MICROINCINERATIONi 



152 



MICROMANIPULATION 



devices for frozen-dehydration are not 

 available, fixation by absolute alcohol 

 plus 10 per cent formalin yields reason- 

 ably good pictures. This particular 

 fixative is one of the few chemical mix- 

 tures which dissolves the minimum of 

 mineral from fresh tissue and adds none 

 to it. Tissues are passed through re- 

 peated changes of absolute alcohol to 

 dry them and are then infiltrated with 

 paraffin in the usual manner. 



The alternative method, that of 

 frozen-dehydration, is the most suit- 

 able for preparation of tissues for micro- 

 incineration. (See Altmann-Gersch 

 and Cryostat.) This technique j^ields 

 tissues which, except for the ice crystal 

 formation, have not been altered, to 

 any perceptible degree, either physi- 

 cally or chemically. Dehydration at 

 sufficiently low temperatures maintains 

 an ice-salt equilibrium and no shifting 

 of minerals in the cell results. If the 

 paraffin infiltration is done with care, 

 shrinkage and consequent cellular dis- 

 tortion is avoided. 



Methods of examination of the in- 

 cinerated preparations are several. 

 One of the simplest and best for studj' 

 and for photography is the dark-field. 

 Of the several types of dark-field, the 

 cardioid condenser probably gives the 

 most uniform results. Illumination 

 from above with the incident light fall- 

 ing on the slide at an angle of 30° is 

 advised by PoUcard. This has some 

 advantages over the dark-field but 

 makes the use of higher magnifications 

 diflacult if not actually impossible. 

 Cellular details are, therefore, to be 

 observed best by using the cardioid 

 dark-field. 



Identification of minerals . Some good 

 results can be achieved by the use of 

 ultraviolet light and with the subse- 

 quent fluorescence of minerals. Stu- 

 dents should consult reference and text- 

 books on mineralogy for details of 

 identification. 



Calcium and magnesium are char- 

 acterized in the dark-field by their 

 dense white ash residues. Iron is oxi- 

 dized during the incineration process 

 and appears as varying tints of red. 

 The amount of this element present can 

 be correlated with the color intensity. 

 Silicon is definitely crystalline in char- 

 acter and is recognizable by its prop- 

 erty of double refraction in polarized 

 light. This may at times be confusing 

 since all minerals blend to some extent 

 with the glass. Lead and other ele- 

 ments which yield black sulfides can be 

 detected by treating the section with 

 gaseous hydrogen sulfide. Uranium in 

 pathological tissues fluoresces with a 



unique color under ultraviolet radia- 

 tion. 



Attempts have been made to quanti- 

 tate the ash residue by photographic 

 means and by the use of a photoelectric 

 cell whose output current is properly 

 amplified. Both methods leave much 

 to be desired both in accuracy and be- 

 cause of the utter relativity of the re- 

 sults obtained. The most useful find- 

 ing obtained from microincineration, 

 therefore, is the appi-eciation of the 

 distribution of the total minerals in the 

 cell. Experimental alterations in them 

 can be detected by the technique. See 

 account by Scott in McClung's book 

 and Electron Microscope, Histospectrog- 

 raphy and Ultraviolet Photomicrog- 

 raphy. 



Microinjection. This is an important exten- 

 sion of microdissection whereby various 

 fluids are injected directly into the 

 cytoplasm or nuclei of living cells. It 

 is capable of yielding information on 

 Permeability, Hydrogen Ion Concen- 

 tration, Oxidation-Reduction Poten- 

 tial which cannot be secured in any 

 other way, but in reaching conclusions 

 due allowance must be made for the fact 

 that cells thus treated are of necessity 

 severely injured. Microinjection with 

 glass pipettes but without an expensive 

 micromanipulator can yield worthwhile 

 results as described by Knower (Mc- 

 Clung, pp. 51-61) but for direct work on 

 cells the micromanipulator is essential. 



Micromanipulation. — Written by Dr. Robert 

 Chambers, Dept. of Biology, Washing- 

 ton Square College of New York Uni- 

 versity, New York, October, 1946 — 

 Broadly speaking, this term covers two 

 sorts of operation: delicate free-hand 

 operations in which the only accessory 

 is a dissecting microscope, and, second, 

 operations conducted by means of 

 micrurgical instruments. 



Considerable training is required in 

 using a compound microscope for free- 

 hand operation because of the inversion 

 of the image. This, however, has been 

 corrected by using the so-called erect- 

 ing ocular. A decided help to relieve 

 fatigue from too long holding of, a dis- 

 secting needle, for instance, is to have 

 the shaft of the needle held in the apex 

 of a pyramid of plastic clay, the base of 

 which has been pressed down on the 

 stage to one side of the microscope. 

 The operator's hand encircles the 

 mound of clay which bends as his fingers 

 guide the needle. The tiring fingers 

 can be released at any time while the 

 needle tip remains in position. De- 

 scriptions of excellent methods for in- 

 jecting minute vessels, such as the 

 marginal vein of chick embryos or lym- 



