part 1 biochemical test

Biochemical Tests

Tests designed to demonstrate the physiological and chemical characteristics of microorganisms which enables the microbiologists, by elimination, to specifically identify the isolated microorganisms and demonstrates the enzyme system of a bacterial cell

CARBOHYDRATE UTILIZATION

Use of carbohydrates by microorganisms using common carbohydrates such as glucose, lactose, and sucrose to determine metabolic characteristics

Common carbohydrates used

Glucose, Lactose, and Sucrose

Lactose Fermentation

Carbohydrate determination test that is the most important; differentiates lactose fermenters (LF) from non-lactose fermenters (NLF)

β-galactoside permease (lactose permease)

Transport enzyme that facilitates entry of the lactose molecule across the bacterial plasma membrane

β-galactosidase

Enzyme that hydrolyzes lactose into glucose and galactose

Lactose fermenter (LF)

Organisms that possess both β-galactoside permease and β-galactosidase enzymes

Non-lactose fermenter (NLF)

Organisms that do not possess both β-galactoside permease and β-galactosidase enzymes

Late lactose fermenter (LLF)

Organisms that possess only β-galactosidase enzyme

Test for Carbohydrate Fermentation

Test using liquid or semi-solid meat extract broth to which an indicator and carbohydrates are added to determine fermentation

Bromocresol purple

pH indicator that appears purple in alkaline conditions and yellow in acidic conditions

Andrade’s fuchsin

pH indicator that appears pale yellow in alkaline conditions and reddish pink in acidic conditions

Phenol red

pH indicator that appears red in alkaline conditions and yellow in acidic conditions

Carbohydrate Fermentation using Broth Fermentation Media

Method used to determine the ability of an organism to ferment a specific carbohydrate incorporated in a basal medium with or without visible gas

Purpose of Broth Fermentation Media

Determine the ability of an organism to ferment a specific carbohydrate incorporated in a basal medium with or without visible gas

Medium for Broth Fermentation Media

Peptone medium with Andrade’s indicator

Positive result in Broth Fermentation Media

Change in color with or without gas using durham tube

TRIPLE SUGAR IRON (TSI)

Test that determines whether a gram-negative bacteria ferments glucose and lactose/sucrose and forms hydrogen sulfide (H2S) and gas

TSI chambers

Two chambers consisting of equal length of butt (anaerobic) and slant (aerobic)

TSI composition

Contains lactose (1%), sucrose (1%), glucose (0.1%), and peptone (2%)

TSI pH indicator

Phenol red

TSI H2S indicator

Ferrous sulfate and sodium thiosulfate

TSI manner of inoculation

Stab and streak

Reactions on TSI

Interpretation of biochemical reactions on Triple Sugar Iron agar to determine carbohydrate fermentation, hydrogen sulfide production, and gas production

NO FERMENTATION

Glucose, lactose, and sucrose are not fermented resulting in alkaline slant and alkaline butt (K/K) due to peptone degradation producing alkaline by-products and is common to non-fermentative organisms

K/K (Alk Slant/Alk Butt)

Reaction showing no fermentation of glucose, lactose, or sucrose with alkaline conditions in both slant and butt due to peptone degradation

Peptone degradation

Process that produces alkaline by-products leading to alkaline reaction in TSI when carbohydrates are not fermented

Non-fermentative organisms

Organisms that do not ferment carbohydrates and show K/K reaction in TSI

K/A (Alk Slant/Acid Butt)

Reaction indicating glucose fermentation only, where glucose is fermented producing acid in the butt while lactose and sucrose are not fermented and aerobic peptone utilization after 12 hours produces alkaline slant

GLUCOSE FERMENTATION

Process where glucose is fermented producing acid while lactose and sucrose are not fermented, resulting in K/A reaction

Aerobic peptone utilization

Occurs after 12 hours producing alkaline reaction in the slant due to peptone metabolism in presence of oxygen

A/A (Acid Slant/Acid Butt)

Reaction indicating lactose fermentation where all sugars are fermented producing acid in both slant and butt

LACTOSE FERMENTATION

Fermentation of lactose and other sugars where acid production from additional sugars keeps both slant and butt acidic (yellow) at 18 to 24 hours

H2S Production

Formation of hydrogen sulfide gas from sodium thiosulfate which requires an acid environment and is detected using ferrous sulfate indicator

Sodium thiosulfate

Compound that is reduced by bacteria in an acid environment to produce hydrogen sulfide gas

Hydrogen sulfide (H2S) gas

Colorless gas produced from sodium thiosulfate that requires an indicator for visualization

Ferrous sulfate

Indicator that reacts with hydrogen sulfide gas to form a black precipitate for detection

Ferrous sulfide

Black precipitate formed when hydrogen sulfide reacts with ferrous ions indicating H2S production

Acid environment for H2S production

Condition required for the formation of hydrogen sulfide from sodium thiosulfate

GAS Production (aerogenic)

Production of gas during fermentation resulting in bubbles, splitting of the medium, or displacement of the medium in the butt

No Gas Production (nonasrogenic)

Absence of gas formation during fermentation with no bubbles or displacement in the medium

Kligler iron agar (KIA)

Medium identical to TSI except it does not contain sucrose as a carbohydrate component

Oxidation-Fermentation Tests

Tests used for identification of bacteria based on their oxidation-fermentation pattern

Purpose of Oxidation-Fermentation Tests

Identification of bacteria based on whether they oxidize or ferment carbohydrates

Hugh and Leifson O/F basal medium (OFBM)

Medium used in oxidation-fermentation tests containing same carbohydrate concentration as TSI but lower peptone concentration (0.2%)

Bromothymol blue

pH indicator in OF test that is green when uninoculated, yellow in acidic environment, and blue in alkaline environment

Uninoculated OF medium

Appears green indicating neutral pH

Acid environment in OF test

Appears yellow due to carbohydrate utilization

Alkaline environment in OF test

Appears blue due to non-utilization or peptone metabolism

Closed tube (anaerobic)

Tube overlaid with mineral oil to create anaerobic condition in OF test

Open tube (aerobic)

Tube without mineral oil allowing oxygen exposure in OF test

Fermenter

Organism that produces acid in both open and closed tubes in OF test

Oxidizer

Organism that produces acid only in open tube and not in closed tube in OF test

Non-oxidizer

Organism that does not produce acid in both open and closed tubes in OF test

ORTHO-NITROPHENYL-BETA-D-GALACTOPYRANOSIDE (ONPG)

Test that determines the presence of slow/delayed lactose fermenters (LLF)

ONPG

Colorless molecule structurally similar to lactose that can enter the cell membrane without lactose permease and is converted by beta-galactosidase into galactose and O-nitrophenyl (yellow chromogen)

Slow/delayed lactose fermenters (LLF)

Organisms detected by ONPG test due to presence of beta-galactosidase but absence of lactose permease

Beta-galactosidase in ONPG test

Enzyme that converts ONPG into galactose and O-nitrophenyl producing yellow color

O-nitrophenyl

Yellow chromogen produced from ONPG hydrolysis indicating positive result

IMVIC REACTIONS (Indole, Methyl Red, Voges Proskauer & Citrate)

Series of biochemical tests used to differentiate members of Enterobacteriaceae based on metabolic properties

INDOLE TEST

Test used to identify organisms that produce tryptophanase which deaminates tryptophan forming indole, pyruvic acid, and ammonia

Tryptophanase

Enzyme that converts tryptophan into indole, pyruvic acid, and ammonia

Indole production reaction

Tryptophan amino acid acted upon by tryptophanase produces indole, pyruvic acid, and NH3

Indole test medium

Tryptophan or peptone broth inoculated and incubated at 35°C for 48 hours

Kovac's method

Indole determination method using Kovac's reagent (p-dimethylaminobenzaldehyde and HCl) producing red color as positive result

Kovac's reagent

Reagent composed of p-dimethylaminobenzaldehyde and HCl used for indole detection

Positive indole (Kovac's)

Red color develops after addition of reagent

Ehrlich's method with xylene

Indole determination method using Ehrlich's reagent (Kovac's absolute ethyl alcohol) producing red color and is more sensitive for detecting small amounts of indole and preferred for anaerobes

Ehrlich's reagent

Reagent composed of Kovac's absolute ethyl alcohol used for indole detection

Positive indole (Ehrlich's)

Red color develops after addition of reagent

Spot indole test

Rapid method for detection of indole using PDAMCA reagent

PDAMCA (p-dimethylaminocinnamaldehyde)

Reagent used in spot indole test

Positive spot indole test

Blue or blue-green color within 20 seconds

METHYL RED (MR) TEST

Test that detects organisms producing large amounts of acid from dextrose by mixed acid fermentation lowering pH of broth

Mixed acid fermentation pathway

Process where glucose is converted to pyruvic acid and further to mixed acids lowering pH to about 4.4

Principle of MR test

Glucose is converted to pyruvic acid followed by mixed acid fermentation producing acidic pH resulting in red color with methyl red indicator

pH indicator for MR test

Methyl red

Positive MR reaction

Deep red color at pH ≤ 4.5

Negative MR reaction

Deep yellow color at pH ≥ 4.5

VOGES-PROSKAUER (VP) TEST

Test that detects organism's ability to metabolize fermentation products to 2,3-butanediol via acetoin intermediate

Butylene glycol pathway

Pathway where glucose is converted to pyruvic acid, then to acetoin and diacetyl leading to 2,3-butanediol

Principle of VP test

Glucose is converted to pyruvic acid then acetoin which is oxidized to diacetyl in presence of reagents producing red color

Positive VP reaction

Red color at neutral pH indicating presence of 2,3-butanediol pathway

Negative VP reaction

Yellow color

VP reagents

5% α-naphthol in 95% ethanol (catalyst/color intensifier), 40% KOH in distilled water (provides alkalinity), and 0.5% creatine in distilled water (prevents false negative results)

MR-VP relationship

Bacteria tend to be positive for either MR or VP but not both, though some are negative for both

CITRATE UTILIZATION TEST

Test used to identify organisms capable of using sodium citrate as sole carbon source and inorganic ammonium salts as sole nitrogen source

Principle of citrate utilization test

Bacteria produce citrate-permease converting citrate to pyruvate and use ammonium salts releasing ammonia causing alkaline pH and color change

Citrate-permease

Enzyme that enables utilization of citrate by converting it to pyruvate

Simmons citrate agar

Medium used for citrate test with initial pH 6.9 and green color

pH indicator in citrate test

Bromothymol blue

Positive citrate test

Blue or Prussian blue color with growth on medium

Negative citrate test

No growth and green color

Christensen's citrate medium

Alternative medium containing phenol red indicator and organic nitrogen turning from yellow to pink in alkaline pH

MALONATE UTILIZATION

Test that determines whether organism can use sodium malonate as sole carbon source and ammonium sulfate as nitrogen source

Principle of malonate utilization

Organisms using malonate also utilize ammonium sulfate producing alkaline pH

pH indicator in malonate test

Bromothymol blue

Positive malonate test

Blue color in the medium

Negative malonate test

No color change (green) or yellow due to dextrose fermentation

ACETATE UTILIZATION

Test that differentiates organisms based on ability to use acetate as sole carbon source producing alkaline pH turning medium from green to blue and used to differentiate Shigella spp. from Escherichia coli