82 



III.— INVESTIGATIONS BEAEING ON THE RELATION OF 

 NOSEMA APIS TO ISLE OF WIGHT DISEASE. 



Methods of Examination of Bees fok the Presence of Nosema apis. 



As carried out by John Innes, B.Sc, M.B., Captain R.A.M.C. 



Each bee was examined in a routine manner, and the following notes 

 were made : — 



(1) Activity, as determined by movements in the postal cage — usually 



a match-box. 



(2) Power of flight on exposure in open space. 



(3) Dislocation of wings ; if present or absent. 



(4) Gait ; whether co-ordinated or paretic. 



(5) Response to stimuli, such as handling, etc. 



The chyle-stomach and colon of each bee were examined systematically, 

 and the removal of these parts from the body was carried out in the following 

 manner. The head of the bee was iirmly crushed between fairly stout 

 dissecting forceps, and while held thus, the last segment of the abdomen 

 was grasped with straight fine-pointed forceps, and gentle traction applied. 

 In this way the colon was carefully pulled out and the chyle-stomach also 

 came away with it. These organs were placed on a slide, separated at 

 their anatomical junction, and teased. Cover-glasses were then applied, and 

 the preparation examined microscopically in the wet, unstained condition. 

 If the spores of Nosema apis were present they were readily identified by 

 their shape, size, and refractility. Young, intracellular stages and planonts 

 were also observed in like manner. 



Stained preparations from smears of the colon, and of the chyle- 

 stomach, were made from every sample of bees examined, and the stain 

 which was almost exclusively used was the iron-haematoxylin of Heideinhain. 

 Although requiring longer time for staining, this was found to give the 

 most reliable and most definite results. The fixative used was a hot alcoholic 

 solution of corrosive sublimate. 



Various other stains were tried, such as Giemsa, Romanowsky, Erhlich's 

 triacid, etc., but the varying results led to their abandonment in favour of 

 the more stable hsematoxylin preparation. 



No stain, however, was found which would show up the spores clearly. 

 Various experiments in this connection were tried, but with no success. 

 Heat had very little effect in helping the stain to penetrate the resistant 

 spore capsule. For its identification in stained preparations we could 



