1891 The Lancet  
THOSE who have followed the discussions which have taken place between the partisans of the constancy (Koch, ::lopf' and those of the mutability of pathogenic bacteria -(Davaine, Naegeli, Pasteur, Buchner) know what stress has been placed on the impurity of cultivations the pathogenic properties of which seemed to have altered. It is evident that one of the simplest ways to solve this vexed question would be, instead of studying the mixed products of the iB various media, then to connect
more » ... n to connect certain physical and ,chemical alteration of the various media with stages of -development, modified and unmodified, and finally to find tow the properties of the organism at each developmental ::stage are or are not modified by external circumstances. I had already attempted to carry out this plan by means of i3,diltition method, such M that used by Brefeld and others since, early in 1881, when working at the organisms of suppurating mucous membranes, a work which I gave up owing to the failure of the methods I was using then, and the special difficulties connected with the subject. I was, however, already then able to satisfy myself that phenomena .analogous to those of karyokinesis were of constant occurrence in multiplying bacteria, and gave rise to many appearances which have been observed by others, though enot explained fully yet. About the middle of last year, whilst studying the ,development of certain pathogenic moulds and other parasites, I felt again the need of following closely the development of single organisms. I failed by plate and drop cultivations to obtain the results I wanted, partly owing to <the effects of liquefaction of certain media or of the mobility -of others, paruly also owing to the form assumed by ,drops. I was then led to adopt a new mode of cultivation which, although not perfect in many of its details, has yielded results which so far have been satisfactory, and some of which have been exhibited this year at a meeting of the Pathological Society (May 5th). The principle of the method is to enclose a thin layer of the nutrient medium 'between two parallel plates, so as to force the organism to ;grow in definite directions. 0 wing to the effects of capillarity the most fluid nutrient media become, so to speak, '6'xed, provided evaporation be prevented, and they become as available as the solidified ones. The method can, of course, be varied in many ways ; but one of the simplest ,plans, and one of those which I have used with satisfactory results, is the following. At each end of a glass slide (1in. by 3in a narrow slip of glass is fixed (A A). This, as will be seen later on, is to act as a support. (The surface of the ,slide on which these slips are fixed will be called the upper one hereafter.) Three small drops of sealing-wax are dropped -on the upper surface of the heated slide (any other thick -cement, solid and emitting no antiseptic vapours, at the temperature of the body, may be used instead of sealingwax). These drops will be used to support a coverglass (c) an inch and a quarter in diameter at a certain distance above the slide, and therefore must form the apices of a triangle capable of being inscribed in the 'circumference cf such a cover-glass. Before placing the drop of fluid on it, the slide must be thoroughly sterilised in the flame of a Bunsen burner, or otherwise (the sealingwax does not interfere with this process). Then the slide is inverted or placed under a thoroughly sterilised plate. A cover glass (C) one inch and a quarter in diameter is sterilised also. and the surface which is to be next to the slide is carefully protected from the access of any germ or dust. On this surface a very small drop (D) of sterilised material may be placed, and this drop touched with a wire charged with a few organisms. A number of cover-glasses being prepared in this way, they may be examined over a sterilised plate with a pretty high power, inoculated surface downwards, and not in contact with the supporting slide, which must also be thoroughly sterilised, until a drop is found to contain the number of organisms wanted. Instead of a drop a streak (E E) can be used, according to the nature of the organism investigated. On the upper surface of the sterilised slide a drop of sterilised nutrient or other medium is deposited by means of a perfectly sterilised pipette. The size of the drop depends on the thickness one desires to give to the preparation or the surface one intends to cover. The diameter of the enclosed film should, in order to prevent contamination, never be more than three quarters of an inch when the cover is not more than an inch and a quarter in diameter. (I often use larger covers and slides, but this in most cases B---—_-IV I.-A slide with interlamellar film, ready for incubation. A A, Side rests. B B, Drops of sealing-wax supporting the cover. C C, Cover-glass compressing a drop of nutrient material. D, Very small drop of gelatine.or other material containing a few spores. II. -A cover-glass with very small central drop inoculated (D). HI.—A cover-glass with a small central streak (E E) containing micro-organisms. IV.-Diagrammatic representation of eleven interlamellar cultivations in a moist chamber, showing the disposition which I have adopted both for this method and for other forms of film or plate cultivations. A, Outer basin containing a thin layer of water (w) at the bottom. B, Covering basin. with a flat bottom, allowing a series of moist chambers to be piled one above the other in the incubator. c, Inner bell preventing condensed water falling upon the slide. D, Plate supported by pieces of cork. E's, Slides. has no advantage.) The centre or the side of such a drop may now be inoculated (in case the cover has not been previously inoculated). Then the cover is placed over the drop. It should be well supported by the three drops of sealing-wax, and should not at this stage flatten much the drop underneath. A heated rod is then applied successively over the three drops of sealing-wax until the inoculated fluid has spread evenly over a certain surface under the central parts of the cover ; the preparation is then ready for the incubator. It has, however, to be kept in mind that owing to the free access air has to the surface of the inoculated fluid, it is necessary to keep the preparation in a moist chamber. The extremely small size of these inoculated slides allows of a large number being packed in an extremely small space. Before incubating the preparation it is necessary to select out of the micro-organisms which have been sown into the fluid one or several. the position and relation of which are carefully noted. For this purpose divided cover-glasses or
doi:10.1016/s0140-6736(02)00147-2 fatcat:ielirwupbrcptmdwwmfxyxrtbu