The morphological changes occurring when living cells are fixed in neutralized formaldehyde have been studied in detail with phase-contrast microscopy. The cells used were (i) salamander spermatogonia obtained from the teased testis, and (2) ssnail amoebocytes growing in tissue culture. The cells were mounted on a slide beneath a coverslip ringed with paraffin wax. Various strengths of formaldehyde made up in saline or distilled water were then introduced while the cells were kept under constant observation by phase-contrast microscopy. The morphological changes during the fixation process were observed for periods of at least 24 hours and the results recorded photographically.
The main changes observed with aqueous formaldehyde were:
A. Cell swelling or shrinkage. In general (e.g. with 5 per cent, formaldehyde) the cell tended to undergo (1) an initial short period of shrinkage, (2) a period of re-expansion followed by swelling, (3) a period of secondary shrinkage. The initial shrinkage appeared less in the amoebocytes than in the spermatogonia, but otherwise their volume changes were fairly similar. If the strength of formaldehyde was below 5 per cent, the initial shrinkage was very slight and subsequent swelling great. With 1 per cent, formaldehyde, sudden collapse of the cell followed swelling. With formalde-hyde concentrations above 10 per cent, the initial shrinkage was greater and was followed by little or no swelling.
B. Formation of ‘bubbles’ from the cells. Clear bubble-like structures often emerged from the spermatogonia during fixation. They were most frequently formed in 5 per cent, formaldehyde. Increasing the strength of the formaldehyde decreased both the number and size of the ‘bubbles’. It is suggested that they may represent an escape of substance through a damaged cell boundary. Similar bubble-like swellings formed in the amoebocytes, but they usually seemed to remain within the cell processes.
C. Nuclear changes. Changes in the size of the nucleus ran approximately parallel with those of the cell, but tended to be somewhat less and with different time relation-ships. With swelling the nucleoplasm became more homogeneous and with gross swelling the heterochromatic bodies disappeared. After prolonged fixation, when the nucleus may have undergone secondary shrinkage, pre-existing nuclear opacities became denser and new opacities sometimes appeared in previously homogeneous regions. Bubbles sometimes emerged from the nucleus.
D. Changes in cytoplasmic structure. In general with prolonged fixation a fine granularity or reticular opacities formed in previously homogeneous cytoplasm. Clear vacuoles also appeared in the cytoplasm after fixation in the more concentrated solutions. The cytoplasmic inclusion bodies were usually well preserved and their appearance little altered.
With formaldehyde made up in saline as opposed to water the initial shrinkage was increased and the subsequent swelling reduced. This effect was most pronounced with dilute formaldehyde. The addition of saline seemed to have little influence on changes in nuclear and cytoplasmic texture, and bubbling, though less in degree, still occurred.
The significance of these observations is discussed in the light of modern views on the physico-chemical action of formaldehyde.
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