pH indicator plates
From fresh streaks, make 1 cm2 patches on plates containing 20 mg /L phenol red. (see below). When cells secrete alkali, the cells become increasingly red as does the surrounding medium. When comparing strains, patches can be placed distant from each other on the same plate; however, because alkali diffuses rapidly through the plate, we usually place only a single patch of cells on the plate, in the center of the plate, and compare patches on different plates.
For measuring alkali production under conditions in which sporulation genes are induced, we prepare YNA-2 medium and add 20 mg/L phenol red before autoclaving and mix using stir bar just before pouring plates
For measuring alkali production in respiratory defective mutants (e.g. cit1D) we prepare SPS2-0.3 and add 20 mg/L phenol red before autoclaving
Incubate plates at 30°C and photograph after 1-6 days of growth with light source beneath plate and plate surrounded by black frame.
YNA-2 plates
30 grams potassium acetate
2.5 grams yeast extract
pH to 7.0 with KOH or HCL
to 500 ml ddH20
10 grams agar
autoclave
pour exactly 33ml per plate
SPS2-0.3 plates
10 grams potassium acetate
0.43 grams yeast nitrogen base
1.25 grams ammonium sulfate
pH to 7.0 with KOH or HCL
to 500ml ddH20
10 grams agar
autoclave
pour exactly 33ml per plate
