Materials

  • Nutrient agar powder
  • Spatula
  • Weighing boat
  • Weighing scales
  • Distilled water
  • Measuring cylinder
  • Conical flask
  • Heat resistant gloves
  • Eye protection
  • Non-absorbent cotton wool
  • Aluminium foil
  • Bunsen burner, tripod and mat
  • Universal bottles and lids (for plates)
  • 2 McCartney or Universal bottles
  • Autoclave tape (optional)
  • Water bath
  • Marker pen
  • Autoclave or pressure cooker

Instructions

Wear a lab coat and use eye protection. Wear heat resistant gloves when handling hot equipment.

Label McCartney bottles (for slopes) with name, date and type of medium (use autoclave tape).

Label universal containers (for plates).

Calculate the volume of medium you require (20 cm3 per plate, 12 cm3 per slope).

Calculate the weight of dry medium required to make up that volume (see container to calculate correct weight).

Measure out the weight of nutrient agar powder you have calculated.

Add agar powder to the desired volume of distilled water in a conical flask and swirl around.

cotton wool stopper, not just a lump of cotton wool but folded so that it can be removed from and replaced in the neck Make cotton wool stopper.

Place stopper in neck of conical flask and cover with aluminium foil (this prevents the cotton wool catching fire when over a Bunsen flame).

Place the conical flask containing the agar in a water bath (metal pot) or on a magnetic heater.

Heat in the water bath over a Bunsen flame, gently swirling occasionally (use a heat resistant glove) until it dissolves fully. (N.B. The solution must be heated until the liquid is transparent, otherwise the agar will not set properly).

Add 12 cm3 molten agar to each McCartney bottle.

Pour molten agar into the universal containers leaving a space of 2 – 3 cm at the top.

Put caps on loosely.

Place in basket for sterilisation.

Autoclave for 15 minutes at 121°C.

To form agar slopes, cool McCartney bottles to 55°C, tighten lids then lie bottle at an angle against a marker pen or similar.

To pour plates, cool universals to 55°C in a water bath .

Using aseptic technique, dispense agar from each universal into one sterile plastic Petri dish (see Pouring plates ).