After isolation of bacteria from a specimen, it has to be identified; the following studies are necessary to characterize a bacterial strain.
Smears prepared from the bacterial colony or liquid culture is examined by staining methods. Gram’s staining divides bacteria into Gram-positive and Gram-negative; Albert’s staining shows morphological characteristics of diphtheria bacilli and Ziehl-Neelsen staining differentiates acid-fast and non acid-fast bacilli. A motility preparation in normal saline is helpful in distinguishing motile from non-motile ones.
The appearance of bacterial colony is usually characteristic:
- Color: Pigment and hemolysin cause change in color.
- Shape: Circular, irregular or radiate.
- Surfaces: Smooth, wavy, rough, granular.
- Size: In millimeters.
- Elevation: Flat, elevated, low convex, convex, umbonate.
- Edges: Entire, undulate, crenated, lobate, ciliate.
- Opacity: Translucent, transparent or opaque.
- Consistency: Mucoid, firm, friable, membranous, butyrous.
Growth in liquid media:
In peptone water, nutrient broth and Hiss’s serum, bacterial growth appears in following forms,
- Deposit formation.
- Surface pellicle formation.
Aerobic or anaerobic growth; oxygen and carbon dioxide requirements; production of pigment, toxins and hemolysins all these properties are helpful for identification and classification.
Bacterial species differ in their capacity to attack different carbohydrates, proteins and fats. Most of the biochemical reactions are based on:
- Presence of specific enzymes in bacterial cultures such as coagulase, oxidase, urease, gelatinase, lecithinase, catalase and others.
- Production of metabolic end products of some compounds like sugar present in the culture media, which are the outcome of enzymatic action of bacteria.
Biochemical tests for identification for bacteria:
|Tests for metabolism of||Substrate||Observation|
|Break down of sugars
a. Oxidative (aerobic)
|Glucose, Sucrose,Mannitol, Lactose||Acid production sometimeswith gas.|
|Voges-Proskaur (VP)||Glucose(Glucose phosphate broth)||Acetyl methyl carbinol|
|Methyl red (MR)||Glucose(Glucose phosphate broth)||Sufficient acid (<pH 4.5)|
|Citrate utilization||Sodium citrate||Growth(Sole carbon source)|
|Sulphur containingamino acids(TSI agar)||Hydrogen sulphide|
|Phenylalanine deaminase||Phenylalanine||Phenylpyruvic acid (PPA)|
|Arginine, lysine,Ornithine||Carbon dioxide|
|Tests for detection of Enzymes|
|Nitrate reduction||Potassium nitrate||Nitrite|
|Urease||Urea (Urea broth)||Ammonia|
ANTIGENIC STRUCTURE (SEROTYPING):
The final identification of an organism is made by agglutination or precipitation reactions. Unknown antigen is detected by known specific antibody (high titer serum) first by slide test and confirmed by tube test.
TYPING BACTERIAL STRAIN:
This is of epidemiological value
- Antigenic typing to distinguish different strains of bacteria.
- Bacteriophage typing for intraspecies typing of some bacteria.
- Bacteriocin typing.
Guinea pigs, rabbits and mice are mostly used for animal inoculation tests, which are of great value in anthrax, tuberculosis, diphtheria, plague, gas gangrene, and tetanus. The animals may be inoculated by subcutaneous, intramuscular, intravenous, intraperitoneal or intracerebral routes. Oral route or nasal spray may also be used.
USE OF ANIMALS IN MICROBIOLOGY
Animals have following uses in Microbiology laboratory,
- Isolation and identification of bacteria
- Preparation of immune and diagnostic sera.
- Differentiation between human and bovine types of tubercle bacilli. The animal is susceptible to the bovine variety of tubercle bacilli.
- For small-scale production of diagnostic antisera.
- Guinea pig:
- For isolation of tubercle bacilli, the animal is susceptible to both human and bovine tubercle bacilli
- For virulence test for the diphtheria and tetanus bacilli.
- For obtaining complement for complement-fixation test
- For investigation of rickettsial diseases like typhus fever.
- It may be used for distinguishing P. pseudotuberculosis from Y. pestis; the animal is susceptible to Y. pestis.
- For isolation of Pneumococci from infective material, the animal is very susceptible to Pneumococci.
- For isolating causative agent of relapsing fever and trypanosomiasis.
- For investigation of viruses of influenzae and Chlamydiae.
- RBCs of sheep are used in a wide variety of tests like the complement fixation test and Paul Bunnel test for infectious mononucleosis.
- Sheep blood is used for preparation of blood agar.
- Kidney tissue from monkey is a common source for cell culture used for primary isolation of viruses.
- The animal is used for vaccine trials.
- The serum is used for preparation of certain culture media.
- The animal is used for the production of antisera.
- The nine banded armadillo (Dasypus novemninctus) is highly susceptible to infection with lepra bacilli