Week 5

Question 1

Oxidase Test

Answer 1

Used to identify bacteria that use the enzyme cytochrome c oxidase for cellular respiration

• Cytochrome c oxidase is the final enzyme used in the electron transport chain
• Differentiate Gram Negative Bacilli

Question 2

The Oxidase Test: Oxidase Reagent

Answer 2

• Chromogenic reducing agent- chemical that develops color as it is oxidized
• Tetramethyl-p-phenylenediamine- is the reagent used to test for the enzyme cytochrome c oxidase
• The Oxidase agent is colorless in the reduced state and turns purple when oxidized

Question 3

Chromogenic Reducing Agent

Answer 3

chemical that develops color as it is oxidized

-the oxidase agent is colorless in the reduced state and turns purple when oxidized

Question 4

Tetramethyl-p-phenylenediamine

Answer 4

reagent used to test for the enzyme cytochrome c oxidase

Question 5

Oxidase Test: Oxidase Positive

Answer 5

-Neisseria
-Pseudomonadaceae
(do not use a metal loop to transfer bacteria, the metal will react with the oxidase reagent and give a false positive result

Question 6

Oxidase Test: Oxidase Negative

Answer 6

-Enterobacteriaceae
(do not use a metal loop to transfer bacteria, the metal will react wit the oxidase reagent and give a false positive result

Question 7

Gram Stain

Answer 7

a differential stain
Step: Reagent: Time: Gram+ Gram-
1. Primary Stain–>crystal violet (1 minute)–>purple-> purple
2. Mordant–>Grams Iodine(1 min)–> Purple–>purple
3. Decolorizer–>acetone+isopropanol(brief)–>purple–>colorless
4. Counterstain–>Safranin–>(1 min)–>purple–>red/pink

Question 8

Comparison of Gram + and Gram - Cell Walls

Answer 8

Gram+:

• teichoic acid
• lipoteichoic acid
• thick peptidoglycan layer
• cell membrane of phospholipids

Gram -:

• lipopolysaccharides (LPS)
• Outer Membrane ( contains LPS)
• lipoproteins
• thin peptidoglycan layer
• cell membrane
• contain porin proteins in upper layer-regulate molecules entering and leaving cell

Question 9

Gram + cell wall

Answer 9

• teichoic acid
• lipotechoic acid
• thick peptidoglycan layer
• cell membrane of phospholipids

Question 10

Gram - cell walls

Answer 10

• lipopolysaccharides (LPS)
• Outer Membrane (contains LPS)
• lipoproteins
• thin peptidoglycan layer
• cell membrane
• contain porin proteins in upper layer- regulate molecules entering and leaving cell

Question 11

Steps of the Gram Stain

Answer 11

1. Application of Crystal Violet (purple dye)
2. Application of Iodine (mordant)
3. Alcohol wash (decolorization)
4. Application of Safranin (counterstain)

Question 12

Gram + reaction in the gram stain

Answer 12

retain crystal violet and stain purple

Question 13

Gram - reaction in the gram stain

Answer 13

lose crystal violet and stain red/pink from safranin counterstain