biochemical tests

O/F

purpose

ingredients/reagents

results interpretation

purpose: determine if organism is oxidizer or fermenter or neither, differentiates GN NF strict aerobe from other bact

ingredients: 1% glucose, peptones, bromothymol blue indicator, semi-solid agar, mineral oil

2 tubes both innoculated with bacteria, one topped with mineral oil (anaerobic condition tube). if both tubes yellow = fermenter,

open tube top yellow, bottom green; closed is green = oxidizer

both tubes yellow = fermenter

both tubes green = neither

if tube is blue it means organism used peptones

principle behind lactose fermentation

lactose = glucose + galactose

need 2 enzymes for galactose: beta galactosidase permease and beta galactosidase.

permease permits galactose to enter cell

beta galactosidase is responsible for breaking it down

if have both = LF

if have only pemease = NLF

if have beta galactosidase only = LLF (will mutate to let it in)

if neither = NLF

ONPG Test

purpose

ingredients

result interpretation

purpose: differentiate LLF from NLF by detecting beta-galactosidase activity in organisms with little to no permease.

ingredients: o-nitrophenyl and galactose (in disc form)

o-nitrophenyl is small so doesn't need permease to enter cell.

if beta-galactosidase enzyme present, yellow colour will be produced (due to O-nitrophenyl complex) = LLF

if clear = NLF

TSI test

purpose

ingredients

test interpretation

1. can organism breakdown glucose and sucrose/lactose

2. gas production

3. H2S formation

ingredients: peptones, 0.1% glucose, 1% sucrose, 1% lactose, phenol red indicator

Na thiosulphate in media provides sulfur, will be broken down into H2S by organism (if able). H2S reacts with ferrous sulfate to produce ferrous sulfide (black precipitate) --> favours anaerobic condition

gas production detected via bubbles, cracks, or lifting of media

sugar breakdown: if can break down glucose all tubes will turn yellow initially. if can not break down other sugars, will use peptones on surface (NA/A result = pink/yellow).

if can use other sugars (A/A result = yellow/yellow)

if cant ferment = NA/No change

MRVP test (mixed acid vs 2,3 butanediol)

purpose

ingredients/reagents

result interpretation

purpose: to determine which fermentation pathway organism uses, mixed acid or butanediol.

aliquot 2 tubes one for MR , second for VP

MR does not require reagents bc strong Acid production overcomes broth's buffering ability

--> add phenol red indicator, if red = positive

VP requires reagents:

6 drops 5% alpha Naphthol, 2 drops KOH, shake for oxygenation, --> if red = positive

theory: pathway produces acetoin which produces diacetyl when oxidized that reacts with KOH and naphthol for red complex

Decarboxylation Test

purpose

ingredients/reagents

result interpretation

purpose: see if organism has decarboxylase enzyme for lysine or ornithine

ingredients: bromocresol purple indicator, amino acid (lysine/ornithine), glucose, and mineral oil

amino acids will initially ferment glucose, produces acidic environment, turns tube yellow.

if organism has the enzymes will try to neutralize environment pH and will release them. turns tube purple and turbid

why is the control tube important in decarboxylase test

shows whether the organism is glucose fermenter which is needed to produce an acidic environment. decarboxylase enzymes only released in acidic environments

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arginine dihydrolase test

purpose

inregredients

result interpretation

purpose: detect if organism has ability to break down arginine

ingredients: bromocresol purple, glucose, arginine (amino acid)

arginine broken down by arginine dihydrolase into citrulline, ammonia, inorganic phosphate.

citrulline broken down by ureidase into ornithine and other alkaline products

key enzymes: arginine dihydrolase, ureidase

will turn yellow before purple like decarb, also 3 tubes

purple = positive , yellow = negative

phenylalinine deaminase test (PPD/PPA)

purpose

ingredients

result interpretation

purpose: detect whether orgnanism can make phenylalanine deanimase enzyme as source of carbon/energy

ingredients: NaCl, 10% ferric chloride (reagent), L-phenylalanine, agar

if phenylalanine deanimase enzyme present, will break down phenylalanine into phenylpyruvic acid and ammonia

add the 10% ferric chloride

it will react with phenyl pyruvic acid (if its present) and turn green --> positive result

if yellow --> negative as it wasnt able to break down phenylalanine

Citrate Utilization Test

purpose

ingredients

result interpretation

purpose: is the organism able to use citrate as sole source of energy in presence of oxygen

ingredients: sodium citrate, ammonium salts (source of nitrogen), bromothymol blue indicator

breaking down citrate results in ammonia so alkaline.

NH3 enhances the development of blue colour because its also alkaline. therefore positive = growth and blue, negative = green no growth)

note* doesnt have to change colour, main thing is growth

Gelatin Test

purpose

ingredients

result interpretation

determine if organism has gelatinase enzyme

ingredients: peptone-peptic digest of beef extract, gelatin

stab to innoculate

if has enzyme it will hydrolyze it into smaller amino acids that can be used by the organism. if the gelatin is broken down it will no longer solidify. check everyday for liquification.

immerse in ice bath to confirm media is liquid

Indole test

purpose

ingredients

result interpretation

to see if organism can be tryptophanase enzyme

ingredient: peptone digest (contains tryptophan)

tryptophan broke down by tryptophanase into indole, pyruvic acid, ammonia.

add kovac's reagent which contains paradimethyaminolbenzaldehyde which reacts with indole to produce a red ring

red ring = positive, colourless ring = negative

Sim test

purpose

ingredients

result interpretation

purpose: indole test, motility test, H2S test

ingredients: tryptophan, semi-solid agar, sodium thiosulphate, ferrous sulfate

stab innoculum into tube

read motility results first: if turbid = motile, else non motile

then add kovacs reagent which contains: paradimethylaminobenzaldehyde

and read indole.

nitrate reduction

purpose

ingredients

test interpretation

to detect if organism can break down nitrate using nitrate reductase enzyme

ingredients: peptones, potassium nitrate (nitrate source), durham tube.

reagents: dimethyl alpha napthylamine & sulfanillic acid, zinc dust

interpreting results:

after incubation.

if gas bubble present and non-fermenter --> positive for nitrate reduction

if no gas add reagents dimethyl alpha napthylamine and sulfanillic acid. --> if red then positive

now add zinc dust --> if red then NEGATIVE, if colourless then positive

urea test

to see if organism can break down urea using urease enzyme

. christensens media is most common

ingredients:

urea

glucose

peptone

phenol red

if urease present it breaks down urea to ammonium carbonate and releases ammonia and co2, all alkaline products --> alkaline shift. positive = pink/red/fuchia, negative = orange