Appendix 2: Detection of Respiratory Deficient Mutants by the Tetrazolium Overlay Method

Respiratory-deficient mutants produce white colonies when treated with 2, 3 5-triphenyl tetrazolium chloride (TTC) whereas respiratory-sufficient colonies develop a deep red colour.

Method

  • TTC agar consists of 1·5% (w/v) agar and 0·1% TTC in 0·067 M phosphate buffer at pH 7·0. For best results, a more concentrated TTC solution is autoclaved separately from the agar in phosphate buffer and added aseptically at approximately 55° C to give the required concentration. Molten agar is kept at 55° C prior to use.
  • Molten TTC agar (10 – 20 mls) is poured over MYGP* (YM) plates bearing three or four-day-old colonies (50 – 100 colonies per plate).
  • After about three hours the number of white (respiratory-deficient) and red (respiratory- sufficient) colonies are scored. The precise time required for optimum differentiation will depend on the age of the colonies.

* MYGP Plates – Agar plates containing Malt extract, Yeast extract, Glucose and Peptone

Reference
Ogur, M., St John, R., and Nogai, S., Science. 1957, 125, 928.