Ph Eur:2.6.14
Endotoxin + Ca2+ / MG 2+: activate clotting factor
stadardised, freeze dried lysate redissolve in pyrogen free water
Mix with serial diluted test solution
Incluabe at 37 deg for 60 min
last dilution at which coagulation is observed - corresponds to an endotoxin conc equivalent to the stated sensitity of the lysate
How do you test for inhibition of test solution to LAL coagulation activity?
Extreme pH
Protein denaturants
Enzyme inhibitors
Substrate interfere with human blood clotting
Spike test solution with amount twice stated sensitivity of the lysate
As the solution dilute out - if inhibit, neat solution will have no clot
How do you test for enhancement in LAL test?
Test cleanest sample and compare with pyrogen free water
What are the factors to be considered when looking at microbial contamination of the product?
Type of organisms: pathogenic?
Number of organisms present:
Patient’s susceptibility
Route of administration
What are the ideal characteristics of a preservative?
Broad spectrum
Rapid action
Chemically stable and effective under all pH conditions
Compatible with excipients and packaging materials
Physically undetectable
Safe to use
Cost effective
What are the strategies to preservative system?
Available water
Most organism req over 70% AW = vapour press of product / water
pH
Majority of microorganisms grow best aroudn neutrality
Limited by physiological acceptability and formulation stability
Temperature
Chemical and microbiological stability
Excipient choice
choose excipient resistant to microbial degradation
Chemical
synthetic preservatives
Acids + salts: Benzoic acid + sodium metabisulphite
Alcohols: Benzyl alcohol + ethanol
Hydroxybenzoates: para hydroxybenzoates and ester
Mercurial: phenyl mercuric acetate (not common)
Phenols: Phenol + cresol + chlorocresol
Quarternary ammonium compounds: Cetrimide + benzalkonium chloride
Biguanides: chorhexidine
Natural
Essential oils and perfumes: tea tree oil + thyme oil
Enzymes and proteains: lactoferrin - bind iron + oral use only
Describe PET
per Ph Eur: 5.1.3 Prepare inocula Candida albicans Aspergillus niger E coli Pseudomonas aeruginosa Staph aureus Optional contaminants Soybean Casen Digest - bac + Sabouraud dextrose agar for yeasts/mould - final conc 10^8 organisms per mL Innoculate product Confirm culture counts single culture inocula used Remove residual antimicrobial activity (dilution / filtration / neutraliser) inoculate each of 5 individual containers 0.1mL to 20mL product mix well Incubation of inoculated product incubate at 20-25 deg Examine containers at 7 + 14 + 21 + 28 days for TVC Calculate percentage changes Interpretation of results Concentratin of viable organisms should be reduced by 1-3 logs by 14th daywith no subsequent recoery/increase
Describe common types of disinfectant used in cleanroom.
Phenolic compounds: Chloroxylenol
Adv: Fungicidal + broad spectrum + soluble
Dis: reduced efficacy with hard water + organic maters + natural soap + not sporicidal + not good cleansing agent
QUATs: Cetrimide
Adv: G+ive + stable + solume + compatible with detergents + fungicidal + odourless
Dis: not sporidical + hard water + organic material + inactivate natural and man made materials
Iodophors:
Adv: Quick microbial kill + G+ive + less irritant to skin
Dis: not good cleaners + stain/discolour + mucous membrane irritant + orgnaic mater + >40deg release iodine
Alcohols:
Adv: Broad spectrum + rapid action + easy to use + min residue + not affected by organic matter + cleaning effect
Dis: not sporicidal + costly in large quantities
Chlorine compound: sodium hypochlorite
Adv: Broad spectrum + sporicidal + not affected by hard water + little residue
Dis: inactivated by orgnaics + loss activity on prolonged storage and UV + strong odour + corrosive
Gluteraldehyde: Tegodor
Adv: non staingin + relatively non corrosive + stable + broad spectrum
Dis: not stable in solution + irritating to skin + inactivate by orgnics
What are the main BI use in sterilisation methods?
Steam: Geobacillus stearothermophillus D Value (121): >1.5min Z value: 10 min
Dry Heat: Bacillus atrophaeus D Value (160) 2.5min Z value: 20 min
EtO: Bacillus atrophaeus
H2O2: G.StearothermophillusD Value 2.5min 600mg/L 54 deg 60 RH
Radiation: Bacilus pumilis D Value (>1.9kGy)
What is LRV in the context of filter sterilisation?
LRV = log reduction value
LVR = Log challenge org / Log filtrate org
Normally >10
What are the main filter testing methods?
Bubble point
Wet filter surface with water
water retained in pores by surface tension
Air pressure to displace water from pore = inversely related to the diameter of the largest pore
There pressure indicates the largest pore size
For smaller filters
Flow test
based on the diffusion of gas through the wetted membrane
Require all pores full of liquid
Air flow rate controlled by Fick’s law
Measure of gas diffusion - characterise membrane in relation to pore size and geometry
Gas pressure s ability to retain an applied presure for a detined perio dof time, per unit area of filter at a given pressure
Hydrophilic membrane
Water intrusion test
Hydrophobic membrances
Measure pressure req to blow water into the membrane
What are the ideal properties of a BI?
Inherent resistant to sterilisation method
Stable and reproducible
Efficient recovery
Nonpathogenicity
characteristic growth features - easy to ID
Uncommon in the natural bioburden
Describe sterility testing procedure
Per Ph Eur: 2.6.1
Media
Soya bean casein digest (20-25 deg) + Fluid thoglycollate (30-35 deg)
Direct innoculation
removal of sample
transfer contents to suitable media
incluabe for 14 days
Dis: product may inhibit growth + turbid product - can’t see + Dilution of test media ( system
Adv: large vol no dilution + wash x 3 remove inhibitory bits + any number of test containers can be filtered
Management of microbial failure
To consider:
What is the ID of the organism?
Likely source?
Number of bacteria present?
Type of sample taken?
Other out of limit for the day of sampling or since? Localised issue?
Links to real time data? i.e. non viable + LAF air flows + Press diff + temp + RH?
Activity at time of sampling? Batch in progress? Cleaning? Interventions? Engineering? Media fill?
Go and look at the area in question
Trend data review: sampler + room + specific location + plant
Action taken:
Notify plant immediately
Quarentine product: batches in progress during sampling + any more batches
Additional sampling/cleaning?
Investigate sampling operation and related lab procedures
Investigate operator related issue
How do you use HACCP to enhance microbiological control of medicines?
HACCP - hazard analysis and critical control point Define product and process Identify potential hazards Identify potential control measures Determination of CCP Establish critical limits for each CCP Establish monitoring system for each CCP Establish CAPA plan Establish verification procedure to demonstrate compliance
Describe what you would find in a disinfectant SOP
Type Contact time Application technique Temperature Concentration and how to mix Training Documentation Compatibility Frequencies Rotation
Describe aldehydes used as a disinfectant
Sporocidal action Eg.2%gluteraldehyde Non corrosive, non staining Broad spec Harmful to skin and mucosa
Describe chlorine and sodium hypochlorite as a disinfectant
Sporocidal 0.5% sodium hypochlorite Non foaming Little residue Broad spec Inactivated after long storage and UV Corrosive Skin irritations
Describe two general purpose disinfectants
1-Alcohol eg. 70% IPA Broad spec Easy to use Rapid action Expensive Not sporocidal Flammable
2- quarternary ammonium compounds
eg, BAK or cetrimide
Broad spec
Active against gram + (staph,strep)
Cheap
Fungicidal
Not sporocidal
Affected by hard water
Tell me about resistance of some clinically important microorganism to disinfectants
Most resistant- bacterial spores eg. Bacillus subtilis or clostridium sporogenes
2- mycobacteria: M. Tuberculosis
3- non lipid coated viruses: polio
4- fungal spores: candida spp
5-vegetative bacteria: ps. Aeruginosa, staph aureus, Salmonella
6- lipid coated bacteria : HIV, hep b
