Api 20E
principle
•
The API 20E System is a standardized, miniaturized microtube system consisting of
21 conventional “basic” and 6 supplementary biochemical tests used for the
identification of Enterobacteriaceae and other non-fastidious Gram-negative
bacteria.
•
The API 20E System consists of microtubes containing dehydrated substrates. The
substrates are reconstituted by adding a bacterial suspension.
•
They are then incubated so the organisms react with the contents of the tubes.
Finally, they are then read when the various indicator systems are affected by the
metabolites or added reagents, generally after 18 to 24 hours incubation at 35 37°C. Refer to API 20E package insert for the principles involved in each of the
reactions, reactive ingredients and quantity of ingredients in each tube.
Cupule
tube
Biochemistry
1. ONPG : ortho-nitrophenyl b-D-galactopyranoside.
ONPG is a substrate that have B-galactosidic bond “like lactose” but didn’t
need permease to enter bacterial cell.
ONPG “colourless”
B-galactosidase
Lac operon
Latose fermenter
Delayed fermenter
Non-fermenter
galactose + ortho-nitrophenol “yellow/pale yellow”
B-galactosidase + Permease + B-trans-acetylase
++
++
++
+/-
-
-
+
-
Biochemistry
2. ADH : arginine Dihydrolase
argenine ADH
citrullin + NH3
citrullin + phosphate ion
carbamoil phosphate
Catabolic arginine
carbomoly transferase
Carbamoil kinase
carbamoil phosphate + ornithine
ATP + CO2 + NH3
• This microtube contains phenole red indicator
• orange color consider +ve only in first 24h
6.8
8.4
+
-
Biochemistry
3.
LDC : lysine decarboxylase.
Lysine
LDC
Phenol red
“as LIA”
cadaverine (pH ) +CO2
+
4.
ODC : ornithine decarboxylase.
ornithine ODC
-
“as MIO”
putriscine (pH ) +CO2
Phenol red
+
-
Biochemistry
5.
CIT : testing the ability of bacteria to utilize citrate as a carbon source
sodium citrate Citrate layase
acetic acid + oxaloacetic acid
OAA
decarboxylase
Na +CO2+H2O
ammonium salt
Na2CO3
NH3+NH4OH
CO2+pyruvate
bromothymol blue : yellow (6) > green (6.9) > blue (7.6)
+
-
Biochemistry
6. H2S : H2S production test.
sodium thiosulfate
H2S
Ferrous sulfate
Reduction “H+”
H2S (g)
ferrous sulfide “black ppt”
+
-
+
-
7. URE : urease utilization test.
urea
•
urease
Phenol red
2 CO2 + NH3
urease works at both aerobic and anaerobic
conditions, but it prefers the second.
Biochemistry
8. TDA : tryptophan deaminase.
tryptophan
TDA indole-pyruvic acid “form within 24 h” + NH3
indole-pyruvic acid +10%FeCl3 + HCl + hydrazine
ferric hydrazon
“” Reddish brown ppt ””
HCl added to TDA reagent to break dow the product of the reaction between
FeCl3 and tryptophan to prevent false positive rasults.
+
-
Biochemistry
9. IND : tryptophan utilization test.
tryptophan
indole
tryptophanase
Kovac’s reagent
Isoamyl alcohol + HCl +
para-nitrophenyl
aminobenzaldehyde
indole + pyruvic acid + NH3
pink or red “with or without ring”
+
-
+
-
10. VP : vogous proscauer test