Algae Enumeration via Culturable Methodology

1. Weigh out a 10 g sample of soil that has either been collected moist from the field, or had water added to it so that it remains moist for 2-3 days. Note that the soil should be moist but not saturated.
2. Prepare a 10-fold dilution series by adding the 10 g of soil into 95 mL of Modified Bristol’s Solution (Figure 1). To create Modified Bristol’s Solution, dissolve the following in 1,000 mL of water: 0.25 g NaNO₃, 0.025 g CaCl₂, 0.075 g MgSO^-₄ · 7H₂O, 0.075 g K₂HPO₄, 0.018 g KH₂PO₄, 0.025 g NaCl, and 0.5 mg FeCl_3.
3. Continue the dilution series by the addition of 1 mL of Suspension A to 9 mL of Bristol’s Solution and additional sequential dilutions.
4. Inoculate 5 replicate tubes each containing 9 mL of Modified Bristol’s Solution with 1 mL each of the dilutions 10^-2 to 10^-6 (Table 1).
5. Incubate the capped tubes for up to 4 weeks in an area exposed to sunlight.
6. Observe the tubes for algal growth once every 7 days. Tubes with algal growth appear green.

Figure 1. How to make a 10-fold dilution series.

Tube	Dilution
B	10-2
C	10-3
D	10-4
E	10-5
F	10-6

Table 1. Tubes and dilutions.