Lab Practical #2

Question 0

Kligler’s Iron Agar (KIA)

Answer 0

Medium: KIA slant
Substrate: glucose/lactose
(1.0% lactose and 0.1% glucose)
Slant/Butt(Gas)
End product: acid/gas
Ph indicator: phenol red
Reagent: none
\+=yellow=A
-=red=K
B test
*must be read in 24 hours
*E. coli= A/A

Question 1

Indole

Answer 1

Medium: triptone broth
Substrate: tryptophan
Bacterial enzyme: tryptophanase
End product: indole
Ph indicator: none
Reagent: Kovac's reagent
-10-12 drops
\+= pink
-= no color
A test
E. coli= +

Question 2

H2S Production (2nd Phase of KIA)

Answer 2

Medium: KIA slant
Substrate: cysteine
End product: H2S
Ph indicator: FeSO4 (ferrous sulfate)
Reagent: none
\+=black=H2S+
-=no black
B test
*H2S + FeSO4 --> FeS (black precipitate) + H2SO4

Question 3

Methyl Red (MR)

Answer 3

Medium: MR-VP broth
Substrate: glucose
Bacterial enzyme: glycol pathway
End product: stable acid pH<4.5
PH indicator: methyl red @ pH 4.4
Reagent: methyl red
- 3-5 drops
\+=red
-=yellow
A test
*E. coli = +

Question 4

Vogues-Proskauer (VP)

Answer 4

Medium: MR-VP broth
Substrate: glucose
End product: acetoin
PH indicator: none
Reagent: VP #1 and 2
-VP #1= alpha-naphthol (5 drops & shake)
-VP #2= guanidine (10 drops & shake)
-wait 20 minutes
-acetoin + VP#1 --> diacetyl + guanidine --> red
\+=red
-=no color
A test
*E. coli= -

Question 5

Citrate Utilization

Answer 5

Medium: Simmon's citrate agar slant
Substrate: citrate (inorganic C)
Bacterial enzyme: citrate-permease
End product: alkalinity
PH indicator: bromthymol blue
Reagent: none
\+=blue pH 7.6
-=green pH 6.9
B test