Effect of fixation on demonstration of phosphatases of Eimeria tenella grown in chick kidney cell cultures.
The effects of fixation with various concentrations of glutaraldehyde or formaldehyde, acetone or ethanol, and freeze-drying on 5 phosphatases of Eimeria tenella and chick kidney cell cultures were demonstrated in situ. Gultaraldehyde inactivated the phosphatases more than did the formaldehyde, but the effect of the combination of the 2 (Karnovsky's fixative) was greater than that of either glutaraldehyde or formaldehyde alone. The higher the concentration of aldehyde and the longer the duration of exposure, the greater the inactivation. The order of sensitivity to aldehyde fixation of the enzymes tested was glucose-6-phosphatase greater than thiamine pyrophosphatase greater than 5'-nucleotidase greater than adenosine triphosphatase greater than acid phosphatase. Cytologic detail was preserved more efficiently with glutaraldehyde than with formaldehyde. Optimal preservation of enzyme activity for cytochemistry was with 2% glutaraldehyde for 30 min or 2% formaldehyde for 1 hr for G-6-Pase, TPPase, and 5'-nucleotidase, and with 2% glutaraldehyde or 2% formaldehyde for 2 hr with ATPase and AcPase. Quenching with subsequent fixation in cold acetone or ethanol resulted in complete inactivation of G-6-Pase, TPPase, and 5'-nucleotidase; although cells fixed in this manner yielded large amounts of reaction product for ATPase and AcPase, the distribution was diffuse, and some of it appeared to be artifactual. Quenching with subsequent freeze-drying was unsatisfactory because nearly all of the cell layers rolled off the cover glasses.
D000096:Acetone / Q000494:pharmacology; D000135:Acid Phosphatase / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004539:Eimeria / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D000431:Ethanol / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D005952:Glucose-6-Phosphatase / Q000378:metabolism; D005976:Glutaral / Q000494:pharmacology; D006651:Histocytochemistry; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D013194:Staining and Labeling; D013834:Thiamine Pyrophosphatase / Q000378:metabolism
D000096:Acetone; D005557:Formaldehyde; D000431:Ethanol; D000469:Alkaline Phosphatase; D000135:Acid Phosphatase; D010744:Phosphoric Monoester Hydrolases; D005952:Glucose-6-Phosphatase; D009699:N-Glycosyl Hydrolases; D000251:Adenosine Triphosphatases; D013834:Thiamine Pyrophosphatase; D005976:Glutaral