Culture Technique - CSIR-NET/ICMR/DBT (Life Sciences)

 Culture Technique- Microbiology

I. Culture Techniques

• Microbiology as a science is very dependent on several specialized laboratory techniques. Laboratory steps routinely employed in microbiology are- Inoculation, Incubation, Isolation, Inspection, and Identification.

• To prepare a culture (visible growth specimen), a medium (nutrient substrate) is inoculated (implanted, seeded) with a sample.

• Methods of Isolation: Cells are spread over a large area (streak plate and spread plate) or are diluted in a large volume (pour plate) so that individuals cells are completely separated and can grow into colonies.

• The cultures are incubated, subcultured, observed microscopically, and identified by the morphological, physiological, genetic methods.

                                                   

II. Artificial Media: 

Artificial nutrient media vary according to their physical form, chemical characteristics, and purpose.

A. Physical Subgroups:

• They can be liquid (broth, milk), semisolid, or solid, depending upon the absence or the quantity of solidifying agent (usually agar and gelatin).

B. Chemical Subgroups:

• A synthetic medium is any preparation that is chemically defined, but a medium containing a poorly identified component is nonsynthetic.

C. Functional Subgroups:

1. A large general-purpose medium:  It is used to grow a wide assortment of microbial types. Example: Tryptic soy broth (TSB).

2. Enriched-medium: It is supplemented with blood or tissue infusion to culture fastidious species. Example: Sheep-blood agar, chocolate (heated blood) agar.

3. Selective-medium: It permits preferential growth of certain organisms in a mixture and inhibits others. Highly selective media are used expressly to favor the growth of one organism over another. 

Example: Mueller telluride, Enterococcus faecalis broth, Phenylethanol agar, Tomato juice agar, Salmonella/Shigella (SS) agar, Lowenstein-Jensen. 

• Sabouraud's dextrose agar = pH of 5.6  discourages bacterial growth. Used to isolate fungi.
• Brilliant green agar = Green dye selectively inhibits gram-positive bacteria. Used to isolate gram-negative Salmonella.
• Bismuth sulfite agar = Used to isolate Salmonella typhi. Inhibits the growth of most other bacteria.
• MacConkey agar = used to distinguish and select for Salmonella.

4. Differential medium: It distinguished among different microbes by bringing out their variations in a particular reaction. 

Example: Blood agar, Eosin methylene blue (EMB) agar, Spirit blue agar, Urea broth, Surfur indole motility (SIM), Triple-sugar iron agar (TSIA), XLD agar.

• Mannitol salt agar = used to distinguish and select for Staphylococcus aureus.

5. Reducing medium: It limits Oxygen availability and is useful for cultivating anaerobes.

6. Carbohydrate Fermentation medium: The ability to utilize sugars can be determined with a carbohydrate fermentation medium.

7. Transport medium: To covey fragile microbes, special transport media are needed to stabilize viability.

8. Assay medium: It is used to evaluate the effectiveness of antimicrobial agents.

9. Enumeration medium: Environmental and Industrial surveillance routinely call for enumeration media to determine the number of microbes in food, drinking water, soil, sewage, and other resources.

10. In certain instances, microorganisms have to be grown in animals and bird embryos.