Test Acetamide Utilization Acetate Utinilzation Bacitracin Test

Principle Deamination of acetamide Medium: Green-> Royal blue Breakdown of Na Ac cause pH of the medium to shift toward the alkaline range Medium: green -> blue Effect of bacitracin on an organism

Expected Result Positive: blue color Negative: no color change (green) Positive:Blue (alkalinized) Negative: no color change (green) Positive: w/zone of inhibition Negative:no zone of inhibition

Quality control Positive: Psedomonas aeruginosa Negative: Stenotrophonomas maltophilia Positive: E. coli Negative: Shigella flexneri Positive: Strep. pyogenes Negative: Strep. algalatiae Other beta-hemolytic streptococci usually are negative Positive: Enterococcus faecalis Negative: Strep. mitis Positive: Strep. pneumonia Negative: Enterococcus faecalis

Bile esculin Agar

Bile Solubility test

Combination of esculetin (end product of esculin hydrolysis) w/ ferric ions to form phenolic iron complex Medium: orangish -> black Bile or a soln. Of a bile salt, such as Na desoxycholate rapidly lyses pneumococcal colonies. Lysis depends on the presence of an intracellular autolytic enzyme.

Positive: Blackerning of the agar slant Negative: no blackening of medium Positive: Colony disintergrates; an imprint of the lysed colony may remain w/in the zone Negative: Intact colonies

Butyrate disk

Camp Test

Detection of the enzyme butyrate esterase in identifying Moraxella (branhamella) catarrhalis. If bromo-chlroindolyl butyrate impregnated in the disk is hydrolyzed by the enzyme, a blue colore indigo compound is formed. Production of a diffusible extracellular protein (CAP factor) that acts synergistically w/ the beta-lysin of Staph. Aureus to cause enhanced lysis of RBC

Positive: Development of a blue color during 5minute incubation period Negative: No color change Positive:Enhance hemolysis is indicated by an arrowhead-shaped zone of beta-hemolysis at the juncture of 2 organisms Negative: No enhancement of hemolysis Positive:Copius bubbles produced Negative:No or few bubble produced Note: Some organism (enterococci) produce a peroxidise that slowly catalyzes the breakdown of H2O2 that may cause a weak + result but this rx is not truly + test. Positive: Growth Negative: No growth Positive:growth on medium, with or without color change Negative: absence of growth

Positive: Moraxella catarrhalis Negative: Neisseria gonorrhoeae

Positive: Strep. agalactiae Negative: Strep. pyogenes

Galatase Test

Breakdown of H202 into 02 and H2O

Positive:Staph. aureus Negative: Staph. pyogenes

Cetrimide

Citrate Utilization

Ability of an organisn to grow in the presence of cetrimide (toxic substance for the growth of many bacteria) Ability of an organism to utilize sodium citrate as the only carbon source and ammonium salts as the only nitrogen source. Medium: green -> blue

Positive: Pseudo. aeruginosa Negative: E. Coli Positive: Klebsiella pneumoniae Negative: E. Coli

Coagulase test

Alteration of fibrinogen that precipitates staphylococcal cell, causing the cells to clump when a bacterial susp. is mixed w/ plasma Enyzymatic ability of an organism to decarboxylate (or hydrolyze) an amino acid to form an amine

Positive: Clot of any size Negative: no clot Positive: Alkaline (purple) color change compared w. The control tube Negative: no color change or acid (yellow) colot in test and control tube

Positive: Staph. aureus Negative: Staph. Epidermitis Positive: Lys - Klebsiella pneumonia Ornithine - Enterobacter cloacae Arg - Enterobacter cloacae Negative: Lys - Enterobacter cloacae Ornithine Klebsiella pneumonia Arg Klebsiella pneumonia Base - Klebsiella pneumonia Positive: Staph. aureus Negative: Staph. Epidemitis Positive: Kleb. pneumoniae Negative: Shigella flexneri A. Peptone medium w/ Andrades indicator Positive: w/ gas -> E. Coli Without gas -> Shigella flexneri Negative: Pseudo. Aeruginosa B. Heart infusion broth w/ bromocresol purple indication Positive: Strep. mutans Negative: Strep mitis Positive:Peritrichous -> E. Coli Polar -> Pseudo. aeruginosa Negative: Kleb. Pneumonia Positive: Proteus vulgaris Negative: Enterobacter aerogenes Positive: Pseudo. aeruginosa Negative: Pseudo. fluorescens Positive: Strep. agalactiae Negative: Strep. pyogenes

Decarboxylase test (moellers methods)

DNA hydrolysis Esculin hydrolysis Fermentation Media

Ability of an organism to hydrolyze DNA. Ability of an organism to hydrolyze glycoside esculin

Ability of an organism to ferment a specific carbohydrate that in incorporated in a basal medium, thereby producing acid with or without visible gas.

Positive: medium turns green - > colorness Negative: medium remains green Positive:Blackened medium, loss of fluorescence under the woods lamp Negative: no blackening and no loss of fluorescence Positive:Straw colored -> pink with or without gas formation in durham tube Negative: growth, but no change in color Heart infusion broth w/ bromocresol purple indication (for streptococci and enterococci) Positive: Purple -> yellow Negative: growth, but no change in color

Flagella stain

Presence of flagella

Gelatin Hydrolysis Growth at 42 C Hippurate Test

Ability of an organism to produce proteolytic enzymes (gelatinase) that liquefy gelatine Ability to grown at 42 C Hydrolysis of hippuric acid by hippuricase that produces glycine and benzoic acid. Then, deamination of glycine w/ ninhydrin to form a purple-colored product

Positive: partial or total liquefaction of the inoculated tube Negative:Complete solidification or tube at 4 C Positive: Good growth at both 35 and 42 C Negative: No growth at 42 C Positive: Deep purple color Negative: Colorless or slightly yellow pink color

Indole production

Ability of an organism to split tryptophan to form the compound indole

Positive: pink to wine-colored ring after addition of appropriate reagent Negative: no color change

LAP Test

Litmus Milk

Detection of the enzyme leucine aminopeptidase by impregnated disks w/ Leucine-B-naphthylamide following hydrolysis of the substrate by the enzyme producing B-naphthylamine that produces red color upon addition of cinnamaldehyde reagent Ability of an organism to metabolize litmus milk

Positive: development of a red color w/in 1 minute upon addine cinnamaldehyde reagent Negative: No color change or development of slight yellow color See pg. 231

A. Kovacs method Positive: E. coli Negative: Kleb. Pneumonia B. Erlichs Method Positive:Elizabethkingia menigoseptica Negative:CDC group EO-2 Positive: Enterococcus faecalis Negative: Leuconostoc sp.

Lysine Iron Agar

Decarbxylation or deamination of lysine and formation of hydrogen sulfide

Methyl Red/VogesProskauer (MRVP) test

Ability of an organism to produce and maintain stable acid end products from glucose fermentation and determine ability to produce neutral end products from glucose fermentation

(K/K) - lysine decarboxylation and no fermentation of glucose (K/A) glucose fermentation Presence of black ppt (+) (R/A) lysine deamination and glucose fermentation VP Test (Barritts method) for gram( -) rods Positive: Red color indicates acetoin production Negative: Yellow color Positive:Bright Red color indicate mixed acid fermentation Negative: yellow color

Alkaline:Alcaligenes faecalis Acid: Enterococcus faecium Peptonization: Burkolderia cepacia (K/K), H2S + (Salmonella typhimurium (K/A) (Shigella flexneri) (R/A) (Proteus vulgaris)

Microdase Test

Motility Testing

Detection of the enzyme oxidase which reacts w/ oxidase reagent and cytochrome C to form the colored compound, indo-phenol Presence of flagella

Positive:Development of blue to purple-blue color Negative: no color change A. Hanging drop Positive: motile Negative: Brownian movement B. Semisolid agar deep Positive: organism will spread out in the medium Negative: remain in the same position Positive: gas production indicated by a bubble in durham tube Negative: No gas production Positive: Electric blue fluorescence Negative:Lack of fluorescence See pg. 236

A. Methyl red Positive:E. coli Negative: Enterobacter cloacae B. VP(Barritts Method) Positive: Enterobacter cloacae Negative: E. Coli C. VP Positive: Strep. mutans Negative: Strep. mitis Positive: Micrococcus luteus Negative: Staph. Aureus Positive: E. Coli Negative: Kleb. Pneumonia

MRS Broth

Determines wheter an organism forms gas during glucose fermentation Presence of the enzyme B-D-glucoronidase which hydrolyses B-D-glucopyranosid-uronic acid Ability of an organism to reduce nitrate. Sulfanilic acid and nitrate react to form a diazonium salt that couples

Positive: Leuconostoc spp. Negative: Pediococcus spp. Positive: E.coli Negative: Pseudo. Aeruginosa Positive: NO3, no gas ( E. Coli) NO3, gas ( Pseudo. aeruginosa)

MUG test Nitrate Reduction

Nitrite Reduction

w/ the alpha-naphthylamine to produce red, watersoluble azo dye. Ability of an organism to reduce nitrites to gaseous nitrogen or to other compounds containing nitrogen

Negative: Acinobacter spp. Positive: No color change to red 2 minutes after the addition of reagents and gas production observed in durham tube (zinc dust can be added for more accurate results especially if the broth does not become red and there was no gas production) Negative: broth becomes red after addition of reagents Positive: Yellow Negative: Colorless Positive: w/ zone of inhibition 14mm Negative: no zone of inhibition Positive: development of dark purple color w/in 10 seconds Negative: absence or color Positive:Acid production (yellow) Weak positive: Compare to other test tubes Negative: Red or alkaline No change or neutral: there is growth but no change in color Positive: Green color develops on slant after ferric chloride is added Negative:Remains on original color upon addition of ferric chloride Positive:Bright red color w/in 5 mins Negative: No color change or an orange color Positive: green-> yellow Negative: No color change; yellow-green indicates a weak reaction that should be regarded as negative Positive: Visible turbidity w/ or without color change from purple to yellow Negative: no turbidity and no color change Positive: Development of a blue color w/in 20 secs Negative:No color development or slightly pink color Positive: Alcaligenes faecalis Negative: Alcaligenes piechaudii

ONPG Test

Optochin test Oxidase test (Kovacs Method) Oxidation/Ferme ntation of Medium (CDC method) Phenylalanine deaminase

Ability of an organism to produce B-galactosidase, an enzyme that hydrolyzes the substrate ONPG to form a visible (yellow) product, orthonitrophenol Determine the effect of optochin (ethylhydrocupreine hydrochloride) on an organism Presence of bacterial cytochrome oxidase using oxidation of the substrate tetreamethyl-pphenylenediamine dihydrochloride to indophenols, a dark purple-colored end product Determine whether an organism uses barbohydrates substrate to produce acid byproducts.

Positive: E. coli Negative: Salmonella typhimurium Positive: Strep. pneumonia Negative:Strep. mitis Positive: Neisseria gonorrhoeae Negative: E. coli

Fermenter: E.coli Oxidizer: Pseudo. aeruginos Nonutilizer: Alcaligenes faecalis

Determine the ability of an organism to oxidatively deaminate phenylalanine to phennylpyruvic acid

Positive: Proteus vulgaris Negative: E. Coli

PYR Test

Pyruvate Broth

Presence of enzymes L-pyrroglutamylaminopeptidase that hydrolyzes the L-pyrrolidonyl-B-napththylamide (PYR) substrates to produce a B-napthylamine Determine the ability of an organism to utilize pyruvate

Positive: Enterococcus faecalis Negative: Strep. mitis Positive:Enterococcus faecalis Negative: Enterococcus faecium Positive:Enterococcus faecalis Negative: Strep. mitis Positive:E. coli Negative: Enterobacter cloacae

Salt-tolerance test Spot indole test

Determine the ability of an organism to grow in high conc. of salt. Determine the presence of the enzyme tryptophase. Tryptophanase breaks down tryphtopha to release indole, which is detected by its ability to combine w/ certain aldehydes to form a colored compound Used to determine whether a gram(-) rod utilizes glucose and lactose or sucrose fermentatively and forms hydrogen sulphide. Phenol red and ferrous sulphate serves as indicators of acidification and H2S formation,

Triple sugar Iron Agar (TSI)

(K/NC) = glucose, lactose and sucrose nonutilizer can also be regarder as (K/K) (K/A) = glucose fermentation only (A/A) = glucose, sucrose and/or lactose fermenter

(A/A)= E. coli (K/A H2S) = Salmonella typhi (K/NC) = Pseudo. aeruginosa

respectively.

Urea Hydrolysis

X and V factor test

Determine the ability of an organism to produce the enzyme urease, which hydrolyzes urea. Hydrolysis of urea produces ammonia and CO2. The formation of ammonia alkalinizes the medium, and the pH shift is detected by the color change of phenol red from light orange at pH 6.8 to magenta at pH 8.1. Haemophilus require accessory growth factors in vitro. Some Haemophilus spp. Require X factor (hemin) alone, V factor (NAD. Nicotinamide-adenine denucleotide) alone or a combination of both

(A/A)= Indicates that the organism ferments glucose and sucrose, glucose and lactose or all w/ the production of gas Positive: Change in color of slant from light orange to magenta Negative: No color change(remains light orange)

Positive: Proteus vulgaris Negative: E. coli

Positive: Growth around the XV disk shows requirement for both factors. Growth around V disk , no growth around the X disk, and light growth around the XV disk shows a V factor requirement Negative: Growth over entire surface of the agard indicates no requirement for either X or V factor

Positive: Haemophilus influenza (show a halo growth around the XV disk) Haemophilus parainfluenzae (show a halo growth around XV and V disk) Negative:Haemophilus aphrophilus ( grow over the entire surface of the plate, X,V, XV factors are not necessary for growth)