Microbiology Lab Practical 2

What are 3 general products that you get from different fermentations?

1. acid (phenol red)

2. alcohol (gas bubbles and no acid production)

3. gas (Durham tube - trap gas bubbles)

A

Negative (red broth/no gas in the Durham tube)

C

Positive (red broth/gas in the Durham tube)

B

Positive (yellow broth/no gas in the Durham tube)

D

Positive (yellow broth/gas in Durham tube)

Oxidase test: obligate aerobes

Produce cytochrome c oxidase (positive for the oxidase test)

Oxidase test: facultative anaerobes

Utilize different oxidases (negative for the oxidase test)

Carbohydrate fermentation

Determines if a microorganism can utilize a particular carbohydrate to do fermentation

Oxidase

Determines if a microorganism produces cytochrome c oxidase or not

A

Negative (bacteria doesn’t change color)

B

Positive (purple/pink color)

Why do you use a wooden stick to transfer bacteria to the paper during the oxidase test?

• metals are great conductors of electrons (we don’t know if the loop or enzymes are doing the transferring)

• avoid false positives

Catalase

• determines if the microorganism produces catalase or not

• distinguishes between Staphylococcus and Enterococcus bacteria

A

Negative (no bubbles)

B

Positive (bubbles)

Chemical reaction to catalase

2H2O2 (hydrogen peroxide) → 2H2O (water) + O2 (oxygen bubbles)

Nitrate reduction

Determines if the microorganism utilize (reduce) nitrate or not

A

Positive (tube that had nitrate A and nitrate B added)

B

Positive (tube that had nitrate A and B added -no color change - then zinc flakes were added)

C

Negative (tube that had nitrate A and B added - no color change - then zinc flakes were added)

Pathway for nitrate reduction

NO3 (nitrate) → NO2 (nitrite) → N2/NH3

Zinc flakes

Reduce nitrate to nitrite

What does SIM stand for?

Sulfur, Indole, Motility

What does IMViC stand for?

Indole, Methyl, Vogues Proskauer, Citrate (Simmons citrate)

Enterobacteriaceae

• gram -

• facultative anaerobe

• enteric bacteria (intestinal)

Sulfur reactions

• cysteine = food

• thiosulfate = final electron acceptor

H2S

Produces black precipitate

Motility

Movement from the stab line

A

Positive (black precipitate through the whole tube)

B

Positive (black precipitate only along the stab line)

C

Negative (no black precipitate)

If a bacterium can use tryptophan, what is one of the products of this reaction?

Indole

Indole

Tryptophan = food

Methyl red

Mixed acid fermentation

Vogues Proskauer

Butanediol fermentation

A

Indole Positive

B

Indole Negative

A

Positive (after incubation the tube is cloudy away from the stab line)

B

Negative (after incubation and only growth long the stab line)

Mixed acid fermenter

• lactic acid

• succinic acid

• acetic acid

• formic acid

Butanediol fermenter

• acetoin

• acetyl-CoA

• ethanol

• lactic acid

• formic acid

Which group of fermenters would result in a lower pH?

Mixed acid fermenter

A

MR Negative (2/3 amount of incubated broth mixed with MR reagents)

B

MR positive (2/3 of incubated broth mixed with MR reagent)

C

VP Negative (1/3 of incubated broth mixed with Barritt’s reagents)

D

VP Positive (1/3 of incubated broth mixed with Barrit’s reagents)

Citrate reaction

Citrate as sole carbon source

A

Negative (after incubation medium is a green color)

B

Positive (after incubation the medium turns a blue color)

Urea reaction

2 benefits of urea

• allows urea to be used as nitrogen source

• raises pH of environment

A

Negative (bacteria growth on the plate but no color change)

B

Positive (bacteria growth on the plate and a dark pink color on the plate

What are the 3 end products of fermentation?

• acid

• alcohol

• gas

Why must a wooden stick be used to transfer bacteria onto the oxidase paper instead of a wire loop?

Metal can transfer electrons resulting in a false positive

What does the bacteria use nitrate for in the nitrate test?

Final electron acceptor

A fermentation tube detects if fermentation occurs. What is the function of the Durham tube?

Trap air bubbles

What oxygen radical does catalase destroy?

Hydrogen peroxide

What 2 groups of bacteria can be differentiated by using the oxidase test?

• facultative anaerobe

• obligate aerobe

If a bacteria is positive for Methyl Red in an MRVP tube, what type of fermenter is it?

Mixed acid fermenter

What are two metabolic pathways bacteria use to produce hydrogen sulfide, and how are the specific substrates utilized in each process?

• break down cysteine for food

• use thiosulfate as final electron acceptor

Is the bacteria in the picture positive or negative for indole production?

Negative

Is the bacteria in the picture positive or negative for indole production?

Positive

Is the bacteria in the picture positive or negative for motility?

Positive

What product(s) can be transported across the cell membrane if a bacteria contains DNase?

Nucleotides

What is casein?

Large white milk protein that provides nutrients and structure

You can see growth on your casein plate but there is no zone of clearing around the bacteria. What is the bacteria using to grow if it is not casein?

Other sugars and nutrients in the milk plate

What is the function of iodine in the starch test?

To see if starch is present around the bacteria

What product(s) can be transported across the cell membrane if a bacteria is able to break down starch?

• glucose (monosaccharides)

• maltose (disaccharides)