BIOCHEMICAL TESTS

- These are tests designed to demonstrate the physiological and chemical characteristics of
microorganisms which enables the bacteriologist, by elimination, to identify the microorganisms
specifically.
- Demonstrates the ENZYME SYSTEM of a bacterial cell.
I. Carbohydrate Fermentation
- anaerobic breakdown of carbohydrates resulting often in the production of large amounts of organic
acids
- Common carbohydrates used in the media:
• Glucose – monosaccharide
• Lactose – disaccharide
• Sucrose - disaccharide
- 2 Enzymes used in Lactose Degradation
1. β-galactoside permease (Lactose permease)
2. β-galactosidase
• LF: possess both of the enzymes
• NLF: do not possess both of the enzymes
• LLF: possess only β-galactosidase
- Medium:
• Liquid or semisolid meat extract broth to which an
indicator and carbohydrates are added (glucose,
lactose, sucrose, and mannitol)
- pH indicators used:
- CHO fermentation using Broth fermentation media
• Purpose: Determine the ability of an organism to
ferment a specific carbohydrate that is
incorporated in a basal medium, with or without
visible gas
• Media used: Phenol red Broth: contains peptone, phenol red (a pH indicator), a Durham tube, and
one carbohydrate (glucose, lactose, or sucrose)
• Positive result: Indicator change to yellow with or without gas formation in Durham tube
II. Triple Sugar Iron
- TSI & KIA: dispensed in tubes with equal butt & slant
- Contents
• CHO:
✓ TSI – glucose (0.1% or 1 part), lactose (1% or 10 parts), and sucrose (1% or 10 parts)
✓ KIA – glucose (0.1% or 1 part), lactose (1% or 10 parts)
• Others: Peptone (2%)
- pH indicator: phenol red
- H2S indicator
• Sodium thiosulfate: forms H2S upon interaction with the organisms
• Ferrous sulfate: binds to H2s = forming a black precipitate
- Medium: Two reaction chamber with an aerobic slant portion and an anaerobic deep portion
- Results:
• Yellow in acidic pH
• Red in alkaline pH
• Black precipitate (+) H2S production
• Bubbles/ crack in medium (+) gas formation
- K/K
• Glucose/Sucrose/Lactose not fermented
• This reaction is typical among the Non-Fermentative organisms
✓ P. aeruginosa
- K/A, H2S (+)
• Glucose fermented
• Lactose/sucrose not fermenters
✓ Salmonella spp.
✓ Proteus
✓ Arizonae
✓ Citrobacter
✓ Edwardsiella
- K/A, H2S (-)
• Glucose fermented
• Lactose/Sucrose not fermented
✓ Yersinia
✓ Escherichia coli
✓ Serratia
✓ Shigella
✓ Providencia
✓ Morganella
✓ Citrobacter
- A/A H2S (+)
• Glucose fermented
• Lactose/Sucrose fermented
✓ Salmonella
✓ Proteus
✓ Citrobacter
- A/A, H2S (-)
• All sugars are fermented
✓ Escherichia coli
✓ Klebsiella
✓ Enterobacter
✓ Serratia
III. ONPG (O-nitrophenyl-beta-D-
galactopyranoside)
- Determines the presence of late or slow lactose
fermenting strains
- ONPG molecule – structurally similar with
lactose
• Can enter the bacterial cell without the Lactose
permease
• In the presence of Beta-galactosidase, ONPG (colorless), is converted into galactose and O-
nitrophenyl which is a yellow chromogen
IV. Indole-MViC
a. KOVAC’S METHOD
- Medium: Tryptone broth or trypticase broth
- Indole indicator: Kovac’s reagent (p-
dimethylaminobenzal-dehyde)
- Positive Result: Development of a pink to deep red
color
b. EHRLICH’S METHOD
- Medium:
• Tryptone broth or trypticase broth
• With Xylene or Ether
- Indole indicator: Ehrlich’s reagent (p-dimethylaminobenzal-dehyde)
- Positive Result: Brilliant red ring between the solvent and the medium.
c. SPOT INDOLE TEST
 - This is a rapid method for the detection of indole.
• Inoculate the bacterium to be tested on an agar medium that contains tryptophan. Trypticase
soy agar or sheep blood agar can be used. Incubate for 18 to 24 hours at the appropriate
temperature to allow for growth.
- Indole Indicator: 1% p-dimethylaminocinnamaldehyde
- Positive Result: blue or blue green color
V. I-Methyl Red-ViC
- Methyl Red Test (Mixed Acid Fermentation
Pathway)
• Principle: Some organisms produce large
amounts 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.
• Medium: MR-VP or the Clark and Lubb’s dextrose broth
• pH indicator: Methyl Red
• Results:
✓ Positive: Red color (pH 4.5 or below)
✓ Negative: Yellow color (above 4.5)
VI. IM-Voges Proskauer-iC
- Voges-Proskauer Test (Butylene glycol pathway)
• 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 guanidine
compounds present in the broth to give a red-colored complex.
• Reagents:
✓ 5% alpha-naphthol in 95% ethyl alcohol
✓ 40% KOH in distilled H2O (provides alkalinity)
✓ 0.5% creatine in distilled H2O (prevents false negative results)
VII. IMVi-Citrate
- Principle: Some organisms can utilize CITRATE
as their sole source of carbon producing acetate
and other alkaline carbonate end products in the
process. These product change the color of the
indicator from green to blue.
- Medium:
• Simmons Citrate Agar/Christensen citrate
medium
• acid: pH 6 (Yellow)
• uninoculated: pH 6.9 (Green)
• alkaline: pH 7.6 (Blue)
- Carbon source: Sodium citrate: the only carbon source
- pH indicator: Bromthymol blue
- Results
• Positive:
✓ Growth with an intense prussian blue color on the slant
✓ Ex. Enterobacter and Klebsiella
• Negative:
✓ Absence of growth and no color change in the medium (remains green)
✓ Ex. Escherichia coli
VIII. Urease
- Principle: This is based upon the ability of some
bacteria to hydrolyze Urea into 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 Pink.
- Medium:
• Christensen’s Urea Agar tubes, or Stuart Urea broth
• Original color: yellow
- pH indicator: Phenol red
- Positive result:
• Color yellow changes to PINK
• Useful in the identification of RAPID UREASE PRODUCERS (w/in
2-4 hours)
✓ Proteus, Providencia, Morganella (PPM)
• WEAK UREASE PRODUCERS within 4 hours
✓ Citrobacter, Klebsiella, Enterobacter, Yersinia, and Serratia
(CKEYS)
IX. SIM (Sulphide, Indole, Motility)
- Motility test
• Medium:
✓ SIM medium or any semisolid motility medium
✓ Can be added with 1% triphenyltetrazolium chloride to aide
visualization (optional)
• Results:
✓ (+) motile organisms spread out/ grow away from inoculation
line
✓ (-) nonmotile organisms grow only along the stab line
- Nonmotile @ 37C: Shigella, Klebsiella, Yersinia
X. Nitrate reduction test
- Principle: Some organisms possess nitrate reductase that can
reduce nitrate (NO3−) to nitrite (NO2−)
- Medium: Peptone Broth with KNO3
- Reagents:
• Reagent A: Sulfanilic acid
• Reagent B: alpha-napthylamine
• Metallic zinc: to confirm negative nitrite results
XI. Decarboxylase Test
- Purpose: Detect the organism’s ability to decarboxylate amino acids (removal of carboxyl group;
amino acid to amine) which results in an alkaline pH.
- Moeller decarboxylase broths containing:
• 1% Lysine
• 1% Ornithine
• 1% Arginine
- pH indicator: Bromcresol purple
- Result
• (+) Purple: alkaline
• (-) Yellow: acid
- CHO source: Glucose
- Triple (+): Plesiomonas shigelloides
- Triple (-): Pantoea agglomerans
XII. Lysine Iron Agar
- Principle: Determine the ability of organisms to deaminate or decarboxylase lysine and produce H2S
gas
- Medium:
• Lysine Iron Agar (Slant/butt)
• CHO: Glucose
• Amino acid: Lysine
- H2S indicator: Ferric Ammonium Citrate
- pH Indicator: Bromcresol Purple
- Interpretation:
• Purple/Purple: Deam - , Decarb +
• Purple/Yellow: Deam -, Decarb -
• Red/Purple: Deam +, Decarb +
• Red/Yellow: Deam +, Decarb -
• Blackening: H2S +
XIII. Phenylalanine Deaminase Test
- Medium used: Phenyl Ethyl Alanine Agar
- Contents:
• 0.2% Phenylalanine
• 10% Ferric Chloride
- Proteus, Providencia, Morganella are the only genera positive for Phenylalanine deaminase.
XIV. Miscellaneous Biochemical Tests (done by request)
a. Gelatin Hydrolysis
- breakdown of gelatin to amino acid
- Gelatin causes liquids to solidify at temp. below 28C
- Hydrolyzed gelatin fails to gel and remains liquid even at temp. below 28C
b. Gelatin stab method
- employs nutrient gelatin deep tubes that contain 12% gelatin
- incubated for at least 48 hours, and then placed into the refrigerator for approximately 30
minutes.
- (+) tube remained liquid
- (-) solidification of the medium
c. Gelatin strip method
- employs strips of clear blue plastic covered with a gray-green coating of gelatin
- (+) gelatin coating is slowly hydrolyzed and the blue plastic strip becomes visible
- (-) no blue plastic visible on the gelatin strip
- Some organisms may produce gelatinase in rather small quantities. Thus, a negative gelatin
strip tube should be reincubated for up to two weeks.
NOTE: Serratia is the only member of the Enterobacteriaceae capable of hydrolyzing gelatin
d. Starch Hydrolysis
- based on the production of amylase to hydrolyze starch
- (+) a reddish color of clear zone will be formed around the bacterial growth.
• B. subtilis
- (-) blue or black area will be formed indicating the presence of starch
• E.coli