Aerobic colony count, Enterobacteriacea, E.coli

Question 1

Enterobacteriaceae

- detection: which media to use

Answer 1

• EE broth, VRBG agar

Question 2

Enterobacteriaceae

- EE broth

Answer 2

EE broth (Enterobacteria Enrichment broth)

• composition: brilliant green, bile, glucose
• Reaction: positive = turbidity + color change
• negative: green color
• positive: yellow color

Question 3

Enterobacteriaceae

- VRBG agar

Answer 3

VRBG (Violet Red Bile Glucose agar)

• composition: violet red, glucose, bile
  reaction:
• turns red –> means its acidic
• pink red coonies with precipitation haloes
• colorless olonies with mucoid haloes
• select a well isolated colony for conformation

Question 4

Enterobacteriaceae

- conformation: which test to use

Answer 4

• Oxidase test, glucose fermentation test

Question 5

Enterobacteriaceae

- Oxidase test

Answer 5

• enterobacteriacea is oxidase negative
• positive reaction: development of dark purple color (indophenols) within 10 sek.
• negative reaction: absence of color

Question 6

Enterobacteriaceae

- glucose fermentation test

Answer 6

• Enterobacteriaceae are positive for this test
• negative reaction: test stays with the original red color (phenol red = alkaline)
• positive reaction: the red color turns yellow (acid. bottom - fermentation), +/- gas production.

Question 7

Escherichia coli

- first step in the detection of E.coli:

Answer 7

differentiation from other coliforms!

• coliforms are Enterobacteria which can ferment lactose with the production f acid and gas when incubated at 30*C
• Coliforms: Escherichia (E.coli), Klebsiella (K. oxytoca), Citrobacter (C.freundii), Enterobacter (E. aerogenes)
• 1st step of differentiation: culture in brilliant green-bile-lactose borth at 44*C for 48hr
• cloudy, gas prodution 1/10 of durham tube
• differentiation –> IMViC!

Question 8

Escherichia coli

- IMVic tests

Answer 8

are used for identification/differentiation of coliforms and E.coli 
E.coli:
- Indol: +
- Methyl red: +
- Voges-proskauer: -
- Citrate: -

Question 9

Escherichia coli

- indole test

Answer 9

• E.coli is indole positive
• positive reaction: red layer at the top of the tube after adding Kovac´s reagent
• negative reaction: lack of color change/yellow ring after ading Kovac´s reagent.

Question 10

Escherichia coli

- Methyld red test

Answer 10

• E. coli is methyl red +. used to detect mixed acid fermentation
• positive reaction: development of red color after addition of methyl red reagent (E.coli, Citrobacter)
• negative reaction: no color change after adding methyl red (Klebsiella, Enterobacter)

Question 11

Escherichia coli

- Voges-Proskauer test

Answer 11

E.coli is Voges-Proskauer negative

• it is used to detect butanediol fermentation (E.coli = no butanediol)
• negative test: lack of color change
• positive test: red-pink color

Question 12

Escherichia coli

- Citrate utilization test

Answer 12

E.coli is citrate negative

• negative test: no color change or growth, remains green
• positive test: blue color and growth

Question 13

Escherichia coli

- determination of b-glucoronidase positive E.coli

Answer 13

• the best confirmation, very specific, no other confirmation needed
• TBX agar plate
• positive reaction: bluish green

Question 14

Escherichia coli

- which media to use

Answer 14

• TBX agar, MMG broth,

Question 15

Escherichia coli

- TBX agar

Answer 15

TBX agar (Tryptone, bile, X-glucuronide)

• composition: casein pepton, bile salt, x-b-glucoronide
• positive reaction: the released chromophore is colored and biulds up within the cells, causing the E.coli colonies to be blue-green

Question 16

Escherichia coli

- MMG broth

Answer 16

MMG broth (mineral modified glutamate)

• composition: lactose, amino acids, vitamins, minerals, sodium glutamate, ammonium sulphate, bromecresol purple
• positive reaction: the color turns yellow from the original purple (bacteria use lactose as carbon source)