The following points highlight the seven steps for identification of bacteria isolated from a specimen. The steps are: 1. Morphology and Staining 2. Cultural Characteristics 3. Biochemical Reactions 4. Antigenic Characters 5. Typing of Bacteria: Bacteriophage Sensitivity 6. Animal Pathogen City 7. Antibiotic Sensitivity.
Identification of Bacteria: Step # 1. Morphology and Staining:
Serve as preliminary criteria. The Gram stained smear shows the Gram reaction, size, shape, groupings of the bacteria and intracellular position of the endospore. Special staining reaction can reveal the presence of capsule.
Hanging drop wet preparation can be used to study the motility of bacteria. An unstained wet film is examined under dark ground illumination microscope to observe the exact morphology of delicate spirochaete. A smear is stained by Ziehl Neelsen method to demonstrate the acid fast staining reaction.
Identification of Bacteria: Step # 2. Cultural Characteristics:
The growth requirement and the appearance of colonies on media to the naked eye are further criteria to assist the identification of bacteria. A culture is a growth of bacterium on artificial nutrient medium or culture medium prepared in the laboratory.
Attempts are made to grow (to cultivate or culture) the bacteria on media of different compositions (glucose, inorganic salts mixture, meat extract or meat infusion with blood) incubated under a variety of conditions (different temperatures, pH) in the presence of atmospheric oxygen (aerobically).
The ability or inability to grow on medium containing a selective inhibitor (e.g. bile salt, optochin, tellurite, bacitracin, malachite green, low pH, high pH) may also be useful to identify the bacteria.
The growth of bacteria in liquid culture medium (e.g. nutrient broth) may show:
(1) A uniform turbidity;
(2) Little deposit at the bottom;
(3) Surface growth (pellicle formation). (Fig. 7.1a).
The appearance of the discrete masses of growth or colonies that can be grown from isolated bacteria on the surface of the solid medium (nutrient agar) can be used to study the size of the colonies (diameter in mm), their outline (whether circular, entire, indented or wavy or rhizoid), their elevation (low convex, high convex, flat, plateau-like, umbonate, or nodular)—Fig. 7.1b), their transparency (clear and transparent) or opaque, whether they are colourless (white or pigmented) or whether they produce any change in the medium (e.g. haemolysis in the blood agar medium).
Identification of Bacteria: Step # 3. Biochemical Reactions:
E.g. fermentation of various sugars (carbohydrates). Morphology and cultural characters may not be able to distinguish some species of bacteria; but these same species may exhibit distinct differences in their biochemical reactions e.g. typhoid and paratyphoid bacilli (glucose and mannitol are fermented without gas production by typhoid bacilli, whereas paratyphoid bacilli produce acid and gas).
Certain serotypes of the salmonella group may resemble one another in fermentation properties.
The growth of the bacteria in liquid medium will ferment particular sugars (glucose, lactose, mannitol) with the production of acid, which is detected by the changes of colour of Andrade’s indicator dye incorporated in the medium; the gas production is detected by the collection of air bubble in a small inverted tube (Durham’s tube) immersed in the medium.
Other tests are used to find out the ability of a bacterium to produce particular end products e.g Indole, hydrogen sulphide, nitrite and certain enzymes (oxidase, catalase, urease, gelatinase, collagenase, lecithinase, lipase) in culture media.
Identification of Bacteria: Step # 4. Antigenic Characters:
Species or types of bacteria can be easily and distinctly identified by “specific” antibody reactions observed in serological tests performed on a glass slide. This specific antibody (antiserum) is obtained from the animal (rabbit) immunized against a particular type of microorganisms which agglutinates with the same antiserum.
An unknown bacterium may thus be identified by demonstrating its reaction with one out of a number of standard known antisera.
Similarly, the serum of a person suffering from a bacterial infection may contain specific antibody. The nature of the infection may thus be diagnosed by demonstrating that the patient’s serum agglutinates one out of a number of known antigens of laboratory cultures, e.g. Widal test in Typhoid fever.
Identification of Bacteria: Step # 5. Typing of Bacteria: Bacteriophage Sensitivity:
A single bacterial pathogenic species may include different types of strains which are distinguishable in minor characters. Recognition of the type of a strain isolated from a patient may be of great importance in epidemiological studies related to the source and the spread of the infection in the community.
The typing of stains may be done by special biochemical or serological tests. Another important method of typing is by testing the susceptibility of the culture to lysis by each of a set of type specific, lytic bacteriophages.
Identification of Bacteria: Step # 6. Animal Pathogen City:
Final identification of a toxigenic strain of tetanus bacillus may be done by injecting the toxin liberated by tetanus bacillus into the base of the tail of two mice, one of them has already been protected by prior injection of specific antiserum to tetanus toxin (a soluble poisonous protein secreted by the tetanus bacilli).
The unprotected mouse shows the symptoms of tetanus, whereas the protected one without any tetanus symptoms identifies the culture, as an organism producing toxin, as the injected antiserum neutralized toxin liberated by tetanus bacilli. Similarly, diphtheria bacillus is also identified by inflammation and necrosis of the skin of guinea pig brought by diphtheria exotoxin.
Identification of Bacteria: Step # 7. Antibiotic Sensitivity:
The organism is tested for its ability to grow on artificial nutrient media containing different antibiotics and chemotherapeutic agents in different concentrations. In disk diffusion test, the culture to be examined is inoculated confluently with swabs over the surface of an agar plate and six to ten paper disks containing different antibiotics are placed in different areas of the plate.
Antibiotic diffuses outwards from each disk into surrounding agar. On incubation, the bacteria grow on areas of the plate except those around the antibiotic disks to which they are sensitive. The width of each growth-free “zone of inhibition” is a measure of the degree of the sensitivity of the drug.
Information about the sensitivity patterns of strains (anti-bio-grams) isolated from the patient is required as a guide to the drug of choice for therapy and may also be used as an epidemiological marker in tracking hospital cross-infection.