536 METHODS FOR INVERTEBRATES. 



further twenty to thirty minutes. Pour the contents of the dish into 

 the mixture in the cylinder and make up the total volume to 200 c.c. 

 Filter into a cylinder and add at once solution No. II., which is prepared 

 as follows : 1 grm. water soluble eosin (yellow shade) in 200 c.c. dis- 

 tilled water. Mix and allow to stand for half an hour, and collect the 

 resulting precipitate on a filter. Dry the precipitate at 60 C. In order 

 to prepare the stain dissolve 0-2 grm. of the dried precipitate in 50 c.c. 

 pure acetone-free methyl alcohol. The method of staining is identical 

 with that of Leishman and Wright. 



1012. GIEMSA'S Method. In many ways this is the easiest and best 

 of the Komanowsky stains ; it is best bought ready prepared, either 

 as a solution or the solid products of Burroughs, Wellcome & Co. For 

 use, films are fixed in absolute methyl alcohol, alcohol-ether or absolute 

 alcohol. They are then placed film side down in the staining fluid, 

 which is prepared as follows : 



Giemsa's stain ... . . .1 drop. 



Neutral distilled water . . .1 c.c. 



They should be stained for about one hour in this stain, washed, dried 

 and examined. 



It is highly important that the water used should be neutral. This 

 is best secured by boiling fresh distilled water for fifteen minutes, and 

 then rapidly cooling and storing in a syphon vessel over soda-lime. 



1013. M. TAYLOR'S Method for Amoeba Cultures (Nature, April, 

 1920). Water from such places as the drainage cuttings in birch, 

 alder, and willow woods, or from the margins of ordinary pools and 

 ponds, together with the filamentous algss and the brown scum, and 

 included diatoms overlying the dead leaves and the other decaying 

 organic matter forming the floor of such places, is gathered in 

 autumn or in early spring. This is allowed to stand in tap- water 

 for some time, until a rich brown scum appears on the top. The 

 surface water, with the scum, is poured off into another glass vessel, 

 and wheat is added (1 gram to a litre of water). In February, 

 minute amoebae begin to make their appearance ; these become 

 fully grown in May and June, and will then divide rapidly, forming 

 a luxuriant culture until the late autumn, when encystment of most 

 individuals again takes place. 



Once started, amoeba cultures require no further attention than 

 a supply of water to compensate for evaporation, and the addition 

 of wheat from time to time. 



Dr. J. Bronte G-atenby informs me that using Sr. Monica Taylor's 

 method he has made successful cultures in boiled and unboiled London 

 tap-water, in London rain-water and in spring-water. Sub-culturing 

 is easily managed. Simply take about a pint of fresh rain-water or 

 boiled tap -water ; about eight wheat seeds are just brought to the 

 boil in a test tube of water, the latter poured away and the seeds shaken 

 into a flat dish containing the rain-water. The dish is then inoculated 



