Unit 4 Differential testing Flashcards by Wanda Tran

how is an octal profile generated

+ive test value of 1, 2, and 4 for 3 grouped tests. add only the positive numbers up generates 7 digit ID

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result of +ive and -ive for methyl red (MR)

+ive is red indicates mixed acid fermentation less than pH 4.4
-ive is yellow pH greater than 6.0

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3 rapid spot tests

catalase, oxidase and spot indole

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appearance of +ive and -ive for catalase

+ive bubbling H2O2 react w/catalase to produce water and O2

-ive no bubbling

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reason for false +ive for catalase

false +ive: nichrome from using loop (use wooden stick), RBC from agar has catalase, other enzymes break down catalase weak bubbling

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reason for false -ive for catalase test

old cultures lose enzyme, too slow in adding reagent

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appearance of +ive and -ive for oxidase (cytochrome)

+ive blue (oxidized)

-ive is colourless (reduced)

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reason for false +ive for cytochrome oxidase test

false +ive: auto-oxidation after 10 secs, nichrome from using loop (use wooden stick)

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reason for false -ive for cytochrome oxidase test

media w/nitrate, tellurite or fermentable CHO; don’t use growth w/MacConkey, more than 24 hrs old

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why is the dye TMPD used for cytochrome oxidase

acts as redox indicator, gives e-

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purpose of spot indole test

org break down tryptophan (casein peptone) resulting in production of indole, pyruvic acid and ammonia

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appearance of +ive and -ive for spot indole

+ive blue/green

-ive no colour change

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reason for false +ive spot indole test

mixed culture plates –> indole diffuses into culture. don’t trust if both are +ive

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reason for false -ive spot indole test

if media has glucose, drops pH, may inhibit indole production.

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practices to remember for indole test

media must supply tryptophan for testing, don’t use reagent if brown/signs of deterioration, avoid plates w/indicator dyes e.g. MAC

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purpose of 6.5% salt tolerance test

most org, high [NaCl] interfere w/membrane permeability and osmotic eqbm

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appearance of +ive and -ive for 6.5% NaCl test

+ive turbid

-ive not turbid

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reason for false +ive 6.5% NaCl test

over-inoculation

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principles of Adonitol test

adonitol added to phenol red broth (a complete medium w/o CHO). Fermentation of adonitol carried out by adonitol dehydrogenase forming acidic end products

appearance of +ive and -ive for Adonitol test

+ive yellow or turbid

-ive red, no change, turbid

what else can be added in Adonitol test and why

Durham tube checks gas production from fermentation. fastidious org +ive reactions is 3-5 days. 30 days to confirm -ive result. if org cannot grow in phenol red broth, turbidity not present

principle for Bile Esculin Agar (BEA)

selective b/c 40% bile: inhibits gp except for enterococci and Group D streptococci. differential b/c esculin

appearance of +ive and -ive for BEA

+ive translucent growth with blackening

-ive no growth/no blackening

what is the indicator is BEA

Fe3+ (from the indicator ferric ammonium citrate). blackening of media

notes to remember for BEA test

some staphylococci grow in presence of bile producing weak iron complex (black) growth is opaque on slant, -ive BEA. Listeria monocytogenes is +ive for esculin hydrolysis and may do so in presence of bile, thus important to do catalase and colony smear. BEA ≠ esculin test; the latter does NOT contain bile (is non-selective)

principle of Citrate test

Simmons Citrate Agar contains ammonium dihydrogen phosphate, only source of nitrogen and sodium citrate, only source of carbon. org capable of using ammonium dihydrogen phosphate and citrate will grow

appearance of +ive and -ive for citrate test

+ive Growth on the slant ** +/- blue colour | -ive: No growth on the slant, no colour change

describe pH indicator of citrate test

Bromothymol blue causes medium to change from green (neutral) to blue (alkaline) with increasing pH (pH >7.6)

notes to remember for citrate test

inoculating from broth culture and heavy inoculum= false positive results. org can grow on citrate medium but won't produce a color change still +ive result. if screw cap used, loosen to provide aerobic condition

principle of decarboxylase test (moeller's method)

first fermentation of dextrose lower ph (purple to yellow) second decarboxylation of amine (yellow to purple)

procedure of decarboxylase test

inoculate 3 broth: lysine, base and orthinine decarboxylase. added oil

why results invalid if oil not added for decarboxylase test

air causes alkalinsation due to DEAMINATION of peptones

what does MIL stand for

motility, indole and lysine

MIL tests for what

H2S production, indole formation and motility of enteric org

+ive and -ive test for sulfide

+ive blackening | -ive no change

reagents of MIL

SIM medium w/o TTC (dye to determine motility) and Kovac's reagent (indole test)

purpose of TSI agar

diff of gnb on CHO utilization (oxidative or fermentative) and H2S production

what is in TSI

peptones, CHO, sodium thiosulfate, ferrous NH4 sulfate, salt, 1.3% agar, phenol red

principles of TSI

glucose fermented: pink on slant yellow butt (k/a) if lactose or sucrose ferments: all yellow (ala) nf gnb: k/nc h2S blackening fermentation causes gas bubbles

urease test principles

for org hydrolyzing urea from ammonia turns medium alkaline. phenol red. yellow to red/pink

+ive and -ive test for urease

phenol red +ive pink -ive no colour yellow

false +ive of urease

utilization of peptones or proteins may pH to 7+ due to protein hydrolysis

principle of MR/VP test

tests further fermentation of pyruvate. glucose fermentation

+ive and -ive test for VP test

+ive red colour | -ive yellow rusty brown

notes for MR/VP test

MR not preformed at BCIT (48hrs incubation)