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:

EMB agar plate of a bacterium

0.85% NaCl solutions (5ml)
sterile transfer pipets
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 (a young pure

culture) into the 0.85% NaCl solution.
2. Make sure that the suspension is homogenous and without clumps of floating
bacteria.
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 (about 5ml).
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.
3. Record results on the diagram handed out to you in lab (1, 2, or 4 points for +
reaction, 0 points for - reaction). The oxidase test reaction should be negative,
and is added as the last test result.
4. Three test reactions are added together at a time to give a 7-digit number, which
can then be looked up in the codebook.

Additional tests such as motility, growth on MacConkey agar,

nitrate reduction and O-F glucose can increase the chances of
a good identification. Your instructor may have you run or
may not. The basic tests are really all that are absolutely
necessary for an identification.

(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

LABORATORY REPORT SHEET

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?

Revised 3/2015, Jackie Reynolds, Richland College