Gray's method 



WHOLEMOUNTS IN RESINOUS MEDIA 



67 



Preparation of Minute Fresh- water Organisms by the Method of Gray 1932 



The technique here given, which is 

 abridged from the original description of 

 Gray 1932 (11360, 52:370), permits one 

 to prepare permanent mounts of individ- 

 ual microscopic organisms. It may be used 

 equally well to mount an individual i)ro- 

 tozoan or an individual alga. It consists 

 essentiall}' of utiUzing a special fixative 

 which renders a layer of albumen on the slide 

 intensely sticky so that the selected object, 

 immediately after fixation, adheres to it. 



The range of possible application of this 

 method is very wide, the notable excep- 

 tions to its use being for rotifers, stalked 

 ciliates (wliicli cannot satisfactorily be 

 narcotized), and nematode worms which 

 cannot be mounted in balsam without 

 great distortion. Almost anything else can 

 be mounted, provided that its size lies be- 

 tween a total length of about three milli- 

 meters, and that of the smallest object 

 which can be seen under a wide-field bi- 

 nocular microscope. 



The reagents required, wloich can most 

 conveniently be kept in drop bottles, are 

 70% alcohol, ether, 40% formaldehyde, 

 glacial acetic acid, and the stock fixative 

 solution (Chapter 18, F 3500.1010 Gray 

 1932) which consists of 1% each of picric 

 acid and mercuric chloride in 90% alcohol. 

 Before commencing to mount a series of 

 specimens, it is also necessary to have 

 some Mayer's albumen (Chapter 28, V 

 21.1 Mayer 1889), some clean shdes, sev- 

 eral small specimen tubes or vials for the 

 preparation of the fixative, some' strips of 

 filter paper, a writing diamond, and, 

 lastly, one or more coplin jars of 70% alco- 

 hol in which the mounts may be accumu- 

 lated as they are made. 



The required fixative is made up in 

 small quantities according to the speci- 

 mens which one desires to mount. When 

 examining a sample of water without 

 knowing what to expect, it is as well to 

 accumulate three mixtures, each for spe- 

 cific organisms. These are: 



A. for 'protozoans 



stock fixative 10 



ether 3 



acetic acid 2 



40% formaldehyde 5 



B. for heavily cidicularized forms (e.g. 

 Gastrotricha) 



stock fixative 10 



ether 1 



acetic acid 4 



40% formaldehyde 5 



C. for delicate larvae {e.g. Miracidia) 



stock fixative 10 



ether 2 



acetic acid 1 



40% formaldehyde 5 



The parts given are by volume and the 

 author usually uses drops in making up 

 these mixtures since only a very small 

 quantity is required even in making many 

 slides. 



Half a dozen clean slides are now taken 

 and smeared with a quantity of Mayer's 

 albumen in the center of each. The layer 

 should be considerably thicker than that 

 which would be applied were one prepar- 

 ing to attach paraffin ribbons. The collec- 

 tion is now examined and any small object 

 which it is desired to mount is taken up 

 in a pipet with the least possible quantity 

 of water and placed on the patch of 

 albumen, beyond the limits of which the 

 water should not run. The surplus fluid 

 is then drawn off, sufficient, however, be- 

 ing left for the animal to swim naturally. 

 The animal is watched until it is in a fairly 

 normal position and a large drop of fixa- 

 tive then allowed to fall on it from above. 

 Immediately the fixative has reached the 

 water, diffusion currents of almost explo- 

 sive intensity result, and considerable care 

 nuist be taken to keep the rapidly moving 

 object within the field of the dissecting 

 microscope. If the object leaves the area 

 of albumen, it must be guided back with a 

 fine glass needle, the point of which will 

 collect a capillary droplet of the fluid. 

 When the animal is in the desired position 

 over the albumen smear, all surplus fluid, 

 which by now will have collected into 

 drops moving slowly over the surface 

 of the slide, is removed l)y the filter |)aper. 

 The object is now closely watched under 

 the dissecting microscope until the droplet 

 of fluid, which will have collected round it, 

 has so far evai)orated as clearly to show 

 the outlines of the object. When evapora- 



