Nutrient stabs and slants preparations

Aim: Subculturing of bacteria and fungus. Nutrient stabs and slants preparations.

Requirement:

  • Chemicals: Nutrient Broth, Nutrient agar, Sabouraud dextrose broth, agar- agar powder.
  • Microorganism: Any bacterial and fungul broth culture
  • Apparatus: 500 ml conical flask, Cotton plug, Stirrer, big size test tubes, weighing balance.

Instrument: Autoclave, Incubator

PROCEDURE:

1] Clean the glassware like a conical flask, Petri dish, test tubes with running water, dry it, wrap it in brown paper and sterilize it in a hot air oven at 100°C for 30min.

2] Weigh the required amount of Nutrient broth (Fluid media for bacterial growth) and Sabouraud dextrose broth (Fluid Media for fungal growth) in the different flasks and dissolve it in water. Heat if necessary to dissolve the media (Precautions must be taken that media should not burn).

3] Weigh the required amount of Nutrient Agar (Solid media for bacterial growth) and Sabouraud dextrose broth and Agar-agar powder (Solid Media for fungal growth), dissolve it in water. Heat if necessary to dissolve the media (Precautions must be taken that media should not burn).

4] Plug the mouth of all flasks using an airtight cotton plug.

5] Sterilize all media by autoclaving at Temperature:121°C, Pressure: 15 lbs, Time: 15 min.

6] Slant culture: Aseptically transferred the sterilized Nutrient agar and Sabouraud dextrose agar media in separate Test tubes (About 10mL each) and store the test tubes in a slanted position to solidify the media. After solidification of agar, aseptically transfer one loop full of microorganism from broth culture and inoculate on slant agar (Bacteria on Nutrient agar slant and Fungi on Sabouraud dextrose agar) using inoculating loop in zig-zag streaking manner.

7] Stab culture: Aseptically transferred the sterilized Nutrient agar and Sabouraud dextrose agar media in separate Test tubes (About 15mL each) and keep the test tubes in the vertically standing position to solidify the media. After solidification of agar, aseptically transfer one loop full of microorganism from broth culture and inoculate in stab agar (Bacteria on Nutrient agar slant and Fungi on Sabouraud dextrose agar) using inoculating loop by dipping method.

8] Nutrient broth: Aseptically transferred the sterilized Nutrient broth and Sabouraud dextrose broth in Test tubes (About 15mL) and keep in a test tube stand in vertical position. After reducing the temperature of broth, aseptically transfer one loop full of microorganisms from broth culture and inoculate in broth by dipping method.

9] Incubate bacterial test tubes for 24hr at 37℃, fungal test tubes for 48hr at 25℃, and then observe the result.