Biochemical Tests
Objectives
Tests to know
Indole
Methyl Red/Voges Proskauer
Citrate
H2S production in SIM
Urea hydrolysis
Sugar fermentation
Lactose fermentation
Oxidase test
Catalase test
Indole Production Test
The amino acid tryptophan can be broken down by enzyme
tryptophanase to form indole.
Tryptophanase differentiates indole-positive enterics, such as
Escherchia coli and Proteus vulgaris from indole-negative
enterics, such as Serratia marcescens.
Media and Reagents: SIM with tryptophan and Kovac’s reagent.
Method: Inoculate medium and incubate at 37°C for 24-48 hours.
After incubation, add five drops of Kovac’s reagent to the surface.
Do not stir or shake the tube.
Expected Results:
Positive test : Kovac’s reagent combines with indole and turns the surface
red.
Negative test: No red color development
Results for indole test
Methyl Red/Voges Proskauer
(MR/VP)
How to Perform Tests: Inoculate 2 glucose broths
with inoculating loop. After 48 hours of incubation, add a
few drops of MR to one tube, and VP reagents to the other
tube.
Properties they test for: Both tests are used to help
differentiate species of the family Enterobacteriaceae.
MR—tests for acid end products from glucose fermentation.
VP—tests for acetoin production from glucose fermentation.
Media and Reagents Used:
Glucose Broth
Methyl Red indicator for acid
Voges Proskauer reagents—A: Alpha-Naphthol, B: Potassium
Hydroxide (KOH).
MR/VP continued
Reading Results:
MR— a + result is red (indicating pH below 6) and a – result is yellow
(indicating no acid production)
VP—A + result is red pinkish after VP reagents are added (indicating
the presence of acetoin) and a – result is no color change.
Methyl Red: left – and right + VP: left + and right –
Citrate Utilization
Citrate is an organic molecule that can be utilized by bacteria
that produce the enzyme citrase. Citrase is produced by some
bacteria such as E. aerogenes but not by others like E. Coli
Media and Reagent: Simmon’s Citrate Agar. It has citrase as
the only carbon source and PH indicator bromothymol blue
Method: Inoculate the slant and incubate at 37°C for 24-48
hours.
Expected results:
Positive test: Growth and color changes to blue
Negative test: No growth and color remains green
Results for Citrate Test
Negative
Positive
H2S Production
Bacteria use enzyme cysteine desulfurase to hydrolyze the
amino acid cysteine, forming hydrogen sulfide as end-product.
Media and Reagent: SIM with cysteine and ferrous sulfate
(detects H2S)
Method: Inoculate the media and incubate at 37°C for 24-48
hours.
Expected Results:
Positive Test: H2S production = Black
Negative Test: No H2S production = No blackening of medium
Results of H2S production
Negative Positive
Urea Utilization
Some bacteria produce urease, an enzyme capable of
breaking down urea and produce alkaline end
products. This distinguishes Proteus from other
bacteria
Media and Reagent: Urea Broth with phenol red
Method: Inoculate the media with a loop and
incubate at 37°C for 24 hours.
Expected Results:
Positive test: production of alkaline end products = pinkish
red color
Negative test: No color change
Results for Urea Test
Positive
Negative
Glucose Fermentation & Gas
Production
How to Perform Test: Inoculate broth with inoculating loop.
Property it tests for: This tests for the bacteria’s ability to
ferment glucose and produce gas and/or an acid end-product..
Media and Reagents Used: Glucose broth contains phenol
red as indicator to indicate an acid end product. A Durham tube is
added to indicate gas production.
Results
A positive result for acid is yellow after indicator is added
(indicating glucose fermentation)
A positive result for gas is a bubble in the Durham tube.
A completely negative result has no color change or reddish
color and no bubble.
Sugar Fermentation Tests
Tube 1: Negative acid /Negative gas
Tube 2A: Must incubate longer (ambiguous result)
Tube 2B: Positive acid /Negative gas
Tube 3A: Positive acid/ Positive gas
Lactose Fermentation
MacConkey Agar differentiates lactose-fermenting bacteria,
such as E. Coli from non-lactose fermenting bacteria.
Media and Reagent: MacConkey Agar and neutral red dye
Method: Streak MAC plate and incubate at 37°C for 2 days.
Expected results:
Positive test: Lactose fermentation = Growth and color change to pink
Negative test: No lactose fermentation = May or may not grow and no
color change
Results of Lactose Fermentation
- Oxidase test:
Some bacteria produce
Oxidase enzyme
(Pseudomonas )
Detection by adding few
drops of colorless oxidase
reagent
Colonies turn deep
purple in color (positive)
Catalase Test
The Catalase test determines if the organism produces the
enzyme “Catalase”, which breaks down hydrogen peroxide
(H2O2) to water and oxygen (O2).
Catalase
2 H2O2 2 H2O + O2 (gas)
and may be seen in aerobic and facultatively anaerobic
organisms like Staphylococcus bacteria.
Addition of H2O2 lead to production of gas bubbles
(O2 production)
Examples of Biochemical Tests
API 50 Test
