MICROMANIPULATION 



202 



MICROMANIPULATION 



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 

 Tradescantia 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 microfiame 

 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. The 

 shaft about 2 mm. from the tip, is bent 

 in the microfiame 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- 

 manipulator so that the tip extends 

 over the microscope stage into a moist 

 chamber. The bent-up tip is adjusted 

 with the screws of the instrument until 

 the tip lies in a hanging drop of fluid 

 suspended from a glass cover-slip serv- 

 ing as the roof of the moist chamber 

 and in the field of the microscope. 



Mechanical contrivances for drawing 

 out the end of a glass capillary into a 

 tapering tip are available. The most 

 elaborate one is that of de Fonbrune, 

 the microforge. This is a highly am- 

 plified mechanism with a built in micro- 

 scope and coarse adjustment parts for 



holding, one, a glass capillary and the 

 other a platinum loop. The microscope 

 is adjusted to view the loop of an elec- 

 trically incandescing fine platinum wire 

 to which one end of the glass capillary 

 is approached and then withdrawn as 

 the glass begins to melt. Then prop- 

 erly done the tip of the capillary is 

 drawn out to a tapering tip. 



The principle of the microforge is 

 based on a much simpler device long 

 ago devised by S. L. Schouten and de- 

 scribed by him in 1934. This can be 

 readily built in any laboratory equipped 

 with an ordinary microscope and micro- 

 manipulator. 



A fairly good mechanical device for 

 drawing microtips is being supplied by 

 the Gamma Instrument Company. It 

 depends upon springs to which the two 

 ends of a glass capillary (1-2 mm. diam.) 

 are fastened while the middle of the 

 capillary passes through several coils 

 of a fine platinum or nichrome wire. 

 When the wire is caused to incandesce 

 electrically the glass softens and the 

 taut springs pull the capillary in two, 

 each part with a tapering tip. A more 

 precise needle puller based on the same 

 principle is the Livingston Micro Pi- 

 pette Puller made by Otto K. Hebel, 

 Swarthmore College, Pennsylvania. 



There are several possible ways by 

 means of which a person with some 

 ingenuity should be able to devise from 

 the usual laboratory equipment a simple 

 mechanical device for drawing needle 

 tips. If the glass capillary is of tubing 

 the microtips can be used for micro- 

 capillaries. 



Injections are performed bj^ breaking 

 the tip of a micropipette against the 

 undersurface of the coverslip while the 

 tip is in view under the microscope. 

 Capillarity draws fluid into the shaft 

 of the pipette when the open tip is in- 

 serted into a hanging drop of fluid, be 

 it oil or any given solution. For micro- 

 injection, the pipette holder, mounted 

 on the instrument, is attached to a 

 looped, capillary brass tube of which 

 the other end is attached to the nozzle 

 of a syringe. Before mounting the 

 micropipette, the syringe is filled with 

 water previously boiled to be air-free 

 and, by means of the plunger, the water 

 is driven into the brass tubing and the 

 pipette holder after which the micro- 

 pipette is inserted. Thus, we have a 

 water-filled system extending from the 

 syringe to the base of the micropipette 

 the shaft of which may contain air. 

 Micro-amounts of any given solution 

 are then drawn into or ejected from the 

 tip of the micropipette by a delicate 

 handling of the plunger of the syringe. 



