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
A)Basic tests
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-alactosidase + 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
Phenole red
“as LIA”
cadaverine (pH ) +CO2
+
4.
ODC : ornithine decarboxylase.
ornithine ODC
-
“as MIO”
putriscine (pH ) +CO2
Phenole 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 : Voges Proskauer test.
sodium pyruvate
acetoin
2,3- Butanediol
Barrit’s reagent + creatine
VP “1” : 40% KOH
VP ”2” : 5% alpha-naphthol
red color “within 10 min”
arrangement of reagents application is not necessary
because of the absence of peptone
Biochemistry
11. GEL : gelatin liquefaction test.
gelatin + cool chart gelatinase black ppt
+
12-20 : sugar fermentation/oxidation tests.
sugar fermentation or oxidation
acid product
+
Bromothymol blue
+ve “yellow”
21. Oxidase test : performed on external fresh culture.
-
note
Fermentation (Enterobacteriaceae, Aeromonas, Vibrio)
1. Fermentation of the carbohydrates begins in the most anaerobic portion
(bottom) of the tube. Therefore, these reactions should be read from the
bottom of the tube to the top.
2. A yellow colour at the bottom of the tube only indicates a weak or delayed
positive reaction.
Oxidation (Other Gram-negatives)
1. Oxidative utilization of the carbohydrates begins in the most aerobic
portion (top) of the tube. Therefore, these reactions should be read from
the top to the bottom of the tube.
2. A Yellow color in the upper portion and a blue color in the bottom portion
of the tube indicate oxidative utilization of the sugar. This reaction should
be considered positive only for non-Enterobacteriaceae Gram-negative
rods. This is a negative reaction for fermentative organisms such as
Enterobacteriaceae.
Biochemistry
B)Supplementary tests
“needed only with multitaxon code”
1. Before addition of reagents (sulfanillic acid & αnaphthyl amine), observe GLU tube (positive or
negative) for bubbles. Bubbles are indicative of
reduction of nitrate to the nitrogenous (N2) state.
2. A positive reaction may take 2-3 minutes for the red
color to appear.
3. Confirm a negative test by adding zinc dust. A pinkorange color after 10 minutes confirms a negative
reaction. A yellow color indicates reduction of nitrates
to the nitrogenous (N2) state.
NO2
Reduction of nitrate
to nitrite only
N2 Gas
Complete reduction
of nitrate to N2 gas
or amines
MOB
Observation of motility by semisolid agar
McC
Growth on MacConkey agar
OF-O
Oxidative utilization of glucose
(OF-open)
OF-F
Fermentative utilization of
glucose (OF-closed)
oxidation
test tube
•1% glucose
•Nutrient agar
•Bromothymol blue
fermentation
test tube
•1% glucose
•Nutrient agar
•Bromothymol blue
•Sterile mineral oil
Biochemistry
B)Supplementary tests
Note: if these tests used, you have to delay the reading of all result for 48h
material
• API 20 E Strips
• Incubation boxes “ tray and lids”
• Report sheets
• Sterile syringe and needle
• Disposable plastic inoculating loop
• 5 ml sterile normal saline
• Sterile Mineral Oil
• MacConkey agar plate
procedure
procedure
procedure
procedure
procedure
procedure
Interpretation
• On the report sheet, the test are separated into groups of three and
number 1 , 2 or 4 is allocated for each test. By adding the numbers
corresponding to the positive reaction within each group, a7- digit profile
number is obtained for 20 tests of the API 20E strip.
• The 7- digit profile is then compared with the numerical profile in the API
20 E analytical profile index book to obtain the organism identification.
Exercise: find the code
Thank you