There are millions upon millions of bacteria in our world. The microbiologist searching for in any particular bacterium uses a number of techniques to distinguish one species from another.
The starting point for most bacteriologists is the gram stain. This simple differential staining technique involves four steps. A heat fixed smear of the bacteria in question is first stained with a solution of methyl violet followed by Lugols iodine, which acts as a mordant fixing the stain in gram-positive bacteria. A quick rinse in acetone removes the stain from any background material and all gram-negative bacteria. The final step is a red counterstain such as safranin. The whole process only takes a few minutes.
This is not the only differential stain used by bacteriologists. The Ziehl-Nielsen stain detects and distinguishes acid-fast bacilli such as the Mycobacteria causing tuberculosis and leprosy from other bacteria such as E. coli. The Albert stain allows for the differentiation of diphtheria causing Corynebacteria species from the microscopically similar but normally harmless diptheroids found on everyone’s skin. Corynebacteria contain metachromatic granules and these show up in an Albert stained slide.
To study bacteria it helps to be able to grow them as isolated colonies. The use of agar allows a nutrient gel suitable for the growth of many bacteria to be made. When looking for pathogenic bacteria additions to this media allow for a rapid screen of pathogens from non-pathogens.
Horse blood forms a common additive to nutrient agar. The Beta hemolytic streptococci produce a clear zone of hemolysis around the bacterial colony allowing an easy detection of these from amongst any other organisms present.
Many bacteria ferment different sugars producing acid in the process. By the incorporation of a suitable sugar and a colored pH indicator into a suitable agar a differential media for such bacteria is produced. The commonest sugar use for this is lactose this is use to separate the non-lactose fermenting Salmonellas and Shigellas causing gastroenteritis from the Lactose fermenters such as E. coli commonly found in fecal samples. Media incorporating lactose includes MacConkey CLED and XLD. E. coli O157 a cause of food poisoning and hemolytic uremic syndrome does not ferment sorbitol whereas other E. coli strains do ferment this sugar. A special formulation of MacConkey agar with sorbitol replacing lactose allows for easy identification of potential E. coli O157 strains.
Once a bacteriologist has found a potential pathogen full identification requires further testing. There are a number of methods for this some requiring the inoculation of numerous tubes containing different sugars for fermentation while others allow for automation. The commonest method used in many microbiology laboratories is the Bio-Merieux API system. The API 20E allows a simple identification of most coliform type bacteria such as E. coli, Proteus species, Salmonella and many others.
The API 20E system consists of 20 small plastic cupules on a plastic coated strip. Each cupule contains the dehydrated requirements for either a sugar fermentation or a biochemical test. The bacterium under investigation is inoculated into either sterile distilled water or isotonic saline and the cupules filled with the aid of a sterile pipette. Some of the cupules are half filled and others completely according to the manufacturer’s instructions. Some tests, such as amino acid decarboxylation tests, require anaerobic conditions so liquid paraffin is used to cover those cupules.
After 18 to 24 hours incubation at 37°C the strip is examined. Some tests require the addition of further reagents to specific cupules. The tests are grouped into groups of three. If the first test in the group is positive is scores as one the second as two and the third as four. In this way, a seven-figure number is produced with no digit larger than seven. This number is then put into the API database to obtain an identification.
The API database is available as a computer program, in book form or over the phone using a keypad phone. The database gives the most likely identification of the bacteria along with the percent probability that it is that bacterial species. The API system provides a convenient method of identifying many common pathogenic and some non-pathogenic bacteria. From personal knowledge, I can tell you it is very much quicker than inoculating individual sugar tubes.
Dept. of Bacteriology University of Wisconsin-Madison