Cynthia H. Pierce

THE ENUMERATION OF VIABLE TUBERCLE BACILLI IN CULTURES AND INFECTED TISSUES

This paper is not directly concerned with the chemotherapy of tuberculosis, but the method of counting viable tubercle bacilli which is described should be useful for in vilro testing of drugs and for the accurate quantitative study of such problems as the development of drug resistance, the effect of chemotherapy on experimental tuberculosis, and so on. It was developed in the belief that the study of the mycobacteria, which has remained somewhat out of the main current of general bacteriology, would be rendered easier and more fruitful if standard bacteriological techniques were increasingly applied to it. Studies on the bacteriology of the tubercle bacillus, and on the pathogenesis of tuberculosis in experimental animals, have been hindered greatly by the lack of satisfactory methods of counting the number of viable organisms in bacterial cultures and in suspensions of animal 0rgans.l Many methods have been proposed and some are quite useful for particular limited investigations, but there has been no method sufficiently simple and accurate to displace even the current method of measuring infective doses in terms of fractions of a milligram of microorganisms. Early experiments on the spread of tubercle bacilli through the animal body gave irregular results.2* 3 p 4 , More consistent and accurate figures have been obtained in recent studies of airborne infection in but the cultural methods used have not been suitable for proper evaluation of the range of variation inherent in the technique. There is general agreement that where it can be used, the surface plate count is the method of choice for making bacterial count^.^ I n general, the important requirements for accurate counts are as follows: (1) A method of obtaining completely dispersed bacteria with no chains or clumps. (2) The use of a diluent in which bacterial growth will not occur and in which the bacteria are protected from the effect of toxic substances, whether present as trace contaminants of laboratory apparatus or derived from animal tissues. (3) A solid, preferably transparent, medium on which single bacterial cells will regularly give rise to recognizable colonies in a reasonable period of time, and which can be prepared in such a way that sufficient replicate counts can be made to allow for the variable distribution of organisms in aliquots of a suspension. (4) Satisfactory techniques for the preparation of dilute suspensions and for the measurement of aliquots of these suspensions for the inoculation of plates. ’*