DIRECT STAINING OF THE TWO TYPES OF NUCLEOPROTEINS INESCHERICHIA COLI'

PHILIP E. HARTMAN AND JOHN I. PAYNE

Department of Microbiology, Medical School, University of Pennsylvania, Philadelphia, Pennsylvania

Received for publication February 22, 1954

This report describes a technique for the differ-ential staining of the two types of nucleopro-teins in bacteria. The staining procedure isadapted from that described by Jacobson andWebb (1952) for other cells. In several respectsit is similar to techniques previously used in bac-terial cytology. Visualization of the two nucleo-proteins individually is possible after treatmentwith specific nucleases.

MATERIALS AND METHODS

Basic staining procedure. In this procedureribonucleoprotein is stained blue and desoxyri-bonucleoprotein is stained red.

(1) Impression smears are made on alcoholcleaned coverslips from squares cut out of agarplates on which bacterial cells are grown directlyon the agar surface or are placed there fromliquid culture and retained until the excessmoisture has soaked into the agar.

(2) The coverslip preparations are immersedimmediately in absolute methanol2 previouslychilled on dry ice (Blank et al., 1951) and left ondry ice overnight.

(3) Coverslips are placed in absolute methanolat room temperature for a few seconds. They aretransferred to a 1:5 dilution in methanol of asaturated methanol solution of methylene blueeosinate and stained for 10 minutes.

(4) Coverslips are rinsed quickly in distilledwater (adjusted to pH 7.0 with NaOH) to rehy-drate and wash off excess dye.

(5) Preparations are placed in freshly pre-pared aqueous Giemsa solution (1:10 dilution,pH 7.0) for 10 minutes.

(6) The excess dye is removed from thecoverslips by a brief rinse in distilled water,pH 7.0.

1 This work has been aided by a grant from theUnited States Atomic Energy Commission, AECcontract no. AT(30-1) 1342.

2 Methanol, Baker analyzed reagent, ACS.

(7) The coverslip preparations are dehydratedrapidly by 4 quick dips in acetone at room tem-perature, 4 dips in a 1 :1 mixture of acetone andxylol, and then transferred through two changesof xylol before draining (blotting should beavoided), and mounted on alcohol cleaned glassslides. Final dehydration may be achieved alsothrough the use of overnight dehydration inacetone previously chilled and maintained ondry ice; the coverslips then are immersed a fewseconds in acetone at room temperature beforethe transfers through xylol.

Reagents used. It is recommended that allreagents be replaced after several hours use.

(a) Fixative. Fixation in absolute methanol atroom temperature frequently results in extremeshrinkage, in "budding" cells, or vacuolization(figure 1, a-c). The use of cold methanol keepsthe production of these gross fixation artifactsto a minimum although limited shrinkage andslight peripheral vacuolization of the cells stilloccur in some preparations. Also, where cells arespread thickly, distortion by adjacent cells mayresult (figure 1, d).A number of agents were used for prefixation

of specimens prior to immersion in methanol.All produced extensive shrinkage at least insome areas of the slide. Better results withalcohol containing reagents were obtained whenfixation was carried out at -5 C (Wolman andBehar, 1952) rather than at room temperature.None of the methods gave as good preservationof the cells as did absolute methanol cooled ondry ice.

(b) Methylene blue eosinate. Saturated solu-tions of May-Greenwald stain3 and Leishman'sstain3 4 were made up in absolute methanol and"aged" for one week or more prior to use.

(c) Giemsa stain. The Giemsa stain or varioussubstitutes were diluted 1:10 in distilled water

3Harleco synthetic resin, Hartman-LeddonCo., Inc., Philadelphia 39, Pennsylvania.4Coleman Bell and Co., Norwood, Ohio.

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immediately before use. A ready-made solution(Gradwohl Laboratories, Cert. no. GGe-13) anda solution prepared from powder (Giemsa stain,original azure blend type, Cert. no. LGe-16)3were satisfactory.

(d) Mounting media. Harleco synthetic resin(HSR)3 was used in most of the work, but"technicon" and "clarite" mounting medialikewise were found to be suitable. In Canadabalsam, pronounced fading occurred in a fewdays. Although fading was generally unequalthroughout the slide, the methylene blue eosinatewas always the first to fade, revealing theGiemsa stained material with clarity. Mostpreparations mounted in Harleco syntheticresin do not fade (during a period of a year orlonger); in those few areas where fading is de-tectable, the blue stain has left the cytoplasmand has become adsorbed to the nuclear struc-tures.

(e) Enzymes. Preparations were immersed in0.4 mg per ml ribonuclease in distilled water atpH 6.0 or 6.8 for 15 minutes or longer at 37 Cafter fixation with cold methanol or with Cha-baud's solution. Osmium tetroxide fixation greatlyinhibited ribonuclease action. Two samples ofcrystalline ribonuclease (General Biochemicals

and Armour and Company) gave identical re-sults.

Absolute methanol fixed coverslip prepara-tions were exposed one hour to crystalline des-oxyribonuclease (Worthington) according to theproceduire of Murray and Whitfield (1953).Similarly fixed specimens were exposed to 0.4mg per ml crystalline lysozyme (Delta) inM/15 phosphate buffer, pH 6.6. Diluent controlswere included with each series.

Optics and photomicrography. Bausch and Lomblight and phase contrast optics with 12.5 X or15 X eyepieces and 97 X objectives were used.Photographs were taken on Kodak Micro-film35 mm and Kodak Panatomic X film. Filters,to increase contrast in the desired stain, wereused in the tungsten filament and zirconium arclighting systems. One set of filters consisted ofeither Kodak wratten red filters A25 and F29or Bausch and Lomb interference filter 630m,u, while the other set included either wrattenblue filter H45 and green filter B58 or Bauschand Lomb interference filter 520 m,u.

Organisms studied. Most of the studies wereperformed with log phase broth cultures of Es-cherichia coli, strain B. However, the standardstaining procedure has been applied to 18 hour

All figures are reproduced here at a final magnification of 3,675 X. All preparations are of uncrushedspecimens and represent log phase broth cultures of Escherichia coli, strain B.

Figure 1. Stained by complete procedure described in Methods and Materials and photographed toshow the staining throughout the cell. (a) Extreme shrinkage, often found in preparations fixed inmethanol at room temperature, (b) "budding" cell, found in preparations fixed in methanol at roomtemperature, (c) vacuolization, found mainly in cells fixed at room temperature but, also, occasionallyin those fixed in cold methanol, especially if air drying is not avoided, (d) distortion of adjacent cells,occasionally found in the more thickly spread areas of the coverslip.

Figure 2. Living cells placed on agar from a log phase broth culture and photographed with phasecontrast.

Figure S. Cells fixed in cold methanol, placed in Leishman's stain for 10 minutes, rinsed in distilledwater, dehydrated through acetone and xylol and mounted in Harleco synthetic resin (HSR). The cyto-plasm is stained blue; nuclei are unstained or only very faintly stained blue.

Figure 4. Stained by the Bouin-thionin procedure (Murray et al., 1950).Figure 5. Stained by the complete procedure described in Methods and Materials. Photographed to

show the blue cytoplasmic staining. However, as comparison with figure 3 will show, the optical densityof the nuclear staining (red) prevents visualization in black and white photographs of the nuclear areas.

Figure 6. Same cells as figure 5, this time photographed to show more clearly the red nuclei. Again,the contrasting stain cannot be removed completely by the use of filters.

Figure 7, a-c. As figure 6, except treated with ribonuclease prior to staining.Figure 8, a, b. As figure 6. Preparations in which some fading of the cytoplasm has occurred. Photo-

graphed to show nuclear structure.Figure 9. Piekarski-Robinow nuclear stain prepared as described in Murray and Whitfield (1953)

and photographed in wet mount. Hydrolyzed in 1 N HCl at 60 C for 8 minutes.Figure 10, a, b. DeLamater (1953a) azure A-thionyl chloride stain after fixation in vapors of 2 per

cent osmium tetroxide for one minute. Hydrolyzed in 1 N HCI at 60 C for 8 minutes.Figure 11. As figure 10, except fixed overnight in cold methanol and rehydrated before hydrolysis.

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DIRECT STAINING OF NUCLEOPROTEINS IN E. COLI

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PHILIP E. HARTMAN AND JOHN I. PAYNE

and log phase cultures of 14 representative gramnegative genera. Although other gram positivegenera were examined, the organism used formost of the experiments involving a gram posi-tive organism was the strain of Bacillus mega-terium used by DeLamater and co-workers.

RESUTS

Figure 2 shows living log phase E. coli, strainB, cells photographed with the phase contrastmicroscope. Staining with methylene blue eosin-ate produces blue cells with intensely stainedpolar and central areas and lighter regions inter-mediate (figure 3). This distribution of stain issimilar to that produced by the Bouin-thioninprocedure for pentosenucleic acid (compare withfigure 4) but more closely resembles the grada-tion of densities observed in the living cells.The areas of lesser density are the sites of thebacterial nuclei.The completed staining process is shown in

figure 5. The cells are photographed to show thelocalization of eosinated methylene blue stainedmaterial in the cytoplasm although the opticaldensity of the Giemsa stained component pre-vents an adequate demonstration by black-and-white photographs. Cell size and shape are pre-served by the methanol fixation.

Ribonuclease completely removes the methyl-ene blue eosinate-staining substances (figure7, a-c). These are removed also by alkaline ex-traction with M/30 Na2HPO4 at 60 C for 3 hours(Welsch and Nihoul, 1948). Nuclear areas takeon a red coloration with Giemsa and fail to stainwith the eosinated methylene blue. The cyto-plasm also loses its staining affinity after hydroly-sis in 1 N HCl at 60 C for 7 to 8 minutes; how-ever, in this case, unlike the previous treatments,the nuclei now stain with both eosinated methyl-ene blue and Giemsa.

Figure 6 shows cells photographed with filtersenhancing the contrast between the red Giemsastained material and the blue cytoplasm. Whilenuclei may be discerned in these cells, their finestructure is obscured in part by the cytoplasmicstaining. Finer details of nuclear structure arevisible after ribonuclease digestion (figure 7, a-c)and cytoplasmic fading in situ (figure 8, a, b).The nuclei are composed of discrete elements withchromosomal configurations. Small extranucleargranules (corresponding to the centrioles ofDeLamater, 1953a, b, c) are present near some

nuclei. The centrioles are found adjacent only tothose nuclei which have configurations charac-teristic of dividing stages, as if they becameachromatic or much smaller during other nuclearstages.

Nuclear staining by the Feulgen technique(as modified by Beutner et al., 1953) is too faintto allow definition of fine structure. On the otherhand, in Piekarski-Robinow wet mounts (figure9) the intense nuclear staining hinders study ofthe nuclear components as entities. DeLamaterstaining with either osmium (figure 10, a, b)or methanol (figure 11) fixation shows the chromo-somal elements more clearly.During infection of E. coli with T2 bacteri-

ophage, the distribution of the Giemsa stainedmaterial undergoes relocalization, in accord withthat previously reported for the desoxyribo-nucleic acid of infected cells (Murray et al.,1950; Beutner et al., 1953).

Finally, exposure of the cells to desoxyribo-nuclease eliminates the Giemsa stained substancebut does not alter the staining of the cytoplasmwith the methylene blue eosinate.Metaphosphate (volutin) stains red with the

basic staining procedure in those genera whichcontain large amounts of this inclusion, e.g.,Corynebacterium diphtheriae, Mycobacterium spp,Spirillum spp. The red coloration of metaphos-phate, in addition to the usual methods for itscharacterization, differs from the red nuclearstaining in three respects: (1) it is produced bymethylene blue eosinate in methanol; (2) it isnot readily soluble during alcohol extraction;(3) in some cells, e.g., Corynebacterium, Myco-bacterium, it is removed with ribonuclease pre-treatment (although in others, e.g., Spirillumserpens, it is not).

In a few gram negative organisms (e.g.,Klebsiella pneumoniae) there is some slightGiemsa staining of the cell wall. These cellsalso decolorize slowly in the gram staining pro-cedure. Gram positive organisms stained witheosinated methylene blue alone present the samestaining picture as gram negative species. Ribo-nucleoprotein, as determined by removal withribonuclease, accounts for most of the staining.However, upon subsequent staining with theGiemsa solution there is an intense red colorationof the cell wall. This staining is prevented withlysozyme digestion for one or two minutes. It isnot prevented by either ribonuclease or desoxy-

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ribonuclease pretreatment of the cells althoughribonuclease does destroy the gram positivity ofthe cells.

DISCUSSION

Jacobson and Webb (1952), in extensive ob-servations on the specificity of the May-Green-wald and Giemsa stain for other cels, have shownthat desoxyribonucleoprotein only stains redwith Giemsa in their technique; desoxyribonuc-leic acid, ribonucleoprotein, and ribonucleic acidstain blue. Therefore, the above results suggestthat the substance of E. coli stained by eosinatedmethylene blue in methanol is ribonucleoprotein(and ribonucleic acid) and, further, that the affin-ity of the bacterial nucleus for the Giemsa stainis due to desoxyribonucleoprotein. This commenton the specificity of the Giemsa stain is directedtoward the gram negative bacteria only and, inparticular, toward E. coli.Giemsa staining without prior exposure to

methylene blue eosinate produces dense colora-tion throughout the cell with only a faint indi-cation of internal structural differentiation.Previous staining with methylene blue eosinatein methanol is thus essential for the subsequentdifferentiation and localization of the Giemsamixture. This may not be necessary for all micro-organisms at all stages of growth. The methyleneblue eosinate and Giemsa procedure does notdemand modification during the phases of thegrowth cycle.For comparison with this technique, prepara-

tions of the same log phase cultures were madeusing other nuclear staining methods (figures9, 10, 11). The nuclear pattern is the same asthat found in uncrushed DeLamater prepara-tions, especially when fixed with cold methanol(figure 11).

Specific staining of desoxyribonucleoproteinin intact cells (figure 7, a-c) shows the presenceof perinuclear granules analogous to the centri-oles of DeLamater. Although these granules areusually, but not exclusively, found near the cellperiphery, they are not "growth points" orsepta.Removal of ribonucleic acid by ribonuclease

is obtained following fixation in cold absolutemethanol or in Chabaud's fixative; osmium fixa-tion greatly inhibits ribonuclease action. Thismay be responsible for the altered appearancesnoted by several workers (Peters and Wigand,1953; Murray, 1953).

The use of acid hydrolysis in nuclear stainingprocedures may be open to criticism; someshrinkage of osmium fixed celLs occurs duringacid treatment. However, the nuclear configura-tions found in (nonhydrolyzed) cells by theeosinated methylene blue-Giemsa method(figures 6, 7, a-c) resemble closely those de-scribed by DeLamater (1953a) for acid hy-drolyzed cells. In addition, wet mounts observedbefore dehydration and after staining withDeLamater's (1953a) procedure show that thenuclear structures are present and consequentlycannot be the product of the dehydrationprocess. Gross alterations in structure havenever been observed following the method out-lined by DeLamater (1953a) either in hydrolyzedspecimens prepared with care or on whole cells(Methods and Materials).Mitotic nuclear configurations of E. coli are

not due to fat globules pressing on the bacterialnuclei since the cells contain no free lipid andonly a small amount of bound lipid localized inperipheral granules (Davis et al., 1953).These facts do not of themselves validate the

interpretations of DeLamater (1953a, b, c), for amitotic cycle, which he has outlined as an initialbasis for further investigation in E. coli. We mayconclude, however, that the chromosomal con-figurations and divisional stages thereof de-scribed by DeLamater may be obtained withtechniques differing in many important respects.Hence, they are not merely products of oneprocedure. Though some modification may benecessary in the future, results now available areconsistent with the cycle he has proposed.As reported elsewhere (refer to figures 1 and 2

of Mudd, 1953) the nuclei and perinucleargranules (centrioles), stained red in the intactcell by the eosinated methylene blue and Giemsaprocedure, are distinct from the cytoplasmicorganelles (mitochondria) of E. coli, strain B.The ribonucleoprotein is distributed throughoutthe cytoplam. The dark and light patterning ofcells stained in Leishman's alone (figure 3) indi-cates that the concentration of ribonucleoproteinin the nucleus must be low in comparison withthat of the cytoplasm.

ACKNOWLEDGMENTS

The authors are indebted to Dr. Warren R.Stinebring for calling their attention to the use

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of the May-Greenwald and Giemsa technique intissue culture studies.

SUMMARY

The methylene blue eosinate and Giemsatechnique of Jacobson and Webb has beenadapted for use on certain gram negative bac-teria. Specific differential staining of ribonucleo-protein (blue) and desoxyribonucleoprotein (red)has been obtained. Ribonucleoprotein is con-

centrated largely or exclusively in the cytoplasmwhere its distribution appears homogeneous andresembles the pattern of densities seen in livingcells under phase contrast. Desoxyribonucleo-protein is confined to the cell nucleus and oc-casional small perinuclear granules.

REFERENCESBEUTNER, E. H., HARTMAN, P. E., MUDD, S., AND

HILLIER, J. 1953 Light and electron micro-scopic studies of Escherichia coli-coliphageinteractions. I. Preparative method. Com-parative light and electron microscopiccytology of the E. coli B-T2 system. Bio-chim. et Biophys. Acta, 10, 143-152.

BLANK, H., MCCARTHY, P. L., AND DELAMATER,E. D. 1951 A non-vacuum freezing-de-hydrating technic for histology, autoradiog-raphy, and microbial cytology. StainTechnol., 26, 193-197.

DAVIS, J. C., WINTERSCHIED, L. C., HARTMAN,P. E., AND MUDD, S. 1953 A cytologicalinvestigation of the mitochondria of threestrains of Salmonella typhosa. J. Histochem.Cytochem., 1, 123-137.

DELAMATER, E. D. 1953a Aspects of bacteria ascells and as organisms. Part II. Intern.Rev. Cytol., 2, 158-177.

DESMATER, E. D. 1953b Structure and di-vision of the bacterial nucleus. In Sym-posium: Bacterial cytology. 6th Inter-national Congress of Microbiology, Rome,Italy. Rend. Ist. Super. SanitA, Supple-ment, pp. 108-135.

DELAMATER, E. D. 1953c The mitotic mecha-nism in bacteria. Cold Spring Harbor Sym-posia Quant. Biol., 19, 99-100.

JACOBSON, W., AND WEBB, M. 1952 The twotypes of nucleoproteins during mitosis.Exptl. Cell Research, 3, 163-183.

MUDD, S. 1953 The mitochondria of bacteria.In Symposium: Bacterial cytology. 6th In-ternational Congress of Microbiology, Rome,Italy. Rend. Ist. Super. Sanit&, Supplement,pp. 67-81.

MURRAY, R. G. E. 1953 The problem of fixationfor studies of bacterial nuclei. In Sym-posium: Bacterial cytology. 6th Interna-tional Congress of Microbiology, Rome,Italy. Rend. Ist. Super. Sanita, Supplement,pp. 136-138.

MURRAY, R. G. E., AND WHITFIELD, J. F. 1953Cytological effects of infection with T5 andsome related phages. J. Bact., 65, 715-726.

MURRAY, R. G. E., GILLEN, D. H., AND HEAGY,F. C. 1950 Cytological changes in E8cher-ichia coli produced by infection with phageT2. J. Bact., 59, 603-615.

PETERS, D., AND WIGAND, R. 1953 Enzyma-tisch-elektronen-optische Analyse der Nu-cleins&ureverteilung dargestellt an E. colials Modell. Z. Naturforsch., 8b, 180-192.

WELSCH, M., AND NIHOUL, E. 1948 A proposede la mise en 4vidence du noyau bacterien.Compt. rend. soc. biol., 142, 1449-1452.

WOLMAN, M., AND BEHAR, A. 1952 A method offixation for enzyme-cytochemistry and cy-tology. Exptl. Cell Research, 3, 619-621.

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