Tests/Agar Flashcards
Staphylococcus
Blood agar
Mannitol salt
Chromogenic media
- SAIDE ID
- Chromagar MRSA
- BioMerieux
- Oxoid chromogenic
- ChromID MRSA
Mannitol salt
7.5% salt = selective
Mannitol + phenol red = differential
Oxoid CHromogenic MRSA Agar
Chromogens detect phosphatase activity in S. aureus -> this will produce denim-blue colonies
Cefoxitin (methicillin) will inhibit MSSA colonies
This allows for the accurate detection of MRSA
BioMérieux Chrome ID
SAID -> S. aureus ID
Chromogens detect alpha glucuronidase in S. aureus
This forms green colonies i.e. S, aureus = green colonies
ChromID MRSA agar
MRSA stains indicated by green colonies
Results from alpha-glucuronidase producing colonies in the presence of cefoxitin
Staphylococcus confirmatory tests
Coagulase test
DNase test
Vitek
MALDI
Slide coagulase test
Detects bound coagulase only
Mix bacterial saline suspension with rabbit plasma (source of fibrinogen), observe agglutination
Tube coagulase test
Detects free and bound coagulase
Mix 9 drops of 3hr incubated test suspension with 1 drop rabbit plasma -> incubate over night -> observe clot
Staphaeurex Plus
Rapid latex agglutination test for the identification of S. aureus
In addition to coagulase, protein A is also found on the surface of 95% of human strains of S. aureus
Certain MRSA possess a capsule that masks both protein A and the coagulase
With Staphaurex, rapid agglutination will occur using any three of these components:
- Reaction between fibrinogen and coagulase
- Binding between Protein A and the Fc portion of IgG
- Binding between MRSA capsular polysaccharide and specific IgG
DN’ase test
Deoxyribonuclease Agar
Nutrient agar that has 0.2% DNA added
Spot inoculate DNase agar and incubate overnight
Flood with 1 molar HCL
HCL precipitates DNA which turns the medium cloudy
DNase-positive cultures -> DNA is unavailable for precipitation so there will be a clear zone around the inoculum e.g. S. aureus
DNase negative will have no zone of clearing e.g. CNS
Novobiocin test
Nutrient agar with novobiocin discs
Lawn inoculum
S. aureus/CNS are novobiocin susceptible
S. saprophyticus are novobiocin resistant
Methicilin suscpetibility for MRSA
Cefoxitin disk
N. meningitidis tests
Direct specimen microscopy
Latex agglutination (gpB, C, Y and W)
Blood agar
Chocolate agar
Chocolate broth
TAXO sugars
N. gonorrhoea tests
Thayer Martin medium or GC/NYC medium
TAXO sugars
Gold standard = commercial PCR deterction on day 1
TAXO sugars
Carbohydrate utilisation tests - discriminatory test
Neisseria use carbohydrates oxidatively - not fermentation tests
Test ability to oxidise glucose, maltose, lactose and sucrose
Media e.g. Choc or GC agar minus antibiotics
Lawn inoculum and apply Taxo sugar discs
Incubate overnight in 5-10% CO2
Add phenol red indicator - yellow = positive oxidation reaction
M. catarrhalis
Blood or chocolate (but not fastidious will grow on nutrient agar)
TAXO sugars (no fermentation)
DN’ase
Growth (nutrient agar)
Tributyrin hydrolisis
Catarrhalis disc test
Organism produces butyrate esterase which hydrolyses tributyrin to release indoxyl which reacts with oxygen to turn blue-green
Rub test colony on disk and observe for a blue/green colour within 2 minutes
M. catarrhalis is positive for tributyrin hydrolysis
Neisser is negative
Haemophilus tests:
Latex agglutination for Hib
X and V factors (chocolate agar for isolation)
Pasteurella Multocida
(3)
Fermentation of glucose and sucrose
Indole production
Ornithine decarboxylase breakdown
B. pertussis
Bordet-Gengou agar
Citrate utilisation
Antigen detection (direct fluorescent antibody test)
Bordet-Gengou agar
Sheep blood in potato glycerol agar
+/- antibiotics
Indole test
Peptone media is a source of the amino acid trytophan
Hydrolysis of trytophan, leads to accumulation of indole
Add Kovacs reagent
Cherry red colour on surface is positive
Red colour seen in pasteurella
Ornithine decarboxylase
Decarboxylase broth contains nutrients, dextrose, pH indicator (purple) plus a single amino acid e.g. ornithine, lysine
Carboxylase breaks down ornithine - alkaline by-products-pH drop-indicator- purple colour change
Control tube - only dextrose ferments dextrose - pH decrease = yellow colour
P.multicoda positive for ornithine decarboxylase
Group B strep (S. agalactiae)
Lancefield test (group B)
CAMP positive
Hydrolysis of hippurate
Does not hydrolyse aesculin
May grow on MacConkey
Citrate
Citrate slope (green) - sole carbon source
Utilise citrate -> pH increased - blue colour
B. pertussis = citrate positive
Hippurate hydrolysis test
(4)
Used to differentiate S. agalactiae from other Beta Haemolytic streps
Hippurate is hydrolysed by hippuricase into Benzoic acid and glycine
Ninhydrin reagent is used as an indicator to detect Glycine
Ninhydrin reacts with glycine to form a deep blue or purple colour
CAMP test
(4)
Detects the production of extracellular protein CAMP factor
CAMP factor acts synergistically with the beta lysin produced by S. aureus
A zone of enhanced lysis of sheep or bovine blood
CAMP positive = Group B strep
CAMP negative = Not GBS
S. pyogenes
Lancefield group A
B-haemolysis
MacConkey -> no growth
Bacitracin -> susceptible
PYR positive
What is PYR
Pyrrolidonyl arylamidase
S. pneumoniae
Optochin susceptibility -> susceptible
Bile susceptibility -> susceptible
Vitek
Lancefield group A
Enterococcus
Lancefield grouping - D
MacConkey - growth
Bile Aesculin - growth
Molecular detection of Van A or Van B genes (VREs)
Chromogenic VRE medium
Enterococcus on VRE Medium
Bluish green = E. faecalis
Violet = E. faecium
Shigella tests:
(6)
Direct detection on faeces using real time PCR (Shigella Ipa gene)
XLD agar
DCA agar
Chrome GIT agar
Biochemical tests
UHG reference lab - serotyping
DCA agar
Shigella and Salmonella agar
Selective factors include sodium deoxycholate and sodium citrate
Differential factors are lactose and sodium ferric chloride
Lactose fermenters = commensals = pink
Shigella = NLF = pale
Salmonella = NLF = pale with H2S positive black centre
XLD agar
Shigella and Salmonella agar
Selective factor is sodium deoxycholate
Differential factors are Xylose, lysine, H2S
Lactose fermenters = commensals = yellow
Shigella = xylose lysine = red
Salmonella = red with black centre
Chrom GIT agar (shigella and salmonella)
Teal coloured colonies
Non-H2S producing salmonellas will look the same
Biochemical tests for Shigella
Indole negative
Methyl red positive
Vogues negative
Citrate negative
urease negative
Lysine decarboxylase negative
Only MR positive
Salmonella tests:
Direct detection of Inv gene (RTPCR)
Selective enrichment on selenite broth
XLD -> red with black centre
DCA -> pale with black centre
Chromagar GIT -> teal coloured
Bismuth Sulfide agar -> black colonies
Biochemical tests
Selenite broth
Salmonella enrichment broth
Selenite broth
Enhances culture yield by reducing growth of faecal coliforms and faecal streptococci.
Take sub-cultures of broth from the upper third of the broth column should be at least 5 cm in depth.
Better for enrichment of Salmonella Typhi
37 not exceeding 18 hours
Bismuth Sulfite agar
Bismuth sulfite agar (BS) is a selective as well as a differential medium for isolation and presumptive identification of Salmonella spp, especially Salmonella Typhi
Uses brilliant green indicator so colonies are black (H2S) or green
Why does Salmonella turn black on chromagar
H2S production
Salmonella biochemical results
I : negative
M: positive
V: negative
iC: positive
urease negative
lysine decarboxylase positive
Neg, pos, neg, pos …
Lysine decarboxylase test
Glucose
Lysine
Bromocresol Purple
Yersinia:
Real time PCR detection
CIN agar
Biochemical tests
CIN agar
Cefsulodin
Irgasan
Novobiocin agar
For Yersinia
CIN agar
Cefsulodin
Irgasan
Novobiocin agar
For Yersinia
Yersinia biochemical tests
Indole positive
Methyl red negative
Vogues positive
Citrate negative
Urease positive
Lysine negative
Ornithine decarboxylase positive
pos, neg, pos, neg, pos, neg …
Proteus:
Swarming on agar
No swarm on MacConkey (NLF)
Brown on chrom UTI agar
Biochemical tests
Proteus biochem tests
Indole negative
MR positive
VP negative
Citrate positive
Urease positive
Lysine decarboxylase negative
Dulcitol negative
Lactose negative
Phenylalanine postiive
Klebsiella:
Mucoid colonies
Biochemical tests
Klebsiella biochem tests
Indole negative
MR negative
VP positive
Citrate postiive
Urease V positive (K. pneumo)
Lysine decarboxylase positive
Dulcitol positive
Enterobacter tests
MacConkey: LF
Biochemical tests
Biochemical results for enterobacter
Indole negative
MR negative
VP positive
Citrate postiive
Urease negative
Lysine decarboxylase negative
Dulcitol negative
Biochemical results for citrobacter
I : produces indole -
M: methyl red positive +
V: Voges-Proskauer negative -
iC: cannot use citrate as sole carbon source +
Urease -
Lysine Decarboxylase -
Dulcitol –
Lactose + LF
EHEC/STEC
Direct detection of shiga toxin genes by RTPCR - EntericBio RTPCR
MacConkey for all E. Coli
STEC
- STEC Chromagar
- CTSMAC agar
CTSMAC agar
For shiga toxin producing E. COLI -> EHEC
Cefixime tellurite sorbitol Mac Conkey
O157:H7 STEC = non sorbiol fermenter = colourless
Non o157:H7 = sorbitol fermenter = pink
Detects most common Shiga-toxin E. Coli serotypes -> mauve colonies
Other enterobacterales are coulerless, blue or inhibited
Biochemical results for E. coli
I: produces indole
M: methyl red positive
V: negative
iC: negative
Indole test
A
decompose the amino acid tryptophan to indole
accumulates in the medium
Inoculate a peptone water
Incubate 24 hrs @ 37
When indole is combined with Kovac’s Reagent
yellow to cherry red
an oily layer at the top of the broth
Methyl red test
Methyl Red Test
detection of end products from the
metabolism of glucose.
Inoculate a buffered glucose
the fermentation of glucose.
MR Positive: When the culture medium turns red
after addition of methyl red
pH at or below 4.4
Voges-Proskauer test
Voges-Proskauer Test
detection of end products from
the metabolism of glucose.
produce acetoin as the chief end
product of glucose metabolism
Heavy inoculum
Add 40% KoH and alpha Napthol
Shake vigorously
Colour development within 15 mins
Voges-Proskauer negative
Simmons Citrate
Simmons Citrate Test
use citrate as sole carbon source
Growth usually results in the bromothymol blue
indicator, turning from green to blue.
The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6
E. Coli biochem tests
produces indole
methyl red positive
Voges-Proskauer negative
cannot use citrate as sole carbon source
Lactose Fermenter
produces lysine decarboxylase
ferments sorbitol except E. coli O157 H7
produce β-glucuronidase except E. coli O157 H7
Chromagar GIT
E. coli - dark pink to red
Enterococcus - turquoise blue
Klebsiella, Enterobacter, Citrobacter - metallic blue
Proteus - brown hali
Pseudomonas - cream/translucent
S. aureus -> golden/opaque
S. saprophyticus -> pink small
Pseudomonas
Blood agar (metallic sheen and sweet odour)
Chocolate agar
Moderately selective media - MacConkey media (NLF)
Selective agar - Cetramide agar
Growth at 42 and 4 degrees
Cetramide agar
Pseudomonas
P, aeruginosa = enhanced pigment = bright green colour
It enhances the production of Pseudomonas pigments such as pyocyanin and pyoverdine which show a characteristic blue-green and yellow-green colour
