TECHNIQUE OF COUNTING BACTERIA 81 



the bacteria to the red cells is known, it is easy to estimate the number of bacteria in 

 the suspension. Owing mainly to the impossibihty of obtaining a perfectly homogeneous 

 distribution of the red cells and bacteria on the sUde, this method is subject to a very 

 considerable experimental error. 



(c) Microscopical Examination of the Organisms in a Counting Chamber. — Though an 

 ordinary haematocytometer may be used and the bacterial suspension stained with a 

 suitable dye, it is much better to employ a Helber chamber (1904). The Helber slide 

 itself should be 2 mm. thick, the depth of the chamber 002 mm., and the area of each 

 small square 00025 sq. mm. ; it should be provided with a cover sUp 1-2 mm. thick. 

 The organisms are examined unstained, under dark-ground illumination, a special con- 

 denser being provided for this purpose. A f inch objective and a x 25 compensating 

 ocular afford a suitable combination for counting most organisms. This method is un- 

 doubtedly the most accurate we possess. Provided a number of technical points are 

 attended to, there is no reason why counts should vary by more than ^ 10 per cent, from 

 the real value. 



(d) The Opacity Method.- — This is essentially an indirect method of bacterial enumera- 

 tion. The opacity of the suspension to be estimated is compared, either by the naked 

 eye or by means of a nephelometer (see Liese 1926, Strausz 1930, Milatz and Rottier 1936), 

 photometer (Mestre 1935), or photo-electric cell (Pulvertaft and Lemon 1933, AljDer and 

 Sterne 1933, Longsworth 1936), with a control suspension of standard opacity, the number 

 of organisms in which has been counted by one of the direct methods just described. Though 

 very rapid and of great value for many purposes, this method suffers from the disadvantage 

 that the opacity to which bacteria give rise appears to be determined not only by their 

 numbers, but also by their size and their optical density. Since different strains of the 

 same species, and even organisms of the same strain at different periods of growth (Wilson 

 1926), vary in size, the opacity method cannot do more than afford an approximate estimate 

 of the numbers of bacteria in a given suspension. According to Liese (1926), who worked 

 with spherical organisms, the opacity is determined by the surface area of the organisms 

 (4 nr'^) and therefore varies with the square of the radius. Thus a suspension containing 

 500 million cocci per ml. with an average diameter of 2 /< would be four times as dense as 

 that of a suspension containing the same number of cocci with an average diameter of 1 //• 

 According to Strausz (1930), however, it is doubtful whether the relationship is quite so 

 simple as this. Considering that some of the light must pass through the organisms them- 

 selves, it is difficult to avoid the conclusion that volume and density probably play a part in 

 determining the opacity of a bacterial suspension. Though unsatisfactory for the exact 

 enumeration of bacteria, the opacity method is very useful, on account of its rapidity, 

 for approximate estimations, and is peculiarly well adapted for the standardization of 

 vaccines, in which the important factor is not the number of individual organisms, but 

 the total amount of bacterial protoplasm per ml. (For very useful information on the 

 micrometric measurement of bacteria, and for tables giving the relationship of volume 

 to surface area, see Skar 1934.) 



(e) The Centrifugal Method. — This is again an indirect method. It consists briefly in 

 centrifuging the suspension in a capillary tube, measuring the height of the column of 

 deposited bacteria, and estimating their number by calculations based on their average 

 diameter or their specific gravity (see Schmidt 1926, Schmidt and Fischer 1930). As an 

 approximate method of ascertaining the bacterial content of a suspension, particularly 

 when the organisms are clumped, this method is often useful, but the doubtful vaUdity 

 of some of the assumptions made in the calculations, and the various factors that may 

 interfere with the sedimentation of the bacteria under a given centrifugal force, are such 

 as to render the final result subject to a considerable error. 



(2) Viable Count. 



(a) The Dilution Method.- — ^This method, which is based on Lister's original method of 

 dilution to extinction, consists in diluting the suspension to a point beyond which unit 

 quantities are sterile. In practice several consecutive dilutions are made, and from each 



