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
