KNOWLEDGE & SCIENTIFIC NEWS. 



[October, 1906. 



Conducted by F. Shillington Scales, b.a., f.r.m.s. 



The Staining of Blood Films. 



The Bn/is/i Medical Journal gives a summary- ol 

 a comparative study by Alphonse Huisman of the value 

 of different methods which have been recommended for 

 the staining- of blood fihns. He has tried seventeen 

 different formula;, following^ the directions given by 

 their different authors, but finds that, with the excep- 

 tion of Jenner's stain, they do not readily yield good 

 results, and often involve a long and complicated 

 technique. With Jenner's method he is always able to 

 obtain satisfactory preparations, but he thinks he can 

 improve upon it by the introduction of certain modifica- 

 tions of his own. The stain he uses is a mixture of 

 equal parts of a 1.175 per cent, solution of solid azure 

 liiue in absolute methyl alcohol and a 0.825 per cent, 

 solution of pure eosin B.\ (Hochst) in the same medium. 

 He stains for two minutes without previously fixing the 

 film. By this method the nuclei appear a violet-blue, 

 whilst the basophile protoplasm takes a light blue 

 stain which gives a good differentiation from the rest 

 of the preparation. The red corpuscles are rose- 

 coloured. The neutrophile granules are rose-violet, 

 violet, or \iolet-blue; basophile granules are blue, the 

 metachromatic basophile granules are violet-red, and 

 the oxyphile granules are red. The author also ob- 

 serves that some of the lymphocytes exhibit, when 

 stained by this formula, evidence of a fuie, blue, or 

 metachromatic granulation. 



Mounting Protozoa. 



\'ery little attention is given in most text-books to 

 methods of mounting Protozoa, either temporarily for 

 the purpose of study or permanently. A brief account 

 of some suitable methods, based on a contribution by 

 Mr. C. W. Hargitt, may accordingly be of some ser- 

 \ ice. With regard to living specimens, in the case of 

 .\m(eba, or the more or less sedentary Protozoa, little 

 pri!caution in the way of supporting the cover-glass, or 

 rendering the specimens quiescent, is necessary. Hut 

 for Paramcecium, Stylonichia, and others of similar 

 activity, some method of restricting their movements is 

 desirable. Narcotics, such as chloral hydrate, cocaine, 

 and nicotine, or gelatinous substances, are apt to set 

 up pathological conditions sooner or later. A few 

 shreds of absorbent cotton under the cover-glass, so 

 tangled as to greatly limit the movements, have proved 

 very satisfactory, without interfering with the vitality 

 of the specimens, and fresh water can be easily added 

 without fear of losing the specimen. 



Intra-vitam staining may be satisfactorily done with 

 many of the Protozoa, and with many stains, especiallv 

 methylene blue, methylene green, methylene violet, iS:c., 

 in very dilute solutions. These stains are used to 

 differentiate the organism during life, and must be 

 watched throughout accordingly. 



For killing and fixing, a rapid re-agent is necessary 

 in order that the least possible distortion may result. 

 This can be done by the method of irrigation under the 

 cover-glass — the most difficult, but the least likely to 



lose the specimen — or by placing all the specimens 

 bodily in the re-agent. Lang's ffuid, used hot, applied 

 to the edge of the cover-glass and drawn in with blotting 

 paper applied to the other edge, is one of the most 

 suitable fluids for the irrigation method. It must be 

 removed by washing out w ith water in a similar way. 

 The stains must be applied also, their excess removed, 

 dehydration by alcohols of increasing strength will 

 follow, and finally Canada balsam must be draw n in l)y 

 similar methods. 



Killing by heating over a lamp or burner, as is done 

 with bacteria, is quite useless. 



Perhaps the best method, when the supply of material 

 permits of it, is to pipette the infusoria into a large 

 watch-glass, carefully draw off the surplus fluid A\itli 

 a finer pipette, if necessary, under a microscope or 

 lens, and to finish withdrawal of the last few drops by 

 a thread syphon. The killing may be done by means 

 of a saturated solution of mercury perchloride, to 

 which has been added one per cent, acetic acid. This 

 makes an excellent killing fluid, though weak chromic 

 acid, 2 per cent, osmic acid, and potassium iodide have 

 also their advocates. Formalin is generally rather un- 

 satisfactory. Certain organisms, however, such as the 

 \'orticellida', prove very difficult to kill in an expanded 

 condition, and recourse must, therefore, be had to 

 some narcotizing method, such as the gradual addition 

 of chloral hydrate, &c. 



When fixed, the organisms may be washed in \\ ater 

 or dilute alcohol, as required, stained by one of the 

 usual methods, dehydrated, cleared with cedar oil, clove 

 oil, &c., and mounted in balsam. Glycerine or 

 glycerine jelly, of course, requires a modified process. 

 With each stage of the operations similar precautions 

 must be adopted in transferring from one medium to 

 another as were used in transferring from water to the 

 killing medium. By the foregoing methods beautiful 

 and permanent mounts may be made of Amoeba, Para- 

 mcecium, X'olvox, &c., in all their life stages. 



Mounting Small Insects. 



.\n excellent method for mounting small insects and 

 Coleoptera was given some time ago by Mr. E. L. 

 Fulmer as follows : — Drop the specimens into absolute 

 alcohol and leave for an hour or more to dehydrate; 

 then transfer to xylol or other clearing agent for a few 

 minutes, and then mount direct in balsam. If it is 

 desired to make opaque objects transparent, they 

 should be boiled for a moment in caustic potash and 

 then treated as above described. If it is desired to 

 clear and mount small and fragile specimens with little 

 handling, this may be accomplished by dropping them 

 in water-free carbolic acid to kill, dehydrate, and clear. 

 .After remaining in this an hour or more they are 

 mounted in balsam. 



The smaller beetles, after being chloroformed, are 

 often mounted directly in balsam. They are cloudy for 

 a time when mounted in this manner, but in the course 

 of four or five weeks they become clear, so that this 

 simple method may be used to advantage with small 

 insects and their parts if they are given time to clear up 

 perfectly before being studied. 



In working with scale-insects (Coccida;), the insects 

 are first picked out of the scales and placed upon a 

 slide where it is desired to mount them. A few drops 

 of a 5 per cent, solution of caustic potash are applied, 

 and the specimens boiled in this for two or three 



minutes by holding the slide over a Bun.sen burner. 



The specimens are then washed two or three times by 

 being covered with a few drops of absolute alcohol, 

 after which they are cleared and balsam applied. 



