MICROMANIPULATION 



153 



MICROMANIPULATION 



phatic vessels of frog tadpoles, are as 

 follows: H. McE. Knower, Chapter in 

 McClung's Handbook Microscopical 

 Technique, New York: Hoeber, 1937; 

 A. L. Brown, Anat. Rec, 1922, 24, 295. 



Micromanipulation in its more re- 

 stricted sense applies to the use of 

 special devices for controlling the move- 

 ments of the tips of microneedles and 

 micropipettes in the field of high powers 

 of the compound microscope. A full 

 account is given in McClung's Hand- 

 book. 



Several instruments are now being 

 built. The ones in most general use in 

 this country are those of Chambers, 

 P^terfi and Emerson, although others 

 are first-class. The essential condition 

 of an instrument is that the movements 

 be sufficiently smooth and controllable 

 under the highest magnifications of the 

 compound microscope. De Fonbrune 

 of Paris has recently produced an im- 

 proved form of the one already de- 

 scribed in McClung's Handbook. 



Micrurgical instruments lend them- 

 selves to several types of operations: 

 (1) Microdissection and injection of 

 animal or plant cells and tissues for 

 studies in cell anatomy and physiology, 

 also cy to -chemistry in which chemical 

 reactions can be obtained by applying 

 chemical agents not only to individual 

 cells but to localized regions within a 

 given cell. (2) Chemical reactions in 

 micro-drops. A very useful method is 

 to deposit the droplets with a micro- 

 pipette in a drop of an inert oil. This 

 prevents evaporation and the sphericity 

 of the droplets permits quantitative 

 determination. Application of the 

 technicjue to certain phases of micro- 

 chemistry are given by Benedetti- 

 Pichler in his book. (3) Isolation 

 studies for obtaining pure line cultures 

 (of bacteria, protozoa, etc., breaking of 

 asci and isolation of the liberated 

 spores, etc.). A good isolation tech- 

 nique is given by Reyniers, J. A., J. 

 Bact., 1933, 26, 251. 



The movements of the instruments 

 can be controlled in any of three dimen- 

 sions; the horizontal permits circus 

 movements in one plane. Circus move- 

 ments are best managed with the de 

 Fonbrune and Emerson instruments. 

 The vertical movement is operated by 

 a separate controlling screw. Since 

 the operations are performed under a 

 single high-power objective, the only 

 criterion for ascertaining different 

 levels is whether the object being oper- 

 ated on lies above, below or in the focus 

 of the objective. 



The manufactured instruments are 

 supplied with instructions as to their 



use. Emerson supplies two types, one 

 which is cheaper for coarser move- 

 ments although it is possible to use the 

 cheaper model for remarkably fine 

 operations. The only way to select an 

 instrument is to know what is wanted 

 and then to decide after having the 

 instrument demonstrated to him. All 

 require the use of a good mechanical 

 stage to move the moist chamber which 

 carries the drops containing the tissue 

 to be operated on. All in all, micro- 

 manipulation requires not only ability 

 but mechanical aptitude on the part of 

 the would-be operator. It is one thing 

 to have an instrument and a good micro- 

 scope. It is another matter to build 

 the many accessories, with cement, out 

 of wood, glass or plastic, which the 

 operator may need for his special pur- 

 poses. Any gadget built may well 

 mean a new discovery. 



Tissues and cells to be operated on 

 often require special means for holding 

 them in place. Actively moving pro- 

 tozoa can be kept quiet by immersing 

 them in egg albumen or a solution of 

 hemi -cellulose. Strips of the epidermis 

 of onion or tulip bulbs, immersed in 

 varying concentrations .of cane sugar, 

 offer good objects for operation on pro- 

 toplasts under different degrees of 

 plasmolysis, likewise stamen hairs of 

 Tradescautia which show mitotic 

 figures. Similar studies may also be 

 made on the epidermis of the tails of 

 tadpoles. For these, the operator 

 should use frogs' Ringer solution to 

 maintain the proper balance of elec- 

 trolytes in the medium. Muscle fibers 

 stripped from the semitendinosus of the 

 frog are good material. Urodeles fur- 

 nish excellent material. An effective 

 means of obtaining red cells undergoing 

 mitosis is to bleed a Necturus or other 

 member of the same order and take a 

 sample of blood after a week or so. 



The microneedles and micropipettes 

 are usually made from glass capillary 

 rods or tubes. Serviceable sizes with 

 an outside diameter of 1-2 mm. can be 

 drawn out in a bunsen flame. The 

 needle tips are made over a microflame 

 by heating and pulling the shaft of a 

 capillary held at both ends with the 

 two hands. A serviceable gas micro- 

 burner for this purpose is a hypodermic 

 needle. When successful, the drawn- 

 out tips taper to a point rapidly enough 

 so that the invisibly, fine tip is sup- 

 ported on a relatively rigid shaft. Tne 

 shaft about 2 mm. from the tip, is bent 

 in the microflame to about a right angle. 

 The other end of the capillary is then 

 inserted into a specially constructed 

 needle-holder and mounted in a micro- 



