Commercial Diagnostic Kits

Around the beginning of 1970s with the advancement of miniaturized microbiological techniques and concepts de-

Gram Positive Egg Shaped Cell

Figure 1. Morphology of Clostridium perfringens under 1,000X light-phase-contrast microscopy. This is a gram-positive, rod-shaped, anaerobic, spore-forming bacterium that causes large numbers of foodborne outbreaks and cases in the world. The dark rod-shaped cells are vegetative bacteria, and the bright refractile oval objects are spores developed in the vegetative cells.

Figure 1. Morphology of Clostridium perfringens under 1,000X light-phase-contrast microscopy. This is a gram-positive, rod-shaped, anaerobic, spore-forming bacterium that causes large numbers of foodborne outbreaks and cases in the world. The dark rod-shaped cells are vegetative bacteria, and the bright refractile oval objects are spores developed in the vegetative cells.

Isotricha Prostoma

Figure 2. Morphology of Isotricha prostoma, under 100X light-phase-contrast microscopy. This is an anaerobic protozoan an important member of the ruminal flora in cattle. Source: Courtesy of T. G. Nagaraja of Kansas State University.

veloped by Fung and colleagues and other scientists, many diagnostic kits appeared on the market mainly for clinical microbiology. These methods gradually found their ways into food microbiology laboratories and sometime into industrial and environmental laboratories. Currently commercially successful diagnostic kits are marketed toward clinical settings. This may change if the need arises in the near and far future. There are two major types of diagnostic kits. One type depends on growth of the pure culture and biochemical reactions obtained in the media. This type of test is typically slow, requiring overnight incubation and anaerobic incubation of cultures. Another type is by monitoring enzyme systems of the pure culture. In this system, anaerobiosis is not necessary and the results can be obtained in about 4 hours. Kelly and Schipp (49) made a detailed analysis of the approaches and designs of anaerobic diagnostic kits, including discussions on substrate concentration, organism concentration, pH of reaction, buffering capacity, additives, incubation time, and growth medium for the identification of anaerobes. In that article, important variables affecting system performance and issues concerning comparative analysis of data reported in journal articles were also studied. To compare data reported in the literature, one needs to address the issues such as source of strains, database version, the "gold standard," number of species/strains tested, need of supplementary tests, and accuracy. In general, a diagnostic kit providing 90 to 95% accuracy compared with the conventional method is considered good. Any system with less than 85% agreement with the conventional method is considered unacceptable.

The major growth-dependent commercial kits for anaerobes are API 20A, marketed by bioMerieux Vitek Inc., Hazelwood, Mo., and Minitek Anaerobe system, marketed by BBL Microbiology Systems, Cockeysville, Md. The API 20A kit consists of 20 microtubes with dehydrated sub strates. A liquid suspension of a pure culture is made and then introduced into each microtube. The kit is then incubated anaerobically, and after 1 to 2 days the reactions are read and data recorded in a convenient chart. The results are converted into a number system. From a code book the identity of the culture can be ascertained. These types of results provide a statistical probability of the unknown culture by comparing the attributes in the computerized database. Thus, an organism may be identified as C. perfringens at 0.9500 level. Sometimes the printout may include requests for additional tests, and in some cases the organism may be given two identifications with probability of each listed. This general format of identification is used by all commercial diagnostic kits.

Minitek Anaerobe II uses paper disks saturated with substrates. Thirty-five disks are available. After a suspension of the pure culture is made, the liquid is dispensed into wells of the units containing the appropriate disks. The assemblage is incubated anaerobically for 2 days. Reactions are read by color changes and recorded. The identity of the unknown can be obtained by following the procedure outlined for the API system.

Use of enzyme test systems include Rapid ID 32A (bioMerieux), AN-Ident (bioMerieux), RapID ANA II (IDS Inc., Norcross, Ga.), Vitek ANI Card (bioMerieux), and MicroScan Rapid Anaerobe Panel (Baxter Diagnostic, Sacramento, Calif.). All these methods are incubated aerobi-cally because enzyme activities will not be influenced by the presence of air. Rapid ID 32A and RapID ANA II are similar in that the cultures are made into a liquid suspension, and the liquid is inoculated into microwells. After 4 hours, the color reactions are read and the identities are ascertained from a code book. Vitek ANI Card involves injection of liquid sample into a plastic card that is the size of a credit card with 30 wells. After 4 hours of incubation, the results are read and data are entered into the ANI program on the VITEK computer. Identification is made by the computer. In the MicroScan Rapid Anaerobe Panel system, the culture is inoculated into 24 substrates and two color interpretation controls. After 4 hours of aerobic incubation and addition of reagents, the panels are read either manually or with the aid of a Touchscan light box. Data are then interpreted by computer for identification.

It should be emphasized that the aforementioned systems are designed for clinical bacteriology. Identifications of unknowns from environmental, food, and industrial sources may not be as accurate because the databases of these systems are from clinical isolates.

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