Further Investigations on the Nature of Ultra-Microscopic Viruses and their Cultivation

Abstract

XIV. Summary: A survey has been made of the different theories put forth to explain the nature of ultra-microscopic viruses, and it has been concluded as probable that they are representatives of some precellular forms of life. The writer's views have been given, and the probabilities have been expounded regarding the constitution of these forms, their evolution towards a composite cell, the conditions under which they exist, the means by which they obtain the necessary energy for their vital processes, and their food requirements. It has been suggested that at times some unit of a cell may undergo a partial or complete reversion to the independent precellular form. The possibility of the existence of other primitive forms of life, belonging to another biological world, has also been considered. The theories discussed have been analysed in relation to the known facts concerning virus diseases, transmissible bacteriolysis and cancers. The reorganisation of the laboratories and the fitting up of various electrical and timing equipment, which were necessary to test the theories experimentally, have been explained, and a description has been given of some of the special pieces of apparatus made in the workshop for the experiments. Experiments were carried out on an extensive scale with animal and vegetable materials containing known viruses, with muds, etc., presumed to contain precellular forms of life, and with bacteriolytic agents. Certain delicate bacteria, isolated from porcelain filtrates, were used as controls. Attention was first given to indicators for growth, and it was found that minute quantities of selenium oxychloride incorporated in media resulted in many bacteria growing with a bright red colour. From an extensive series of experiments on nutritive requirements, it was determined that beef extracts usually act detrimentally on bacteria, and they should not be used. The detrimental effect was marked with the bacteriolytic agents. Good agar media were gradually evolved. Rain water proved to be better than water from a town supply. A watery extract from certain clays supplied sufficient nourishment for many bacteria, but the media were improved by the addition of watery extracts of bracken ash and of the fronds of certain ferns, notably those belonging to the Asplenium group. The associated conditions used during cultivation were varied in many ways, particular attention being given to the influence of solar rays and the energy emitted from projector lamps. In different experiments the wave energy was modified by reflection or filtration, different bands being tested both consecutively and intermittently. The materials used consisted of lenses, prisms, natural rocks and crystals, as well as animal, vegetable and chemical filters. Many bacteria were found to tolerate solar rays when cultivations were made on suitable media, particularly if the action was intermittent. They were least tolerant to the region of the spectrum around 4000 Å. A spore-bearing bacillus has been obtained from cultures of a mud nitrate on a leaf mould extract medium, placed under the intermittent influence of filtered solar rays. A group of the colonies developed a homogeneous waxy change, which was proved to be associated with a dissolution of the spores. It is suggested that the change is due to the presence of a “sporolytic agent”, although it has not been found possible to demonstrate the agent in a porcelain filtrate. In a number of cases pleomorphic bacilli have been cultivated from old porcelain filtrates obtained from various diseased plants and muds. Many were found to be very sensitive as to their nutritive requirements. Most of them grew well in subdued daylight and under the influence of a 100-watt projector lamp. Other porcelain filtrates produced granules on the surface of certain experimental media. When subcultures were made, granules associated with a marked milky opalescence of the medium occurred along the line of inoculation. No bacterium could be found in films made from the granules. The phenomenon appeared only on tubes of media which were autoclaved, set and inoculated on the same day. The milky opalescence can be produced without the presence of an unheated filtrate. The significance of the granules is being investigated. Many comparative experiments have been carried out with suspensions of dead micrococci, either uninoculated or inoculated with living micrococci or a bacteriolytic agent or with both. It has been shown that suspensions of dead bacteria undergo more marked clearing when placed under the influence of solar or other rays than when placed in darkness. The effect is increased when the suspensions are prepared from cultures grown on tubes of media which have been autoclaved, set and inoculated on the same day, and it is particularly marked when the suspensions have been inoculated with living micrococci. The significance of these and other results has been considered, and attention has been called to the utilisation of the factors concerned for the purpose of freeing intracellular toxins in the preparation of various vaccines. In conclusion I should like to record my appreciation of the valuable services rendered by my only assistant, Mr H. Frankham, the laboratory attendant and caretaker of the Institution.