Unit 4 Differential testing Flashcards
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
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
3 rapid spot tests
catalase, oxidase and spot indole
appearance of +ive and -ive for catalase
+ive bubbling H2O2 react w/catalase to produce water and O2
-ive no bubbling
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
reason for false -ive for catalase test
old cultures lose enzyme, too slow in adding reagent
appearance of +ive and -ive for oxidase (cytochrome)
+ive blue (oxidized)
-ive is colourless (reduced)
reason for false +ive for cytochrome oxidase test
false +ive: auto-oxidation after 10 secs, nichrome from using loop (use wooden stick)
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
why is the dye TMPD used for cytochrome oxidase
acts as redox indicator, gives e-
purpose of spot indole test
org break down tryptophan (casein peptone) resulting in production of indole, pyruvic acid and ammonia
appearance of +ive and -ive for spot indole
+ive blue/green
-ive no colour change
reason for false +ive spot indole test
mixed culture plates –> indole diffuses into culture. don’t trust if both are +ive
reason for false -ive spot indole test
if media has glucose, drops pH, may inhibit indole production.
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
purpose of 6.5% salt tolerance test
most org, high [NaCl] interfere w/membrane permeability and osmotic eqbm
appearance of +ive and -ive for 6.5% NaCl test
+ive turbid
-ive not turbid
reason for false +ive 6.5% NaCl test
over-inoculation
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)
