Api 20e biochemical test strip

Biol 2420 Lab: API 20E

Objectives:

Learn how to perform and interpret the miniaturized, multi-test technique for bacterial identification.

Resource:

Lab manual: http://delrio.dcccd.edu/jreynolds/microbiology/2420/files/API%20%20RAPID%2020E.pdf

BioMerieux, Inc: https://apiweb.biomerieux.com

API 20E Instructions: http://biomanufacturing.org/uploads/files/587872707301898351-api20einstructions.pdf

Pearson MicroLab Tutor Video: API 20E

Introduction:

API-20E test strip (from bioMerieux, Inc.) is a standardized multi-test identification kit for Enterobacteriaceae family members which uses 21 miniaturized biochemical tests and a database (online software or codebook).

API 20 E (API = Analytical Profile Index)

Manufacturer: BioMérieux

Principle:

API test strip consists of 20 microtubes (wells) containing dehydrated testing media.

An unknown bacterial suspension is used to rehydrate and inoculate the media and the strip is incubated.

During incubation, biochemical reactions produce color changes, which are either spontaneous or revealed by the addition of reagents.

The test reactions are read according to the reading table, all + and - test results are compiled to obtain a profile number, which is then compared with the profiles in the codebook or uses online software to determine the identification of the bacterial genus and species.

(oxidase is not part of the strip but counts for the 21st test of API 20E - Enterobacteriaceae members are all negative)

Materials needed:

Setting up API 20E for inoculation and incubation:

Unknown bacterial specimen in agar plate

API 20E test strip

Sterile saline solution to make bacteria suspension

Sterile mineral oil to create anaerobioses for some tests

Reagents for reading API 20E after incubation:

Barrett's reagents A and B for VP test

Kovac's reagent for indole test

10% FeCl3 for TDA test

Pearson MicroLab Tutor Video: API 20E Oversized video in the file “API 20E.mpg” Please play in the file “API 20E.mpg” separately.

https://youtu.be/PXIis18qN9k

https://www.youtube.com/watch?v=ew3amo02_b0

More API videos:

Procedure:

1. Prepare a suspension of unknown bacteria in the saline tube.

Procedure continued:

2. Inoculate the API strip:

All wells - filled with bacteria suspension to the neck.

CIT, VP, and GEL - filled with more bacteria suspension to the top.

ADH, LDC, ODC, H2S, and URE - filled with sterile mineral oil to the top to create anaerobioses for the tests.

Procedure continued:

3. Incubate the strip in its chamber (tray):

Fill the divots of the tray with water to ensure the humidity for the microtubes not to dry out.

Place the strip into the tray.

Cover the chamber with the lid, and label it.

Incubate at 37oC for 18-24 hours.

4. Read test reactions according to the reading table after Incubation:

Add the test reagents to the wells:

1 drop of Barritt's A and B to VP (wait ~ 10 minutes to read)

1 drop of Kovac's to IND

1 drop of FeCl3 to TDA

Read all other tests without reagents.

Record all the results on the report sheet:

20 test reactions (+ or - ) from the strip

21st oxidase negative (-)

Procedure continued:

Case#1: Read the test reactions (+ or -) and record them on the report sheet for No.1 unknown bacterium. bacterium:

(answer key shown on the next page)

Interpretation:

Calculate 7-digit profile number based on all the 21 test results recorded:

Each three-test is grouped as section to calculate a single digit, assigning positive and negative results with specific scores, accordingly:

1st positive =1

2nd positive =2

3rd positive =4

negative =0

Add the three scores together as the single digit for this section.

For example:

“+ - +” section’s digit as 5 (1 + 0 + 4 = 5)

“+ + +” section’s digit as 7 (1 + 2 + 4 = 7)

21 tests are grouped into seven (21/3=7) sections, giving 7 digits in total as the 7-digit profile number for the bacterium.

No1. unknown bacterium’s

7-digit profile number: 5144572

Interpretation continued:

Input the 7-digit profile number onto BioMérieux website to identify the genus and species of the unknown bacterium.

No1. unknown bacterium’s 7-digit profile number: 5144572

Result:

Identification of No1. bacterium with 7-digit profile 5144572 is: Escherichia coli ---------- very good identification!

(genus) (species)

Case#2: No. 2 unknown bacterium’s 7-digit profile number: 5146472

Result:

Identification of No2. bacterium with 7-digit profile 5146472 is:

doubtfully narrowed to 3 possible organisms

----- need additional test(s) for the final identification.

Case#3: No. 3 unknown bacterium’s 7-digit profile number: 5537352

Result:

Identification of No3. bacterium with 7-digit profile 5537352 is:

unacceptable profile ----- error.