568 PROTOZOA 



Here again it has been found that when a suitable bacterium is 

 present, Dimastigamoeha will grow well on the agar and Peter's 

 saline without the addition of a glycero-phosphate as indicated 

 for Colpidium (see p. 567, footnote). 



The material containing the amoeba to be cultivated is placed 

 on the surface of the agar in a drop of the saline, and the amoebae 

 as they grow will generally move away from this centre of inocula- 

 tion. The bacteria or other organisms present serve as food, so 

 long as they are not deleterious or too numerous. To guard 

 against drying of the surface of the medium — tubes should be 

 covered with a rubber cap or kept in a moist chamber — ^plates are 

 generally inverted and some water placed in the lid of each. 



After a period of division the amoebae encyst. On transference 

 to new media, the cysts will usually hatch and give rise to a new 

 culture, even though they have been kept on the old medium for 

 months or even years. The common coprozoic amoeba, Dimastig- 

 amceha (Ncegleria) gruberi, multiplies under suitable conditions for 

 five or six days, and then encysts. It should be sub-cultured every 

 week or so to prevent overgrowth by other organisms. 



Pure cultures of an amoeba may be obtained from a single 

 active organism or a cyst by removing one of these by the Barber 

 technique {Philippine J. Sci., B, ix, 1914, p. 307). The following 

 simple method, devised by Drew, may be used to start a culture 

 from a single cyst provided it be large enough to be seen with a 

 low magnification. 



1114. Separation of a Single Cyst with a Capillary Pipette. To 

 make the pipette, take a capillary glass tube about 6 cm. long 

 and 1 mm. in diameter. Flame the centre and draw out quickly 

 to the fineness of a hair. Break off the two outer pieces, about 

 5 cm. from each end, to obtain two short pipettes, consisting of a 

 wider portion and a very fine capillary portion. 



Next select a culture of the amoebae, rich in cysts, add a drop 

 of sterile water, and rub a portion of the growth into this with a 

 sterile platinum wire. Allow a minute portion of this emulsion 

 to run into the capillary end of the prepared tube, and then run 

 in sterile water till about 0-5 cm. of the broad portion of the tube 

 is filled. Mix the contents of the tube by vigorous rotation. 



Now prepare an agar film on a microscope slide, by melting 

 the medium in one of the stock tubes and pouring a few drops 

 on to a slide. Allow this to set. Place on the microscope stage 

 and focus the upper surface with an inch objective. Tap out on 

 to filter paper some of the liquid in the capillary tube, and then, 

 whilst looking through tlie microscope, gently touch the film with 

 the fine end of the pipette. A small volume of the suspension of 

 cysts will run on to the jelly and will spread out in an area which 

 is quite visible, and which occupies only a small portion of the 

 field. If no cyst is present, or if there should be more than one, 



