A methodological study for the quantification and the control of the physico-chemical processes occurring during cell fixation. I. The development of a new technology

Abstract

Fixation in a traditional sense means the immersion of biological material into a chemical fluid. For permanent preservation the fixative is always "offered" (1) in excess of the cell sample, and the process of fixation is influenced by (2) chemical impurities of the fixative fluid. Both factors influence the succeeding dyeing of cells. In order to avoid these uncontrolled criteria, a new technology for controlled cell fixation has been developed, whereby freshly prepared formaldehyde and methanol gas in an "inert" gas-flow of helium was applied to thin membranes by aid of a capillary flow-in technique. The instrumental equipment consists of (1) an ultra-high vacuum flow-apparatus with a total-pressure measuring unit, (2) a gas-supply device, (3) a mass spectrometer including a pump system, and (4) a Teflon and/or glass-gas chamber for the treatment of synthetic (Hostaphan foils) or biological membranes (mesenterium) with formaldehyde as the fixative gas. The amount of "offered", adsorbed, absorbed, diffused, and desorbed fixative gas could be absolutely estimated after the saturation of the membranes with an "on-line" operating "inert" mass spectrometer of the Omegatron type. The gas treatment of the Hostaphan foils with formaldehyde showed that nearly all adsorbed gas molecules could be desorbed. In contrast to native membranes the greatest proportion of the gas molecules adhered to the biological surface, and only a small quantity were desorbable. Physisorption or physisorption and chemisorption occured depending on the adsorber surface property. A monolayer of formaldehyde of 5.10(14) to 1.10(15) molecules per 10(16) A2 surface area can be postulated on the basis of these preliminary results. This value corresponds to a mass of about 5.10(-8) g CH2O. It resulted in an area-coverage ratio of CH2O molecules per cell of 10(9):1. The membrane surface facing the gas side always amounted to 1 cm2. A fixative gas concentration of 10(6) molecules/cm3, and therefore a degree of coverage of less than 1/1000 monolayer can be estimated absolutely. For a precise determination of the degree of fixation, further experiments and the evaluation of additional physico-chemical parameters are necessary.