**5.3.7 API method**

The API-20E test kit for the identification of enteric bacteria (bioMerieux, Inc., Hazelwood, MO) provides an easy way to inoculate and read tests relevant to members of the Family *Enterobacteriaceae* and associated organisms. A plastic strip holding twenty mini-test tubes is inoculated with a saline suspension of a pure culture (as per manufacturer's directions). This process also rehydrates the dessicated medium in each tube. A few tubes are completely filled (CIT, VP and GEL as seen in the photos below), and some tubes are overlaid with mineral oil such that anaerobic reactions can be carried out (ADH, LDC, ODC, H2S, URE).

After incubation in a humidity chamber for 18-24 hours at 37°C, the color reactions are read (some with the aid of added reagents), and the reactions (plus the oxidase reaction done separately) are converted to a seven-digit code which is called the Analytical Profile Index, from which name the initials "API" are derived. The code can be fed into the manufacturer's database via touch-tone telephone, and the computerized voice gives back the identification, usually as genus and species. An on-line database can also be accessed for the identification. The reliability of this system is very high, and one finds systems like these in heavy use in many food and clinical labs.

Note: Discussion and illustration of the API-20E system here does not necessarily constitute any commercial endorsement of this product. It is shown in our laboratory courses as a prime example of a convenient multi-purpose testing method one may encounter out there in the "real world."

In the following photos:

	- The amino acids tested are (in order) arginine, lysine and ornithine. **Decarboxylation** is shown by an **alkaline** reaction (red color of the particular pH indicator used).
	- The carbohydrates tested are glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin and arabinose. **Fermentation** is shown by an **acid** reaction (yellow color of indicator).

In the first set of reactions:


The API-20E test kit for the identification of enteric bacteria (bioMerieux, Inc., Hazelwood, MO) provides an easy way to inoculate and read tests relevant to members of the Family *Enterobacteriaceae* and associated organisms. A plastic strip holding twenty mini-test tubes is inoculated with a saline suspension of a pure culture (as per manufacturer's directions). This process also rehydrates the dessicated medium in each tube. A few tubes are completely filled (CIT, VP and GEL as seen in the photos below), and some tubes are overlaid with mineral oil such that anaerobic reactions can be carried out (ADH, LDC,

After incubation in a humidity chamber for 18-24 hours at 37°C, the color reactions are read (some with the aid of added reagents), and the reactions (plus the oxidase reaction done separately) are converted to a seven-digit code which is called the Analytical Profile Index, from which name the initials "API" are derived. The code can be fed into the manufacturer's database via touch-tone telephone, and the computerized voice gives back the identification, usually as genus and species. An on-line database can also be accessed for the identification. The reliability of this system is very high, and one finds systems like these in heavy use in

Note: Discussion and illustration of the API-20E system here does not necessarily constitute any commercial endorsement of this product. It is shown in our laboratory courses as a prime example of a convenient multi-purpose testing method one may encounter out there

Note especially the color reactions for **amino acid decarboxylations** (ADH through

Hydrogen sulfide production (H2S) and gelatin hydrolysis (GEL) result in a black color

 A positive reaction for tryptophan deaminase (TDA) gives a deep brown color with the addition of ferric chloride; positive results for this test correlate with positive phenylalanine and lysine deaminase reactions which are characteristic of *Proteus*,

 Culture "5B" (isolated from an early stage of sauerkraut fermentation) is identified as *Enterobacter agglomerans* which has been a convenient dumping ground for organisms now being reassigned to better-defined genera and species including the new genus *Pantoea*. This particular isolate produces reddish (lactose +), "pimply" colonies on MacConkey Agar which exude an extremely viscous slime as may be seen here; this appearance is certainly

 Culture "8P44" is identified as *Edwardsiella hoshinae*. The CDC had identified this culture (in 1988) as the ultra-rare Biogroup 1 of *Edwardsiella tarda* which may not be in the API-20E database (*Figure 15)*. This system probably would not be able to differentiate between these two organisms. Note that 8P44 shows H2S production

atypical of organisms identified as *E. agglomerans* or *Pantoea* in general.

 The amino acids tested are (in order) arginine, lysine and ornithine. **Decarboxylation** is shown by an **alkaline** reaction (red color of the particular pH indicator used). The carbohydrates tested are glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin and arabinose. **Fermentation** is shown by an **acid**

ODC) and **carbohydrate fermentations** (GLU through ARA).

reaction (yellow color of indicator).

**5.3.7 API method** 

ODC, H2S, URE).

many food and clinical labs.

throughout the tube.

In the first set of reactions:

*Morganella* and *Providencia*.

in the "real world." In the following photos: which is probably typical of *Edwardsiella tarda* Biogroup 1. Clinical laboratories usually run this test in Triple Sugar Iron Agar in which the organism's fermentation of sucrose (with consequent high acid production) tends to negate the H2S reaction, and – as a result – the organism is mis-characterized throughout the literature as H2S negative even though it shows a positive reaction in KIA and other H2S-detecting media.

Fig. 15. API Test Result for *Salmonella* 
