Miscellaneous Biochemical Tests

Miscellaneous Biochemical Tests
Module 1.6
LEARNING OBJECTIVE
 Understand and apply the principles or various biochemical tests.

 Perform various biochemical tests correctly using the appropriate
techniques.
 Presumptively identify some of the organisms using different
biochemical tests.
 Analyze and interpret the results of different biochemical tests.
Catalase Test
Purpose
The Catalase Test is used to identify organisms that produce the enzyme catalase. It
differentiates staphylococci from streptococci.
Reagent: 3% H202
Catalase
2H2O2 2H2O + O2
Types:
a. Aerobic catalase test – using 3% H202
b. Anaerobic catalase test – using 15% H202

How to do the Test
1) Slide method (detects only bound coagulase).

a) Add one drop of 3% Hydrogen peroxide on a clean glass slide.
b) Aseptically take a loopful of the test organism and emulsify in the

H2O2 drop.
2) Tube method (detects the presence of either bound or free).

a) Inoculate the test organism on agar slant and incubate for 24 hours.
b) Allow 1 mL of 3% hydrogen peroxide to flow over the slant.

Limitations
 Colonies which will be used for this test should not be taken from BAP.
Don’t use bacteria from old culture because the enzymes activity drops by time.
 Pseudocatalase reaction – Aerococcus, Enterococcus and Rothia

Coagulase Test
Purpose
The Coagulase Test is typically used to differentiate Staphylococcus aureus from other
Gram-positive cocci
Reagent: rabbit plasma
Positive result: clot/coagulum

Notes
When the slide test is employed, all negative slide reactions
must be confirmed by the tube test .
1) The slide agglutination technique may lead to false-positives:
 since some strains produce clumping factor resulting in a positive slide test
and a negative tube coagulase test.
2) spontaneous agglutination may occur when rough cultures are used.
The tube test is more reliable than the slide test.

The slide test should be read very quickly, as false
positives can occur.
The slide test should not performed with organisms

taken from high-salt media such as Mannitol Salt
Agar, as the salt content can create false positives.
Oxidase Test
 Determines the presenc of bacterial enzyme cytochrome oxidase.
 Based on the ability of the enzyme to produce the dye indophenol blue from the oxidation of
tetra-methyl-1,4-phenylenediamine.
Reagents: 1% Kovac’s reagent (tetramethyl-p-phenylenediamine dihydrochloride) or 1%

Gordons and Mcleods reagent (Dimethyl-p-phenylenediamide dihydrochloride)
 Quality Control
• Positive: Pseudomonas aeruginosa
• Negative: Escherichia coli
Limitations
• Iron Loop, nichrome loop, Stainless steel – surface oxidation products fromed while
doing flame.
• Mac Conkey agar – the pink violet color mau be due to carry over from the medium
Modified Oxidase Test
 To differentiate gram-positive, catalase-positive cocci (micrococci from staphylococci).
Reagent: 1% tetramethylparaphenylenediaminedihydrochloride in dimethylsulfoxide
Indicator: Bromthymol blue (alkaline – blue; acidic: yellow)
 Expected Results
• Positive: Development of blue to purple-blue color
• Negative: No color change
 Quality Control:
• Positive: Micrococcus luteus
• Negative: Staphylococcus aureus
Urease Test
 To determine the ability of the organism to split urea forming two molecules of ammonia by
Urease with resulting alkalinity.
Urea ----- Urease ---- (NH4)2 CO3 + NH3 + CO2
 Medium: Christensen Urea Broth/Slant
 pH indicator: Phenol red
 Quality control: Positive: Proteus vulgaris
Weak positive: Klebsiella pneumoniae
Negative: Escherichia coli

Limitations
 Alkaline reactions may appear after prolonged incubation and may be the result of
peptone or other protein utilization raising the pH.
Rapid Urease – after 2 hours

- “PPM”
Slow Urease – after 4 hours

- “CKEYS”
Dnase Test
 To differentiate organisms based on the production of deoxyribonuclease.
 It is used to distinguish Serratia sp. (positive) from Enterobacter sp., Staphylococcus aureus
(positive) from other species, and Moraxella catarrhalis (positive) from Neisseria sp
 Medium: DNA medium
 Reagent: Methyl green dye
 Quality Control
• Positive: Staphylococcus aureus
• Negative: Escherichia coli.
Two methods
a. Toluidine Blue – (+) = pink zone

Methyl green – (+) = clear zone
b. HCL precipitation – no precipitation after 1N HCL when Dnase (+)

- (+) clear zone ; (-) no clearning
Phenylalanine Deamination
 To detect if the bacteria has the ability to remove the amino group (-NH2) of phenylalanine.
 The genera Morganella, Proteus, and Providencia can be differentiated from other members of
the Enterobacteriaceae family.
 Medium: Phenylalanine medium
 Reagent: FeCl3
 Quality Control
• Positive: Proteus mirabilis
• Negative: Escherichia coli
Nitrate Reduction Test
 To differentiate members of Enterobacteriaceae on the basis of their ability to produce nitrate reductase
enzyme that hydrolyze nitrate to nitrite.
 Medium: Nitrate broth (Heart infusion broth containing 0.2% potassium nitrate)
 Reagents: A: a-napthylamine and Acetic acid
B: Sulfanilic acid and acetic acid
 Quality control:
• Posittive control: E.coli
• Negative control: Acinetobacter baumanni
Result
 Positive: Development of red color w./in 30 seconds
 Negative: No color change
 Negative reaction (True): Nitrates not reduced
 Negative reaction (False): Reduction of products other than nitrites
Development of red colour after the addition of zinc dust indicates presence of residual
nitrate and confirms true negative reaction.
Decarboxylation Test
 To determine the enzymatic ability of an organism to decarboxylate or hydrolyze an amino acid

to form an amine with the liberation of carbon dioxide.
 Indicator: Bromcresol purple
 Medium: Moeller’s medium
• Positive control: Lysine – Klebsiella pneumonia; Ornithine - Enterobacter aerogenes;
Arginine - Enterobacter cloacase

• Negative control: Lysine – Enterobacter cloacae; Ornithine – Klebsiella pneumonae;
Arginine = Klebsiella pneumoniae

Interpretation
Positive: Alkaline (purple)

color change compared with
the coloured tube (pH 6.8)
Negative: No color change or

acid (yellow) in test and
control tube (pH 5.2)
Motility Testing
 To determine whether an enteric organism is motile. An organism must have flagella to be

motile.
Medium: SIM or MIO
 Result:
Positive: Motile organisms will spread out into the medium from the site of
inoculation
Negative: Nonmotile organisms remain at the site of inoculation
• Positive: Escherichia coli
• Negative: Staphylococcus aureus
Motility test using the
MIO medium
Motility test using the SIM medium.
Malonate Utilization Test
 To determine the ability of an organism to utilize sodium malonate as its sole source of
carbon.
Result:
Positive: Deep Blue color
Negative: Green
• Positive: Enterobacter aerogenes
• Negative: Yersinia ruckeri
AMA (Acetate, Malonate,Acetamide) Utilization
Test
 To detect bacteria the utilizes acetate/malonate/acetamide as a sole source of

carbon.
 Indicator: Bromthymol blue
 Incubation: 37 degrees Celsius for 5 to 7 days
 Results: (+) Blue; (-) Green
Positive control Negative Control

Acetate Escherichia coli Shigella flexneri
Malonate Salmonella Shigella
Acetamide P.Aeruginosa S.maltophilia
Gelatin Hydrolysis Test
 Test to detect if bacteria is able to produce the enzyme gelatinase.
Result:
Positive: Gel liquefaction
Negative: Gel solidification
• Positive: Bacillus subtilis/ Proteus vulgaris
• Negative: Escherichia coli/ Enterobacter aerogenes
• Uninoculated control tube: medium becomes solid after refrigeration

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Miscellaneous Biochemical Tests

 Understand and apply the principles or various biochemical tests.

a. Aerobic catalase test – using 3% H202

b. Anaerobic catalase test – using 15% H202

1) Slide method (detects only bound coagulase).

b) Aseptically take a loopful of the test organism and emulsify in the

2) Tube method (detects the presence of either bound or free).

b) Allow 1 mL of 3% hydrogen peroxide to flow over the slant.

 Pseudocatalase reaction – Aerococcus, Enterococcus and Rothia

Reagent: rabbit plasma

Positive result: clot/coagulum

When the slide test is employed, all negative slide reactions

must be confirmed by the tube test .

1) The slide agglutination technique may lead to false-positives:

2) spontaneous agglutination may occur when rough cultures are used.

The tube test is more reliable than the slide test.

The slide test should not performed with organisms

 Determines the presenc of bacterial enzyme cytochrome oxidase.

Reagents: 1% Kovac’s reagent (tetramethyl-p-phenylenediamine dihydrochloride) or 1%

 To differentiate gram-positive, catalase-positive cocci (micrococci from staphylococci).

Reagent: 1% tetramethylparaphenylenediaminedihydrochloride in dimethylsulfoxide

Indicator: Bromthymol blue (alkaline – blue; acidic: yellow)

Urea ----- Urease ---- (NH4)2 CO3 + NH3 + CO2

 Medium: Christensen Urea Broth/Slant

 pH indicator: Phenol red

 Quality control: Positive: Proteus vulgaris

Weak positive: Klebsiella pneumoniae

Negative: Escherichia coli

Rapid Urease – after 2 hours

Slow Urease – after 4 hours

 To differentiate organisms based on the production of deoxyribonuclease.

 Medium: DNA medium

 Reagent: Methyl green dye

a. Toluidine Blue – (+) = pink zone

b. HCL precipitation – no precipitation after 1N HCL when Dnase (+)

 Medium: Phenylalanine medium

 Reagents: A: a-napthylamine and Acetic acid

B: Sulfanilic acid and acetic acid

 Negative: No color change

 Negative reaction (True): Nitrates not reduced

 Negative reaction (False): Reduction of products other than nitrites

 To determine the enzymatic ability of an organism to decarboxylate or hydrolyze an amino acid

 Indicator: Bromcresol purple

 Medium: Moeller’s medium

Arginine - Enterobacter cloacase

Arginine = Klebsiella pneumoniae

Positive: Alkaline (purple)

Negative: No color change or

 To determine whether an enteric organism is motile. An organism must have flagella to be

Positive: Deep Blue color

 To detect bacteria the utilizes acetate/malonate/acetamide as a sole source of

 Indicator: Bromthymol blue

 Incubation: 37 degrees Celsius for 5 to 7 days

 Results: (+) Blue; (-) Green

Positive control Negative Control

 Test to detect if bacteria is able to produce the enzyme gelatinase.

Positive: Gel liquefaction

Negative: Gel solidification

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