Page:The Journal of Tropical Medicine, volume 6.djvu/229
June 15, 1903.]
THE JOURNAL OF TROPICAL MEDICINE. 197
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posed to replace Ehrlich’s triacid for the representation
of the granulations of leucocytes. Stimulated by this
communication I made experiments which proved that acetone may be used to stain malaria parasites, more especially their chromatin.
A 1 per cent. methylene blue med. solution, of which
1 cem. had been neutralised by 0-9 eosin (A—G) could
be used as follows: Methylene blue 1:0 + acetone
2-0 + eosin 0°9 to 1:7 stained in from five to fifteen
minutes. When more eosin was used the time taken
was longer; when less was used it was shorter. When the correct time had been reached a punctiform sedi- ment formed.
This method, however, is not reliable, for a month later I tried to stain some preparations by it, and they were a failure. I found it was not worth while to find out the cause, for I discovered a much more reliable method: Distilled water ad 10:0 are added to 1:0 methylene blue + 2-0 acetone, and afterwards the eosin is added, 0°9 to 0°7. The addition of eosin in the neutralised condition is most reliable, but one and a half times as much (1°3—1°4) is applicable, although every stain is not so good with more eosin. The mixture is
poured over the preparation, which should be inverted during the process. _ With a definite methylene blue (Med. Hachst) solution, two months old, prepared according to Ziemann, I was able (on addition of 1°7 eosin) to stain blood- | corpuscles lilac in twenty minutes, and nuclei of leuco- | eytes a good violet ; after a period of thirty-five minutes | the blood corpuscles were eosin red, the chromatin was still more deeply stained, and at sixty minutes deposits of colour set in. _ With another solution, one month old (again with the addition of 17 eosin) the chromatin was only slightly /Stained in twenty, thirty-five and sixty minutes. A methylene blue vrectif. Erhlich solution also led to fauure.
In one word, not only did different sorts, but even the same kind of methylene blue, gave unstable results. ing of the solutions is a factor that cannot
ye forgotten. I proved that warming the solutions weakened their power of staining and therefore pre- I ed a cold solution as follows: 1-0 g. methylene blue s placed in a mortar and moistened with a little water, and then rubbed to a homogeneous cream ; this is diluted with water and filtered, and the mortar and filter washed out with water until the water amounts to is solution yielded the neutral tint with methylene ue 1: eosin 0°9; but mixed with acetone and water —as above—and 1-7 com. eosin, a sufficient colouring of the chromatin was not obtained in twenty, thirty-five E sixty minutes. The experiment, however, with the ddition of only 0° ccm. eosin, shows that this solution ly requires different treatment. In fifteen minutes the taining was complete, the central parts being only some- that weak. Similarly ¢ /a Ziemann: Two methylene + 1°6 eosin yielded a good chromatin stain in thirty- five minutes. The maturing of the methylene blue solu- m has never been satisfactorily explained, notwith- nding all that has been written on the subject. I Ould not discover whether exclusion of the air or the
effects of the atmosphere favoured this maturing. It may possibly be decomposition. Heating means sterili- sation, and this consideration should teach us to pre- pare cold solutions. The above-mentioned cold solution improves considerably in seventeen days.
Ziemann’s method, in the proportion of | methylene blue to 0°6 eosin, obtained a complete staining of the chromatin in ten minutes, and with acetone water and 0°9 eosin this result was obtained in five minutes. It is known that by the addition of alkalines the power of methylene blue solutions to stain chromatin is increased. Usually the blood corpuscles, in these preparations, assume a bluish-violet tone, which renders the discovery of small parasites very difficult. On standing, this became deeper still, so that I always, ex tempore, added a 2 per cent. borax solution. The following mixture yielded a strong chromatin stain in twenty- five minutes, but the blood corpuscles were somewhat dark:1 per cent. methylene blue, 1:0 + 2 per cent. borax, water 0°5 + distilled water ad 10:0 + 1 per cent. eosin (A—G) 1:2. The preparation becomes somewhat paler if plunged into weak eosin water several times, yet is is not satisfactory. By adding more eosin the stain becomes lighter and more reddish. Borax, methylene blue @ la Ziemann (with the addition of eosin without dilution) gave unsatis- factory results.
I also tried lime-water and calcium carbonicat. pre- cipit. As much as will go on the tip of a knife, shaken up with a | per cent. methylene blue solution, filtered, yielded good results d /a Ziemann, but did not do with acetone. I also tried metals (pluwmbum oxidatum and plumbum carbonicum without success), and [ found that soluble Credé’s silver, called collargol, yielded by far the best results. I add 0°5 g. collargol to 100°0 ccm. of 1 per cent. methylene blue and shake all well up; 1 then filter after a few hours. This solution can, how- ever, be used unfiltered if care is exercised not to shake up the sediment. This arg. methylene blue was neutral- ised in the proportion of 1 methylene: eosin 0'8, and for staining d /a Ziemann in the proportion of | methy- ene to 0°6 eosin poured over it, is more practical than the best Ziemann solution. The stain is excellent in fifteen minutes, and in older solutions a shorter time suffices. When | ccm. of arg. methylene blue is diluted with distilled water ad 10:0, and then 0°6—0°7 ccm. 1 per cent. eosin (A—G) is added, double time is required for staining. With the addition of acetone the following procedure is recommended: Arg. meth. blue 1:0 + acet. 2°0 + distilled water ad 10°0 te eosin ('8—1°5. Eosin (0°8) is also the most reliable in this method. Time required: half an hour—less in older solutions. When sediments occur the time is to be lessened, or a fresh solution is to be prepared.—Deuwtsche Medizinische Wochenschrijt, April 25, 1903. a Summeper-Fatt Frevers.—Mr. Krauss (Amer. Med. Assoc., May 5th, 1903), states that some of the summer- fall fevers were typhoid, some were malarial and a few of them were neither, but the writer was opposed to the belief in an x-lever.