1 70 Phylum N emathelminthes 



Order telogonia, Family ancylostomidae 



THE GROWTH OF HOOKWORM LARVAE ON PURE 

 CULTURES OF BACTERIA* 



OVA of the dog hookworm, Ancylostoma caninum, have been ob- 

 tained free from feces and sterilized. These sterile ova have been 

 inoculated onto agar cultures of various bacteria, and the larvae have 

 hatched normally and grown to the infective stage with bacteria as their 

 sole source of food. 



The method employed for freeing the ova from the feces consists in 

 thoroughly mixing up about 25 grams of freshly passed feces from a 

 heavily infested dog in 500 cc. of water. The mixture is then washed 

 through a series of copper-wire sieves ranging up to a mesh of 100 wires 

 to the inch. The larger particles in the feces are caught in the sieves but 

 the ova readily pass through with the filtrate. This filtrate is allowed to 

 stand in a large sedimenting cone for about an hour while the ova and 

 heavy debris settle to the bottom. The supernatant fluid is then poured 

 off; the sediment is transferred to a 50 cc. centrifuge tube and repeatedly 

 washed with water, the solid matter being thrown to the bottom each 

 time by centrifuging at a speed of 1,000 revolutions per minute. After 

 the supernatant fluid from the washing has become practically clear, 

 saturated salt solution is poured into the tube and the contents are again 

 centrifuged at the same speed. This time the ova come to the surface and 

 may be collected by removing the surface film with the open end of a 

 piece of large glass tubing. If the material is centrifuged four or five 

 times, a majority of the ova present may be recovered. If much solid 

 material comes to the surface with the ova, it may be necessary to refloat 

 the ova in saturated salt solution a second or even a third time in order 

 to get rid of the foreign material. This method is tedious and time- 

 consuming, but if the feces of a heavily infested dog are used, large quanti- 

 ties of ova may be obtained almost entirely free from fecal material. 



Ova collected by this method may be sterilized by treatment with a 

 5% antiformin solution in 10% formalin. From 10 to 50% of the ova 

 remain viable after this treatment. The ova are washed several times 

 with sterile distilled water and are then ready for inoculation onto the 

 agar cultures. During the process of sterilizing and washing, the ova 

 are best kept in a sterile 50 cc. centrifuge tube closed with a cotton plug. 



Cultures were made up in 250 cc. Erlenmeyer flasks stoppered with cot- 

 ton plugs, and consisted of 20 cc. of ordinary bacteriological agar which 

 had been diluted with three parts of water. The flasks were autoclaved 

 and inoculated with bacteria 24 hours before the ova were introduced. 



* Reprinted, with slight changes, from Science 69:74, 1929, by Oliver R. McCoy, 

 School of Hygiene and Public Health. 



