Biochemical Tests

Laboratory Objectives
 Tests To Know
 Case Study Tests
Indole
Methyl Red/Voges Proskauer
Citrate
H2S production
Urea hydrolysis
Motility
Lactose fermentation
Sucrose fermentation
Glucose fermentation & gas production
Coagulase
Catalase test
 Indole Test
 How to Perform Test: Inoculate Tryptone broth with inoculating loop.
 Property it tests for: This test is performed to help differentiate
species of the family Enterobacteriaceae. It tests for the bacteria
species’ ability to produce indole. Bacteria use an enzyme,
tryptophanase to break down the amino acid, tryptophan, which
makes by-products, of which, indole is one.

tryptophanase
Tryptophan NH3 + pyruvic acid + indole
 Media and Reagents Used: Tryptone broth contains tryptophan.
Kovac’s reagent—contains hydrochloric acid,
dimethylaminobenzaldehyde, and amyl alcohol—yellow in color.
 Indole (Cont.)

Reading Results: Kovac’s reagent reacts with indole and creates

a red color at the top part of the test tube.
Indole +ve ------ E. coli & P. vulgaris
Indole -ve ------ S. marcescens & E. aerogenes
 Methyl Red (MR)
 How to Perform Tests: Inoculate glucose broths with
inoculating loop. After 48 hours of incubation, add a few drops of
MR reagent
 Properties they test for:
MR—tests- Some microbes produce “mixed acids” (lactic,
formic, succinic, acetic acids) when glucose is fermented. If
these acids are stable they will reduce the pH of the media to
below 5.
 Media and Reagents Used:
Glucose Broth
Methyl Red indicator for acid
 MR (cont.)
 Reading Results:
MR— a + result is red (indicating pH below 6) and a – result is yellow (indicating
no acid production)

left – (E. aerogenes) right + (E. coli)

 Voges Proskauer (VP)
 How to Perform Tests: Inoculate glucose broths with
inoculating loop. After 48 hours of incubation, add a few drops of
VP reagents
 Properties they test for:
VP—tests- Some microbes instead of producing stable acids
from glucose fermentation produce neutral products such as
2,3 butanediol (alcohol). In this chemical reaction acetoin is an
intermediate product. Microbes that produce acetoin and 2,3
butanediol are positive in the Voges-Proskauer test.
 Media and Reagents Used:
Glucose Broth
Voges Proskauer reagents—A: (Barritt’s) 5% Alpha-Naphthol,
& ethanol, B: Potassium Hydroxide, & Deionized Water.
 VP (cont.)
 Reading Results:
VP—A + result is cherry red after VP reagents are added (indicating the
presence of acetoin) and a – result is no color change.

left + (E. aerogenes) and right – (E. coli)

 Citrate Utilization
 How to Perform Test: Inoculate slant with inoculating loop.
 Property it tests for: This test is used to help differentiate
species of the family Enterobacteriaceae. It is selective for
bacteria that has the ability to consume citrate (citrase) as its
sole source of carbon and ammonium as sole nitrogen source.
 Media and Reagents Used: Simmon’s Citrate Agar contains
sodium citrate (carbon source), ammonium ion (nitrogen source),
& pH indicator—bromthymol blue (green at neutral pH and
“prussian blue” in alkaline pH. ).
 Reading Results:
A +ve result is blue (meaning the bacteria metabolized
citrate) and a –ve result remains green
 Citrate (Cont.)

left + (E. aerogenes) and right – (E. coli)

 H2S Production in SIM
 How to Perform Test: Stab SIM media with inoculating needle.
 Property it tests for: This test is used to help differentiate species of
the family Enterobacteriaceae. This test is used to determine the
ability to reduce sulfur into H 2S.
 Media and Reagents Used: SIM media contains the sulfur containing
amino acid, cysteine, sodium thiosulfate, & peptonized iron or ferrous
sulfate.
 Reading Results: H2S will react with the iron or ferrous sulfate and
produce a black precipitate. A positive result has a black precipitate
present and a negative result has no black precipitate.
 Urea Hydrolysis
 How to Perform Test: Inoculate Urea broth with
inoculating loop.
 Property it tests for: This test is done to determine a
bacteria’s ability to hydrolyze urea to make ammonia
using the enzyme urease.
 Media and Reagents Used: Urea broth contains a
yeast extract, monopotassium phosphate, disodium
phosphate, urea, and phenol red indicator.
 Reading Results: Urea broth is a yellow-orange color.
The enzyme urease will be used to hydrolyze urea to
make ammonia. If ammonia is made, the broth turns
a bright pink color, and is positive. If test is negative,
broth has no color change and no ammonia is made.
 Motility Test
 How to Perform Test: Stab motility media with inoculating
needle.
 Property it tests for: This test is done to help differentiate
species of bacteria that are motile.
 Media and Reagents Used: Motility media contains tryptose,
sodium chloride, agar, and a color indicator.
 Reading Results: If bacteria is motile, there will be growth
going out away from the stab line, and test is positive. If bacteria
is not motile, there will only be growth along the stab line. A
colored indicator can be used to make the results easier to see.
Motility

From left to right:

+ – +
 Lactose Fermentation
 How to Perform Test: Inoculate lactose broth with inoculating
loop.
 Property it tests for: This tests for the bacteria’s ability to ferment
lactose.
 Media and Reagents Used: Lactose broth contains beef extract,
gelatin, peptone, and lactose. A phenol red indicator is added to
indicate acid production from fermentation.
 Results
A positive result is yellow after indicator is added (indicating
lactose fermentation)
A negative result will have no color change or will be redish.
 Sucrose Fermentation
 How to Perform Test: Inoculate sucrose broth with inoculating loop.
 Property it tests for: This test is done to help differentiate species of
the family Enterobacteriaceae. This tests for the bacteria’s ability to
ferment sucrose and production of acid end-product
 Media and Reagents Used: Sucrose broth contains beef extract,
gelatin peptone, and sucrose. Phenol red indicator is added to
indicate an acid end-product.
 Results
A positive result is yellow after indicator is added (indicating
sucrose fermentation)
A negative result has no color change or is reddish.
 Glucose Fermentation & Gas
Production
 How to Perform Test: Inoculate broth with inoculating loop.
 Property it tests for: This test is done to help differentiate species of
the family Enterobacteriaceae. 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 beef extract,
gelatine peptone, and glucose. A phenol red indicator is added to
indicate an acid enproduct. 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
 Mannitol Salt Agar (MSA)
 How to Perform Test: Inoculate an MSA plate using streak plate
method and incubate 24-48 hours.
 Property it tests for: This tests for the bacteria’s ability to
tolerate 7% salt concentration and ferment mannitol. The media
is selective because it selects for salt tolerant bacteria. The
media is also differential because it differentiates the salt tolerant
organisms on their ability to ferment mannitol.
 Media and Reagents: MSA media contains nutrient agar,
mannitol, 7% sodium chloride and phenol red indicator.
 MSA Results
 Reading Results:
If the organism is tolerant to salt it will grow.
If the organism is not tolerant to salt it will not grow.
If the salt tolerant organism can ferment mannitol then there will be yellow zones
around the colonies.
If the salt tolerant organism cannot ferment mannitol then the media will remain pink.

Growth with no mannitol fermentation.

Growth with + mannitol fermentation.
 Mstaph broth
 How to Perform Test: Inoculate broth and incubate for 24-
48 hours.
 Property it tests for: This tests for the bacteria’s
ability to tolerate 7% salt concentration and ferment
mannitol. The media is selective because it selects for
salt tolerant Staphylococcus.
 Media and Reagents: This media contains nutrients
appropriate for growing Staphylococcus and 7% salt.
 M-STAPH Results
 Reading Results:
If the organism is tolerant to salt it will grow.
If the organism is not tolerant to salt it will not grow.

Tolerates Salt.
Does not tolerate salt.
 Coagulase
 How to Perform Test: Inoculate rabbit plasma with one
single colony. Break up colony and stir until blended in plasma.
Incubate at 37 degrees C for 24 hours.
 Property it tests for: This tests for the bacteria’s
ability to clot blood plasma using the enzyme
coagulase. If the organism has coagulase it will clump
rabbit plasma.
 Media and Reagents: This media contains rabbit
plasma dissolved in buffer.
 Coagulase Results
 Reading Results:
If the organism is has coagulase it will clump the plasma.
If the organism does not have coagulase it will not clump the plasma.
 CATALASE TEST
 Place a drop of 3% H2O2 on glass slide.
 Select colonies to be tested
 Mix colonies in drop of H2O2 (Hydrogen
Peroxide)

 H2O2 catalase
H2O + O2

Catalase +ve