Micro TBL Pre-Reading Flashcards
what do many microorganisms require?
B group vitamins
this requirement satisfied by yeast extract
at what temperature does agar form a firm gel?
37 degrees C
at what temperature is agar used as a liquid and how this useful?
45 degrees C
this is at sufficiently low temp without killing microorganisms
this = imp in pour plate counting methods
solid media designed for the growth of anaerobic organisms usually contain non-toxic reducing agents.
give examples of these?
- sodium thioglycollate
- sulphur containing amino acids
give example of redox indicators?
- methylene blue
- resazurin
why may redox indicators be incorporated into anaerobic media?
to confirm the sufficiently low redox potential has been achieved
media for yeasts and moulds have … pH than bacterial culture media?
lower
pH of yeasts & moulds: 5.5-6.0
pH of bacterial culture media: 7.0-7.4
why is lactic acid used to impart a low pH?
it is not inhibitory to fungi at concentrations used
in bacteria, the patterns of binary fission take place how often?
every 25-30 mins under optimal conditions of laboratory cultivation
how do moulds grow?
through extension & branching of hyphae to produce mycelium
how may anaerobic organisms be grown?
on petri dishes given incubated in anaerobic jar
what are anaerobic jars made of?
rigid plastic with airtight lids
- petri dishes placed in them together with low temp catalyst
- catalyst made of palladium-coated pellets/wire
- this causes oxygen in jar to = combined with hydrogen generated by additions of water to NaBH4
what is the periphery of a colony?
part which = actively growing and usually non-pigmented
what is the term planktonic used to describe?
freely suspended cells
bacteria attached to a substrate (surface in the body) are known as what?
sessile
they = said to exhibit biofilm/micro colony mode growth
what are planktonic cells routinely used for?
testing procedures designed to assess activity of antimicrobial chemical & processes
there are several situations where it is necessary to measure the number of microbial cells in culture, sample or specimen.
give examples of these?
- when measuring levels of microbial contamination in raw material/manufactured medicine
- when using microorganisms in making of therapeutic agents
- when evaluating effects of antimicrobial chemical/decontamination process
- when assessing nutrient capability of growth medium
in what case is it necessary to know the total number of microbial cells present?
- in vaccine making (dead & living cells may both cause immune response)
- pyrogen testing (dead & living cells induce fever when injected into body)
what is a viable count?
counting living cells only
when might MPN (most probable number) counts be used?
when anticipated count is relatively low
what do MPN counts involve?
- inoculating multiple tubes of culture medium (3/5)
- with 3 different volumes of sample
- there should be proportion of tubes receiving inocula where no microorganism present
- these remain sterile after incubation
where are MPN counts used?
water, food, dairy industries
MPN counts are highly…
innacurate
…. measurements are the most common means of estimating the total numbers of bacteria present in sample
turbidity
how do you take turbidity measurements?
- use spectrophotometer/colorimeter
- reading conc from calibration plot
why cannot fungi undergo turbidity measurements?
cannot be readily handled in this way because suspension may not = uniform / may sediment in spectrophotometer cuvette
what might direct microscopic counting be an appropriate method for?
- bacteria
- yeasts
- fungal spores
the traditional methods of viable counting all suffer from the same limitations.
what are they?
- labour intensive
- not easy to automate
- slow, because need incubation period for colonies to develop/liquid cultures = turbid
- need large volumes of culture media
give examples of the operating principles most common in detection of living cells?
epifluorescent techniques:
- dyes exhibit diff colours in living & dead cells / appear colourless outside cell
- become fluorescent when absorbed
living cells generate ATP:
- readily detected by enzyme assays e.g. luciferin emits light when exposed to firefly luciferase
resistance, capacitance/impedence of culture medium changes as result of bacteria/yeast growth & metabolism
manometric techniques = appropriate for monitoring growth of organisms that consume/produce large amounts of gas during metabolism
give examples of viable count methods?
- pour plate (counting colonies in agar)
- surface spread/surface drop methods
- membrane filter methods
- MPNs
give examples of total count methods?
- direct microscopic counting
- turbidity methods
- dry weight determination
- nitrogen, protein/nucleic acid determination
give examples of rapid methods (indirect viable counts)?
- epifluorescence
- ATP methods
- impedance
- manometric methods
what are the advantages of the pour plate method?
- doesn’t need pre-drying of agar surface
- will detect lower conc than surface spread methods
what are the disadvantages of the pour plate method?
- very small colonies of strict aerobes at base of agar may = missed
- colonies of diff species in agar appear similar (difficult to detect contaminants)
what are the advantages of surface spread/ surface drop methods?
- gives larger colonies than pour plates (easier to count)
- easier to find contaminants by appearance of colonies
what are the disadvantages of surface spread/ surface drop methods?
- agar surface needs pre-drying to absorb sample
- possibility of confluent
growth, particularly with moulds, making individual colonies
what are the advantages of membrane filtration methods?
- will detect lower conc than other methods
- antimicrobial chemicals in sample = physically removed from cells
what are the disadvantages of membrane filtration methods?
- viscous samples won’t go through membrane
- particulate samples = block membrane —> restricting filtration capacity
apart from adding preservatives, give another factor that can potentially attenuate the preservative activity?
oil-water partitioning
with tests involving liquid systems, how can the early growth of viable cells be assessed?
by some light scattering processes
what might quantitative PCR be useful in demonstrating?
presence/growth of microorganisms that are slow/difficult to culture under usual lab conditions e.g. viruses
why must determination of MIC values be conducted under standardised conditions?
deviation from standard test conditions can result in considerable variation in data
what have the historical gradient plates, ditch-plate and cup-plate techniques been replaced with?
more quantitative techniques:
- disc diffusion
- broth & agar dilution
- e-tests
describe briefly what a disc test is?
filter-paper discs impregnated with antimicrobial replace antimicrobial-filled cups / wells
disc test process?
- standard suspensions of log-phase growth cells prepared and inoculated on to surface of appropriate agar plates to form lawn
- paper discs with known conc of antimicrobial agent placed on dried lawn
- plates incubated aerobically at 35 degrees C for 18 hours
when do problems arise with the disc test?
when inoculum density is e.g. too low —> indistinct edge to inhibition zone after incubation
as the distance from the disc …., there is a logarithmic …. in the antimicrobial concentration
increases
reduction
what is the most convenient and presently accepted method of determining bacterial MICs?
E (epsilometer) test
give examples of antimicrobial-resistant bacterial pathogens?
- vancomycin-resistant enterococci (VRE)
- methicillin-resistant staph. aureus (MRSA)
- vancomycin intermediate staph.aureus (VISA)
standard methodology may fail to detect the resistant phenotype.
give the factors that cause this??
- heterogenous expression of resistance e.g. VISA, MRSA
- poor agar diffusion of antimicrobial
- slow growth of resistant cells
give the conditions where expression is enhanced?
- lower temperatures
- higher salt conc
how can you improve the clarity of inhibition zone edges? (tests for fungistatic and fungicidal activity)
adding methylene blue
what is the MFC?
the lowest drug conc showing no growth / fewer than 3 colonies per plate to obtain 99-99.5% killing activity
give examples of bacterial species that are cocci and gram pos?
- e.faecium
- s.aureus
- s.epidermidis
- s.pneumoniae
give examples of bacterial species that are bacilli and gram pos?
- b.subtilis
- c.difficile
- l.monocytogenes
- c.sporogenes
give examples of bacterial species that are cocci and gram neg?
- h.influenzae
- m.catarrhalis
- n.gonorrhoeae
- v.parvula
give examples of bacterial species that are bacilli and gram neg?
- p.aeruginosa
- s.enterica
- p.mirabilis
- e.coli
