Microbio lab 2

Exoenzymes

Synthesized inside cell, secreted outside the cell to breakdown substrates (usually macromolecules). Some act as a virulence that contribute to disease.

What biochemical tests use exoenzymes?

Starch Agar, Milk Agar, DNA Agar, Blood Agar, and Gelatin tube.

Hemolysin

Exotoxin produced by some bacteria which destroy RBCs Sheep blood agar allows a person see hemolysis

beta hemolysis

Complete destruction of RBCs (A)

alpha hemolysis

Partial or incomplete lysis of RBCs (B)

gamma hemolysis

No hemolysis (C)

What enzyme hydrolyzes starch?

Amylase

What positive reaction indicates that starch was hydrolyzed when iodine is added to a starch plate?

Clearing around growth

What negative reaction indicates starch wasn't hydrolyzed when iodine is added to a starch plate?

Blue/black color right up to growth

What is the substrate in a milk agar?

Casein

What enzyme breaks down casein in a milk agar?

Caseinase

Positive result for milk agar showing casein was hydrolyzed

Clear zone around growth. (right)

Negative result for milk agar indicating casein wasn't hydrolyzed

No change in milk agar (left)

What enzyme breaks down DNA in a DNA agar?

DNAse

What is the product of a DNA agar?

Nucleotides

What is the indicator when bound to DNA at pH 7.5?

Methyl green

Positive reaction for a DNA agar

Yellow around growth (right)

Negative reaction for DNA agar

No change in green color around growth (left)

What enzyme breaks down gelatin?

Gelatinase

What is the indicator for gelatin tube?

No additional indicator, just a cold temperature.

Positive gelatin tube reaction

Liquid media when cooled in ice bath

Negative gelatin tube reaction

Solid media after cooled in ice bath

What biochemical tests test for sugar fermentation?

Phenol red sugar broth, Mac Conkey's Media, Triple Sugar Iron Agar (TSIA) slant tube.

What type of media is a MAC plate?

Selective (for gram -) and differential (lactose fermentation)

What is the indicator for a MAC plate?

Neutral Red

What is the indicator for phenol red sucrose broth?

Phenol red pH indicator

What else besides sugar fermentation does TSIA detect?

H2S production, gas production

What is the indicator in a TSIA slant?

Phenol red and ferric iron

What is the incubation time for TSIA slant?

24hrs

What is the inoculation method for TSIA slant?

Stab and streak

What sugars does TSIA contain?

0.1% glucose, 1% sucrose, 1% lactose, and peptone.

"A" for acid in Phenol red lactose broth

yellow

"NC" for no change in phenol red lactose broth

red

"AC" for acid and gas in phenol red lactose broth

yellow and air bubble in durham tube

Positive lactose fermentation on MAC plate

Left

Negative lactose fermentation on MAC plate

A/A, G TSIA

E

K/A, g TSIA

A

K/A, H2S+ TSIA

D

K/K TSIA

What two types of amino acid degradation are there?

Deamination and Decarboxylation

Deamination

NH2--\>NH3

Decarboxylation

COOH--\>CO2

What happens in a lysine decarboxylase test?

Cadaverine is respired under aerobic conditions. Oil is added to maintain anaerobic environment.

What indicator is used in a lysine test?

Bromcresol purple indicator. Detects increase in pH if cadaverine is present.

Positive lysine result shows decarboxylation reaction

A

Negative lysine result

C

What enzyme catalases use of nitrogen in urea?

Urease

What indicator is used in urea degradation?

Phenol red indicator

Why is urea a defined media?

Because urea is the sole source of nitrogen. Urea--\>NH3_CO2

How do you inoculate a urea broth?

Light stab with a needle

What indicator is used in a phenylalanine malonate broth?

Bromthymol blue pH indicator

What is utilized in a phenylalanine malonate broth?

Malonate is utilized and turns into an alkaline product

Positive phenylalanine malonate broth

Left

Negative phenylalanine malonate broth

Right

Besides malonate utilization, what else can phenylalanine broths detect for?

Deamination

What indicator shows deamination in phenylalanine broth?

3-5 drops of FeCl3

Positive phenylalanine deamination

dark green precipitate

Negative phenylalanine deamination

Left. Yellow precipitate

If urea is degraded, what will raise the pH causing color change?

Ammonia

Positive urea broth

1

Negative urea broth

2

What is an IMCiC test?

Standard series of tests (enteric bacteria) Indole, Methyl Red, Voges-Proskauer, Citrate

What type of broth is used in an indole test?

1% Tryptone broth

What enzyme catalyzes tryptophan into indole+pyruvic acid+NH3?

Tryptophanase

What is in an MRVP broth?

High amounts of glucose. Test tells how glucose is utilized by the bacteria.

+MR

Glucose--\>acid

+VP

Glucose--\>acetoin, butanediol. Right. Red/pink

What does a Simmon's Citrate slant tube determine?

A bacterium's ability to transport citrate (carbon source) from outside of the cell to the inside.

Why is citrate a defined media?

It is the sole source of carbon.

What is the indicator detecting pH change in a citrate test?

Bromthymol blue indicator

Positive citrate test

Left

Negative citrate test

How do you inoculate a citrate test?

Stab and streak

What is the incubation time for a lysine test?

1-4 days

What is the reagent in an Indole test?

20 drops of kovac's reagent

Positive indole test

left

Negative indole test

How does an MRVP broth work?

Take 1mL of MRVP broth and place it in tube labeled VP. In original tube, add 10 drops of methyl red to find MR result. In VP tube, add 4 drops of KOH and 12 drops of alpha-napthol.

-VP

No color change. Yellowish

-MR

No color change. Yellowish

Reduction of O2 and H2O results in what toxic intermediates

Superoxide (O2-) and hydrogen peroxide (H2O2)

What must an organism posses to survive reduction (oxygen toxicity)?

Superoxide dismutase, catalase, or peroxidase

What is required to undergo respiration?

Electron transport chains, an electron acceptor (O2 in aerobic, something else in anaerobic), cytochromes

Cytochromes

Major component of respiration that are proteins containing iron

What is the most common terminal electron acceptor used in anaerobic respiration?

Nitrate (reduced to nitrite)

Denitrification

Loss of nitrogen to the atmosphere. NO3\- --\>NO2\- --\> N2O --\> N2 (nitrogen gas)

What is added to a nitrate reduction test as an indicator?

10 drops of Rgt. A & B.

If nitrate broth turns red in first round of tests, what is it's result?

Positive for nitrate reduction to nitrite

If nitrate broth remains colorless in first round of tests, what is added?

Zinc

Once zinc is added to a colorless nitrate broth, and it is still colorless, what is the result?

Presence of gas. Positive

Once zinc is added to a colorless nitrate broth, and it turns red, what is the result?

Negative.

How do you perform a cytochrome oxidase test?

Use oxidase reagent as an artificial electron acceptor. Add loopful of organism.

Organism has oxidase

Right. Blue in less than 30 seconds. Accepts e-

Organism doesn't have oxidase

No color change. Doesn't accept e-

What enzyme neutralizes hydrogen peroxide into whater and oxygen gas?

Catalase

Positive catalase test

Bubbling

Negative catalase test

No bubbling

What biochemical tests detect for respiration and oxygen toxicity?

Nitrate reduction, oxidase, catalase

How do you check if an organism is pure?

Perform a gram stain, and reassure that preliminary testing matches up.