Isolation and Identification of Bacteria
Dr. Sudheer Kher
Culture Methods Isolate bacteria in pure cultures. Demonstrate their properties. Obtain sufficient growth for preparation of antigens & for other tests. Typing bacterial isolates. Antibiotic sensitivity. Estimate viable counts. Maintain stock cultures.
Types of culture methods Streak culture or surface plating Lawn or carpet culture Stroke culture Stab culture Pour plate method Sweep plate method
Streak Culture Routinely employed for isolation Platinum / Nichrome loops
Identification methods Microscopy – Morphology & staining reactions Cultural characteristics Biochemical reactions Antibiotic sensitivity Serology Molecular methods like PCR Bacterial typing
Microscopy - Morphology Shape – spherical, rod shaped, comma shaped or spiral shaped Size – 0.2 to 1.5 in diameter & about 3-5 in length.
Microscopy - Morphology Arrangement – pairs, tetrads, groups, chains, chinese letter pattern. Motility – wet/ hanging drop preparation
Microscopy - Morphology Spores – shape & position
Capsules
Staining reactions Differential & special stains. Gram’s stain
Staining reactions Ziehl-Neelsen stain
Fluorescent staining - Mycobacteria
Staining reactions Special stains. Albert’s stain
Cultural characteristics On solid media Shape – circular, irregular Size in mm Elevation – elevated, convex, concave, umbonate or umbilicate
Cultural characteristics On solid media Edges – entire, crenate, fimbriate
Cultural characteristics On solid media Surface – smooth, wavy, rough, glistening Colour
Cultural characteristics
On solid media Structure – opaque, translucent or transparent Consistency – membranous, friable Emulsifiability
Cultural characteristics On solid media – Hemolysis on blood agar (BA)
Cultural characteristics In stroke culture Degree of growth – scanty, moderate or profuse. Nature – discrete or confluent, spreading Elevation, surface, edges, colour, odour, consistency & changes in the medium. Broth culture Degree of growth, turbidity, deposit, pellicle, colour, odour.
Resistance Resistance to heat & to disinfectants. E.fecalis resistant to heat at 60°C for half an hour. Clostridial spores resistant to boiling.
Biochemical properties
1. Sugar fermentation – acid & gas production 2. Hydrogen sulphide production
Biochemical properties Indole production
Citrate utilisation
Biochemical properties Methyl red (MR) test
Voges-Proskauer Test (VP)
Biochemical properties Urease test
Nitrate reduction
Biochemical properties
Serology Specific serological reactions can help in the identification of species. Employed to detect the presence of antibodies in the serum that can react only with specific micro-organisms. e.g. Widal test - Typhoid
Molecular Methods Polymerase Chain Reaction (PCR) Detection of nucleotide sequences Help in rapid detection Can also detect bacteria which cannot be grown or are slow growing detect as few as 2 to 10 bacteria per ml of specimen
Typing of Bacteria Bacteriophage typing – bacteria show differential susceptibility to lysis by bacteriophages - widely used to discriminate among the strains of salmonellae & staphylococci. Bacteriocin typing – naturally occurring antibacterial substances, produced by most bacterial species, that are active mainly against strains of the same genus as the producer strain
Rapid Identification Tests – Automated methods API – Analytical Profile Index
