API 20E

This API-20E test strip (from bioMerieux, Inc.) is used to identify the enteric gram negative
rods (although API makes a variety of other test strips for yeast, Staph, anaerobes, etc.) 20
separate test compartments are on the strip, all dehydrated. A bacterial suspension is used to
rehydrate each of the wells. Some of the wells will have color changes due to pH differences:
others produce end products that have to be identified with reagents. A profile number is
determined from the sequence of + and - test results, then looked up in a codebook having a
correlation between numbers and bacterial species.

OBJECTIVE:

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

MATERIALS NEEDED:

agar plates of bacterial species

0.85% NaCl solutions (5ml)
sterile Pasteur pipettes + bulbs
sterile mineral oil
API 20E test strip (for oxidase - gram negative rods)
API test strip incubation chamber AFTER INCUBATION: 10% FeCl3, Barrett's reagents A
and B, Kovac's reagent

PROCEDURE:

Prepare a suspension of the bacteria in the saline tube

1. Inoculate a large colony (2-3mm diameter)of the bacterium (pure culture) into the
0.85% NaCl solution, making sure that the suspension is
homogenous and without clumps of floating bacteria.
2. Use a McFarland barium sulfate standard #3 to quantitate the
suspension and to produce a standard inoculums size..

Inoculate the API strip

1. Holding the strip at a slight angle up from the table top, you will
now inoculate the bacterial suspension into each well with the
sterile pipette.
 2. Touch the end of the pipette to the side of the cupule,
allowing capillary action to draw the fluid into the well as
you slowly squeeze the bulb. This should eliminate any
bubbles forming in the wells. Each well should be filled
up to the neck (see diagram).
3. CIT, VP, and GEL have boxes around their names.
These test wells will be filled all the way up to the top of
the well.
4. LDC, ODC, ADH, H2S, and URE are filled as described
in step B, but they will then be filled up to the top with
sterile mineral oil.

Incubate the strip in its chamber

1. The bottom of the incubation chamber has small indented wells in the bottom: fill it with
water just enough to fill these indentations.
2. Place the strip into this bottom. There should not be so much water that it slops onto
the API strip.
3. Place the top of the incubation chamber over the bottom, and label it.
4. Place the strip at 37o C for 18-24 hours.

INTERPRETATION:

1. Add the proper reagents to the

compartments:
o 1 drop of Kovac's to the IND (read within
a couple of minutes)
o 1 drop of Barritt's A and B to VP (a +
reaction may take up to 10 minutes)
o 1 drop of FeCl3 to TDA
2. Read all other tests as described (chart below) without reagents using the table
below..
3. Record results on the diagram handed out to you in lab. The oxidase test reaction
should be negative, and is added as the last test result.
4. Take your result sheet to the computer to INPUT YOUR REACTIONS: The API 20E
website database will provide you with potential organisms.

A more specific 9-digit number can be obtained with a few

more tests:, but NOT DURING THIS LAB.

(pictures from the API 20E documentation, from BioMerieux)

READING THE API 20

TESTS SUBSTRATE REACTION TESTED - RESULTS + RESULTS

ONPG ONPG beta-galactosidase colorless yellow
ADH arginine arginine dihydrolase yellow red/orange
LDC lysine lysine decarboxylase yellow red/orange
ODC ornithine ornithine decarboxylase yellow red/orange
CIT citrate citrate utilization pale green/yellow blue-green/blue
H2S Na thiosulfate H2S production colorless/gray black deposit
URE urea urea hydrolysis yellow red/orange
TDA tryptophan deaminase yellow brown-red
IND tryptophan indole production yellow red (2 min.)
VP Na pyruvate acetoin production colorless pink/red (10 min.)
GEL charcoal gelatin gelatinase no diffusion of black black diffuse
GLU glucose fermentation/oxidation blue/blue-green yellow
MAN mannitol fermentation/oxidation blue/blue-green yellow
INO inositol fermentation/oxidation blue/blue-green yellow
SOR sorbitol fermentation/oxidation blue/blue-green yellow
RHA rhamnose fermentation/oxidation blue/blue-green yellow
SAC sucrose fermentation/oxidation blue/blue-green yellow
MEL melibiose fermentation/oxidation blue/blue-green yellow
AMY amygdalin fermentation/oxidation blue/blue-green yellow
ARA arabinose fermentation/oxidation blue/blue-green yellow
OX oxidase oxidase colorless/yellow violet

QUESTIONS:

1. What is the purpose of the water in the tray?

2. What is the function of the mineral oil?

3. What are the advantages of this test (compared to regular biochemical tube media)?

4. What are the disadvantages of this test?

LAB MANUAL: TABLE OF CONTENTS

8/2009, Jackie Reynolds, Richland College