TOPIC I: BIOCHEMICAL TEST PROG MT

MICROBIOLOGY 401
Triple Sugar Iron (TSI) / Kligler’s Iron Agar (KIA) Media Used:
●​ Moeller Decarboxylase Broth:
Principle: Determines ability to ferment glucose, lactose, sucrose, -​ 1% Lysine
and to produce gas or H₂S. -​ 1% Ornithine
-​ 1% Arginine
Medium Contents: Glucose (0.1%), Lactose (1%), Sucrose (1%) ●​ pH indicator: BROMCRESOL PURPLE
●​ (+) Purple: alkaline
Phenol Red (pH indicator) ●​ (-) Yellow: acid
Sodium Thiosulfate + Ferrous Sulfate (H₂S indicator) ●​ CHO Source: Glucose
●​ 1 control tube - without amino acid
CHO Content:
●​ TSI Lysine Iron Agar
-​ Glucose (0.1% or 1 part)
-​ Lactose (1% or 10 parts) Principle: Determine the ability of organisms to deaminate or
-​ Sucrose (1% or 10 parts) decarboxylase lysine and produce H₂S gas
●​ KIA -​ Useful in identification of Salmonella, members of
-​ Glucose (1%) Proteus group (Proteus, Providencia, and Morganella)
-​ Lactose (1%) which are deaminase (+)
-​ Selective Medium
Results:
●​ A/A - Yellow/Yellow Medium: Lysine Iron Agar (Slant/Butt)
●​ K/K - Red /Red
●​ K/A - Red/Yellow Contains:
●​ A/K - not possible ●​ Protein, Glucose, Lysine
●​ H2S (+) - Black Precipitate ●​ Ferric Ammonium Citrate - H₂S INDICATOR
●​ Gas (+) - Bubbles/Crack in Medium ●​ Bromcresol Purple - pH INDICATOR
-​ Yellow if pH <1.2
Summary of TSI Reactions -​ Purple if pH >6.8
●​ K/K, H2S (-)
-​ Glucose/Sucrose/Lactose not fermented typical IMViC Reaction
among the non-fermentative organisms
●​ K/A, H2S (+), Gas (+) Indole, Methyl Red, Voges Proskauer, and
-​ Glucose fermented with acid and gas Citrate
-​ Lactose/Sucrose non-fermenters
➢​ Salmonella Indole
➢​ Proteus
➢​ Citrobacter I.​ KOVAC’S METHOD
●​ K/A, H2S (-), Gas (-) ●​ Medium:
-​ Glucose fermented, with acid, no gas -​ Tryptone/Trypticase Broth
-​ Lactose/Sucrose not fermented ●​ Indole Indicator:
➢​ Shigella -​ Kovac’s reagent (pdimethylaminobenzal-dehyde)
➢​ Providencia ●​ Positive Result:
➢​ Serratia -​ Red color
➢​ Anaerobic [Link]
●​ A/A, H2S (-), Gas (+) II.​ EHRLICH’S METHOD
-​ All sugars are fermented with acid and gas ●​ Medium:
➢​ Escherichia -​ Tryptone Broth or Trypticase Broth with
➢​ Klebsiella Xylene
➢​ Enterobacter ●​ Indole Indicator:
●​ A/A, H2S (+), Gas (+) -​ Ehrlich’s reagent
-​ Glucose fermented with gas (pdimethylaminobenzal-dehyde)
-​ Lactose/Sucrose fermented ●​ Positive Result:
➢​ Citrobacter freundii -​ Red color

Decarboxylase Test Benzaldehyde: Pink to Purple

Cinnamaldehyde: Green to Blue
Principle: Degradation of amino acids and their specific end
products III.​ RAPID INDOLE TEST
●​ Lysine (amino acid) → Lysine Decarboxylase ●​ Rapid method for the detection of indole
Cadaverine (amine) + CO2 ●​ Smeared onto filter paper that has been moistened with
●​ Ornithine → Ornithine Decarboxylase → Putrescine pdimethylaminocinnamaldehyde
-​ Deaminase (+): RED ●​ Indole Indicator:
-​ Decarboxylase: Alkaline Purple -​ p-dimethylaminocinnamaldehyde
●​ Positive Result:
-​ blue or blue-green color in the filter paper

BMLS GROUP 5 1
 Methyl Red and Voges-Proskauer -​ WEAK UREASE PRODUCERS within 4
hours
Methyl Red Test (Mixed Acid Fermentation ➢​ Citrobacter, Klebsiella, Enterobacter,
Pathway) Yersinia, and Serratia (cKEYS)

Principle: Some organisms produce large amounts of acid from Motility Test
dextrose while others produce less. This test is based upon the final
hydrogen ion concentration (acidity) reached by a culture. The test ●​ Medium: SIM medium or any semisolid motility
will be positive if the pH is 4.5 or lower, and negative if above pH medium
4.5. -​ Can be added with 1% triphenyltetrazolium
chloride to aid visualization (optional)
●​ Medium: -​ SIM has iron (reacts w/ H2S gas) & sodium
-​ MR-VP or the Clark and Lub’s medium thiosulfate
●​ pH indicator: ●​ Results:
-​ Methyl Red -​ (+): motile organisms spread out/ grow away
●​ Results: from inoculation line
-​ (+): Red color (pH 4.5 or below) -​ (-): nonmotile organisms grow only along the
-​ (-): Yellow color (above 4.5) stab line
●​ Nonmotile @ 37C: Shigella, Klebsiella, Yersinia
Voges-Proskauer Test (Butylene Glycol Pathway)
Carbohydrate Fermentation
Principle: Some bacteria have the ability to produce acetoin
(acetylmethylcarbinol) from glucose. In an alkaline pH, acetoin is ●​ Anaerobic breakdown of carbohydrates resulting often in
oxidized to diacetyl (dimethylcarbinol), which reacts with the production of large amounts of organic acids
guanidine compounds present in the broth to give a red colored ●​ 2 Enzymes used in Lactose Degradation
complex. -​ β-galactoside permease (Lactose permease)
-​ β-galactosidase
●​ Medium: ●​ LF: possess both of the enzymes
-​ MR-VP or the Clark and Lub’s Medium ●​ NLF: do not possess both of the enzymes
●​ Reagents: ●​ LLF: possess only β-galactosidase
-​ Alpha-naphthol
-​ 40% KOH or NaOH CHO Fermentation Using Broth Fermentation Media
●​ Results:
-​ (+): Red color (pH 4.5 or below) Principle: Determine the ability of an organism to ferment a
-​ (-): Yellow color (above 4.5) specific carbohydrate that is incorporated in a basal medium, with
or without visible gas
Citrate ●​ Media:
-​ Peptone Medium: Andrade’s indicator
Principle: Some organisms can utilize SODIUM CITRATE as -​ Heart Infusion Broth: Bromcresol Purple
their sole source of carbon producing acetate and other alkaline Indicator
carbonate end products in the process. These products change the ●​ Expected result:
color of the indicator from green to blue. -​ (+): Indicator change to yellow
●​ Medium: -​ (-): Growth, but no change in color
-​ Simmons Citrate Agar/Christensen Citrate -​ Medium remains purple
Medium
-​ Na acetate - only carbon source ONPG (O-nitrophenyl-beta-D-galactopyranoside)
-​ Bromthymol Blue - pH indicator
-​ Uninoculated Simmon Citrate agar is an ●​ Determines the presence of late or slow lactose
intermediate green color fermenting strains
●​ ONPG molecule – structurally similar to lactose
Urease Test -​ Can enter the bacterial cell without the lactose permease
-​ In the presence of β-galactosidase, ONPG (colorless), is
Principle: Urea splits urea molecule into NH3, CO2, and H2O. converted into galactose and O-nitrophenyl which is a
Ammonia reacts in the solution to form an alkaline compound, yellow chromogen
ammonium carbonate, which results in an increased pH of the ●​ Expected Result
medium and a color in the indicator to pink-red. -​ (+): Yellow (presence of β-galactosidase)
-​ (-): Colorless (absence of enzyme)
●​ Medium:
-​ Christensen’s Urea Agar tubes/ Stuart Urea Nitrate Reduction Test
broth
-​ Original color: Yellow Principle: Some organisms possess nitrate reductase that can
-​ pH indicator: Phenol Red reduce nitrate (NO3−) to nitrite (NO2−)
●​ Positive Result:
-​ Color yellow changes to PINK ●​ Medium: nutrient broth with 0.1% potassium nitrate (can
-​ Useful in the identification of RAPID have Durham Tube)
UREASE PRODUCERS (within 2-4 hours) ●​ Reagents:
➢​ Proteus, Providencia, Morganella -​ Alpha-napthylamine
(PPM) -​ Sulfanilic acid
-​ Zinc dust

BMLS GROUP 5 2
 Phenylalanine Deaminase Test

Principle: Determines whether an organism possess the enzyme

that deaminates phenylalanine to phenylpyruvic Acid

●​ Medium: agar slant containing 0.2% concentration of

Phenylalanine
●​ Reagent: 10% Ferric Chloride (FeCl3)
●​ Expected Result:
-​ (+): Green color develops in slant after ferric chloride is
added
➢​ Proteus, Providencia, Morganella
-​ (-): slant remains original color after addition of ferric
chloride

Gelatin Hydrolysis

Principle: determines whether an organism producegelatinase

capable of hydrolyzing gelatin

Expected Result:
-​ (+): partial or total liquefaction of the inoculated tube
(the control tube must be completely solidified at 4C
w/in 14D
-​ (-): complete solidification of the tube at 4C

BMLS GROUP 5 3