quiz 2 microbio

Standard plate count PURPOSE

isolate colonies of bacteria from a large population and determine the titer of the stock culture

(Standard plate count) Titer

the number of cells/mL

(Standard plate count) CFU = colony forming unit

the number of colonies that develop on the plate

(Standard plate count) Original cell density (OCD) equation?

CFU / Final Dilution factor (FDF)

\*units will be CFU/mL

(standard plate count) steps of using a micropipette

1. twist dial to desired volume
2. add disposable pipette tip
3. press plunger to first stop
4. insert pipette tip into solution to be transferred
5. slowly release plunger to retrieve liquid
6. move pipette tip into desired tube
7. press plunger past first stop to second stop to transfer liquid, watch liquid stick to wall of tube; remove tip, then release plunger
8. eject tip

What is the max and min for p1000?

Max: 1000

minimum: 100

What is the max and min for p200?

Max: 200

Min: 20

What is the max and min for p20?

Max: 20

Min: 2

(standard plate count) Should you take out the agar deep all at once?

No, do not take out nutrient agar deep all at once or they will polymerize (solidify) before poured on the plate.

\*pour near flame and do not move it\*

(standard plate count) Why are the colonies different sizes?

because the bacteria that is growing aerobically (on top of the agar where oxygen is available) grows faster than bacteria growing within agar. Bacteria in the middle of agar aren’t getting oxygen and grow slower (anaerobically aka with no oxygen) and only make for fermentation 2 ATP per glucose when they grow aerobically so with oxygen they get 30 and 32 ATP per glucose.

Endospore stain PURPOSE

to stain and visualize endospores and differentiate spore forming cells from non-spore forming cells (a differential stain)

When do endospores form?

when conditions become harsh

What is the process called when an endospore is formed?

sporulation (harsh conditions)

(Endospore stain) what are examples of harsh conditions?

* temperature
* pH
* dehydration
* nutrient loss
* UV

What is an endospore made of?

keratin (found in skin)

Why are endospores resistant to heat and chemicals?

because of a tough outer covering made of the protein keratin

What is the process when the endospore goes back to a vegetative cell?

germination (favorable conditions)

(Endospore stain) What is steamed?

steam malachite green for 5 minutes

(Endospore stain) Bacillus anthracis

causes anthrax

(Endospore stain) B. polymyxa

\-green is the endospore

\-red/pink is vegetative cell

(Endospore stain) what are two genus of bacteria that are most commonly gonna have endospores?

* Bacillus (aerobic)
* Clostridium (anaerobic)

^both gram +

Acid fast stain PURPOSE

to stain acid fast bacterial cell walls and differentiate acid fast bacteria from non acid fast bacteria (differential stain)

(acid fast stain) Mycobacterium

has a cell wall made of 60% mycolic acid (an adherence factor)

(acid fast stain) sticky/waxy to protect from:

* phagocytic digestion
* dehydration
* mycobacterium can survive on surfaces up to 6 months
* chemicals (antibiotics, disinfectants, etc.)

(acid fast stain) What color will Mycobacterium be? S. epidermidis?

* Mycobacterium will be pink (fuschia)
* S. epidermidis will be blue

(acid fast stain) Mycobacterium tuberculosis

causes lung TB (tuberculosis)

(acid fast stain) M. smegmatis, S. epidermidis

* blue = acid fast (-)
* pink = acid fast (+)

Phenol red broth PURPOSE

to determine if bacteria can ferment a specific carbohydrate

(phenol red broth) What is the purpose of the Durham tube?

it is used to detect production of gas

(phenol red broth) what is the substrate?

carbohydrate

(phenol red broth) what is the enzyme?

various endoenzymes

(phenol red broth) what are the products?

acids, alcohol, +/- gases

(phenol red broth) What are the results (colors) and their meaning?

* yellow (turbid) = + for sugar metabolism, (A/G or A/-)
* red/pink (turbid) = - for sugar metabolism (bacteria use peptones), (K) → alkaline
* no growth (no turbidity) = no result → inconclusive (media is undefined)

\*turbid means bacteria grew

(phenol red broth) why must the readout be done between 24-48 hours after inoculation?

because when bacteria are given the choice between metabolizing proteins or sugars if they can’t metabolize the sugar, the sugar will be the preferred food source, and as they do that, they will lower the pH (yellow acidic (+))

essentially bacteria metabolize sugar first and lowers the pH

Oxidation/fermentation (O/F) test PURPOSE

determining whether bacteria oxidize and/or ferment glucose

(oxidation/fermentation (O/F) test) what is the substrate?

glucose

(oxidation/fermentation (O/F) test) what are the enzymes?

various endoenzymes

(oxidation/fermentation (O/F) test) what are the products of aerobic (oxidized with oxygen; no oil)?

acids

(oxidation/fermentation (O/F) test) what are the products of anaerobic(fermented without oxygen; mineral oil)?

acids, alcohols, +/- gases

Indole production test PURPOSE

to determine whether bacteria can produce indole from tryptophan (an amino acid)

MRVP purpose

to determine whether bacteria ferment glucose into mixed acids or acetoin

(methyl red) what is the substrate?

glucose

(methyl red) what are the enzymes?

various endoenzymes

(methyl red) what are the products?

mixed acids (pH 4.4)

(Voges-Proskauer) what is the substrate?

glucose

(Voges-Proskauer) what are the enzymes?

various endoenzymes

(Voges-Proskauer) what are the products?

acetoin

Citrate utilization test PURPOSE

to determine whether bacteria can utilize citrate as a sole carbon source

(citrate utilization test) what is the substrate?

citrate

(citrate utilization test) what are the enzymes?

citrate permease

(citrate utilization test) What are the results of a positive test?

* blue (because pH went up)
* green with growth

(citrate utilization test) what are the results of a negative test?

* green with no growth

what were the IMViC results?

Indole

* pink/red = positive
* yellow = negative

MR

* pink/red = positive
* not pink/red (clear ish) = negative

VP

* pink/red = negative
* clearish = positive

Citarte

* green with no growth = negative
* blue or green with growth = positive