1. Specimen
1.1. Depends on the type of infection. (Pus, joint aspirate, blood, sputum, stool. Urine ).
2. Inspection
2.1. Macroscopy : size, shape..
2.2. Microscopy : Smear prepration
2.2.1. loop full of distilled water (in solid media) pick a colony and mix drying Fix
2.2.1.1. Gram staining: Fix by methanol Crystal violet (primary stain) Gram’s iodine (mordant) Acetone-alcohol (decolorizing agent) Safranin (counter stain)
3. Culture
3.1. Based on their consistency a) solid medium:– contains 2% agar Colony morphology, pigmentation, hemolysis can be appreciated. Eg: Nutrient agar, Blood agar b) liquid medium:– no agar. For inoculum preparation, Blood culture, for the isolation of pathogens from a mixture. Eg: Nutrient broth c) semi solid medium:– 0.5% agar. Eg: Motility medium
3.2. Based on the constituents/ ingredients a) simple medium b) complex medium c) synthetic or defined medium d) Special media:-
3.2.1. Enriched media: Substances like blood, serum, egg are added to the basal medium. Used to grow bacteria that are exacting in their nutritional needs. Eg: Blood agar, Chocolate agar
3.2.2. Enrichment media: Liquid media used to isolate pathogens from a mixed culture. Media is incorporated with inhibitory substances to suppress the unwanted organism.
3.2.3. Selective media: The inhibitory substance is added to a solid media. Eg: Mac Conkey’s medium for gram negative bacteria
3.2.4. Indicator media: These media contain an indicator which changes its colour when a bacterium grows in them.
3.2.5. Differential media: A media which has substances incorporated in it enabling it to distinguish between bacteria. Eg: Mac Conkey’s medium Distinguish between lactose fermenters(pink) & non lactose fermenters.
3.2.6. Sugar media
3.2.7. Transport media
3.2.8. Media for biochemical reactions
3.3. Based on Oxygen requirement - Aerobic media - Anaerobic media: use Anaerobic
3.4. Capnophiles require high CO2: use -Candle jar - CO2-packet
4. Identification
4.1. Biochemaicl test
5. Isolation
5.1. Streak plate technique: streaking.
5.1.1. pure colonies
5.2. Slant inoculation technique: dip & zig zag.
5.3. Liquid medium inoculation technique: scrath well
5.4. Semisolid medium inoculation technique: dip
5.4.1. Motile bacteria and NON-motile bacteria