IT appears to be the general consensus of opinion that initial cooling or

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1 25 1 The Initial Cooling of Tissues in the Freezing-drying Technique By I. ZLOTNIK, PH.D., B.V.SC, M.R.C.V.S. (From the Moredun Research Institute, Gilmerton, Edinburgh) With one plate (fig. 2) SUMMARY When a mixture of dry ice and pentane is subjected to high vacuum for one hour the temperature of the mixture is reduced to 125 C. The low temperature of the dry ice/pentane mixture persists for about h and rises only very slowly thereafter. The chilled dry ice mixture when used for the initial cooling of tissues produces good cytological preservation. INTRODUCTION IT appears to be the general consensus of opinion that initial cooling or freezing of tissues must be carried out as rapidly as possible in order to prevent great distortion of tissue structure caused by slow formation of ice crystals. The practice in recent years has been to immerse small pieces of fresh tissue in a liquid such as isopentane cooled to about 165 C by means of liquid nitrogen (Hoerr, 1936). More recently, however, Bell (1952) stressed the importance of still lower temperatures and advocated the use of propane cooled to C instead of isopentane. Lacy and Davies (1959) objected to isopentane on the grounds that its mixture with liquid nitrogen is explosive and suggested the use of freon-12. The above variations concern the cooling liquid, while the use of liquid nitrogen was accepted. In the present writer's experience, however, it is the liquid nitrogen that presents the major difficulty, especially for routine work when a number of blocks of tissue have to be removed from an animal and chilled in quick succession. Apart from the actual dangers from the use of liquid nitrogen, the temperature of the cooling mixture is very variable and rises very rapidly with the quick volatilization of the liquid gas. In order to maintain a low temperature, repeated addition of liquid nitrogen is usually required, a procedure which is very tiresome; and even so the rapid transfer of the quickly frozen pieces of tissue to the freezing-drying apparatus is still necessary in order to prevent their thawing. In view of the difficulties involved in the routine use of liquid nitrogen mixtures a method has been evolved in which the very low but rather unstable temperature of the liquid nitrogen mixtures has been replaced by a higher temperature maintained for a much longer period. The results obtained have been compared by cooling pieces of tissue from the same organ in the following three ways: (a) liquid nitrogen; (b) mixture of isopentane and liquid nitrogen; and (c) CO 2 /pentane mixture. Histological examination of sections proved [Quarterly Journal of Microscopical Science, Vol. 101, part 3, pp , Sept. I960.]

2 252 Zlotnik Initial Cooling in Freezing-Drying that while liquid nitrogen by itself caused considerable distortion of tissue, especially in tubular organs such as kidneys, the CO 2 /pentane mixture produced preservation of tissue as good as the isopentane / liquid nitrogen combination. In the large neurones of the brain, however, the distortion of the Nissl substance was less marked in the CO 2 /pentane mixture than in isopentane chilled with liquid nitrogen. PROCEDURE AND RESULTS The effect of initial cooling and subsequent freezing-drying was studied on various tissues from 12 sheep, 3 guinea-pigs, 3 rabbits, and 3 mice. The animals were killed by complete bleeding and the organs were removed in quick succession. The following organs were usually taken: brain, spinal cord, pituitary gland, thyroid, adrenal, pancreas, kidney, liver, spleen, and lymph glands. Pieces of tissue varying in length from 2 or 3 mm to 10 or 20 mm and about 2 mm in thickness were cut with a very sharp knife from each organ immediately after its removal from the carcass, and were immersed in the cooling liquid. As a rule the whole procedure of destroying the animal, removing the required organs, cutting out about 30 pieces, and initial freezing them did not exceed 25 min. The actual initial cooling was carried out in pentane (boiling-point 30 0 to 40 0 C), chilled to C by means of CO 2 /dry ice in the following apparatus. A wide-mouthed vacuum jar, 210 mm high and 90 mm in diameter, was filled with finely broken 'dry ice' (CO 2 ), to about 40 or 45 mm from the bottom. A cylinder of perforated zinc, 65 mm high and 40 mm in diameter, was placed on top of this, and the space between it and the wall of the vacuum jar was filled with finely broken dry ice (fig. 1). Enough pentane was then poured in to damp all the dry ice and to fill the perforated zinc cylinder. The vacuum jar with its contents was next placed in the large drying tube of the Edwards Bone-and-Artery 'Freeze-Dryer', model BA2, and subjected to high vacuum for 1 h. After the pump had been stopped and the vacuum jar removed from the drying tube, blocks of tissue placed on tinfoil were very rapidly immersed in the pentane inside the perforated zinc cylinder. The temperature of the pentane ranged between C and C and remained so for about 10 to 12 min, and thereafter rose by about i C every 3 or 4 min. On the average the temperature of the pentane after \ h (the vacuum flask being kept on the bench at room temperature of 22 0 C) was C, after 1 h 112 C, and after 80 min C. After the initial cooling frozen blocks were transferred to small tissue baskets partly filled with solid CO 2, and these were placed in chilled glass specimen tubes situated inside the drying tube of the Edwards BA2 at 30 0 C for subsequent freezing-drying. It is worth noting that the Edwards BA2 was slightly modified by the fitting of an additional moisture trap mounted above the rotary pump, and of an isolation valve in the pumping line. This enabled P 2 O 5 to be renewed in the additional moisture trap without breaking the vacuum. Depending on the size and number of the tissue blocks, the drying

3 FIG. 2 I. ZLOTNIK

4 Zlotnik Initial Cooling in Freezing-Drying 253 time varied between 48 and 72 h. After freezing-drying it has been found to be an advantage to place the thoroughly frozen-dried blocks in a desiccator at FIG. I. Vacuum jar for initial cooling of tissues. room temperature in vacuum over calcium chloride for a further 1 or 2 days. Embedding was carried out in a vacuum bath at 56 C for 4 min. Sections were cut on an ordinary rotary microtome and mounted on slides either dry for fluorescent microscopy or on 3 % formalin solution in 1 % calcium chloride for cytology. Various staining techniques have been used for testing the efficiency of the freezing-drying procedure, but it was found that staining for mitochondria by the method described by Chang (1956) was the most reliable. The results obtained with all the tissues proved very good and consistent. FIG. 2 (plate). Sections of tissues, stained by Chang's method. A, convoluted tubule in kidney of sheep. B, adrenal cortex of sheep. c, liver of guinea-pig, showing fatty changes. D, villus of small intestine of mouse.

5 254 Zlotnik Initial Cooling in Freezing-Drying Good preservation of histological detail in blocks measuring up to 8 mm by 12 mm, without the appearance of zones (fig. z, A-D), was the rule. In most organs mitochondria were easily stained and the formation of large ice crystals was not noticeable. Larger blocks of tissue showed less perfect histological preservation, especially in the centre of the section. In the central nervous system good histological pictures were obtained without any perineural shrinkage, but in the very large neurones some distortion of the Nissl substance was obvious. The author wishes to thank Mr. J. C. Rennie for his technical assistance and also Mr. D. Watson for preparing the photomicrographs. REFERENCES BELL, L. G. E., Int. Rev. Cytol., 1, 35. CHANG, J. P., Exper. Cell Res., 11, 643- HOERR, N. L., Anat. Rec, 65, 293. LACY, P. E., and DAVIES, J., Stain Tech., 34, 85.