Algemene microbiologie 1/6 dr. Freya Cools & dr. Magalie Kerkhofs
Inleidingsles practicum 6/03/2020
General introduction
- specially cleaned lab coats so you don’t need to bring your own
- everyday 2h of virology, 2h of bacteriology
- attendance list, don’t forget it!
- write your group numbers on all your materials (Agar plats e.g.)
- gloves not for bacteriology because of Bunsen burners -> nasty scars
Dag 1:
- bacteriologie: measuring concentration of sample
- identificatie: selective agar plates
- inhibitie microbial growth: AB: MIC, AB: Kirby Bauer test
Dag 2:
- inhibitie microbial growth: AB: MBC
- identificatie: staining techniques + API test
- human microbiome: pharynx and nose swab
- check results day 1
Dag 3:
- identificatie: API test (continued)
- fungi
- check results day 2
Waste
- glass that is contaminated -> first in the bleach before you throw it away to decontaminate
Bacteriology
- Tryptic soil broth (TSB) = liquid growth medium (yellow)
- Physiologically Buffered Saline (PBS) 0.9%NaCl (see through color)
- RBS = decontamination solution
Measuring concentration
Identification of unknown sample
Plastic pipettes -> one use only, no need to put them in bleach
-> plastic pipettes used for dilution series (one use only), exact volume!
Pasteur pipettes -> one use only, contaminated? -> bleach first -> then throw it away
-> used for putting sample on Agar plate (2 drops e.g.)
-> you use Pasteur here because they’re cheaper
Inoculation loops
-> we’re using throw aways
-> to spread, picking colony for staining
-> pick up colony to put on new Agar plate
5 main parts:
1. introduction: measuring concentration of sample
2. identification: unknown sample (different techniques)
3. inhibition of growth using AB
4. Human microbiome -> swabbing nose and throat
5. Fungi
Measuring concentration
- talking about millions/billions of bacteria so you need to make dilution series in tubes
- then we transfer certain amount on agar plate -> incubate over night to give bacteria chance
to grow
- usually dilution of 10x times more, 10x times more and so on…
- 37°C T to incubate overnight -> one bacterium will give rise to one colony -> one colony
contains couple of million bacteria, will be able to see them then as dots that you can count
- you dilute to see the spread of dots so you can count the colonies
- left is highly concentrated, more to the right 10x diluted, and so on -> second to last (35) is a
,Algemene microbiologie 2/6 dr. Freya Cools & dr. Magalie Kerkhofs
Inleidingsles practicum 6/03/2020
good one to count
-> below 10 is error quite big so 10-100 CFU’s is ideal
-> >100CFU’s, colonies can start to merge again -> underestimation of your real value
So 35 colonies, all originated from of one bacteria -> one bacteria gives rise to 1 colony so on this
plate before they were growing there were 35 bacteria present, diluted 10.000 times
->35 bacteria, 1:10.000 dilution -> 350.000 bacteria in original volume you put on plate
-> if you put 0.1mL on plate -> you have 3.5 million bacteria in sample per 1mL
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So first we make dilution in liquid, we use 0.1mL of original and not 1mL and 0.9mL PBS, you
don’t need growth medium here because we don’t want them to grow, we just want them to
survive so PBS is sufficient as we transfer them on Agar plates and they do have growth medium
to grow
-> not in water because it’s hypo?tonic -> death
-> PBS is isotonic so ideal -> happy cells!
- only count countable plates!
Identification
You’ll get sample on Agar plate only with group number on top, don’t know which bacterium -> we
need to test!
- staining, we won’t do that (no time for that) but Margaux is already staining them as we speak
- use of selective Agar plates with ingredients and growth factors, also possible inhibitors to
prevent certain bacteria from growing or better growth than general
3 different Agar plates:
1. if nothing is written on your plate it’s a Tryptic Soy Agar plate (general)
2. enriched Agar plate (blood Agar)
- if you add blood to Agar plate, bacteria who struggle to grow under normal conditions,
will be able to grow because of the high nutrients -> mostly sheep blood
3. selective Agar plates, inhibitors, pH indicators,…
Viable plate count test -> using TSA plate
Enriched plate
Blood agar plate
-> blood is enriched so more bacteria will grow, some bacteria will show hemolysis
pattern = way of bacteria to deteriorate the blood cells that are inside the plate
-> the way they do that, you get different colonies
-> different kinds: - alfa: (around every colony is yellow, greenish circle (halo))
= partial hemolysis, no complete lysis but Hb will oxidate -> green halo
- bèta: blood cells completely destroyed, complete lysis, see trough
plate because bacteria destroy red blood cells + yellow color
- gamma: no hemolysis, bacteria not destroying RBC
Selective agar plate
Cetrimide Agar plate
- Pseudomonas is resistent to this plate, everything else will die
- Pseudomonas makes this color (blue-yellow pigmentation), nothing to do with Agar itself
McConkey Agar: selective agar for GRAM-, 4 selective agents: lactose, bile salts, neutral red
and crystal violet
- crystal violet with bile salts will inhibit all GRAM+ to grow
- of the GRAM- bacteria, some will be able to ferment lactose -> you get acid byproducts
=> pH will drop
- neutral red is pH indicator -> will switch to acid color (dark purple)
- drop in pH makes bile salts start precipitate -> halo around colonies
- TSA is yellow, not blue by itself -> just blue light behind it to make it more clear
s22
,Algemene microbiologie 3/6 dr. Freya Cools & dr. Magalie Kerkhofs
Inleidingsles practicum 6/03/2020
1. Staph (GRAM+) & E.coli (GRAM-)
- on TSA plate they look quite similar
2. on McConkey plate -> Staph won’t grow anymore due to being GRAM+
3. E.coli & Salmonella
- E.coli able to ferment lactose, Salmonella is growing (GRAM-) but will not ferment lactose so pH
won’t drop -> neutral red won’t shift to red color (purple here) -> precipitation of bile salts won’t
happen
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MSA plate
- high concentration of salt: only halo tolerant, hydrophilic bacteria able to grow bv. Staph. Aureus
- selective for Staph but also other halo tolerants able to grow
- mannitol (sugar) can be fermented by some bacteria but not all, if fermented -> pH drop,
phenolred -> shift color to acid color -> yellow
- Staph will be able to ferment mannitol -> pH drop -> switch red to acid yellow
- other Staphs will not be able to ferment mannitol
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Nickerson Agars, bit different
- slightly lower pH, selective for yeast so not bacteria
- most common yeast is Candida
- Bismuth sulfite erin dus will change colony color to brown
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Chromogenic agar, quite new (10y)
- selective depending on ingredients but chromogenic mix is unknown (protected by patents)
- you’ll get a better selection than with regular selective agar plates
- e.g. identification of MRSA (Multiple Resistent Staph Aureus)
- so you’ll be able to show if you have Staph A.
- so a chromogenic agar plate of the right type will show you whether or not that specific SA is
a multiresistent form
- slight change of protocol because agar didn’t arrive in time: throat swaps will be done with
regular blood plates and not chromogenic agar plate
Identification - staining
- LM usage, bacteria approximately 1µm in size
- difference GRAM+ and GRAM- weten
- cells are a lot bigger than bacteria, so 100x magnification! => immersion oil
- using lenses, light will always diffract and your lense is trying to capture as much light as
possible
- the more you start magnifying, the bigger the diffraction is, the more waves you are losing
-> here you have 5 light waves, 2 already outside the lense to capture (you’ll only see 3 of
them so bad for quality of image
-> you can solve this with thin layer of immersion oil that replaces thin air layer
-> breaking index is a lot smaller than of air so the 5 light beams will drop nicely into lense
- so to not lose resolution, you can adjust the pin
- you’ll be replacing air with oil, avoidance of light
- oil is very toxic for lenses so clean microscope because lenses are super expensive
-> they’ll check if you did!
- GRAM- is pink, GRAM+ is purple
So you have information regarding GRAM+ or GRAM- of unknown sample and you also have a lot
of information out of the selective agar plates that should confirm with what you saw in the GRAM
stain => those 2 results combined will allow you to chose an API test
, Algemene microbiologie 4/6 dr. Freya Cools & dr. Magalie Kerkhofs
Inleidingsles practicum 6/03/2020
Identification - API
API test (brand) contains a lot of selective media -> collection of all those different tubes with
different medium -> you can identify your unknown sample
- here you are testing if you have an enzyme that can ferment mannose, here cellulose,
here glucose and so on…
- tests on the presence on a lot of different enzymes -> dus of het enzyme aanwezig is of niet
leidt tot identification
- lot of different API tests so you need background info: GRAM+/-, is it a yeast,…
- catalase test: if catalase is present, H2O2 -> H2O + air -> you’ll start to see bubbles, if not no
bubbles catalase
- oxidase test: if oxidase is present you’ll have a color change from light to darkish purple blue
-> so you need to chose API test based on these criteria, she’ll check if you selected the
right one (on Tuesday)
- make your inoculum in water or PBS
- she’ll always give your right API test just to be sure you don’t use a false one
Fill tube = not filling the whole tube, fill cupules = full filling, en dan nog een waarbij je tube met
bacteriën vult en dan olie erbovenop (to make it anaerobic) moet toevoegen en dus moet je air
bubbles vermijden!
Inhibition of microbial growth
Kirby Bauer test of Disk Diffusion Test, quite simple
- you take a plate, put bacteria on and put AB disks (little papers impregnated with AB)
- let them grow, after growth you measure dm’s of inhibition
- n2: bacteria been able to grow all the way up until AB -> so for sure resistant to AB
- here you see inhibition
- assess diameters -> lists you use, and compare to table f.ex. if it’s bigger than 10mm’s
your E.coli is susceptible to the AB, if it’s smaller than 8mm’s it’s resistant
MIC & MBC are more quantitative so you’ll get real value
MIC = minimal concentration AB which inhibits growth bacteria
- you make dilution series of AB, you add same amount of bacteria, let them grow
- if you dilute your AB, at certain point your bacteria will be able to grow
=> MIC is 1.5mg/mL here, otherwise bacteria will grow
MBC = minimal concentration of AB where bacteria are actually dead
=> maybe bacteria just didn’t grow or maybe they died (difference, right)
=> to see if they’re alive or not, take small fraction of the tubes that not display growth en
put them on agar plate, AB still present will start to dilute inside agar plate and
therefore lose function
=> bacteria still there, that are growth inhibited but not dead, get the chance to grow
=> plate with lowest concentration of AB that did not display growth, is the MBC
Practicals: you’re using Vancomycine, you get stock solution with certain concentration -> make
1:2 dilution series so 2x times more, 2x times more and so on.
- add bacteria to every tube and incubate to induce growth, the day after you’ll see this
- lower AB concentration, growth, higher AB concentration, bacteria growth
inhibited
- then you put these tubes, that did not show growth, on agar plate to give bacteria the
chance, if they’re still alive, to start growing on agar plate
- you’re doing both but you need to know which is MIC which is MBC
- in the lab, it depends on the sort of drug (AB), which one you’re using -> some AB are
bacteriostatic by nature so you’ll not find an MBC
- for some AB it depends on concentration: if you start increasing C, it’ll become
bactericidal, if decreasing, it will become bacteriostatic
- the tubes will show if they’re growth inhibitor but you will not know if they’re dead or not
- tubes, you can only measure MIC, if you put them on plate and give bacteria chance to
grow you’ll be able to determine MBC
- first you put them in tubes, then put them on Agar plates
- so on Monday you compare tubes, Tuesday you compare Agar plates
