Practicum II

Question 1

Hydrolysis of Starch

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 1

• Medium: Starch agar plate
• Substrate: Starch (Polysaccharide)
• Enzyme/Pathway: Hydrolysis of Amylase
• pH Ind./Reagents: Iodine
• End Products: Maltose (Disacharide)
• (+) appearance: Light/Golden area around colonies
• (-) appearance: Dark around colonies

Question 2

Hydrolysis of Gelatin

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 2

• Medium: Nutrient gelatin tall
• Substrate: gelatin (protein)
• Enzyme/Pathway: Hydrolysis of gelatinase
• pH Ind./Reagents: N/A
• End Products: amino acids
• (+) appearance: Liquid
• (-) appearance: Solid

Question 3

Fermentation of Carbohydrates

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 3

• Medium: CHO Broth (1 CHO, Basal Medium, Phenol Red, Durham Tube)
• Substrate: 1x CHO (eg. glucose)
• Enzyme/Pathway: Fermentation
• pH Ind./Reagents: N/A
• End Products: A: ATP + Organic Acid, AG: ATP + Organic Acid + CO2 gas, SA: ATP + Organic Acid
• (+) appearance: A: Yellow, AG: Yellow + Bubble. SA: Orange
• (-) appearance: -: Red, B: Magenta, SB: half red/half magenta

Question 4

Fermentation

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 4

• Medium: Litmus Milk
• Substrate: Lactose
• Enzyme/Pathway: Fermentation
• pH Ind./Reagents: N/A
• End Products: A: Organic Acid + ATP, AC: Acid + ATP + Hard Curd (Protein), AC: Acid + ATP+ Hard Curd + CO2 gas
• (+) appearance: A: Pink milk, AC: Pink milk + Curd, AC: Pink milk + curd with gas cracks
• (-) appearance: Purple milk like control, blueberry shake with no solid

Question 5

Alkanization

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 5

• Medium: Litmus Milk
• Substrate: Casein
• Enzyme/Pathway: Alkanization
• pH Ind./Reagents: N/A
• End Products: Polypeptides (colloid) + basic amines
• (+) appearance: B: Blue milk (blueberry shake)
• (-) appearance: Purple milk (like control), pink milk

Question 6

Peptonization

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 6

• Medium: Litmus Milk
• Substrate: Casein & Lactalbumin (colloid) / Proteins
• Enzyme/Pathway: Peptonization (hydrolysis)
• pH Ind./Reagents: N/A
• End Products: Amino Acids (crystalloid) / Amino Acids + Rennet Curd
• (+) appearance: P: Juice / PC: Juice + Flakes
• (-) appearance: Purple milk like control, blueberry shake, pink milk

Question 7

Reduction

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 7

• Medium: Litmus Milk
• Substrate: Litmus
• Enzyme/Pathway: Reduction (Anaerobic Respiration)
• pH Ind./Reagents: N/A
• End Products: Leucolitmus (reduced litmus + ATP)
• (+) appearance: R: bleached (bottom)
• (-) appearance: Litmus colors (purple, pink, blue)

Question 8

Reduction of Nitrates

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 8

• Medium: Nitrate Broth
• Substrate: Nitrate
• Enzyme/Pathway: Reduction of Nitrate / Anaerobic Respiration
• pH Ind./Reagents: Pour out 1 mL, 3 drops of sulfanillic acid, 2 drops of dimethyl alpha naphthylamine solution
• End Products: ATP + Nitrite
• (+) appearance: Orange/Cherry Red
• (-) appearance: Baby pink, beige

Question 9

Production of Decarboxylase

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 9

• Medium: aa broth (1 aa, brom cresol purple, oil, basal medium)
• Substrate: aa (ornithine, arg)
• Enzyme/Pathway: decarboxylation/decarboxylase
• pH Ind./Reagents: N/A
• End Products: basic amine + CO2 gas
• (+) appearance: Purple, Violet, Silvery
• (-) appearance: Burgundy, Yellow

Question 10

Hydrolysis of Urea

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 10

• Medium: Urea Broth (Phenol Red)
• Substrate: Urea
• Enzyme/Pathway: Hydrolysis of Urease
• pH Ind./Reagents: N/A
• End Products: Basic ammonia + CO2 gas
• (+) appearance: Magenta
• (-) appearance: Red, Yellow

Question 11

Oxygen Requirements

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 11

• Obligate aerobe
• Obligate Anaerobe
• Facultative Anaerobe
• Microaerophiles

Question 12

Production of Hydrogen Sulfate

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 12

• Medium: SIM tall (contains Fe2+)
• Substrate: Cysteine
• Enzyme/Pathway: Production of H2S/ Anaerobic Respiration
• pH Ind./Reagents: N/A
• End Products: ATP + H2S gas
• (+) appearance: Black Colloid (FeS)
• (-) appearance: Like Control

Question 13

Production of Indole

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 13

• Medium: SIM tall (containes Fe2+)
• Substrate: Tryptophan
• Enzyme/Pathway: Production of Indole
• pH Ind./Reagents: Add 10 drops of Kovac’s Solution
• End Products: Indole
• (+) appearance: Red Kovac’s at top
• (-) appearance: Yellow Kovac’s

Question 14

Motility

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 14

• Medium: Sim or Motility Medium
• Substrate: N/A
• Enzyme/Pathway: N/A
• pH Ind./Reagents: N/A
• End Products: N/A
• (+) appearance: feathery spread across stab line
• (-) appearance: straight spread across stab line or close to stab line

Question 15

Methyl Red Test

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 15

• Medium: MRVP Broth
• Substrate: Glucose
• Enzyme/Pathway: Fermentation
• pH Ind./Reagents: Shake, Add 1mL to Clean test tube, 10 drops Methyl Red
• End Products: ATP + Organic Strong Acids (4.4 or less)
• (+) appearance: Red
• (-) appearance: Yellow

Question 16

Voges-Proskauer Test

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 16

• Medium: MRVP Broth
• Substrate: glucose
• Enzyme/Pathway: fermentation
• pH Ind./Reagents: Shake, add 1mL to clean test tube, 10 drops alpha naphthol, 10 drops potassium hydroxase, creatine, stand for 20-25 minutes
• End Products: ATP + AMC (acetyl methyl carbinol) + organic acids
• (+) appearance: Ring of red at top of test tube
• (-) appearance: no ring/yellow test tube

Question 17

Utilization of Unusual Nitrogen

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 17

• Medium: Ammonium Phosphate broth (brom cresol purple)
• Substrate: Ammonium Phosphate
• Enzyme/Pathway: Use of unusual Nitrogen/ammonium phosphate test
• pH Ind./Reagents: N/A
• End Products: Phosphoric Acid + Nitrogen is used
• (+) appearance: Dirty Yellow
• (-) appearance: Burgundy, Violet

Question 18

Utilization of Unusual Carbon

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 18

• Medium: Na Citrate Broth
• Substrate: Citrate
• Enzyme/Pathway: Use of unusual carbon/Na citrate test/Citratase
• pH Ind./Reagents: N/A
• End Products: Growth
• (+) appearance: Cloudy, “shiggle” tube and see flakes like snowglobe, thick layer at top
• (-) appearance: teeny bit at the bottom of the test tube (not bowl worth)

Question 19

Detection of Catalase

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 19

• Medium: Slant (any except blood)
• Substrate: H2O2 (hydrogen peroxide)
• Enzyme/Pathway: Catalase
• pH Ind./Reagents: H2O2 FLOOD!
• End Products: H2O + O2 gas
• (+) appearance: bubbles
• (-) appearance: no rxn

Question 20

MSA/Staph

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 20

• Medium:
• Substrate:
• Enzyme/Pathway:
• pH Ind./Reagents: N/A
• End Products:
• (+) appearance:
• (-) appearance:

Question 21

Coagulase Production

Answer 21

• Medium:
• Substrate:
• Enzyme/Pathway:
• pH Ind./Reagents: N/A
• End Products:
• (+) appearance:
• (-) appearance:

Question 22

Latex Agglutination

Medium, Substrate, Enzyme/Pathway, pH Ind./Reagents, End prod., +/- app.

Answer 22

• Medium:
• Substrate:
• Enzyme/Pathway:
• pH Ind./Reagents: N/A
• End Products:
• (+) appearance:
• (-) appearance:

Question 23

Hemolysin Production

Answer 23

• α: partial lysis of hemoglobin, leaving greenish/brown discoloration around colonies
• β: Total lysis of hemoglobin and RBCs, leaving clear colorles zones surrounding colonies
• γ: Plasma proteins are used for growth, red (no change in medium)