78 



THE ART OF MAKING MICROSCOPE SLIDES 



Hydra 



are required, it is desirable to cut the 

 anthers into pieces before placing them in 

 the fluid. Maceration will usually be com- 

 plete in about eight hours so that it is con- 

 venient to start the preparation in the 

 evening and to examine individual pieces 

 at intervals in the course of the next 

 morning until one has determined that 

 maceration is complete. The completion 

 of maceration may be judged by the fact 

 that the materials should be flabby, but 

 not completely disintegrated. 



At this stage the microsporocytes are 

 extracted by gently squeezing either the 

 anther, or each individual piece of anther, 



into a small drop of water. The micro- 

 sporocytes themselves will not appear to 

 be distinct but will appear as a gelatinous 

 mass. This mass should be accumulated in 

 a drop of water on a single slide and then 

 small drops taken from it and made into 

 smears on other clean slides. These smears 

 need not be fixed, but may be permitted 

 to dry on the shde to which they will ad- 

 here so well that they can be stained by 

 any nuclear staining method. Aceto-car- 

 mine is in general use for temporary prep- 

 arations, and a description of the use of 

 this stain in plant material is given in 

 Chapter 20. 



Preparation of a Dissociated Hydra 



It is a little pathetic that the majority 

 of elementary textbooks of biology should 

 include an illustration showing the types 

 of cell to be found in hydra and that in- 

 structors should then issue to the student 

 a series of sections in which these cells are 

 not visible. The illustrations have mostly 

 been taken from older textbooks dating 

 from the period when disassociation tech- 

 niques for animal tissues were common. 

 It would surely be more reasonable to 

 show students shdes wliich agree with the 

 illustrations in the books they use. 



Fixation is necessary before dissocia- 

 tion, and the only question to be settled is 

 whether or not the finished slide should 

 show muscle cells in an expanded condi- 

 tion, in which case the hydra will have to 

 be narcotized before fixation, or whether 

 it will be sufficient to kill the hydra with- 

 out narcotization. It seems better, how- 

 ever, to show cells in the expanded condi- 

 tion and the hydra should therefore be 

 collected from the tank or pond where 

 they are growing, and accumulated in a 

 watch glass of water which is kept in a 

 cool place in subdued fight so that the 

 hydra may expand. A drop of 2% chloral 

 hydrate is then added for each five milfi- 

 liters of water in the watch glass. This 

 should be mixed with the water by suck- 

 ing in and out of a pipet. This is likely to 

 cause partial retraction of the hydra but 

 they will expand again. After about 10 

 minutes two or three more drops per five 

 milUliters of water may be added and 

 mixed in, as the hydra are usually by this 

 time sufficiently narcotized not to con- 



tract. The hydra should then be watched 

 until touching with a hair causes no retrac- 

 tion. The watch glass is then very care- 

 fully picked up and placed in a fingerbowl. 

 The reason for this is that hydra can most 

 satisfactorily be fixed in large cjuantities 

 of hot fixative. The solution of Perenyi 

 (Chapter 18, F 6000.0040 Perenyi 1888) is 

 excellent for this purpose. Tliis fixative, 

 which has few other uses, is made by dis- 

 solving % of a gram of chromic acid in 135 

 milHliters of water and then adding to 

 this 100 millihters of ethanol. Seventeen 

 and a half millihters of strong nitric acid 

 are then added and the solution placed on 

 one side until it has turned violet. The 

 fixative should be heated to 70°C. and 

 then flooded suddenly over the narcotized 

 hydra which should remain in the fixative 

 for about two days before being removed 

 for dissociation. An individual dissociated 

 hydra may be prepared as a smear, but it 

 is presumed in this case that a number of 

 shdes are being made for class issue, so 

 that it is better to proceed by a different 

 technique. The hydra are taken from the 

 fixative (it is unnecessary to wash them) 

 and placed in a few drops of the selected 

 dissociating agent in the bottom of a small 

 tube. The selection among the acid, dis- 

 sociating media given in Chapter 19 under 

 the heading AF 41.1 is not important, but 

 the writer has been successful in the pres- 

 ent preparation by the method of Hopkins 

 as quoted by Roberts [Chapter 28, VJ41.1 

 Hopkins (1895)]. This method requires 

 first, 20% nitric acid, second, a saturated 

 solution of potassium alum. 



