part 2 biochemical test

LYSINE IRON AGAR (LIA) SLANT

Medium that determines the ability of an organism to deaminate or decarboxylate lysine and produce H2S and is most useful with TSI in screening stool specimens for enteric pathogens

Purpose of LIA

Determines lysine deamination, lysine decarboxylation, and H2S production and is used with TSI for screening enteric pathogens

LIA composition

Contains lysine, peptones, and a small amount of glucose with an aerobic slant and anaerobic butt

Glucose fermentation in LIA

Causes the butt to become acidic (yellow)

pH indicator in LIA

Bromocresol purple

H2S indicator in LIA

Ferric ammonium citrate and sodium thiosulfate

Manner of inoculation in LIA

Stab-streak-stab

K/K (Purple Slant/Purple Butt)

Lysine deaminase negative and lysine decarboxylase positive

K/A (Purple Slant/Yellow Butt)

Lysine deaminase negative and lysine decarboxylase negative

R/A (Red Slant/Yellow Butt)

Lysine deaminase positive and lysine decarboxylase negative

Lysine decarboxylation

Occurs in the anaerobic butt where lysine is converted by lysine decarboxylase to cadaverine causing the butt to revert to purple

Cadaverine

Product of lysine decarboxylation that neutralizes organic acids from glucose fermentation and returns the butt to alkaline purple

Negative lysine decarboxylation

Decarboxylase is not produced and the butt remains acidic (yellow)

No deamination

Slant remains purple when deamination does not occur

Lysine deamination

Occurs in the aerobic slant producing a reddish purple color

H2S production in LIA

Occurs only in an alkaline environment and may mask the purple color in the butt with black precipitate

Black precipitate in LIA

Indicates H2S production and is also a positive result for decarboxylation

DECARBOXYLASE TEST (MOELLER'S METHOD)

Test used to detect amino acid decarboxylase enzymes using Moeller's decarboxylase broth

Moeller's decarboxylase broth

Medium containing 1% lysine, 1% ornithine, 1% arginine, glucose, and peptones with mineral oil overlay for anaerobic conditions

Mineral oil overlay

Creates anaerobic environment in decarboxylase test

pH indicators in decarboxylase test

Bromocresol purple and cresol red

Principle of decarboxylase test

Detection of carboxylases (LDC, ODC, ADH) that remove carboxyl groups from amino acids

Lysine decarboxylase (LDC)

Converts lysine to cadaverine

Ornithine decarboxylase (ODC)

Converts ornithine to putrescine

Arginine dihydrolase (ADH)

Converts arginine to citrulline then ornithine then putrescine

Positive decarboxylase test

Color change from yellow to purple

Negative decarboxylase test

No color change and remains yellow

PHENYLALANINE DEAMINASE TEST (PAD)

Test that determines ability of organism to oxidatively deaminate phenylalanine to phenylpyruvic acid

Indicator in PAD test

10% ferric chloride (FeCl3) added after incubation

Positive PAD test

Green color on slant after adding FeCl3

Negative PAD test

Slant remains original color after adding FeCl3

Positive tryptophan deamination

Brown color

UREASE TEST

Test that determines whether microorganism can hydrolyze urea forming water, CO2, and ammonia causing alkaline pH

Medium for urease test

Christensen's urea agar or Stuart urea broth

pH indicator in urease test

Phenol red

Positive urease test

Bright pink coloration

Negative urease test

No color change (yellow)

Rapid urease producers

Organisms that hydrolyze urea within a few hours turning the entire tube pink including Proteus spp., Citrobacter, Providencia, Klebsiella, Morganella

Slow/weak urease producers

Organisms that hydrolyze urea slowly and only turn the slant pink including Enterobacter, Yersinia, Serratia

MOTILITY

Test that determines whether an organism is motile or non-motile

Medium for motility test

SIM medium or any semisolid motility medium

Streaking method for motility

Straight stab

Triphenyltetrazolium chloride

Indicator added to aid visualization of motility

Motile organisms

Show spreading away from stab line with hazy appearance

Non-motile organisms

Remain at the stab line

Non-motile Enterobacteriaceae exceptions

Shigella, Klebsiella, and Yersinia (at 25°C)

SULFIDE INDOLE MOTILITY (SIM) MEDIUM

Medium used to detect motility, indole production, and H2S production

Motility in SIM

Cloudiness spreading from line of inoculation indicates positive result

H2S production in SIM

Black precipitate indicates positive result

Indole in SIM

Pink to red color after adding Kovac's reagent indicates positive result

MOTILITY INDOLE ORNITHINE (MIO) AGAR

Medium used to detect motility, indole production, and ornithine decarboxylase activity

Motility in MIO

Clouding or spreading growth from inoculation line

Ornithine decarboxylase in MIO

Purple color indicates positive result

Indole in MIO

Pink to red color after adding Kovac's reagent indicates positive result

NITRATE & NITRITE REDUCTION TEST

Test that determines whether an organism produces nitrate reductase which reduces nitrate to nitrite detected by adding α-naphthylamine and sulfanilic acid

Nitrate reductase

Enzyme that reduces nitrate to nitrite in the nitrate reduction test

Reagents for nitrate reduction test

α-naphthylamine and sulfanilic acid used to detect nitrite

Positive nitrate reduction

Red coloration indicating reduction of nitrate to nitrite

Further reduction of nitrite

Reduction of nitrite to nitrogen gas, nitric oxide (NO), or nitrous oxide (N2O) which does not produce color with reagents

Absence of color in nitrate test

May indicate nitrate not reduced (negative) or nitrate reduced beyond nitrite to nitrogen compounds not detected by reagents

Durham tube in nitrate test

Inserted upside down in broth to trap produced nitrogen gas

Zinc dust

Reduces nitrate to nitrite and is added to determine true-negative results or if reduction occurred beyond nitrite

True negative nitrate test

Red coloration after adding zinc dust indicating nitrate was not reduced by organism

Positive for nitrite reduction beyond nitrite

No color before zinc but no red color after zinc indicates reduction beyond nitrite

DNASE TEST

Test used to differentiate organisms based on production of DNase enzyme

Principle of DNase test

Medium contains DNA-methyl green complex appearing pale green and hydrolysis of DNA causes fading of green color producing colorless zone around colonies

Positive DNase test

Colorless zone around colonies indicating DNA hydrolysis

DNase positive organisms

Staphylococcus aureus, Moraxella, and Serratia

OXIDASE TEST

Test that determines presence of cytochrome oxidase system that oxidizes reduced cytochrome with molecular oxygen

Oxidase indicator

0.5% or 1% tetramethyl-p-phenylenediamine dihydrochloride (Kovac's) or p-aminodimethylaniline oxalate

Positive oxidase test

Dark purple color

Negative oxidase test

Absence of color

GELATIN HYDROLYSIS TEST

Test that determines ability of bacteria to produce gelatinases that hydrolyze gelatin and liquefy solid gelatin medium

Gelatinase

Extracellular proteolytic enzyme that hydrolyzes gelatin

Principle of gelatin hydrolysis

Gelatin solidifies at 28°C but hydrolyzed gelatin fails to solidify even below 28°C

Gelatin hydrolysis positive organisms

Serratia and Proteus

Gelatin stab method

Method using nutrient agar tube with 12% gelatin incubated for 48 hours then refrigerated for about 30 minutes

Positive gelatin stab test

Partial or total liquefaction of medium

Negative gelatin stab test

Gel state with no change

Gelatin strip method

Method using strips of clear blue plastic coated with gray-green gelatin

Positive gelatin strip test

Hydrolyzed gelatin causing blue plastic strip to become visible

Negative gelatin strip test

No blue plastic visible on gelatin strip