450 PROTOZOOLOGY 



vere amoebic infections may have resulted from already existing 

 dormant infections aided by the newly formed association with bac- 

 teria. 



The cysts remain viable for a considerable length of time outside 

 the human intestine, if environmental conditions are favorable. Since 

 information regarding the viability and longevity of the cyst is 

 highly important from the epidemiological standpoint, many papers 

 have dealt with it. In testing the viability of the cyst, the following 

 two tests have been used by the majority of investigators. 



(a) Eosin-staining test. Kuenen and Swellengrebel (1913) first 

 used a dilute solution of eosin (1:1000). It has since been used by 

 Wenyon and O'Connor, Root, Boeck, and many others. Solutions 

 used vary from 1:10,000 (Root) to 1:100 (Boeck). A small amount 

 of fresh cyst-containing material and a drop of eosin solution are 

 mixed on a slide, then dead cysts will appear stained reddish under 

 the microscope, while living cysts remain unstained. Whether or not 

 unstained cysts might be dead or uninfectious is unknown. But as 

 Wenyon and O'Connor wrote, "if we accept the eosin test as a 

 criterion and regard all unstained cysts as living, the error in judg- 

 ment will be on the safe side." Root found neutral red in 1:10,000 

 dilution to give a slightly larger proportion of stained cysts than 

 eosin. Frye and Meleney's (1936) comparative study leads one to 

 look upon this method as a fairly dependable one. 



(b) Cultivation test. Improved cultural technique now brings 

 about easily excystment of viable cysts in a proper culture medium. 

 For example, Yorke and Adams (1926) obtained in 24 hours "a 

 plentiful growth of vegetative forms" from cysts in Locke-egg-serum 

 medium (p. 887). Snyder and Meleney (1941) note recently that the 

 excystation does not take place in various culture media unless liv- 

 ing bacteria were added or oxygen concentration of the media was 

 decreased. Animal infection method has not been used much, as 

 experimental animals (cats) show individual difference in suscepti- 

 bility. Some of the published results are summarized below. The 

 testing method used is indicated by: a for eosin test or 6 for cultiva- 

 tion test and is given after the name of the investigators. 



1. Cysts in faeces kept in a covered container. All cysts disap- 

 peared in 3 days at 37°C; at 27-30°C. half of the cysts found dead 

 by the 4th and all dead by the 9th day (Kuenen and Swellengrebel ; 

 a). Alive for 3 weeks (Thomson and Thomson; a). Remain un- 

 changed for several weeks if kept "cool and moist" (Dobell). All 

 dead within 10 days at 16-20° or 0°C. (Yorke and Adams; b). 



2. Cysts kept in water emulsion. All alive on the 9th, but almost 



