MIC 230 Lab Exam 2

what are hydrolysis reactions?

water reacting with larger molecules within an active site of an enzyme

What are exoenzymes

a protein catalyst produced inside a cell and then secreted into the external environment to function outside the cell body

Starch Hydrolysis

Medium:

Inoculation:

Incubation:

Substrate:

Exoenzyme:

Products:

Indicator:

Results:

Medium: starch agar

Inoculation: single streak

Incubation: 48hr

Substrate: starch (carbohydrate)

Exoenzyme: amylase

Products: glucose

Indicator: iodine till you cover the medium

Results: area around growth: yellow (+), dark (-)

Casein Hydrolysis

Medium:

Inoculation:

Incubation:

Substrate:

Exoenzyme:

Products:

Indicator:

Results:

Medium: milk agar

Inoculation: single streak

Incubation: 48hr

Substrate: casein (protein)

Exoenzyme: caseinase

Products: polypeptides and amino acids

Indicator: clear or not clear around bacteria

Results:: clear (+), not clear (-)

Gelatine Hydrolysis

Medium:

Inoculation:

Incubation:

Substrate:

Exoenzyme:

Products:

Indicator:

Results:

Medium: gelatin deep

Inoculation: 3 full loops, ½ down deep

Incubation: 7 days

Substrate: gelatin (protein)

Exoenzyme: gelatinase

Products: polypeptides and amino acids

Indicator: Ice 10 min

Results: liquid (+), solid (-)

Hemolysis

Medium:

Inoculation:

Incubation:

Target:

Extracellular Protein:

Indicator:

Results:

Medium: blood agar

Inoculation: 4-Q streak

Incubation: 48 hr

Target: red blood cells

Extracellular Protein: hemolysins

Indicator: 5% sheep blood

Results: Alpha - partial destruction (brown); Beta - total destruction (clear); Gamma - no destruction (red)

Indole

Medium:

Inoculation:

Incubation:

Substrate:

Enzyme/Protein:

Products:

Indicator:

Results:

Medium: Tryptone broth

Inoculation: loop or culture

Incubation: 48 hr

Substrate: Tryptophan

Enzyme/protein: Tryptophanase

Products: Indole, Pyruvic acid, and Ammonia

Indicator: Kovac's reagent (20 drops)

Results: cherry-red ring (+), yellow or color-neutral (-)

Urea Hydrolysis

Medium:

Inoculation:

Incubation:

Substrate:

Enzyme/Protein:

Products:

Indicator:

Results:

Medium: urea broth

Inoculation: loop of culture

Incubation: 24-48 hr

Substrate: urea

Enzyme/Protein: urease

Products: ammonia and CO2

Indicator: Phenol red

Results: pink (+), orange (-)

Citrate Permease

Medium:

Inoculation:

Incubation:

Substrate:

Enzyme/Protein:

Products:

Indicator:

Results:

Citrate Permease

Medium: Simmons citrate agar

Inoculation: drag loop across slant

Incubation: 48 hr

Substrate: sodium citrate

Enzyme/Protein: citrate permease

Products: Pyruvate, CO2, and ammonia

Indicator: Bromothymol blue

Results: growth and blue: (+), no growth and green/teal (-)

Sugar Fermentation

Medium:

Inoculation:

Incubation:

Substrate:

Process:

Products:

Indicator:

Results:

Medium: Phenol red broth

Inoculation: loopful of culture

Incubation: full 48 hr

Substrate: Lactose, sucrose, or mannitol

Process: fermentation

Products: Acid and gas

Indicator: observe color and Durham tube

Results: Red = No Change, Yellow = acid, air in durham tube = Gas

(AG, A, or NC)

MacConkey

Medium:

Inoculation:

Incubation:

Substrate:

Process:

Products:

Indicator:

Results:

Medium: MacConkey agar

Inoculation: 4-Q streak

Incubation: 24-48 hr

Substrate: lactose

Process: fermentation

Products: acid

Indicator: Neutral red

Results: growth = gram negative if pink color = (+) (does ferment lactic acid)

3 Sugar Fermentation

Medium:

Inoculation:

Incubation:

Substrate:

Process:

Products:

Indicator:

Results:

Medium: TSI agar

Inoculation: stab and streak with a needle

Incubation: 48 hr

Substrate: Glucose, lactose, and sucrose

Process: fermentation

Products: acid, gas, H₂S

Indicator: phenol red

Results: Slant - pink (k) or yellow (A), Butt - pink (k) or yellow (A), gas production - cracks or bubbles (G), H₂S - black (H₂S)

For 3 sugar fermentations, tell me what K/A means or A/AG means

Is A/K possible? Why?

K/A: ferments glucose only

A/A: ferments glucose and sucrose and/or fructose and/or sucrose

A/K is NOT possible because you can’t ferment sucrose or others without being able to ferment glucose

Cysteine degradation

Medium:

Inoculation:

Incubation:

Substrate:

Process:

Products:

Indicator:

Results:

Medium: TSI agar

Inoculation: stab and streak using a needle

Incubation: 48 hr

Substrate: Cysteine

Process: hydrolysis/degration

Products: Hydrogen sulfide, Pyruvate, and Ammonia

Indicator: ferric iron

Results: black H₂S (+)

Methyl Red

Medium:

Inoculation:

Incubation:

Substrate:

Process:

Products:

Indicator:

Results:

Medium: MR-VP broth

Inoculation: loopful into broth

Incubation: 48 hr

Substrate: glucose

Process: mixed acid fermentation

Products: mixed organic acids

Indicator: 10 drops of methyl red indicator

Results: red/pink (+), yellow (-)

Voges Proskauer

Medium:

Inoculation:

Incubation:

Substrate:

Process:

Products:

Indicator:

Results:

Voges Proskauer

Medium: MR-VP broth

Inoculation: loopful of culture

Incubation: 48hr

Substrate: glucose

Process: 2,3-butanediol fermentation

Products: 3-butanediol, acetoin, diacetyl

Indicator: 4 drops of 5% α-naphthol and 12 drops of 40% KOH

Results: pink( (+), yellow (-)

Nitrate Reduction

Medium:

Inoculation:

Incubation:

Substrate:

Process:

Products:

Indicator:

Results

Nitrate Reduction

Medium: nitrate broth

Inoculation: loopful of culture

Incubation: 48 hr

Substrate: nitrate

Process: anaerobic respiration using NO₂ or NO₃ as a terminal electron acceptor

Products: NO₂ (nitrite), N₂ (nitrogen gas)

Indicator: 10 drops of Nitrate reagent A (Sulfanilic acid) and B (alpha-naphthylamine); add zinc if still yellow

Results: if it turns pink when adding reagents A and B, (+); if still yellow and you add zinc then it turns pink, (-)

Oxidase

Medium:

inoculation:

Incubation:

Substrate: ?

enzyme:

Products:

Indicator:

Results

Oxidase

Medium: filter paper

Inoculation: loopful

Incubation: none; just that the culture is pure and at room temp

Substrate: ?

enzyme: Cytochrome c oxidase

Products: Indophenol blue

Indicator: oxidase reagent

Results": turns blue/purple (+), no change in color (-)

Catalase

Medium:

inoculation:

Incubation:

Substrate:

enzyme:

Products:

Indicator:

Results:

Catalase

Medium: slide

Inoculation: loopful

Incubation: none, but need pure culture and room temp

Substrate: Hydrogen peroxide

Enzyme: catalase

Products: water and oxygen

Indicator: look to see if there are bubbles

Results: bubbles (+), no bubbles (-)

how do you find the frequency of mutation in cells?

# of colonies on plate / # cells spread on plate

how do you find the number of cells spread on a plate?

concentration x volume plated

what is a screen for mutations?

plate culture on a plate and see if they are any mutations which we can see on the plate

what is a selection for mutations?

plate a culture and only allow the bacteria with the correct mutation grow.

What are we testing metabolically when we do a starch hydrolysis test?

testing to see if the cell has the enzyme amylase and can break down starch to produce smaller sugars like glucose

What are we testing metabolically when we do a casein hydrolysis test?

testing to see if the cell can produce caseinase and can break down larger molecules

What are we testing metabolically when we do a gelatin hydrolysis test?

testing to see if they produce gelatinase which is used to break down large molecules

What are we testing metabolically with a hemolysis test?

testing to see if the cell can break down or rupture red blood cells for nutrients

What are we testing metabolically with an indole test?

to metabolically break down (catabolize) the essential amino acid tryptophan

What are we testing metabolically with a urea hydrolysis test?

presence of the urease enzyme and a microorganism's ability to use urea as a sole nitrogen source

What are we testing metabolically with a citrate permease test?

whether a bacterial organism can use citrate as its sole source of carbon and energy.

What are we testing metabolically with a sugar fermentation test?

a microorganism's ability to metabolize a specific sugar anaerobically to produce energy

What are we testing metabolically with a MacConkey test?

a bacterium's ability to metabolize and ferment the carbohydrate lactose

What are we testing metabolically with a 3-sugar fermentation test?

Evaluates whether a bacterium can ferment:

• Glucose (present in a low concentration of 0.1%)

• Lactose (present in 1% concentration)

• Sucrose (present in 1% concentration)

Detects the formation of carbon dioxide or hydrogen gas during sugar fermentation

Tests the ability to reduce sulfur

What are we testing metabolically with an MRVP test?

if they use mixed acid fermentation or 2,3-Butanediol fermentation

What are we testing metabolically with a nitrate reduction test?

a bacterium's anaerobic respiration metabolism—specifically its ability to use nitrate (NO₃⁻) as a terminal electron acceptor in the absence of oxygen

What are we testing metabolically with an oxidase test?

whether a bacterium produces the enzyme cytochrome c oxidase, which plays a key role in the electron transport chain during aerobic respiration

What are we testing metabolically with a catalase test?

an organism's ability to produce the catalase enzyme to defend itself against oxidative stress and tolerate oxygen environments

what is the most common bacteria DNA vector

plasmids

what does transformation of a bacteria mean?

uptake of new DNA bacteria

what does it mean when a bacteria is competent

when they can uptakes extracellular DNA naturally