180 BEVIEW — TEOPICAL MEDICINE, ETC. 



Small-pox. De Korte' gives the following technique for demonstrating the 

 organisms found by Funk and himself in variolous matter and in human vaccine lymph, and 

 which he named provisionally Amceba variolm vel vaccinia: — 



To be satisfactorily seen, a haugiug-drop proparatiou of variolous or vaccine lymphs must be made. Avoid 

 all manipulations of whatsoever kind. All ordinary methods of staining, fixing, or drying, the pressure of the 

 cover slip and desiccation invariably lead to rupture of the ectosarc, and to disintegration of the parasites; 

 for these reasons, in smear preparations, only the detritus of the parasites is to be found. To obtain sterile 

 variolous and vaccine lymphs, wash the pock with an antiseptic without rupturing its walls, remove any 

 excess of disinfectant, and force a sterile capillary tube into the interior of the vesicle ; by depressing the 

 free end of the tube the vesicular contents will gravitate into the tube. For storing purposes the ends must 

 be carefully sealed in a flame. The lymph being thus collected, in the case of human vaccinia on the ninth 

 day of eruption, centrifugalise the tube to concentrate the parasites, as there are comparatively few amoebae 

 present. Prom the centrifugal end of such a tube allow a small drop to exude on to a cover-slip, and put 

 up as a hanging-drop on a warm stage. At 98° F. the amcelia puts out pseudopodia and is actively motile. 

 The spores, being excessively minute and not very refraotile, can be seen, but only with difficulty, at the edge 

 of the specimen. Small-pox matter should be collected about the fifth day of eruption or earlier, and similarly 

 treated, but it is unnecessary to centrifugalise, as the parasites are present in enormous numbers. In glycerinated 

 calf lymph the parasites and spores are best seen suspended in a drop of normal saline solution, the method 

 of procedure being identical in other respects with that indicated for variolous matter. All these parasites 

 can be stained in vivo by suspending them in a normal saline solution tinted with aqueous safraniu, Lceffler's 

 blue or Bismarck brown. The following method of making permanent preparations, unsatisfactory though it 

 is, may be partially successful when absolutely fresh material is procurable ; after keeping in vitro the parasites 

 wiU not admit of this procedure. Spread on a cover-slip a small drop of variolous or human vaccine lymph 

 (that from a monkey is more satisfactory) with the edge of a second cover-slip, avoiding pressure as far as 

 possible ; immerse immediately in equal parts of spt. rect. and ether for ten minutes, and then stain with 

 Loeffler's blue for five minutes. The parasite of variola stains irregularly, the nucleus probably not at all. 

 Mount in Canada balsam. Glycerinated calf lymph spread in a thin film in the above manner can be fixed 

 with the fumes of 2 per cent, osmic acid for five minutes, stained with dilute aqueous safranin for ten 

 minutes; it can then be mounted permanently in equal parts of glycerin and water in a shallow cell, 

 thus avoiding the pressure of the cover-slip, which would destroy the organism. Permanent preparations are, 

 however, distinctly unsatisfactory, but anyone taking the trouble to follow the directions laid down will not 

 have the least difficulty in seeing the Ammba variolce vcl vaccinice in a hanging-drop preparation. 



It is an amceboid protozoon measuring about 1/2500 of an inch in diameter, having 

 the form of an oblate spheroid, containing intra-cellular spores, and in the case of the 

 parasite found in human vaccine lymph, actively amcjeboid. In human vaccine lymph it is 

 to be found on or about the ninth day of vaccination, after which it disappears spontaneously. 



In a later paper- he describes the organism more fully, saying : — 



Let a smear be made on a cover-glass of some variolous lymph at the seventy-second hour of the eruption ; 

 let the preparation be fixed with heat or other fixative (except Leishman's or Jenner's stain) and stained with the 

 ordinary dyes. The specimen will present a granular detritus with few, if any, cellular structures. Take, 

 however, some of the lymph from the same vesicle, and make a hanging-drop preparation. The latter will now 

 present a very different appearance. It will be seen to contain a large number of morphological elements 

 resembling pus cells, containing highly refractile grains within the cell substance. Among them will be seen 

 large cells having a definite nucleus and karyosome, and limited by a thick-walled ectosarc ; this form I have 

 termed the encysted parasite. Again, as more readily obtainable, fix and stain a cover-slip preparation of fresh 

 active glycerinated calf lymph ; beyond some epidermic cells, nothing but a granular detritus is visible on 

 examination. Yet this same lymph suspended in normal saline solution, will display, in addition to the refringent 

 grains constituting the granular detritus, a large number of circular elements varying in size, some having thick 

 ectosarcs, also some less numerous segmented bodies ; these may consist of a few or of a considerable number of 

 segments ; this body I have termed the morula, and it probably represents amitotic multiplication of the germs. 



If the vesicular contents of a human vaccine vesicle be examined as a hanging-drop with the highest powers 

 of the microscope and suitable adjustment of the light, disregarding any gross cell element which may be present, 

 the fluid will be seen to contain a vast number of exceedingly small grains, which appear to be motile on a 

 warmed stage ; their shape and size, because of their smallness, is practically indeterminable. These grains, I think, 

 are spores; they are larger and more distinct in variolous matter and always normally situated within the cell ; 

 they are motile in calf lymph, immobile in glycerinated calf lymph. The various bodies thus far described will 

 be seen to consist of four elements — spore, sporidium or amceba, encysted parasite, and morula body found in 

 glycerinated calf lymph. Two objections have been raised to the parasitic nature of these bodies. It has been 

 affirmed on the one hand that they are degenerate epithelial cells, and on the other hand that they are nothing 

 but leucocytes. Both arguments are refuted by the experiments already described. There is no difficulty in 

 fixing epithelial cells or leucocytes. An object cannot both be present and absent from one situation at one time, 

 yet at a period when all are agreed on the absence of leucocytes from the vesicular contents of the pock, the 

 bodies above described are seen to be present in large numbers in hanging-drop preparations. If the variolous 

 matter be gathered with antiseptic precautions and stored in a capillary tube, at the end of twelve months the 

 parasites will be found to be present provided they be sought for in a hanging-drop preparation, not otherwise. 

 It is extremely improbable, that leucocytes will remain intact for this period outside the living body in whatsoever 

 manner they "may be kept. It may be objected that variolous lymph has a specific conservative action on 

 leucocytes, and that they may live in this fluid for a very long time. For this assumption there is no warrant ; for if 



^ De Kort^, W. E. (November 19th, 1904), "The Parasites of Small-pox and Vaccinia," in Report of 

 Meeting of the Pathological Society. British Medical Journal, Vol. II. 



= De Korte, W. B. (December 1st, 1906), " The Virus of Small-pox and Vaccinia." £/-i7isA MaiicalJournal, 

 Vol, 11. 



