API RAPID 20E

RapiD 20 E is a standardized system for the identification of Enterobacteriaceae in 4 hours,

which uses 20 miniaturized biochemical tests chosen for their highly discriminant value and
adapted to rapid interpretation. Each of the 20 microtubes contains dehydrated substrates, to
which a bacterial suspension is added to reconstitute the media. During incubation, metabolism
produces color changes that are either spontaneous or revealed by the addition of reagents.
The reactions are read and identification is obtained by referring to the Analytical Profile Index
or using the identification software.

The organism should be oxidase negative and a gram negative bacillus. The agar plate
culture used should be well-isolated and pure, and grown on a lactose-containing
medium (MacConkey’s, EMB, etc.)

Materials Needed:
Micropipettor and tips
0.85% saline tubes
API strip
Sterile mineral oil
Pure culture isolation plate of bacterium
0.5 McFarland standard

Preparation of the inoculum

1. You will need a tube of 0.85 % physiological saline (2ml) to make the bacterial suspension.
2. Use an inoculation loop to pick 1-4 well-isolated colonies of identical morphology from the
agar plate by successive touches. Only young cultures should be used (18-24 hours
old).
3. Carefully emulsify to obtain a homogeneous suspension with a turbidity equivalent to 0.5
McFarland.
NOTE : For accuracy, it is imperative that the inoculum density be adjusted to 0.5
McFarland. In particular, a weaker inoculum may lead to false negative results.

Inoculation of the strip

1. Remove the strip from its individual packaging and place
the strip in the incubation box.
2. With the micropipette, distribute the 50 μl of bacterial
suspension into the tubes of the strip. Note the following:
a. To avoid the formation of bubbles at the base of the
tubes, tilt the strip slightly forwards and place the tip
of the pipette against the side of the cupule.
b. The accuracy of the filling is very important. Tubes insufficiently filled or overfilled
may be the source of false positive and false negative results.
c. For the CIT test, 50 μl will fill the smaller tube and lower portion of the cupule.
3. For the underlined tests (LDC, ODC and URE), completely fill the cupule with mineral oil.
4. Place the lid on the box and incubate at 37°C for 4 - 4 ½ hours (place in fridge if not reading
at 4 hrs).
READING AND INTERPRETATION
Reading the strip
1. After incubation, read the strip by referring to the Reading Table.
2. FIRST, record all spontaneous reactions (before adding reagents) on the result sheet. For
the fermentation tests (sugar substrates), a green color indicates the onset of acidification
and should be considered as a positive reaction.
3. Reveal the VP and IND tests by adding the corresponding reagents :
- VP Test : add 1 drop of each of VP 1 and VP 2 reagents. Wait 5-10 minutes. A red
color indicates a positive reaction to be recorded on the result sheet.
- IND Test : add 1 drop of JAMES reagent. The reaction takes place immediately. A
pink color indicates a positive reaction to be recorded on the result sheet.
NOTE : Do not put the lids back on the strips during the reading of the results.
Interpretation
1. Identification is obtained with the numerical profile.
2. Determination of the numerical profile :
On the result sheet, the tests are separated into groups of 3 and a value 1, 2 or 4 is indicated
for each. By adding together the values corresponding to positive reactions within each
group, a 7-digit profile number is obtained. The oxidase reaction constitutes the 21st test and
has a value of 4 if it is positive.
3. Identification : This is performed using the database (V3.1) with the apiweb TM
identification software. Enter the 7-digit numerical profile manually via the keyboard.
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?

Fall 2011 - Jackie Reynolds, Richland College, BIOL 2421