The object of soil bacteriologists is to discover means of favouring the activity of soil bacteria, especially those activities that are useful to the higher plant. Knowledge is therefore needed of the changes in numbers and activities of the soil bacteria, and of the influence of soil conditions on them. The necessity of studying these changes has required the development of a quantitative technique by which the numbers of bacteria in the soil and their activities can be estimated.
The method commonly used in counting bacteria in soil is a modification of the plating method of Koch. In counting bacteria two difficulties have to be overcome—their immense numbers and their small size. The numbers of bacteria in soil are so large that the bacterial population of a gram of soil could not, of course, be counted directly. The method adopted, therefore, is to make a suspension of soil in sterile salt solution, and to dilute this suspension to a convenient and known extent, which will depend on the numbers of bacteria expected. In ordinary field soils it is found convenient, for example, to dilute the soil suspension so that one cubic centimeter of the diluted suspension will contain 1⁄250,000th of a gram of soil. Such a volume will commonly contain a number of bacteria sufficiently small to count. The second difficulty is that the organisms are microscopic, and yet cannot be readily counted under the microscope owing to the presence of soil particles in the suspension. Hence recourse is had to plating. One cubic centimeter of diluted suspension is placed in a petri dish and mixed with a suitable nutrient agar medium, melted, and cooled to about 40° C. The medium sets, and after a few days’ incubation the organisms multiply and produce colonies visible to the naked eye. By counting these colonies we obtain an estimate of the number of bacteria in the one cubic centimeter of suspension, it being assumed that every organism has developed into one colony, and by multiplying this number by the degree of dilution we obtain the numbers per gram of soil. In practice a number of parallel platings are made from one cubic centimeter portions of the diluted suspension and the mean number of colonies per plate is taken. By this means the error due to the random distribution of bacteria in the suspension is reduced, because of the greater number of organisms counted.
In drawing conclusions from bacterial count data, it is necessary to distinguish between the indication which the method gives of the absolute numbers of bacteria in the soil and the accuracy with which it enables the numbers of two soil samples to be compared. The method cannot be used for the former purpose at present. We do not know how far the figures obtained by this counting method fall short of the actual number of bacteria in the soil. One reason for this is the difficulty of effecting a complete separation of the clumps of bacteria into discrete individuals in the suspension. Then again, there is no known medium upon which all the physiological groups of bacteria will develop and produce colonies. And even on a suitable medium some of the individuals may fail to multiply.
In comparing the bacterial numbers in two soil samples, however, the case is different. Within each bacterial group investigated the plate method should give counts proportional to the bacterial numbers in the soil. Thus, by the method one should be able to tell whether the bacterial numbers are increasing or decreasing over a period of time, or whether a certain soil treatment produces an increase or a decrease. With this end in view the technique of the method has been improved by recent workers. It was found that, when carefully standardised, the process of dilution of the soil could be carried out without significant variation in result ([Table IV.]), and that the accuracy of the method is limited mainly by the variation in colony numbers on parallel platings, due in part to random distribution of bacteria throughout the final suspension, and partly to the uneven development of colonies on the medium. The question of the medium was therefore taken up with a view to improving the uniformity of results obtained with it. Lipman, Conn, and others effected an improvement by using chemical compounds as nutrient ingredients, thus making their media more closely reproducible. On most agar media, an important disturbing factor is the growth of spreading colonies, which prevent the development of some of the other colonies. A medium has been devised at Rothamsted on which these spreading organisms are largely restricted.[61] A statistical examination[19] has shown that on this medium errors due to the uneven development of colonies, except in special cases, are prevented, so that in fact the variation in colony numbers between parallel plates is found to be that produced merely by random distribution of bacteria in the diluted suspension (see [Table IV.]). In this case the accuracy of the counts of the bacteria in the diluted suspension depend directly on the number of colonies counted, and can be known with precision.
TABLE IV.—BACTERIAL COUNTS OF A SOIL SAMPLE.
Parallel Plate Counts from Four Sets of Dilutions made by Different Workers.
| Counts of Colonies on each Plate. | ||||
|---|---|---|---|---|
| Plate. | Set I. | Set II. | Set III. | Set IV. |
| 1 | 72 | 74 | 78 | 69 |
| 2 | 69 | 72 | 74 | 67 |
| 3 | 63 | 70 | 70 | 66 |
| 4 | 59 | 69 | 58 | 64 |
| 5 | 59 | 66 | 58 | 62 |
| 6 | 53 | 58 | 56 | 58 |
| 7 | 51 | 52 | 56 | 54 |
| Mean | 60·86 | 65·86 | 64·28 | 62·86 |
| Standard deviation between the four sets = 5·62. | ||||
| Standard deviation between plates within the sets = 7·76. | ||||
The knowledge obtained from counts of soil bacteria is subject to another serious limitation. We do not know which of the bacteria counted are the most effective in bringing about the various changes that take place in the soil. It is not even known which of them are active in the soil and which are in a resting condition. It is thus possible to have two soils containing equal numbers of bacteria but showing widely different biochemical activity, if one soil contains organisms of a higher efficiency. Moreover, as has been pointed out, many important groups of soil bacteria do not develop on the plating media, and so are not counted. These considerations led to the development of supplementary methods by which it was hoped to estimate the actual biochemical activity of the soil microflora. The first of these methods was developed by Remy, who attempted to study the biochemical activity of a soil by placing weighed amounts into sterile solutions of suitable and known composition, keeping them under standard conditions for a definite time and then estimating the amount of the chemical change that was being studied. Thus, to test the activity of the organisms that produce ammonia from organic nitrogen compounds, he inoculated soil into 1 per cent. peptone solution and measured the amount of ammonia produced in a given time. By similar methods the power of a soil to oxidise ammonia to nitrate, to reduce nitrate, or to fix atmospheric nitrogen, is tested. This method has been extensively used and developed by more recent workers. It suffers, however, from the same serious disadvantage that it was designed to avoid, for we cannot be certain that those bacteria that develop in the nutrient solution are the types that are active in the soil, and, moreover, even where the same types do function in the two conditions, we do not know that the degree of their activity is the same in soil and in solution cultures. For instance, Nitrosomonas appears to show very different degrees of activity in soil and in culture.
Another method, therefore, of studying the activity of soil micro-organisms is the obvious one of estimating the chemical changes that they produce in the soil itself. This method has obvious advantages over the unnatural methods developed from Remy’s, but it has a number of limitations that make its actual application difficult. In the first place, we cannot always tell whether changes found to occur in soil are due to the activity of micro-organisms, or are purely chemical reactions unassisted by biological agencies. Then, if we succeed in showing that the changes are due to micro-organisms, it is very difficult to determine which organisms are effecting them. This cannot be definitely tested by isolating suspected organisms and testing their activity in sterile soil, because in sterilising soil its nature and composition is altered. In spite of these difficulties, however, the study of the chemical changes that take place in the soil has produced valuable knowledge, when it has been combined with a study of the changes in the number and variety of the micro-organisms that accompany these reactions. This method of investigation is well illustrated by the work of Russell and Hutchinson on the effects of heat and volatile antiseptics on soil, where a study of the chemical changes such as ammonia production, that occurred in these treated soils, combined with a study of the changes in bacterial numbers, led to the realisation that the soil micro-population was a complex one, containing active protozoa.
A great difficulty in applying quantitative methods to bacteria in the field is the great variation in the density of the bacterial population over a plot of field soil, which may be so great that a bacterial count from a single sample is quite valueless. For example, the distribution of bacterial numbers over a plot of arable soil near Northampton was studied by taking sixteen samples distributed over an area about 12 feet square. The result showed that in some cases the bacterial numbers in samples taken 6 inches apart differed by nearly 100 per cent. Fortunately, under favourable conditions, a remarkably uniform distribution of bacterial numbers over a plot of soil can be found.
On such a plot it is possible to investigate the rapidity with which the numbers of the soil micro-organisms alter in point of time. For example, on the dunged plot of Barnfield, Rothamsted, which has been cropped with mangolds for forty-seven successive years, the area distribution of bacteria has been found to be so uniform that if a number of samples of soil are taken from the plot at the same time, the difference in bacterial numbers between the samples cannot be detected by means of the counting technique (see [Table V.]). The work of Cutler, Crump, and Sandon[16] on this plot showed that the bacterial numbers vary very greatly from one day to the next, and that these fluctuations took place over the whole plot, since two series of samples, taken in two rows 6 feet apart, showed similar fluctuations (see [Fig. 7]). The discovery of these big daily fluctuations in numbers led to an inquiry as to how quickly bacterial numbers change, and samples from Barnfield, taken at two-hourly intervals, showed that significant changes in numbers took place even at such short intervals.