In the course of a cytological investigation of mouse ascites tumors ( 1 :2 ) and of neoplastic cells of chickens(3), it was observed that under certain conditions small cytoplasmic granules could be stained with toluidine blue. The appearance and distribution of these granules were identical with those of the mitochondria as revealed by phase microscopy and supravital staining( 2,3 ) . This paper describes a simple stain for mitochondria based on these observations, and presents a study of the chemical basis of the method employed.Experimental. Tissues. The following tissues were used. Ehrlich, Krebs 2B, sarcoma 3 7. MC 1 J1: sarcoma, TA3 carcinoma, lymphoma # I , 6C3 HED and DBA lymphoma mouse ascites tumors (4)' chicken, rat and mouse liver and spleen, rat pancreas and the RPL-12 chicken lymphoma( 5 ) . Staining. 1. a) Prepare smears of ascites tumors or of cells scraped from the freshly cut surface of tissues. Drop the smears rapidly while still wet, face downwards, into 1070 formalin, freshly saturated with HgC12 at 37°C and fix a t this temperature for at least 10 minutes. b) Fix blocks of tissue (less than 3 mm thick) in the same way for 2 or 3 hours; prepare frozen sections ( 6 p thick), float in 70% alcohol, mount on dried, albuminized slides and allow to dry. 2. Wash the slides in tap water for 10 minutes; if necessary, they can be stored in water for several days. 3. Stain for 5 minutes in 0.01% toluidine blue 0 in McIlvaine buffer of pH 6.0. This solution will keep for at least a month. 4. Wash for one minute in distilled water. 5. Immerse in a freshly prepared mixture of equal parts of 5% aqueous ammonium molybdate and 1 a/o aqueous potassium ferrocyanide for 5 minutes. 6 . Wash for one minute in distilled water. The nuclei may be counterstained with carmalum at this stage. Dehydrate in alcohol, clear in xylol and-_ -*The authors wish to thank Marie J. Warford for her valuable technical assistance and Leslie McWilliam for the photomicrographs. mount in Permount. The mitochondria are stained dark blue, the cytoplasm pale blue; and the chromatin pale purple (Fig. 1-3).t Rationale of the method. 1. Fixation. In order to investigate the role of fixation in demonstrating the mitochondria, smears were fixed in the following solutions: 1070 formalin saturated with HgClz (formol sublimate), 5% glacial acetic acid in saturated aqueous HgC12, 10% formol saline, formol Zenker, acetic Zenker, Bouin and Worcester (6) fluids. A few granules resembling mitochondria were stained capriciously only when mercurial fixatives were used; the mitochondria appeared larger and staining was more intense after fixation in formol sublimate. Fixation was sometimes inadequate at room temperature, and the more rapid fixation of smear preparations produced by raising the temperature of the fixative caused the cells to adhere better to the slide. Extension of the time of fixation in formol sublimate led to an increase in the precipitation of mercurial salts, but had no effect on the number and size-of the mitochondria stained, Prolo...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2025 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.