An in-depth guide on the practicals undertaken in BS5100. The first practical is visualising the cells under light microscopy, and doing catalase and oxidase biochemical tests. The NBT assay is next, which highlights how phagocytosiscan occur and its consequences. The clinical features of CGD is al...
Microbiology: Practical One Discussion of Results 10/11/2020
First thing you did was gram stain. Bacteria A looks like purple yeast (gram pos cocci). Bacteria
B is pink bacillus (gram neg rods). Thick suspension and no gaps (hard to see morphology)-too
much microorganism on the loop. Cocco bacillary is for a fat rod that is gram neg (rare to see
gram neg cocci).
From that, see oxidase test. Pseudomonas is oxidase pos organisms, even more negative
ones. 10 seconds for results; A had no colour change, oxidase neg. B-blue/purple so has
oxidase culture and is oxidase positive. Gram pos cocci we would not bother for a test.
Catalase-similar to oxidase test with just a drop of catalase reagent onto the culture. Look for
release of bubbles now. A-bubbles produced (like a fizzy drink). B-can see bubbles. Both
cultures catalase positive. Already worked out gram stain of organisms, is it worth doing a test
on both gram pos and neg? Useful for gram pos AND neg here because some gram neg are
negative catalase (anaerobic). Virtually all gram neg growing aerobically are catalase positive.
So for gram pos it is v useful, differentiates strep (neg) vs staph (pos). BUT B WAS GRAM POS
AND CATALASE POS. I think she meant was neg though; we got neg.
Gram always first to help guide on what test should be done next.
Staphaurex ext. Gram pos-start with gram stain coccus, then catalase (belongs to strep or
staph); A catalase pos so we know is Staphylococcus group. Next Q is is it aureus or another
coag neg staphylococcus. Aureus is focussed on as it is established in lots of infections (sepsis,
food poisoning, etc). Other staph are more commensal normal flora. Isolated staph, could it be a
pathogen or is it likely just skin flora? ID staph aureus or not then! Staphorex (used to be one
called coagulator) tests coagulator factor and presence of protein A. Colossal factor? Confirms
ID of staph aureus. You just take a loop with culture on toothpick, emulsify the growth on the
suspension on the card. Mix organism into the latex reagent, then rock it for a minute. Look at
spillage for any clunking. A is very lumpy, lots of clumps (so positive). Lots of glutination,
showing staph. Aureus. B shouldn’t really have been tested, we know B is gram neg rod (so not
used for this test). B just to show us a neg result; stayed milky, no lumps/clumps.Classic
negative result with this test! 1 min for the result remember.
We were also given media to look at. Blood plates useful for gram pos organisms, showing
haemolysis (alpha-green, beta-total lysis of blood, gamma-nonhemolytic). Alas, don’t store very
well (whole of blood plate haemolyses). No clumps of blood plate shown now :( as too old.
Usually just incubate overnight. Left-Mac Cockney. Half is A, other half is B. A is red (lots of
growth), B is difficult to see growth as so many colonies that have not changed colour. A
changed colour showing they are a lactose fermenting colony. A is gram negative, and usually
only gram positives grow on MacCockney agar. Most gram positives grow on MacCockney.
Strep would look weedy, no growth at all. Staph are pretty robust and tough, can grow quiet
well.
MSA-mannitol salt agar. Gram pos (A) grows and looks yellow, B is difficult to see (neg). B not
growing because is salt! Yellow-s. Aureus my notes say! Staph live on skin, so can live on salt!
Agar is differential, inhibiting other organisms growth. The yellow colour is from the mannitol,
they fermentate the mannitol. Most other staph cannot ferment mannitol.
CLED-cysteine lactose present (like MacCockney) but different indicator). Yellow/yellow green is
lactose fermenter. Staph aureus is more greeny colour, so lactose fermenter for sure! Electrolyte
deficient! Blue colony (no colour change) then non lactose fermentor. Pseudomonas not really
, seen here (organism B) because given colour same as agar itself. With this it’s good to pinpoint
colonies and see the sheen when shined in the light and physically moved.
Also some susceptibility testing done! Can see zone of inhibition
nicely. Zone of clearing in growth around disc is zone of inhibitoin.
Measure diameter of zone and compare it with zone sizes expected to
interpret if org is susceptible to the antibiotic or not. Chemical has to
diffuse out into the surrounding agar; big molecule, does not diffuse
long distance but small molecules diffuse a lot further. Top left and
right, show susceptibility to the antibiotics, but size of molecules could
differ. Must check the tables online on what diameter of zone should
be for each antibiotic. Bottom antibiotic-can see a tiny zone around the
disc, but resistance to that antibiotic. Halfway house
classification-intermediate (docs dont understand what it meant) is no
more. Now, need to give more antibiotic and prolonged exposure
(higher dose) for a therapeutic effect. Cannot use intermediate
anymore (I for intermediate). I is still being reported, but information is
increased dose/duration of therapy to get the benefit. We cannot see which disc is which for
interpretation sadly. I tried recolouring it. Might want to check diameter still.
Bacteria B zones of inhibition. Isolate-pretty big zones of inhibition
around the antibiotics. Big zones-cannot put too many discs on each
plate. Zones overlap, very tough to measure diameter. Need to
measure against a standard for size. Depth for each of the agar plates
too. Thick agar vs thin agar-diffusion of antibiotic is different (far on thin
plate). Must be tightly controlled and standardized for susceptibility
testing.
Practical objectives: preparing a bacterial smear + Gram staining!
Biochemical tests: catalase, oxidase, coagulase. Plating on selective
and differential media: blood agar, mannitol salt agar, and MacConkey
agar. Antimicrobial susceptibility testing.
Streaking-diluting concentration of microbial growth of the sample until
you get single colonies. Isolation streak (streak plate technique)-four
quadrants, allows grading of relative concentration of organisms.
General purpose isolation technique. For many tests, need pure well
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