Vol. 19, No. 4JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1984, p. 453-4560095-1137/841040453-04$02.00/0Copyright C 1984, American Society for Microbiology

Fluorescent Staining of Intracellular and Extracellular Bacteria inBlood

JAMES D. MANSOUR,* JAMES L. SCHRAM, 'AND THOMAS H. SCHULTEMicrobiology Department, Becton Dickinson and Co. Research Center, Research Triangle Park, North Carolina 27709

Received 29 July 1983/Accepted 15 December 1983

The fluorescent dye ethidium bromide stains Escherichia coli and Staphylococcus aureus in whole blood.The staining is rapid, relatively specific, and does not require fixation of the sample. Furthermore, stainedbacteria can be seen microscopically without the need for a final wash to remove unbound dye. By usinglysostaphin, an S. aureus-specific lytic enzyme, we have demonstrated that S. aureus can be stained withethidium bromide even after phagocytosis. After short periods of incubation with the dye (less than 5 min),bacteria, both intracellular and extracellular, were the predominant fluorescent particles. With increasingtime of incubation, blood cell components, notably leukocyte nuclei, began to fluoresce.

Fluorescence microscopy is being used increasingly inboth clinical and research laboratories. Immunofluorescencemicroscopy in particular is now a well-established techniquein a variety of diagnostic tests, primarily because of the highsensitivity of fluorescence, coupled with the high specificityof antibodies. Although they do not provide the exquisitespecificity of fluorescently tagged antibodies, fluorescentdyes themselves can be relatively specific for particular cellsor cell structures and can be used in rapid and inexpensivestaining protocols. Examples of these applications includethe use of acridine orange to selectively stain bacteria indried smears of clinical blood culture samples (8, 10) and theuse of 4'-6-diamidino-2-phenylindole (16, 15) and Hoechst33258 (2, 14) dyes to indicate viral, chlamydial, or mycoplas-mal contamination of tissue culture cells.Ethidium bromide, a phenanthridinium dye which binds to

nucleic acids, is a fluorescent dye which has been usedextensively to stain both procaryotic (12, 13, 16) and eucary-otic (1, 3, 4, 11, 19) cells. Although ethidium bromide isknown to bind tightly to double-stranded nucleic acids invitro (6, 9), the binding of ethidium bromide to cells may beinfluenced by factors other than nucleic acid content alone.In mutant rhoo yeast cells, staining by ethidium bromideappears not to be solely due to DNA binding (3), and inunfixed cultured L cells, the staining appears to be depen-dent on factors other than total DNA content of the cell (1).To avoid variables involved with stain penetration of cells,most ethidium bromide staining protocols involve fixation ofcells, followed by incubation with dye for 15 min or more. Inthis paper, we report an ethidium bromide staining protocolwhich involves no fixation of bacterial cells and in which weincubated bacteria with dye for less than 5 min beforemicroscopic observation. By using this protocol, we demon-strated rapid and preferential labeling of both intracellularand extracellular Escherichia coli and Staphylococcus au-reus in the presence of whole blood.

MATERIALS AND METHODSEthidium bromide was purchased from Sigma Chemical

Co., St. Louis, Mo. It was found to migrate on silica gel thin-layer chromatography with an ethanol-1 N hydrochloric acid(50:1) solvent system as essentially a single species. Lyso-staphin was also purchased from Sigma and prepared as a

* Corresponding author.

stock solution at a concentration of 240 U/ml in water. Allother chemicals were reagent grade.

S. aureus ATCC 25923 and E. coli ATCC E11775 werepurchased from the American Type Culture Collection,Rockville, Md., and routinely grown in Trypticase soy broth(BBL Microbiology Systems, Cockeysville, Md.) at 37°C tolog phase, washed once with normal saline (0.85% NaCl),and resuspended in normal saline for use. Quantitation ofviable S. aureus and E. coli was accomplished by pour plateanalysis with Trypticase soy broth agar.Human blood was drawn into sodium polyanetholsulfon-

ate (SPS)- or heparin-containing Vacutainer blood collectiontubes (Becton Dickinson and Co., Paramus, N.J.) and usedfor experiments within 2 h. Cell-free plasma was producedby centrifuging heparinized human blood at 3,500 x g for 15min and filtering the supernatant through a 0.2-,um (poresize) filter.

Fluorescent staining of S. aureus and E. coli in whole blood.S. aureus or E. coli (106 CFU) was added in a 0.01-mlvolume to 1 ml of heparinized or SPS-anticoagulated blood.The samples were then either stained immediately or prein-cubated for 30 min at 37°C to allow phagocytosis to occur.Staining was accomplished by incubating 0.7 ml of samplewith 0.2 ml of staining buffer (100 mM sodium borate, 60 mMEDTA, 0.05% formaldehyde, 0.05% Triton X-100, pH 9.2)and 0.1 ml of ethidium bromide (100 ,g/ml in water). Thismixture was then incubated for 1 min at room temperature,and 0.01 ml was withdrawn for microscopic observation.Light microscopy and ethidium bromide fluorescence wereviewed with an Olympus BHA microscope equipped forepiillumination by using a mercury arc lamp with a greendichroic, 590 barrier filter and IF545 and BG36 excitationfilters. For photomicrographic purposes, the Olympus pho-tomicrographic PM-10 system (equipped with an FK 3.3eyepiece) and a PM-CP Polaroid camera back (type 667 film,Polaroid Corp., Cambridge, Mass.) were employed. Themicroscope objective used for all photographs was a x 100-magnification oil immersion lens. Total magnifications aresupplied for each photomicrograph.Treatment of S. aureus with lysostaphin. S. aureus

(106CFU) was added in a 0.01-ml volume to 1 ml of normalsaline, cell-free plasma, or heparinized, or SPS-anticoagulat-ed human blood. The mixtures were incubated at 37°C for 30min and then further incubated with ±2.4 U of lysostaphinfor an additional 10 min at 37°C. Quantitation of viable S.aureus was accomplished by pour plate analysis of the

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FIG. 1. Fluorescence (A) and light (B) photomicrographs of SPS-anticoagulated human blood containing E. coli (wet preparation).The whole blood-bacteria sample was stained with ethidium bro-mide for 1 min at 10 pg of ethidium bromide per ml in staining bufferwithout fixation. Samples were further diluted before photomicrog-raphy. Magnification, x990.

sample with Trypticase soy broth agar. Control experimentsdemonstrated that lysostaphin at 0.025 U/ml in agar had noinhibitory effect on the growth of colonies on pour plates.Samples containing lysostaphin were diluted to levels belowthat concentration during plating.

RESULTSTo characterize the fluorescent labeling of bacteria with

ethidium bromide, washed cultures of either E. coli or S.aureus at concentrations of 106 CFU/ml were added tofreshly drawn human blood anticoagulated with SPS. Themixture was then stained with ethidium bromide as de-scribed above and diluted with normal saline to allow easymicroscopic observation. Figures 1A and 2A are fluores-cence photomicrographs showing the ethidium bromidestaining of E. coli and S. aureus, respectively. These are wetpreparations involving no fixation and incubation with dyefor only 1 min. The transmitted light photomicrographsshowing the same respective fields are presented in Fig. 1Band 2B. A comparison of the fluorescence and light photomi-crographs shows the specificity of ethidium bromide for thebacteria under these conditions. No blood components ap-pear to be fluorescently stained.

Staining of bacteria by ethidium bromide is a very rapidprocess, occurring within a few minutes. Leukocyte nucleiare also stained by ethidium bromide but require longerincubation periods with the dye, and only a relatively smallpercentage stain readily. Five minutes after ethidium bro-

mide addition, 100% of the bacteria and only 10% of theleukocytes appeared to be stained by the dye. By 30 min,20% of the leukocytes were stained, but neither erythrocytesnor platelets appeared to fluoresce.During the course of these experiments, we also noted that

bacteria which appeared to have been phagocytized andwere intraleukocytic were also stained by ethidium bromide.Pursuing this observation further, we established that signifi-cant percentages of both E. coli and S. aureus are suscepti-ble to phagocytosis when incubated at 37°C in freshly drawnheparinized, rather than SPS-anticoagulated, human blood.By using this model phagocytosis system, we could studyethidium bromide staining of these intraleukocytic bacteria.Figures 3A and B are fluorescence and transmitted-lightphotomicrographs showing the apparent intracellular local-izations of an E. coli organism which had been stained withethidium bromide. During the time required to locate andphotograph this leukocyte, the nuclei had also stained bright-ly.By using microscopy alone, it is sometimes difficult to

determine whether a leukocyte-associated bacterium is actu-ally intracellular (phagocytized). The localization of leuko-cyte-associated S. aureus, though, can be more preciselyaccomplished by microscopy in conjunction with the use oflysostaphin, an enzyme which specifically cleaves the pepti-doglycan of S. aureus (17). S. aureus organisms exposed tothe activity of this enzyme are completely lysed, whereas S.aureus organisms which have been phagocytized are largelyprotected from lysis (5, 18). Table 1 shows the activity andspecificity of lysostaphin for extracellular S. aureus. More

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FIG. 2. Fluorescence (A) and light (B) photomicrographs of SPS-anticoagulated human blood containing S. aureus (wet preparation).Staining conditions were as described in the legend to Fig. 1.Magnification, x 990.

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ETHIDIUM BROMIDE STAINING OF BACTERIA IN BLOOD 455

TABLE 1. Effect of lysostaphin on intracellular and extracellular S. aureus

% Bacteria associated Pour plates values (CFU/ml) % Bacteria viableSample with WBC' as scored after enzymeby fluorescence - Lysostaphin + Lysostaphin treatment

microscopyHeparinized blood 25 1.2 x 107 2.6 x 106 22SPS-anticoagulated blood 4 1.9 x 107 1.3 X 106 7Cell-free plasma NAb 4.2 x 107 1.9 X 104 <1Normal saline NA 1.8 x 107 7.9 X 104 <1

a WBC, Leukocytes.b NA, Not applicable.

than 99% of the S. aureus organisms incubated in cell-freeplasma or normal saline and then treated with lysostaphinwere killed by the enzyme. As mentioned above, S. aureusincubated in heparinized blood was susceptible to phagocy-tosis. After a 30-min incubation period at 37°C in heparinizedblood, 25% of the S. aureus organisms appeared by fluores-cence microscopy to be associated with, and presumablyphagocytized by, leukocytes. Lysostaphin treatment of thatpreparation (see Table 1) resulted in ca. 78% reduction inbacterial viability, indicating that 22% of the S. aureuspopulation was protected from the effects of the enzyme.Furthermore, S. aureus incubated in SPS-anticoagulatedblood was less susceptible to phagocytosis as determined bymicroscopy, and there was a corresponding decrease in thenumber of bacteria protected from the effects of lysostaphin(Table 1). Thus, lysostaphin, under the conditions describedhere, appeared to lyse virtually all extracellular S. aureus

but did not appreciably affect intracellular (phagocytized) S.aureus.

Figures 4A and B are fluorescence and transmitted-lightphotomicrographs of S. aureus in heparinized human bloodtreated with lysostaphin and stained with ethidium bromide.There are no free or extracellular S. aureus organisms visiblein this preparation because of the lytic effects of the enzyme.The stained S. aureus organisms seen in the fluorescencephotomicrograph are intracellular and yet clearly stained byethidium bromide.

DISCUSSIONThe addition of ethidium bromide and staining buffer to

human blood containing E. coli or S. aureus has been shownto result in the rapid and preferential staining of the bacteria,

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FIG. 3. Fluorescence (A) and light (B) photomicrographs ofleukocyte-associated E. coli. E. coli was added to freshly drawnheparinized human blood and incubated at 37°C for 30 min to allowfor phagocytosis. A sample was then removed and stained asdescribed in the legend to Fig. 1. Magnification, x990.

FIG. 4. Fluorescence (A) and light (B) photomicrographs ofphagocytized S. aureus. S. aureus was added to freshly drawnheparinized human blood and incubated at 37°C for 30 min. Lysos-taphin (2.4 U/ml) was then added, and incubation continued for anadditional 10 min. Samples were then removed and stained withethidium bromide as described in the legend to Fig. 1. Magnifica-tion, x990.

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456 MANSOUR, SCHRAM, AND SCHULTE

with minimal staining of the blood cells. The method is rapidand simple and does not require a fixative or final wash stepbefore microscopic analysis. The basis for this preferentialstaining of the bacteria is not yet understood, although thefact that intracellularly located bacteria (those ingested byleukocytes) are stained very quickly, often before the nucleiof the surrounding leukocyte, would suggest that the dyeaffinity for bacterial binding sites, rather than permeabilityproperties of cell membranes, may be the primary determi-nant. With increased time of sample incubation with dye,leukocyte nuclei begin to fluoresce. However, within thefirst 5 min after dye addition, bacteria, both extracellular andintracellular, are the predominant fluorescent entities.Erythrocytes and platelets did not stain with ethidium bro-mide under our conditions.

It is difficult to identify phagocytized bacteria by micros-copy alone. Extracellular bacteria bound to a leukocyte canoften appear to be intracellular. To minimize this possibility,we have used both microscopy and resistance to the lyticenzyme lysostaphin as a measure of the phagocytosis of S.aureus. Lysostaphin is a staphylococcus-specific lytic en-zyme widely used in neutrophil functional assays and hasbeen found not to significantly penetrate polymorphonuclearleukocytes (18) or human alveolar macrophages (5). Al-though a recent paper suggests that lysostaphin penetratesneutrophils (20), we feel that lysostaphin did not appreciablyact upon the intracellular, viable S. aureus population in oursystem because of the close correlation between lysostaphinresults and results obtained by microscopy. As indicatedabove, we found that ethidium bromide fluorescently stainseven phagocytized S. aureus. We observed the ethidiumbromide staining of phagocytized E. coli as well, although inthis case phagocytosis was judged by microscopy alone.

For the purpose of this study, E. coli and S. aureus wereused as representative bacteria. We have looked at Pseudo-monas aeruginosa, Klebsiella pneumoniae, and Streptococ-cusfaecalis in blood with this staining method, and they tooshowed similarly specific staining by ethidium bromide. Webelieve this method will be generally applicable to otherbacteria. The whole-blood preparations in this paper wereseeded with 106 CFU/ml. Bacterial concentrations belowthis level make it difficult and time consuming to evaluateresults with a microscope. The concentrations of bacteriaencountered in clinical cases of bacteremia are, of course,generally well below these levels. Potentially, however, thespecificity of ethidium bromide for bacteria could be utilizedin clinical microbiology in several ways. Ethidium bromidestaining, rather than Gram or Wright staining, could be usedfor detection of intracellular or extracellular bacteria in buffycoat preparations (7). The relative lack of background stain-ing would be a great advantage in this area. Another applica-tion might be the use of ethidium bromide staining for thedetection of bacteria in blood cultures. Acridine orange, afluorescent dye which shows a color-specific fluorescentstaining of bacteria (10), has been shown to be of value inthis area. Ethidium bromide may have additional advantagesbecause it can be used directly on a wet preparation andshows minimal background staining.Ethidium bromide in the proper staining buffer preferen-

tially stains bacteria in complex samples. This simple, rapid,and inexpensive staining protocol may be applicable not onlyto the detection of bacteria in blood but also in other systems

or body fluids in which it is necessary to detect bacteriaagainst a background of eucaryotic cells.

LITERATURE CITED1. Bohmer, R. M. 1979. Discrete changes of the fluorescence yield

from cells vitally stained with ethidium bromide (EB), asdetermined by flow cytometry. Exp. Cell Res. 122:407-410.

2. Chen, T. R. 1977. In situ detection of mycoplasma contamina-tion in cell cultures by fluorescent Hoechst 33258 stain. Exp.Cell Res. 104:255-262.

3. Corliss, D. A., and W. E. White, Jr. 1981. Fluorescence of yeastvitally stained with ethidium bromide and propidium iodide. J.Histochem. Cytochem. 29:45-48.

4. Crissman, H. A., P. F. Mullaney, and J. A. Steinkamp. 1975.Methods and applications of flow systems for analysis andsorting of mammalian cells, p. 179-246. In D. Prescott (ed.),Methods of cell biology, vol. 9. Academic Press, Inc., NewYork.

5. Easmon, C. S. F., H. Lanyon, and P. J. Cole. 1978. Use oflysostaphin to remove cell-adherent staphylococci during invitro assays phagocyte function. Br. J. Exp. Pathol. 59:381-385.

6. Hudson, B., W. B. Upholt, J. Devinny, and J. Vinograd. 1969.The use of an ethidium analogue in the dye-buoyant densityprocedure for the isolation of closed circular DNA: the variationof the superhelix density of mitochondrial DNA. Proc. Natl.Acad. Sci. U.S.A. 62:813-820.

7. Humphrey, A. A. 1944. Use of the buffy layer in the rapiddiagnosis of septicemia. Am. J. Clin. Pathol. 14:358-362.

8. Kronval, G., and E. Myhre. 1977. Differential staining of bacte-ria in clinical specimens using acridine orange buffered at lowpH. Acta Pathol. Microbiol. Scand. Sect. B 85:249-254.

9. Le Pecq, J.-B., and C. Paoletti. 1967. A fluorescent complexbetween ethidium bromide and nucleic acids: physical-chemicalcharacterization. J. Mol. Biol. 27:87-106.

10. McCarthy, L. R., and J. E. Senne. 1980. Evaluation of acridineorange stain for detection of microorganisms in blood cultures.J. Clin. Microbiol. 11:281-285.

11. Nicolini, C., F. Kendall, R. Baserga, C. Dessaive, B. Clarkson,and J. Fried. 1977. The GO-Gl transition of WI38 cells. Exp.Cell Res. 106:111-118.

12. Paau, A. S., J. R. Cowles, J. Oro, A. Bartel, and E. Hungerford.1979. Separation of algal mixtures and bacterial mixtures withflow-microfluorometer using chlorophyll and ethidium bromidefluorescence. Arch. Microbiol. 120:271-273.

13. Roser, D. J. 1980. Ethidium bromide: a general purpose fluores-cent stain for nucleic acid in bacteria and eucaryotes and its usein microbial ecology studies. Soil Biol. Biochem. 12:329-336.

14. Russel, W. C., C. Newman, and D. H. Williamson. 1975. Asimple cytochemical technique for demonstration of DNA incells infected with mycoplasmas and viruses. Nature (London)253:461-462.

15. Salari, S. H., and M. E. Ward. 1979. Early detection ofChlamydia trachomatis using fluorescent DNA-binding dyes. J.Clin. Pathol. 32:1155-1162.

16. Steen, H. B., and E. Boye. 1980. Bacterial growth studied byflow cytometry. Cytometry 1:32-36.

17. Strominger, J. C., and J. M. Ghuysen. 1967. Mechanisms ofenzymatic bacteriolysis. Science 156:213.

18. Tan, J.S., C. Watanakunakorn, and J. P. Phair. 1971. Amodified assay of neutrophil function: use of lysostaphin todifferentiate defective phagocytosis from impaired intracellularkilling. J. Lab. Clin. Med. 78:316-322.

19. Taylor, I. W., and B. K. Milthorpe. 1980. An evaluation ofDNAfluorochromes, staining techniques, and analysis for flow cyto-metry. J. Histochem. Cytochem. 28:1224-1232.

20. van den Brock, P. J., F. A. M. Dehue, P. C. J. Leih, M. T. vanden Barselaar, and R. van Furth. 1982. The use of lysostaphin inin vitro assays of phagocyte function: adherence to and penetra-tion into granulocytes. Scand. J. Immunol. 15:467-473.

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