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API Rapid 20E Identification Guide

The document provides instructions for using the API RAPID 20E system to identify Enterobacteriaceae bacteria. [1] The system uses 20 miniaturized biochemical tests in microtubes to identify bacteria in 4 hours. [2] Proper preparation of the bacterial suspension with a turbidity of 0.5 McFarland is important for accurate results. [3] The strip is inoculated and incubated at 37°C for 4-4.5 hours before reading color changes to determine the identification.

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GUIDO ERNESTO VILLOTA CALVACHI
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363 views3 pages

API Rapid 20E Identification Guide

The document provides instructions for using the API RAPID 20E system to identify Enterobacteriaceae bacteria. [1] The system uses 20 miniaturized biochemical tests in microtubes to identify bacteria in 4 hours. [2] Proper preparation of the bacterial suspension with a turbidity of 0.5 McFarland is important for accurate results. [3] The strip is inoculated and incubated at 37°C for 4-4.5 hours before reading color changes to determine the identification.

Uploaded by

GUIDO ERNESTO VILLOTA CALVACHI
Copyright
© All Rights Reserved
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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

Common questions

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The API RAPID 20E system utilizes software to enhance bacterial identification accuracy by employing the apiweb identification software (V3.1) that interprets the 7-digit profile number generated from the test results. This software provides a comprehensive and reliable identification by referencing a detailed database, thus minimizing human error and increasing diagnostic precision .

The results of oxidase and gram-stain tests are critical for using the API RAPID 20E system because they determine the eligibility of the bacterial samples used in the test. The organism should be oxidase negative and a gram-negative bacillus, as the system is specifically designed for identifying Enterobacteriaceae. These preliminary tests ensure the correct identification of the target bacterial group and prevent the inclusion of non-relevant specimens .

When filling the microtubes of the API RAPID 20E strip, care must be taken to avoid bubbles, ensuring full but not excessive filling, especially for the CIT test which requires precise filling of the smaller tube and lower portion of the cupule. Inadequate or excessive filling can lead to false positive or negative results. Accurate distribution is crucial for maintaining the integrity of the results .

In the API RAPID 20E system, mineral oil is used to maintain the integrity of certain biochemical tests, specifically those involving the LDC, ODC, and URE tests. For these underlined tests, the cupule must be completely filled with mineral oil to create an anaerobic environment, which is necessary for the accurate functioning and interpretation of these specific biochemical reactions .

The preparation of the inoculum for the API RAPID 20E test involves several components and steps. First, a tube of 0.85% physiological saline is required to make the bacterial suspension. Using an inoculation loop, 1-4 well-isolated colonies of identical morphology are picked from the agar plate. These colonies must come from young cultures that are 18-24 hours old. The colonies are then carefully emulsified to form a homogeneous suspension with a turbidity equivalent to 0.5 McFarland. It is imperative that the inoculum density is accurately adjusted to 0.5 McFarland to avoid false negative results .

In the API RAPID 20E system, the use of a 7-digit profile number enhances accuracy by standardizing the identification process through precise categorization of results. Each test group is assigned a numerical value (1, 2, or 4), and positive reactions are summed within each group, resulting in a unique 7-digit number. This numerical profile is then used to reference a database (V3.1) via software, allowing for an accurate and reliable identification of Enterobacteriaceae .

The API RAPID 20E system leverages color change as a mechanism for identifying bacterial metabolism by utilizing biochemical tests that generate color changes as a result of metabolic activity. During incubation, the metabolism of the bacterial suspension causes spontaneous color changes or those revealed by the addition of reagents. For example, positive acidification in fermentation tests is indicated by a green color, and other reactions are visually interpreted for identification purposes using the Analytical Profile Index or via software .

Potential errors from incorrect inoculum density in the API RAPID 20E system include false negative results. If the inoculum is too weak, the biochemical reactions may not produce sufficient color change, leading to inaccurate results. Therefore, ensuring the inoculum density is at 0.5 McFarland is necessary to prevent discrepancies in the identification process .

The API RAPID 20E system compares favorably to traditional biochemical tube media in terms of efficiency and ease of interpretation due to its miniaturized and standardized approach. The system enables rapid identification of Enterobacteriaceae within four hours, unlike traditional methods which are more time-consuming. Interpretation is streamlined using a numerical profile, reducing complexity and increasing accuracy. This improves both speed and reliability in microbial diagnostics .

It is important not to put the lids back on the strips during the reading of API RAPID 20E results to allow maximum visibility of the color changes and to prevent any condensation that might obstruct the reading of results. This could potentially impact the accuracy of identifying positive reactions based on color change .

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