11 



II. On a Ciliate. Blepnarisma, Spirostomum or Paramecium. 



A. Successful handling of a ciliate calls for a much smaller drop than 

 was used for an amoeba, since the ciliate moves so rapidly. The smaller the 

 drop the greater the ease in catching one. . 



1. Impale and cut animals against the cover slip. 



2. Attempt cutting animals against the water film. This is done by- 

 raising the needle into the drop above. the animal and lowering it by a quick 

 movement when the animal swims directly beneath it. Which of these ciliates 

 cuts with the greatest ease? 



III. Human tissue cells. 



A. Mount, separate and tear cells obtained from the lining of the cheek. 



1. Compare with other cells as to ease of cutting and reparability. 



2. Are contiguous cells easily separated? 



MICROINJECTION EXPERIMENTS 



I. On Amoebae. 



A. Amoeba dubia. 



1 * A typical oil - Olive oil . Fill a pipette which has been bent 

 nearly to a right angle with oil by the method described above for aqueous 

 solutions. By means of the mechanical stage, center a firmly attached amoeba 

 over the pipette tip. Focus on the under surface of the organism. Slowly bring 

 up the pipette; pierce the under surface and slowly expel a drop of oil of 

 about the diameter of the nucleus. Withdraw the pipette quickly and smoothly. 

 Observe the following points: (a) the immiscibility of the oil with the proto- 

 plasm and (b) the circulation of the globule. (See references number 1 and 2). 



2 * Injec tion of a salt solution ; Fill the pipette with some M/50 CaCl? 

 solution. Inject successively increasing amounts of solution beginning with a 

 volume equal to that of the nucleus. Observe: In what striking way does the 

 reaction differ from that of the injection of pure water? (See reference number 

 v j p • o ( 9 y • 



5 * Injection of pure w ater into dyed amoeba : Isolate a few amoebae 

 in 10 cc of spring water, in Syracuse watch glass and add a drop of 1% neutral 

 red solution. Allow to stand for 5-10 minutes. Transfer into the usual hang- 

 ing drop for operation. Fill the pipette with glass distilled water and inject, 

 notice that the injected liquid disperses the granules at the site of injection 

 leaving a clear zone. This dispersal of particles may be used as a criterion of 

 volume and rate of injection. Vary the quantity of water injected - from a volume 

 equal to that of the nucleus to a volume one-half as large as that of the amoeba. 

 Observe the effects following the injection of increased quantities of water. 

 (bee reference number 3, p. 376 and reference number 4). 



... 4# I njection of pure water into undyed amoeba : Repeat the experiments 

 outlined m section 3 until you gain facility in estimation of injection volumes. 

 , . .. 5 * ^.lectio n of an indicator dve . Phenol Red. Fill the pipette with the 

 dye in the usual manner. Expel a drop on the cover slip and note whether there 

 is a difference in color between the expelled drop and the large drop of dye. If 

 the expelled drop is bluish, it indicates that free alkali has been picked up 

 from the walls of the pipette. If so, empty and fill until no color change is 

 observed in the droplet. Inject as above, gradually increasing the amount. (See 

 reference number 5). This dye has a range of from pH 6.8 - 8.6, with one turnin< 

 P k+ 7° m yellow to oran g Q a* about 6.8, and another from orange to pink at 

 about 7.9. Observe whether the dye permeates the cytoplasm; is picked up by any 

 cytoplasmic inclusions; diffuses through the plasma membrane; or undergoes any 

 color chanees. ' 



