Lab 3

ocular micrometer vs stage micrometer

Ocular micrometer: A ruler etched into ocular lens
Stage micrometer: A ruler etched into the center of a microscope slide only used to calibrate

how do you calibrate an ocular micrometer

using the stage micrometer to determine calibration factor
Calibration factor=number of lines in stage scale x 10mcm(distance b/n lines)/number of lines in ocular scale

Phenol red sugar fermentation test purpose and procedure

*contains phenol red indicator, turns yellow w/acidic pH

Purpose: determines if the bacteria can ferment a particular sugar

• If bacteria ferments sugar, red to yellow

• if bacteria is CO2 producing, air bubble forms in tube

Methyl red test

Purpose: determines if bacteria carry out mixed acid fermentation

• Uses methyl red - Voges-Proskauer (MR-VP)

• methyl red reagent added after incubation

• red=positive test

Voges-Proskauer test

Purpose: to determine if bacteria carry out 2,3, butanediol fermentation

• uses MR-VP broth

• after incubation, 1 mL from tube is transferred to sterile tube

• 2 reagents added to 1 mL (VP reagent A & VP reagent B)

• Tube sits 20-30 min

• red color = positive

Citrate test

Purpose: to see if bacteria have the enzyme citrase

• citrate agar used

• inoculated via streak stab method (needle)

• royal blue = positive

Oxidase test

Purpose: determines if the bacteria produces enzyme (cytochrome) oxidase

• no media required

• 1 drop oxidase reagent placed on filter paper

• bacteria added to wet spot

• dark blue/purple = positive (10-30 sec)

Catalase test

Purpose: determines if bacteria produces catalase

• on TSA slant or microscope slide

• after incubation, add 2-3 drops of H2O2 to slant or 1-2 drops on slide

• bubbling = positive

Nitrate reduction test

Purpose: to see if bacteria has enzymes to convert nitrate to nitrite

• nitrate broth w/durham tube inoculated

• bubble=positive for nitrate reduction

• if no bubble, add nitrate reagents A & B

• red=positive for nitrate reduction

• no change zinc added

• no change=positive

• red=negative

Starch hydrolysis

Purpose: determines if bacteria produces enzyme amylase

• starch agar plate inoculated with 1-2 inch line at center via loop

• after incubation, several dropperfuls of iodine are added

• clear halo = positive

Urea hydrolysis

Purpose: determines if bacteria produces enzyme urease

• urease breaks done urea to ammonia

• urea broth inoculated via loop

• pink = positive

SIM

Sulfur, Indole, Motility

Purpose: determines if bacteria can reduce sulfure, produces enzyme tryptophanase, or is motile

***tryptophanase breaks down tryptophan to indole

• stab center w/needle

• For Indole, Kovac’s regent added after incubation

• Black=positive for sulfur

• Red layer=Positive for tryptophanase

• cloudy=positive for motility

What is the tube inside of the tube called

Durham tube

Types of sugar used in phenol test

• Glucose

• Maltose

• Lactose

• Mannitol