A selective and simultaneous staining method for nuclear apparatus and cytoplasmic membrane of some bacteria has been presented. Nuclear apparatus is stained with basic fuchsin after hydrolysis with I N HCl and cytoplasmic membrane is restained with Victoria blue 4R after treating with saturated mercuric chloride. By this method, the nuclear apparatuses of B. subtilis, Sal. typhi 57 and Staph. aureus were stained red, and the cytoplasmic membrane and septum bluish purple distinctly. Thus this staining method would be of a great advantage in displaying the cellular structures of the bacteria.</p

Kanemasa, Yasuhiro

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Acta Medica OkayamaVolume 16, Issue 1 1962 Article 5FEBRUARY 1962Selective staining of cytoplasmic membraneand nuclear apparatus of bacteriaYasuhiro Kanemasa∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Selective staining of cytoplasmic membraneand nuclear apparatus of bacteria∗Yasuhiro KanemasaAbstractA selective and simultaneous staining method for nuclear apparatus and cytoplasmic mem-brane of some bacteria has been presented. Nuclear apparatus is stained with basic fuchsin afterhydrolysis with I N HCl and cytoplasmic membrane is restained with Victoria blue 4R after treat-ing with saturated mercuric chloride. By this method, the nuclear apparatuses of B. subtilis, Sal.typhi 57 and Staph. aureus were stained red, and the cytoplasmic membrane and septum bluishpurple distinctly. Thus this staining method would be of a great advantage in displaying the cellularstructures of the bacteria.∗PMID: 14030606 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 16, 33-36 (1962)SELECTIVE STAINING OF CYTOPLASMIC MEMBRANEAND NUCLEAR APPARATUS OF BACTERIAYasuhiro KANEMASADepartment of Microbiology, Okayama University Medical School, Okayama(Director: Prof. S. Murakami)Received for publication, January 10, 1962"The submicroscopic structure of bacterial cell has been revealed in detailwith the development of the technique in electron microscopy. And cytochemi-cal observations are contributing in revealing the chemical constituents of eachpart of bacteria, but we have not any satisfactory methods to stain the nuclearapparatus and cell membrane selectively which is required first to distinguishbacterial strain under light microscope.For this purpose attempts were made by some investigators. GUSTAIN 1reported a method to stain the nuclear apparatus of Staphylococcus, Pneumo-coccus, mould, etc. Besides, the simultaneous staining method of both ofnuclear apparatus and cell wall was successfully achieved by CASSEL2, and thisstaining method is generally used because of its usefullness in studying themorphologic structure of bacteria. ROVINOWs attempted to stain selectively thecytoplasmic membrane of Bacillus megatherium by some decoloration pro-cedure after the nuclear staining.So, the author tried to stain the cytoplasmic membrane and nuclear appar-atus of bacterial cells simultaneously but more distinctly. In this paper the resultsobtained by staining a few strains of bacteria with basic fuchsin and Victoriablue 4R are demonstrated.MATERIALS AND METHODSB. subtilis, Sal. tYPhi 57 and Staph. aureus in stock served as ma-terials. These organisms were grown on nutrient agar plate. After 12-hourculture a small agar block, on which the bacteria grew forming a thin film, wascut off 5 mm. in size placed on a glass slide, cells up, and then kept in a closedvessel. These were exposed to osmic tetraoxide vapor for 2 min. at roomtemperature by placing a few drops of 2 per cent OS04 solution into the vessel.This fixed bacterial cell film was then imprinted on a slide glass to make a fixedcell film. The fixed films were well dried up. Then the films were hydrolyzedin 1 N HCl at 60° C for 7 min. and washed with water gently. The films were331Kanemasa: Selective staining of cytoplasmic membrane and nuclear apparatusProduced by The Berkeley Electronic Press, 196234 Y. KANEMASAtreated with saturated mercuric chloride solution for 5 min. and stained againfor 30 sec. in 0.05 per cent Victoria blue 4R. The films were gently washed inwater, mounted in water with a cover slide and examined under an oil immer-sion lens.OBSERVATION AND DISCUSSIONOn the specimens stained with the above-mentioned method, nuclear ap-paratus appeared red being stained by basic fuchsin and cytoplasmic membranepurple by Victoria blue 4R in all strains of the bacteria examined. Observationsreconfirmed the fact described by ROBINOW and otherss that nuclear apparatuseswere stained by basic dyes. Generally, the bacteria are stained red homogene-ously by basic fuchsin. Bacterial cells contain a quantity of DNA and RNAby which the cells are stained uniformly by basic dyes making the localizationof nuclear apparatuses indistinguisable. The result shows that RNA should beremoved to see the nuclear apparatus distinctly by staining with basic dyes.Hydrolyzation by 1 N HCl for 7 minutes at 60 0 C was enough to remove RNAfrom the cells as reported by PIEKARSKI4• The author obtained satisfactory resultsby this hydrolysis on all organisms used in this study. Hydrolysis by chilledperchloric acid, Cassel's method, was also studied and results were as good aswith HCl.Cytoplasmic membrane of bacteria can be stained by Victoria blue 4R asreported by ROVINOW and MURRA yS. This dye belonging to those of phenolmeth-ane group has some affinity to the lipids, probably the phospholipids and stainscytoplasmic membrane, but not the cell wall. But with the lapse of time thedye arrested at the cytoplasmic membrane diffuses into the cytoplasm makingthe picture of the membrane ambiguous. Pretreatment with saturated mercuricchloride solution can appreciably slow down the diffusion of the dye. Watermounting offers an advantage in this process because of the rapidity in observa-tion.Photo 1 shows the picture of B. subtilis which has been stained by themethod mentioned, and two to four nuclear apparatuses can be distinguished ina cell. Also the migration process of nuclear apparatus in division can clearly berecognized. The cytoplasmic membrane is stained bluish purple. The transverseseptum appearing on a dividing cell can also be observed as the similar pictureas the cytoplasmic membrane. Photo 2 is the picture of Sal. typhi 57 showingthe same staining as in B. subtilis. Photo 3 is of Staph. aureus, gram-positivePhoto 1.Photo 2.Photo 3.Photo 4.B. subtilis, Culture and staining, see text.Sal. tyPhi 57, Culture and staining, see text.Staph. aureus, Culture and staining, see text.Plasmolyzed B. subtilis, Culture and staining see text.2Acta Medica Okayama, Vol. 16 [1962], Iss. 1, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol16/iss1/5Cytoplasmic Membrane a Nuclear Apparatus of Bacteria 35........,..,fit~.,­Y' • .,.1.~ .•••. I~~ .....111 dbI3Kanemasa: Selective staining of cytoplasmic membrane and nuclear apparatusProduced by The Berkeley Electronic Press, 196236 Y. KANEMASAcoccus, and shows the similar staining properties as the gram negative bacterialcells studied; i. e., one to two nuclear apparatuses can be seen in a cell and thetransverse septum in the dividing cell is recognized between two nuclei. Photo 4shows B. subtilis cells plasmolyzed by exposing to ether vapor and stained withVictoria blue 4R after treating with mercuric chloride. The cytoplasmic mem-brane is stained bluish purple and the cell wall is faintly visible outside of themembrane, showing that Victoria blue 4R stains cytoplasmic membrane but notthe cell wall.SUMMARYA selective and simultaneous staining method for nuclear apparatus andcytoplasmic membrane of some bacteria has been presented. Nuclear apparatusis stained with basic fuchsin after hydrolysis with I N HCI and cytoplasmicmembrane is restained with Victoria blue 4R after treating with saturated mer-curic chloride. By this method, the nuclear apparatuses of B. subtilis, Sal.typhi 57 and Staph. aureus were stained red, and the cytoplasmic membraneand septum bluish purple distinctly. Thus this staining method would be of agreat advantage in displaying the cellular structures of the bacteriaACKNOWLEDGEMENTThe author wishes to express his appreciation to Prof. M'Jrakami for his guidance and toDr. Okuma and Miss Kuroda for their technical assistances.REFERENCES1. GUSTAIN, M.: Ueber eine allgemeine Methode zur Darstellung des Ektoplasmas der gram-positiven Bakterien. Cent. Bact. Parasit., 93, 393, 19242. CASSEL, W. A.: A procedure for the simultaneous demonstration of the cell walls and chromati-nic bodies of bacteria. ]. Bacterial., 62, 239, 19513. ROBINOW, C. F. and MURRAY, R. G. E.: The differentiation of cell wall, cytoplasmic membraneand cytoplasm of gram positive bacteria by selective staining. Exp. Cell Research. 4, 390,19534. PIEKARSKI, G.: Cytologische Untersuchungen an Paratyphus und Colibacterien. Arch. f.Micrab., 8, 428, 19375. CASSEL, W. A.: The use of perchloric acid in bacterial cytology. ]. Bacterial. 59, 185,19504Acta Medica Okayama, Vol. 16 [1962], Iss. 1, Art. 5http://escholarship.lib.okayama-u.ac.jp/amo/vol16/iss1/5

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