Freeze-dehydration for permanent mounts after aceto-orcein stain.
Fresh smears of mammalian material were stained 5 min (only) by La Cour's (1941) aceto-orcein method at 60°C, briefly rinsed and drained, and then dehydrated in a prechilled equal-parts mixture of absolute alcohol and anhydrous acetone held 2 hr or more at — 20°C or lower by means of dry ice. The preparations were differentiated 30 sec at room temperature in 95% alcohol containing 1% HCl, counterstained (optional) in a 0.01% solution of fast green FCF in absolute alcohol for 10 sec, passed through 2 changes of xylene and mounted in HSR or other synthetic resin.
Enhanced differentiation of Zaprionus chromosomes by prestaining acid hydrolysis.
Two variations of orcein staining have been adapted to salivary gland chromosomes of Zaprionus. Method I: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, stained with 2.5% orcein in 60% acetic add for 15-20 min, and squashed in 60% acetic acid. Method II: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, transferred to a saturated solution of carmine in 45% acetic acid for 1 min, then to a mixture of 50 ml of 1% orcein in concentrated lactic acid and 50 ml of 30% acetic add for 5 min. They are squashed in the same mixture. The unproved differentiation of chromosomes from cytoplasm is attributed to the removal of cytoplasmic ribonucleic add by add hydrolysis.
Methods for the study of the cytology of bacteria and pleuropneumonia-like organisms.
Methods for the demonstration of the various cytological elements of bacteria are described. The specialized techniques applicable to pleuropneumonia-like organisms and L-forms of bacteria are also presented.