This difficulty has, however, been overcome by a further modification of the dilution method, and it is now possible in any soil sample to count both the numbers of cysts and active forms. Also a further advance in technique has made it possible to recognise and enumerate the common species of protozoa, instead of simply grouping them as Ciliates, Flagellates, and Amœbæ, as was done in the past.[7]
Briefly the method consists in dividing the soil sample into equal portions (usually 10 grams each) one of which is counted, thus giving the total numbers of protozoa (active + cystic) present. The second portion is treated over-night with 2 per cent. hydrochloric acid, the HCl used being B.P. pure 31·8 per cent. Previous experiments have shown that such acid kills all the active protozoa, leaving viable the cysts. The number of cysts is therefore found by counting this treated sample, and the number obtained subtracted from the total gives the active number.[F]
[F] The proof of the accuracy of this method will be found in the following papers:—
(1) Cutler, D. W. (1920), Journ. Agric. Sci., vol. x., 136-143.
(2) Cutler, D. W., and Crump, L. M. (1920), Ann. App. Biol., vol. vii., 11-24.
The discovery of this method at once puts into the hands of the investigator a much more efficient instrument for studying the activities of the soil micro-population, especially since at a slightly later date Thornton’s method for counting bacteria was devised.
Early in 1920 Cutler and Crump[6] decided to make a preliminary survey of the protozoon and bacterial populations of one of the Rothamsted field soils (Broadbalk dunged plot). The investigation was continued for 28 days, daily soil samples being taken. The results so obtained showed that an extended investigation of the micro-population of field soil would yield interesting and important results, especially as it was evident that certain views held by soil biologists required modification.
In July of the same year, therefore, it was decided to start an extended investigation of the soil protozoa and bacteria. The method adopted was to make counts of the numbers of bacteria and of six[G] species of protozoa in soil samples taken daily direct from the field (Barnfield dunged plot) and by statistical methods to correlate these counts one with another and with the data for external conditions. Observations at shorter periods than 24 hours could not be made, but it was found possible to continue the research for 365 days.[7]
[G] Actual counts were made of six species, though, as stated on [p. 10], observations were made on seventeen.
