Microbiology Lab- Oxidase, Catalase, Nitrate Reductase Tests

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Oxidase Purpose

to identify bacteria containing cytochrome C

Cytochrome C

an electron carrier in the electron transport chain

Phenylenediamine

artificial electron donor is used to reduce cytochrome C
colorless in reduced state
purple/blue when oxidized

Cytochrome C is present

phenylenediamine becomes oxidized
purple/blue color

Oxidase Procedure

collect bacteria from agar plate
place on blotter paper
apply reagent and observe color

Time for oxidase test

10 seconds

Purple/Blue

Oxidase Positive

Lack of color

oxidase negative

Catalase Test Purpose

to identify bacteria that produce the enzyme catalase

Catalase principle

most bacteria produce hydrogen peroxide, which is toxic to microbial cells. If the bacteria produces the enzyme catalase, which breaks down hydrogen peroxide into water and oxygen gas

Catalase procedure

place H202 on a colony

Gas bubbles

O2
bacterial colonies that produce catalase
catalase positive

No Gas Bubbles

catalast negative

Nitrase Reduction Test Purpose

to determine if an organism can reduce nitrate into nitrite using nitrate reductase

Nitrase Reductase outcome #1

NO3--->NO2
nitrate reduction positive

Nitrate Reductase outcome #2

NO3--->NO2--->other products (NH4, NO, N2, N2O)
nitrate reductase positive

Nitrase Reductase outcome #3

not metabolized
nitrate reduction -

genetics

presence of enzyme

Nitrase Reductase Procedure

10 drops of A and B

Red Color after Reagent A and B

positive result
nitrate reductase
prescense of nitrite

No color change after Reagent A and B

indicates no nitrate (NO2) present
add zinc powder

Nitrate C reagent

zinc powder

Red Color after reagent C

present of nitrate (NO3)
negative result

No color change after Reagent C

no nitrate (NO3) present
nitrates were reduced to nitrites which were then reduced to other products
positive result

API 20E

analytical profile index
20 biochemical tests
E- enterobacteriaceae

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