Culture conditions
37C aerobic blood agar
Gram stain
Gram negative bacilli
Biochemical tests
KOH positive
Catalase weak positive
Oxidase NEGATIVE
Presumptive enterobacterales tests
- Brilliance chromagar
- Utilization of specific substrates - citrate, urea
- Ability to degrade AA - tryptophan (Indole production)
- Fermentation of CHOs
- Commercial API-20E
additional confirmatory tests
MALDI-tof /Vitek
Send salmonella/shigella to SSL ref lab in UHG Galway
Diseases - Lactose fermenters
E.coli - commensal = UTIs, BSI, GI, LRT
Klebsiella - opportunistic = LRT (pneumonia), UTIs, wound infections
Citrobacter - opportunistic = UTIs, wound infections
Enterobacter - opportunistic = LRT, systemic
Disease - non-lactose fermenters
Salmonella = GI (food poisoning), enteric infections, typhoid fever
Shigella = bacillary dysentery
Proteus - commensal = UTIs, supparative infections.
Lactose fermenters
E.coli
Klebsiella
Citrobacter
Enterobacter
Non-lactose fermenters
Salmonella
Shigella
Proteus
Pseudomonas (Oxidase positive)
Brilliance chromagar pink colonies
E.coli
Presumptive enterobacterales tests (11)
- Indole production
- Methyl Red test
- Voges- Proskauer test
- Peptone sugar (dulcitol/mannitol) fermentation
- Lysine decarboxylase broth control
- Lysine decarboxylase broth test
- Phenylalanine agar
- Urea agar
- Citrate slope
- MacConkey agar
- API-20-E
Incubation conditions for 10 biochemical tests
37C aerobic 48hrs.
What do you add to lysine decarboxylase tests following inoculation?
Layer of mineral oil to promote fermentation.
- Indole production test result
*any extra additions?
Add 6-8 drops Kovacs reagent to test.
Cherry red colour on surface
= E.coli
- MR test results
*any extra additions?
Need to add 4-5 drops Methyl red solution
*Positive = red colour remains
= Citrobacter, E.coli
*Negative - yellow colour
= Enterobacter, Klebsiella
- VR test results
*Need to add extra?
Add 5 drops of VP1 and VP2 to test and shake vigorously for 10mins.
*Positive = red colour
= Enterobacter + Klebsiella
*Negative = no colour change
= Citrobacter + E.coli
- Peptone sugar (dulcitol) fermentation results
*Need to add extra?
Nothing to add
*Positive = pink to yellow colour change
= variable (21-79% of strains give positive reaction)
* negative = Peptone remains pink.
= proteus
5+6. Lysine decarboxylase test results
Nothing to add
*positive = control is yellow but test is purple
= E.coli, Klebsiella
*Negative = both are yellow.
= Citrobacter, Proteus , Enterobacterales (most negative but some positive strains)
- Phenylalanine agar test results
Add 4 drops of 10% FeCl to slope
*Positive = Brown colour develops
= proteus
*Negative = no colour develops
- Urea agar test results
No need to add anything
* Urease Positive = Yellow to pink/red
= Proteus , Klebsiella.
*negative = yellow
= Citrobacter, E.coli, Enterobacter
- MacConkey Agar
After inoculating urea agar and before flaming use same inoculate loop to streak out MacConkey agar purity plate. Should contain only one bacterial species - similar to colonies found on test agar plate.
- Citrate slope
After inoculating MacConkey purity plate, flame loop and subculture from original inoculated water and inoculate citrate slope. Incubate 27C for 24hrs.
Positive = Blue + growth
= citrobacter, enterobacter, klebsiella, proteus
Negative = green + no growth
E.coli
E.coli definitive biochemical test results
MR test positive (Red colour)
Citrate slop negative (green colour)
Klebsiella definitive biochemical test results
Urease positive (pink/red agar)
Phenylalanine negative (no brown colour)
Mucoid + metallic colonies
