6 THE AMOEBAE LIVING IN MAN 



the methods which suffice for this purpose are not adequate for obtaining 

 a complete knowledge of the amoebae themselves or of their develop- 

 ment. For example, if an amoeba found in the stools is so degenerate 

 as to be unidentifiable, its species may, in practice, often be deter- 

 mined by the finding of its cysts. But this does not mean that these 

 amoebae are really undeterminable as to their genus or species; nor does 

 this empirical method permit us to dispense with a study of the normal 

 organisms when considering them from a protozoological standpoint. 

 As a matter of fact, I believe all the species of amoebae living in the 

 human intestine are easily determinable if examined in a fresh and 

 normal state, though often undeterminable under the conditions imposed 

 by laboratory practice. Makeshift methods of examination and deter- 

 mination are only of value for purposes of ordinary laboratory diagnosis, 

 which has often to be made from material which is quite unsuitable for 

 zoological study ; and it is hardly necessary to point out that a mere 

 examination of amoebae or cysts in saline or iodine solution — which 

 is usually sufficient for diagnostic purposes — cannot supply that detailed 

 information requisite for a true understanding of the organisms 

 investigated. This information, as I would again emphasize, can be 

 obtained only by studying an abundance of the best material with the 

 aid of all the cytological resources now at the disposal of protozoologists. 



The following special points concerning technique may be noted 

 briefly here, as they have some interest or importance. 



It is absolutely essential to study the living organisms as well as fixed 

 and stained specimens. Every competent protozoologist now recognizes 

 this as a general principle ; but we have already had, nevertheless, 

 numerous descriptions of "amoebae" from man which have never even 

 been seen alive. After studying the living amoebae or cysts, an examina- 

 tion of them in iodine solution* is often very helpful — especially for 

 cysts, in which the iodine-reaction of the contained glycogen often 

 supplies important information. In iodine solution, also, owing to the 

 fixation which takes place, the nuclei become easily visible ; and their 

 structure can thus be studied roughly, and their number counted accu- 

 rately. Any good cytological method of fixation will usually fix amoebae 

 well, but the fixation of cysts is often much more difficult. The best 

 fixative for cysts is, in my experience, Schaudinn's sublimate-alcohol 

 solution to which 4 to 5 per cent, of glacial acetic acid has been added. 

 Cysts — especially those of E. coU — are sometimes difficult to stain : and 

 it is useful to remember that they are, in general, more readily permeable 

 to watery than to alcoholic solutions. Grenacher's borax-carmine, used 

 warm, and acidified with a small quantity of glacial acetic or hydro- 

 chloric acid, will often stain the contents of cysts when all other methods 

 have failed. For ordinary routine purposes I have found Mayer's 

 haemalum by far the most reliable and rapid stain for cysts of all sorts ; 

 though it cannot, of course, replace the finer cytological stains — such as 

 the various iron-haematoxylin methods. My alcoholic iron-haematein 



* I do not know who first introduced this method for the study of intestinal amoebae 

 and cysts. The method is, of course, one of the earliest ever used for studying pro- 

 tozoa. McCarrison (1909) Kuenen and Swellengrebel (1913) and others, have used it for 

 studying the cysts of Entamoebae ; and since its recommendation by Wenyon (191 5) as 

 a routine method, it has come into general use. Iodine should be used as a strong- 

 aqueous solution in potassium iodide — the stronger the better. 



