Differential stains and media can provide preliminary identification of microbes, but to identify Gram- bacteria to the species, a more sophisticated test is required.
* Bacterial Identification *
Differential staining of bacteria can provide preliminary information when a medical technician is attempting to identify an unknown type of infection-causing bacteria. Basic information, such as whether an organism is Gram positive or Gram negative, is used to decide which diagnostic test will be employed to determine the genus and species of the bacterium in question.
* The API 20E by bioMerieux *
If the microbe is a Gram negative rod shaped (bacillus) bacterium, one of the diagnostic tests most frequently employed is the API 20E test strip manufactured by bioMerieux, Inc., a biotech company that produces a variety of different test strips for identification of yeast, Staph, and anaerobic bacteria, to name just a few.
The bioMerieux API 20E system consists of a plastic strip of 20 individual, miniaturized tests tubes (cupules) each containing a different reagent used to determine the metabolic capabilities, and, ultimately, the genus and species of enteric bacteria in the family Enterobacteraceae.
* How the API 20E System Works *
* Loading the Cupules: Each cupule is inoculated with a saline suspension of a pure bacterial culture, rehydrating the dried reagent in each tube. Some of the tubes are to be completely filled (tests CIT, VP and GEL), whereas others are topped off with mineral oil so that that anaerobic reactions (reactions that occur in the absence of oxygen) can be carried out (tests ADH, LDC, ODC, H2S, URE).
* Incubating the API 20E: The strip is then incubated in a small, plastic humidity chamber for 18-24 hours at 37C. Living bacteria produce metabolites and wastes as part of the business of being a functioning cell. The reagents in the cupules are specifically designed to test for the presence of products of bacterial metabolism specific to certain kinds of bacteria.
* Reading API 20E Results: After incubation, each tube (an individual test) is assessed for a specific color change indicating the presence of a metabolic reaction that sheds light on the microbes identity. Some of the cupule contents change color due to pH differences, others contain end products that have to be identified using additional reagents.
* The API 20E Secret Code*
Interpretation of the 20 reactions, in addition to the oxidase reaction (which is done separately), is converted to a seven-digit code, a process that seems much like decoding a message with a super secret spy decoder ring. The technician can then look up the code in a huge manual that has the names of bacterial species associated with each seven-digit string of numbers.
* Sources *
Willey, J., Sherwood, L. and Woolverton, C. (2008) Prescott, Harley & Klein’s Microbiology 7th Edition. McGraw Hill.