Serial dilution

1. Equipment

  • Culture tubes, (sterile) screw-capped or steristoppered (see Note below).
  • Test-tube racks, open mesh.
  • Media — usually dilute e.g. f10, f20.
  • Automatic dispenser (sterile) or 10 mL sterile glass pipettes.
  • Glass pipettes 1 mL or pasteur (sterile), rubber or silicone teat.
  • Bunsen burner or small flame.

2. Method

  • Using aseptic technique, dispense 9 mL of media into each of ten test tubes with sterile automatic dispenser or sterile 10 mL pipettes. Label tubes 10-1 to 10-10 indicating dilution factor.
  • Aseptically add 1 mL of enrichment sample to the first tube (10-1) and mix gently.
  • Take 1 mL of this dilution and add to the next tube (10-2), mix gently.
  • Repeat this procedure for the remaining tubes (10-3 to 10-10).
  • Incubate test-tubes under controlled temperature and light conditions:

(a) temperature and photoperiod — as close to the natural environment as possible.

(b) light intensity — slightly lower than the natural environment.

  • Examine cultures microscopically after 2—4 weeks by withdrawing a small sample aseptically from each dilution tube. A unialgal culture may grow in one of the higher dilution tubes e.g. 10-6 to 10-10. If tubes contain two or three different species then micromanipulation can be used to obtain unialgal cultures.

Note: Sterilizing screw-capped culture tubes

The following procedure is required to remove toxic materials from culture tubes and screw caps.

  • Half-fill culture tubes with distilled water. Plug with steristoppers or non-absorbent cotton-wool. Autoclave.
  • Take screw-caps from culture tubes and place open side down in a glass petri dish. Pour distilled water around caps, replace top of petri dish and autoclave.
  • Under aseptic conditions remove steristoppers or cotton-wool plugs and pour out distilled water. Flame tube and replace steristopper or screw-cap.