WEEK 11-12: BIOCHEMICAL TESTS

BIOCHEMICAL TESTS
Are tests designed to demonstrate the physiological and chemical characteristics of microorganisms which enables the
bacteriologist, by elimination, to identify the microorganisms specifically.

Carbohydrate Fermentation

• Fermentation is the anaerobic breakdown of substance (carbohydrates) resulting often in the production of large amounts of
organic acids.
• Enterobacteriaceae and other glucose fermenters first begin to metabolize glucose, since, glucose utilizing enzymes are present
and it is the simplest sugar from which the bacteria can gain the most energy.
• Carbohydrate Fermentation tests:
A. Carbohydrate Fermentation Medium: The carbohydrate fermentation medium is a liquid or semisolid meat extract broth with an
indicator and a carbohydrate added.

• pH indicators commonly used:

1. Bromocresol purple: Purple (alkaline) to Yellow (acid) at pH 6.3

2. Phenol red: Red (alkaline) to Yellow (acid) at pH 7.9
3. Andrade’s acid fuchsin: Pale yellow (alkaline) to Reddish pink (acid) at pH 5.5
• Carbohydrates commonly used:

1. Glucose
2. Lactose
3. Sucrose
4. Mannitol

B. Durham Tube: Consists of a test tube with a smaller inner tube. The test tube is filled with about 5 mL of the carbohydrate broth,
and the smaller tube, mouth down, is slipped into it. The tubes are sterilized in the autoclave. During this procedure, the broth rises
in the inner tube.

• Principle: Organisms that ferment the carbohydrate in the broth produce acid, thus, changing the color of the medium (depending
upon the indicator used) to the acidic color. Gas formation may accompany this reaction in some microorganisms.
• Procedure:

1. Inoculate broth lightly

2. Incubate overnight at 37°C
3. Examine daily for 4-5 days
• Results:
Positive: Bromocresol purple: Purple (alkaline) to Yellow (acid)
Phenol red: Red (alkaline) to Yellow (acid)
Andrade’s acid fuchsin: Pale yellow (alkaline) to Reddish pink (acid)
Gas formation in the inverted insert tube

Negative: Color of the medium remains the same

No gas formation

Carbohydrate Fermentation with Triple Sugar Iron (TSI) and Kligler’s Iron Agar (KIA)
TSI is a butt/slant medium considered to be a modification of KIA. The only difference between the two is the sucrose is not included
in KIA.

Carbohydrate Content
1. TSI: Glucose (0.1%), Lactose (1%), Sucrose (1%)
2. KIA: Glucose (0.1%), Lactose (1%)
pH Indicator: Phenol red
H2S Indicator: Ferric ammonium sulfate
Principle:

1. Glucose utilization by certain microorganisms occurs both aerobically on the slant where oxygen is available and anaerobically in
the butt. Once glucose is fermented, acids will be produced. These acids in the medium will cause the phenol red to assume a
yellow color. Thus, the butt and slant will appear yellow after 6 hours of incubation.
2. After depletion of the limited glucose, some organisms utilize lactose or sucrose and continue making acid end products. The slant
and butt will remain yellow after 18-24 hours incubation. The reaction is called acid over acid (A/A), fermenting two or all of the
sugars.
3. Some organisms are not able to utilize lactose but instead break down peptone in the medium. The byproducts of this protein
metabolism, which occurs on the surface of the slant, are alkaline and cause the phenol red to revert back to its original red color.
After 18-24 hours incubation, the TSI will thus show a red slant a retained yellow butt. The reaction is called alkaline over acid
(K/A), signifying that only glucose was fermented.
4. Glucose non-fermenters may also produce alkaline products from peptone utilization. Reaction seen will be alkaline over alkaline
(K/K), signifies no sugar was fermented.
5. Some organisms have the ability to produce gas from the fermentation of sugars while others produce large amounts of hydrogen
sulfide gas.
Procedure:

1.

1. Inoculate medium and incubate overnight

2. Record pH changes only after 11-24 hours incubation
Results:

1. Yellow in acid pH, red in alkaline pH

2. H2S production: Presence of gas precipitate
3. Gas production: Bubbles in the medium

Other Methods of Determining Carbohydrate Fermentation

• Russell’s Double Sugar (RDS): Contains glucose and lactose with Andrade’s acid fuchsin as indicator. The positive result is a
change of color from pale yellow (alkaline) to reddish pink (acid).
• With the use of Smith Fermentation Tube

IMVIC REACTION: Indole, Methyl Red, Voges Proskauer, Citrate

A. Indole Test
Principle: Organisms that produce the enzyme tryptophanase are able to degrade tryptophan into indole, which can be detected by
its ability to combine with certain aldehydes to form a colored compound.
Methods:
I. Kovac’s Method

1. Medium: Tryptophan broth/ Trypticase broth

2. Indole indicator: Kovacs reagent (p-dimethylaminobenzaldehyde)
3. Procedure and Result:
o Add 0.5 mL of Kovacs reagent to 2-3 mL of a 48-hour old broth culture
o Gently shake the tube and observe for the development of pink to deep red color which is a positive result

II. Erlich’s Method

1. Medium: Tryptophan broth/ Trypticase broth

2. Indole indicator/ Erlich’s reagent: p-dimethylaminobenzaldehyde
3. Procedure and Result:
•
o Add 1mL xylene to a 48-hour old broth culture
o Shake the tube well and allow to stand for a few minutes until the solvent rises to the surface
o Gently add 0.5 mL of Erlich’s reagent down the sides of the tube and observe for the development of a brilliant red ring between the
solvent and medium

III. Spot Indole Test: Rapid method for the detection of indole

1. Indole indicator: 1% p-dimethylaminocinnamaldehyde

2. Procedure and Result:
•
o Saturate a filter paper in the bottom of a petri dish with the reagent
o With a wooden stick or loop, rub a portion of the colony on the filter paper and observe for the rapid development
of blue or blue green color

B. Methyl Red Test

Medium: MR-VP medium/ Clark and Lubs dextrose broth medium
pH Indicator: Methyl red
Principle: Some organisms produce large amount of acid from dextrose while others produce less. This test is based upon the final
hydrogen ion concentration (acidity) reached by a culture. The test will be positive if the pH is 4.5 or lower, and negative if above pH
4.5
Procedure: Add 5 drops of methyl red to 5 mL of a 48-hour old broth culture and observe the reults
Results: Positive – Red color (pH 4.5 or below)
Negative – Yellow color (pH above 4.5)

C. Voges Proskauer Test

Medium: MR-VP medium
Reagents:

1. 5% alpha naphthol in 95% ethyl alcohol

2. 40% KOH in distilled H2O (Provides alkalinity)
3. 5% creatine in distilled H2O (Prevents false negative results)
Principle: Some bacteria have the ability to produce acetoin (acetylmethylcarbinol) from glucose. In an alkaline pH, acetoin is
oxidized to diacetyl (dimethylcarbinol), which reacts with guanidino compounds present in the broth to give a red-colored complex.
Procedure and Result:

1. Inoculate MR-VP broth and incubate at 35-37°C for 48 hours

2. To 1mL of the culture broth, add 2 drops creatine solution, 6 drops alpha naphthol, and then 2 drops KOH, shaking after the addition
of each reagent
3. Observe for a pink to cherry red color which is the positive result

D. Citrate Test
Medium: Simmon’s citrate slant
pH Indicator: Bromthymol blue
Principle: Some organisms can utilize citrate as their sole source of carbon producing acetate and other alkaline carbonate end
products in the process. These products change the color of the indicator from green to blue.
Procedure and Result:

1. Inoculate simmon’s citrate slant and incubate for 24 hours or up to 4 days at 35-37°C
2. Positive – Prussian blue color (Alkaline)
3. Negative – No color change (Green)
 Catalase Test
Principle: The test is based on the presence of catalase in some microorganisms catalyzing the release of oxygen and water from
hydrogen peroxide.
Procedure:

1. Pour 1mL of hydrogen peroxide over a culture grown on infusion agar slant or pick a colony and mix it with 1 drop 3% hydrogen
peroxide on a clean glass slide
Positive Result:

1. Rapid evolution of bubbles of gas

2. NOTE: The test should not be performed with colonies grown on blood agar (RBC contain catalase thus, gives a positive result)

Oxidase Test
Principle: Some organisms produce cytochrome oxidase which in the presence of air, acts on certain aromatic amines to produce
colored compounds.
Methods:
I. Spot Oxidase Test (Kovac’s Method)

1. Reagent: 1% tetramethyl-p-phenylenediamine dihydrochloride

2. Procedure and Result:
•
o Place a filter paper into a petri dish and moisten it with several drops of the reagent
o With a platinum wire or wooden stick, rub 24-hour old colony on the moistened filter paper
o Observe for a color change to blue or dark purple which is a positive result
II. Oxidase Test (for Neisseria)

1. Reagent: 1% dimethyl-p-phenylenediamine hydrochloride

2. Procedure and Result:
•
o Add several drops of the reagent onto the colony on a solid medium or add 1 drop of the reagent to a filter paper and spread the
colony onto it.
o The colonies become pink, then red and finally purple black
III. Indophenol Method

1. Reagents:
•
o p-aminodimethylaniline hydrochloride (or oxalate)
o Alpha naphthol

1. Procedure and Result:

•
o To an 18-24-hour old culture grown on a nutrient agar slant add 2-3 drops of the above reagents
o Tilt the tube to mix the reagents and flow over the growth
o Intense blue color within 2 minutes (Positive result)

Urease Test
Medium: Christinsen’s urea agar or urea broth
pH Indicator: Phenol red
Principle: This is based upon the ability of some bacteria to hydrolyze urea to ammonia water and CO2 by means of the enzyme
urease. If urea is split to form ammonia, the medium becomes alkaline and the indicator turns from yellow to red.
Procedure: Inoculate medium and incubate at 35°C and observe at 15, 30 and 60 minutes and up to 4 hours for a change in color
Result: Positive – Pink or red
Negative – Yellow

Lysine Decarboxylase Test

Medium: Lysine Iron Agar (LIA) or Mueller’s Medium
pH Indicator: Bromcresol Purple
Principle: Some organisms can decarboxylate the amino acid lysine to form cadaverine leading to the alkalinization of the medium
and subsequent change in color of the indicator from yellow to purple.
Procedure:

1. Inoculate 4 tubes of LIA and overlay each tube with sterile mineral oil
2. Incubate all 4 tubes at 35°C and read daily for not more than 4 days
Result: Positive – Purple
Negative – Yellow

Phenylalanine Deaminase Test

Medium: Phenylalanine agar
Reagent: 10% aqueous ferric chloride
Principle: Organisms like Proteus and Providencia produce phenylalanine deaminase that breaks down phenylalanine to
phenylpyruvic acid which reacts with ferric ion to produce a green color.
Procedure:
1. Inoculate slant medium and incubate overnight at 35°C
2. After incubation let 4-5 drops of reagent run down over the growth
Result: Positive – Green color on the surface of the slant and in the fluid on the slant

Motility Test
Medium: SIM (Sulfide, Indole, Motility) or any semisolid motility test medium
Procedure: With a straight needle stab the medium to a depth of 10 mm and incubate overnight at 35°C under 10% CO2 tension
Result: Positive – Motile organisms spread out or grow away from the line of inoculation
Negative – Non motile organisms grown only along the stab line

Nitrate Reduction Test

Medium: Peptone broth with potassium nitrate
Reagents:

1. Reagent A: Sulfanic acid and acetic acid

2. Reagent B: N-dimethyl-1-naphthylamine and acetic acid
Principle: Some organism possesses nitrate reductase that can reduce nitrate to nitrite. Nitrite combines with an acidified
naphthylamine substrate forming a red colored end product. However, if the organism further reduces nitrite to nitrogen gas, the test
for nitrite will yield a negative (colorless) result.
Procedure:

1. Grow organism in nitrate broth for 24-48 hours or longer for poorly growing organisms
2. A Durham tube may be placed into the broth to detect the presence of nitrogen gas
3. Add 3 drops of reagent A and 3 drops of reagent B into the broth and wait for 30 minutes
Result: Positive – Red
Negative – No color change