Api 20 E principle The API 20 E

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Api 20 E

Api 20 E

principle • The API 20 E System is a standardized, miniaturized microtube system consisting

principle • The API 20 E 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 20 E 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 20 E 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

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 galactose + ortho-nitrophenol “yellow/pale yellow” Lac operon Latose fermenter Delayed fermenter Non-fermenter B-alactosidase + Permease + B-trans-acetylase ++ ++ ++ +/- - - + -

Biochemistry 2. ADH : arginine Dihydrolase argenine ADH citrullin + NH 3 citrullin +

Biochemistry 2. ADH : arginine Dihydrolase argenine ADH citrullin + NH 3 citrullin + phosphate ion carbamoil phosphate Catabolic arginine carbomoly transferase carbamoil phosphate + ornithine ATP + CO 2 + NH 3 Carbamoil kinase • This microtube contains phenole red indicator • orange color consider +ve only in first 24 h 6. 8 8. 4 + -

Biochemistry 3. LDC : lysine decarboxylase. Lysine LDC Phenole red “as LIA” cadaverine (p.

Biochemistry 3. LDC : lysine decarboxylase. Lysine LDC Phenole red “as LIA” cadaverine (p. H ) +CO 2 + 4. ODC : ornithine decarboxylase. ornithine ODC Phenole red - “as MIO” putriscine (p. H ) +CO 2 + -

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

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 +CO 2+H 2 O ammonium salt Na 2 CO 3 NH 3+NH 4 OH CO 2+pyruvate bromothymol blue : yellow (6) < green (6. 9) < blue (7. 6) + -

Biochemistry 6. H 2 S : H 2 S production test. sodium thiosulfate H

Biochemistry 6. H 2 S : H 2 S production test. sodium thiosulfate H 2 S Ferrous sulfate Reduction “H+” H 2 S (g) ferrous sulfide “black ppt” + - 7. URE : urease utilization test. urea • urease Phenol red 2 CO 2 + NH 3 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”

Biochemistry 8. TDA : tryptophan deaminase. tryptophan TDA indole-pyruvic acid “form within 24 h” + NH 3 indole-pyruvic acid +10%Fe. Cl 3 + HCl + hydrazine ferric hydrazon “” Reddish brown ppt ”” HCl added to TDA reagent to break dow the product of the reaction between Fe. Cl 3 and tryptophan to prevent false positive rasults. + -

Biochemistry 9. IND : tryptophan utilization test. tryptophan indole tryptophanase Kovac’s reagent Isoamyl alcohol

Biochemistry 9. IND : tryptophan utilization test. tryptophan indole tryptophanase Kovac’s reagent Isoamyl alcohol + HCl + para-nitrophenyl aminobenzaldehyde indole + pyruvic acid + NH 3 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

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

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 ØNO 2 B)Supplementary tests “needed only with multitaxon code” addition of reagents (sulfanillic

Biochemistry ØNO 2 B)Supplementary tests “needed only with multitaxon code” addition of reagents (sulfanillic acid & αReduction of nitrate 1. Before naphthyl amine), observe GLU tube (positive or negative) for bubbles. Bubbles are indicative of to nitrite only reduction of nitrate to the nitrogenous (N 2) 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 (N 2) state. ØN 2 Gas Complete reduction of nitrate to N 2 gas or amines ØMOB Observation of motility by semisolid agar ØMc. C Growth on Mac. Conkey agar ØOF-O Oxidative utilization of glucose (OF-open) ØOF-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

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

material • API 20 E Strips • Incubation boxes “ tray and lids” •

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 • Mac. Conkey agar plate

procedure

procedure

procedure

procedure

procedure

procedure

procedure

procedure

procedure

procedure

procedure

procedure

Interpretation • On the report sheet, the test are separated into groups of three

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, a 7 - digit profile number is obtained for 20 tests of the API 20 E 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

Exercise: find the code

Thank you

Thank you