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 tests1. 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” galactose + ortho-nitrophenol “yellow/pale yellow”

B-galactosidase

Lac operon B-alactosidase Permease B-trans-acetylase

Latose fermenter ++ ++

Delayed fermenter ++ +/-

Non-fermenter - -

+ +

+ -

Biochemistry2. ADH : arginine Dihydrolase

argenine citrullin + NH3

citrullin + phosphate ion carbamoil phosphate + ornithine

carbamoil phosphate ATP + CO2 + NH3

• This microtube contains phenole red indicator

• orange color consider +ve only in first 24h

ADH

Catabolic arginine carbomoly transferase

Carbamoil kinase

+ -6.8 8.4

3. LDC : lysine decarboxylase. “as LIA”

Lysine cadaverine (pH ) +CO2

4. ODC : ornithine decarboxylase. “as MIO”

ornithine putriscine (pH ) +CO2

Biochemistry

LDCPhenole red

ODCPhenole red

+ -

+ -

5. CIT : testing the ability of bacteria to utilize citrate as a carbon source

sodium citrate acetic acid + oxaloacetic acid

Na +CO2+H2O Na2CO3 CO2+pyruvate

ammonium salt NH3+NH4OH

bromothymol blue : yellow (6) > green (6.9) > blue (7.6)

Biochemistry

Citrate layase

OAAdecarboxylase

+ -

6. H2S : H2S production test.

sodium thiosulfate H2S (g)

H2S ferrous sulfide “black ppt”

7. URE : urease utilization test.

urea 2 CO2 + NH3

• urease works at both aerobic and anaerobic

conditions, but it prefers the second.

Biochemistry

+ -

Reduction “H+”

Ferrous sulfate + -

urease

Phenol red

8. TDA : tryptophan deaminase.

tryptophan indole-pyruvic acid “form within 24 h” + NH3

indole-pyruvic acid +10%FeCl3 + HCl + hydrazine ferric hydrazon

HCl added to TDA reagent to break dow the product of the reaction between FeCl3 and tryptophan to prevent false positive rasults.

Biochemistry

TDA

+ -

“” Reddish brown ppt ””

9. IND : tryptophan utilization test.

tryptophan indole + pyruvic acid + NH3

indole pink or red “with or without ring”

10. VP : Voges Proskauer test.

sodium pyruvate 2,3- Butanediol

acetoin red color “within 10 min”

arrangement of reagents application is not necessary

because of the absence of peptone

Biochemistry

tryptophanase

Kovac’s reagentIsoamyl alcohol + HCl + para-nitrophenyl aminobenzaldehyde

+ -

+ -

Barrit’s reagent + creatineVP “1” : 40% KOHVP ”2” : 5% alpha-naphthol

11. GEL : gelatin liquefaction test.

gelatin + cool chart black ppt

12-20 : sugar fermentation/oxidation tests.

sugar fermentation or oxidation

acid product

+ve “yellow”

21. Oxidase test : performed on external fresh culture.

Biochemistry

gelatinase

+ -

+

-

Bromothymol blue

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.

note

NO2 Reduction of nitrate to nitrite only

1. Before addition of reagents (sulfanillic acid & α-naphthyl 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 pink-orange color after 10 minutes confirms a negative reaction. A yellow color indicates reduction of nitrates to the nitrogenous (N2) state.

N2 Gas Complete reduction of nitrate to N2 gas or amines

MOB Observation of motility by semisolid agarMcC Growth on MacConkey agarOF-O Oxidative utilization of glucose

(OF-open)

OF-F Fermentative utilization of glucose (OF-closed)

Biochemistry B)Supplementary tests“needed only with multitaxon code”

• 1% glucose• Nutrient agar• Bromothymol blue• Sterile mineral oil

• 1% glucose• Nutrient agar• Bromothymol blue

oxidation test tube

fermentation test tube

Biochemistry B)Supplementary tests

Note: if these tests used, you have to delay the reading of all result for 48h

• 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

material

procedure

procedure

procedure

procedure

procedure

procedure

• 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.

Interpretation