prolonged survival ofserratiamarcescens in chlorhexidinechlorhexidine (as soonas it wasreceived in...

Vol. 42, No. 6APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1981, P. 1093-11020099-2240/81/121093-10$02.00/0

Prolonged Survival of Serratia marcescens in ChlorhexidineTHOMAS J. MARRIE' 2* AND J. WILLIAM COSTERTON3

Departments ofMedicine' and Microbiology,2 Dalhousie University and The Victoria General Hospital,Halifax, Nova Scotia, and The Department ofBiology,3 University of Calgary, Calgary, Alberta, Canada

Received 23 February 1981/Accepted 22 September 1981

During an outbreak of Serratia marcescens infections at our hospital, wediscovered widespread contamination of the 2% chlorhexidine hand-washingsolution by S. marcescens. Examination by electron microscopy of the sides ofbottles in which this solution was stored revealed that microorganisms wereembedded in a fibrous matrix. Bacteria, free in the liquid, were morphologicallyabnormal, showing cell wall disruption or cytoplasmic changes. Furthermore,bacteria adherent to the walls of the storage jugs and embedded in this fibrousmatrix also had morphologically abnormal cytoplasm. Despite these changes,viable S. marcescens organisms were recovered from the fluid during a storageperiod of 27 months. The concentration of chlorhexidine required to inhibit thesestrains of Serratia was 1,024 ,tg/ml; however, the organism could survive inconcentrations of up to 20,000 ,tg/ml. Additional studies are needed to define themechanism(s) that allows such bacteria to contaminate and survive in disinfec-tants.

Chlorhexidine (Hibitane; Ayerst Laboratories,Montreal, Quebec, Canada), 1,6-di-(4-chloro-phenyldiguanido) hexane, is an antiseptic that iswidely used as a hand-washing detergent in hos-pitals (13, 16). It has a wide spectrum of anti-microbial activity and has an extremely lowpotential for eliciting dermal reactions (13). Thisagent has been used in our hospital for 7 years.For the past 6 years, approximately 200 patientsper year at our hospital have been infected orcolonized with Serratia marcescens. In Decem-ber, 1978, one of the patients in the cardiovas-cular intensive care unit developed S. marces-cens bacteremia. Subsequent investigations re-vealed contamination of chlorhexidine with S.marcescens. This contamination probably oc-curred in the pharmacy when the plastic stockbottles were filled. Chlorhexidine (Hibitane) issupplied by the manufacturer as a 4% solutionof chlorhexidine gluconate. In our pharmacy, thesolution is diluted to 2% with tap water anddispensed to the wards in 4-liter plastic stockbottles; when empty, they are returned to thepharmacy, where they are rinsed with tap waterand refilled. A random sample of 11 stock bottlesrevealed that 7 were contaminated with S. mar-cescens, and an additional 2 were contaminatedwith S. marcescens, Flavobacter spp., and Pseu-domonas spp.

In this paper, we describe how these bacteriasurvived in stock bottles containing dilutions ofthe commercial skin cleansing product Hibitane.

MATERIALS AND METHODSSurvival of organisms in stock solutions of

Hibitane. Six 4-liter plastic stock bottles containing2% chlorhexidine (Hibitane) from different wards werestored in the laboratory. Colony counts were per-formed on samples from each stock bottle at approxi-mately monthly intervals for 27 months. Organismswere identified according to the criteria of Edwardsand Ewing (6).Determination of MICs. (i) Organisms tested.

Twenty-five isolates of S. marcescens from contami-nated chlorhexidine and five strains of S. marcescensisolated from various clinical sources (three fromurine, one from sputum, and one from blood) weretested. Twenty isolates of other bacteria from clinicalsources, including Escherichia coli (8), Enterobacterspp. (3), Klebsiella spp. (2), Pseudomonas aeruginosa(4), Proteus spp. (1), and Staphylococcus aureus (2),were collected 6 months after disposal of the contam-inated Hibitane.

(ii) Antimicrobial agents. Amikacin powder wasobtained from Bristol Laboratories, Montreal, Quebec,Canada, and gentamicin was obtained from ScheringLaboratories, Pointe Claire, Quebec, Canada.

Serial twofold dilutions of amikacin and gentamicinwere incorporated into Mueller-Hinton agar. Chlor-hexidine gluconate (20%; Ayerst Laboratories) wasdiluted and incorporated into agar in serial twofoldconcentrations from 0.5 to 2,048 pg/ml.

(iii) MIC. Organisms to be tested were grown inbrain heart infusion broth (Difco Laboratories, De-troit, Mich.) for 6 h and diluted to match the densityof a 0.5 McFarland standard (1). A Steers replicator(14) was used to inoculate the surfaces of the agarplates, and the plates were incubated for 18 h at 35°C.

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1094 MARRIE AND COSTERTON

The minimal inhibitory concentration (MIC) was theconcentration which completely inhibited growth orresulted in a light haze.A control strain of Staphylococcus aureus and of

Escherichia coli with known chlorhexidine MICs wereobtained from Harold Baker, Ayerst Laboratories,Montreal, Quebec, Canada. In addition, a strain ofPseudomonas aeruginosa with known amikacin andgentamicin MICs was also used as a control strain.

Electron microscopy studies. (i) Scanning elec-tron microscopy. A 3- by 2-cm piece of plastic wascut from the side of each of the five bottles containingcontaminated chlorhexidine. The bottle containingchlorhexidine that was negative on culture served asa control. In addition, a plastic bottle containing 4%chlorhexidine (as soon as it was received in the phar-macy from the manufacturer) was examined. In allinstances, the plastic samples were taken from thesides of the bottles at 2 to 3 cm below the top of thefluid level.The first piece of plastic was examined after the

contaminated chlorhexidine had been stored in thelaboratory for 20 months. The remaining pieces wereexamined after 26 months of storage.The inner surface of the plastic was sampled just

before the fixation procedure, using a cotton swab.The swab was inoculated onto MacConkey agar, andthe organisms were identified as outlined above. Thepieces of plastic were fixed for 24 h at 20°C with afixative solution consisting of 5% glutaraldehyde andcacodylate buffer (0.067 M; pH 6.2) with 0.15% ruthe-nium red. Each piece of plastic was then "metallized"with osmium tetroxide and thiocarbohydrazide (10),dehydrated in ethanol and Freon-113 before criticalpoint drying (5), and examined with a Hitachi S450scanning electron microscope (Hitachi, Rexdale, On-tario, Canada).

(ii) Transmission electron microscopy. (a)Plastic. Another piece of plastic from the storage jug,containing only Serratia marcescens on culture, wasfixed as detailed above, washed five times in the buffer,postfixed in 2% OS04 in buffer, washed five more timesin buffer, and dehydrated through a series of acetonewashes. All of the solutions used in processing thespecimen, from the washes after glutaraldehyde fixa-tion to dehydration with the 70% acetone solution,contained 0.05% ruthenium red (ruthenium red wasomitted from the 90 and 100% acetone solutions be-cause of its limited solubility in these solutions). Afterfurther dehydration in propylene oxide, the specimenwas embedded in Vestopal (Ladd Industries, Burling-ton, Vt.), sectioned, stained with uranyl acetate andlead citrate (11), reinforced with evaporated carbon,and examined with an electron microscope (AEIModel No. 801; Associated Electronic Industries, Har-low, England) at an acceleration voltage of 60 kV.

(b) Sediment from contaminated chlorhexi-dine. Ten-milliliter samples of contaminated chlor-hexidine from three storage jugs (from which onlySerratia marcescens was isolated) were centrifuged at300 x g for 10 min. Each sediment was washed threetimes in phosphate-buffered saline (pH 7.2) and thenfixed in 5% glutaraldehyde and cacodylate buffer(0.067 M; pH 6.2) with 0.15% ruthenium red for 2 h at

room temperature (-20°C). The remainder of thesteps were performed as outlined above.At the time these samples were collected, the con-

taminated Hibitane had been stored in the laboratoryfor 18 months.

(c) Bacteria from contaminated chlorhexidinegrown on solid medium. A 0.001-mi amount ofcontaminated chlorhexidine was streaked onto brainheart infusion agar plates containing Tween 80 (8) andincubated aerobically for 18 h at 37°C. Three colonieswere then scraped off the plates, emulsified in phos-phate-buffered saline, and centrifuged at 300 x g for10 min. The remaining steps were performed as out-lined above.

(d) Material from the side of storage jug. Thechlorhexidine was discarded from storage jug 7BU(only Serratia marcescens was isolated from the fluid)after a 27-month period. A reddish precipitate wasevident on the sides of the jug. This was scraped offwith a scalpel blade. The material was transferred toa test tube, and 5% glutaraldehyde and cacodylatebuffer (0.067 M; pH 6.2) with 0.15% ruthenium red wasadded. The remaining steps were performed as out-lined above.

(e) An isolate of Serratia marcescens whichhad never been exposed to chlorhexidine. Anisolate of Serratia marcescens (from the urine of acatheterized patient) was inoculated to brain heartinfusion broth, incubated for 6 h, and then centrifugedat 300 x g for 10 min. The sediment was then fixed asoutlined above.Sudan black stain. A 10-ml sample of contami-

nated chlorhexidine from each stock bottle was re-moved and centrifuged at 300 x g for 10 min. Thesediment was washed once in phosphate-buffered sa-line and then smeared onto a glass slide, air dried, andheat fixed. It was then stained with Sudan black B for10 min, washed, cleared with xylol and counterstainedwith 0.5% aqueous safranine for 15 s, washed in tapwater, blotted dry, and examined under oil immersionwith a Zeiss light microscope (4).

RESULTSChlorhexidine from five ofthe six stock bottles

was contaminated with Serratia marcescens,and samples from two of these also grew Fla-vobacter spp. and Pseudomonas sp. Chlorhexi-dine from the sixth container was negative whencultured initially and remained so throughout(Table 1). The concentration of organisms in thefive contaminated bottles was 108 colony-form-ing units per ml initially and declined during the27-month observation period (Table 1). Serratiamarcescens was still recovered from one stockbottle (7BU) in concentrations of 106 colony-forming units per ml after 27 months of storagein the laboratory. Three species of organisms,Serratia marcescens, Pseudomonas sp., andFlavobacter sp., were recovered initially fromtwo stock bottles, 4S and 7B. The total numberof organisms was similar in both (2.4 x 10' and3 x 10' colony-forming units per ml, respec-

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S. MARCESCENS IN CHLORHEXIDINE 1095

TABLE 1. Results of serial cultures of stock solutions of chlorhexidine

No. of CFU/ml isolated at indicated month, day, and year'Source of Organism isolated Aug. 1979 Ag. Jansample' Jan.1979Au. Jn Ap.18

1 8 15 1980 1981Ap.18

1oV Serratia marcescens 1.49 X 108 108 108 10 1o8c

4S Serratia marcescensPseudomonas sp. 3 x 108 ND ND ND ND ND 3 x 106dFlavobacter sp.

7B Serratia marcescensPseudomonas sp. 2.4 x 108 ND ND ND ND ND 2 X 106dFlavobacter sp.

7BU Serratia marcescens 1.6 x 108 10o lo8 107 106 1068A Serratia marcescens 1.3 x 108 1o8 lo8 107 105 NG

BC NG NG NG NG NG NG NGa These bottles were held in the laboratory; samples from 1OV, 7BU, 8A, and BC were cultured at least

monthly.b CFU, Colony-forming units; NG, no growth; ND, not done.c Stock bottle used for electron microscopy studies at this time.d Pseudomonas sp. only.

TABLE 2. Minimal inhibitory concentrations ofchlorhexidine gluconate required to inhibit

indicated organisms

MIC (tg/ml)Organism (source) No.

50% 90% Range

Serratia marcescens (stock 25 1,024 1,024 512-1,024solutions of chlorhexi-dine)

Serratia marcescens (clini- 5 16 128 16-128cal specimens)

Escherichia coli (clinical 8 2 2 2specimens)

Enterobacter spp. (clinical 3 16 16 16specimens)

Klebsiella spp. (clinical 2 16 16-32specimens)

Pseudomonas aeruginosa 4 16 32 16-32(clinical specimens)

Proteus sp. (clinical speci- 1 64mens)

Staphylococcus aureus 2 0.5 0.5 0.5(clinical specimens)

tively). No attempt was made to quantitate eachorganism individually. When sampled again 27months later, only Pseudomonas sp. was re-covered from both in concentrations of 106 col-ony-forming units per ml.The MIC of chlorhexidine gluconate required

to inhibit the various organisms tested is shownin Table 2.

All 25 isolates of Serratia marcescens re-covered from contaminated chlorhexidine wereinhibited by 8 ILg of amikacin per ml. Only 32%were inhibited by 2 ,ug of gentamicin per ml; theremainder required 128 ,ug/ml for inhibition of

FIG. 1. Scanning electron micrograph of the innersurface of a piece of a plastic chlorhexidine stockbottle. The chlorhexidine from this bottle was nega-tive when cultured aerobically. (Note the absence ofbacteria and the woven texture of the surface of theplastic.) The bar indicates 5 ,um.

growth.Figure 1 shows the inner surface of stock

bottle BC, which contained chlorhexidine but

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was negative on culture. (Note the uneven tex-ture of the surface.)Scanning electron microscopy of the inner

surface of the 10V plastic stock bottle is shownin Fig. 2 through 4. Only Serratia marcescenswas isolated from the liquid, sediment, and sidesof this bottle. Figure 2 is a low-power viewshowing bacteria embedded in a matrix of fi-brous material that, in some areas, is heaped upinto distinct masses. Discrete microorganismsare evident adherent to the plastic. In Fig. 3,bacteria are seen within the fibrous matrix, andin Fig. 4, these organisms are seen to be embed-ded in the fibrous matrix to varying degrees.When sections from the remaining plastic stockbottles were examined (1 year later than thepiece of plastic shown in Fig. 2 through 4),


bacteria were still evident embedded in a matrix(Fig. 5); however, this material was not as exten-sive as is shown in Fig. 2 through 4. Figure 6 isa transmission electron micrograph of a bacte-rium adherent to the surface of a piece of plasticfrom the 10V stock bottle (sample taken froman area adjacent to the piece shown in Fig. 2through 4). The ruthenium red-positive natureof the material that covers the bacterium isevident even though this material is radicallycondensed by dehydration.A transmission electron micrograph (Fig. 7) of

the sediment of contaminated chlorhexidineshowed that most of the bacteria free in the fluidwere abnormal. Bacteria from all three contam-inated stock bottles containing Serratia onlyhad the same appearance. As seen in Fig. 7,

FIG. 2. Scanning electron micrograph of the inner surface of a section of a plastic chlorhexidine stockbottle (1OV; Table 1) contaminated with Serratia marcescens. Before examination, the specimen had beenfixed in buffered glutaraldehyde. (Note the many bacteria on the surface of the plastic [lower arrow] and theconfluent mass offibrous material in which bacteria are embedded [two upper arrows].) The bar indicates 5um.


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FIG. 3. High-magnification scanning electron micrograph of the inner surface of a section of a plasticchlorhexidine stock bottle (1OV) contaminated with Serratia marcescens. (Note the association of bacterialcells with the matrix of the fibrous masses.) The bar indicates 1.0 ptm.

FIG. 4. High-magnification scanning electron micrograph of the inner surface of a section of a plasticchlorhexidine stock bottle contaminated with Serratia marcescens. Some bacterial cells are completely orpartially (arrows) submerged in this confluent mass offibrous material. The bar indicates 1.0 ,um.

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Figure 10 is a transmission electron micro-graph of material scraped from the surface ofstorage jug 7BU (Table 1). Only Serratia mar-cescens was isolated from this container. Thecell walls of the bacteria in this section are wellresolved, but intracellular detail is still poor.There are no discernible ribosomes, and thecytoplasm resembles that of the bacteria seen inFigure 8A. (Note the continuum of the finefibrous matrix.)

DISCUSSION_- Contamination of disinfectants in hospitals is

not a new problem. In 1967, Burdon and Whitby(3) described contamination of chlorhexidineand savlon (chlorhexidine-cetrimide mixture) byPseudomonas spp. The addition of 4% (vol/vol)isopropyl alcohol to aqueous preparations ofthese disinfectants reduced the incidence of con-

FIG. 5. Scanning electron micrograph of a piece ofplastic from a chiorhexidine stock bottle contami-nated with Serratia marcescens (7BU; Table 1).Many of the bacteria are embedded in a matrix. Thewoven background of the plastic is not seen in mostof the micrograph, suggesting that it is covered by thematrix. The bar indicates 5 jm.

many of the bacteria were disrupted, whereasothers contained large vacuoles in their cyto-plasm. When examined by light microscopy,these vacuoles were seen to stain with Sudanblack. Although the cell -walls of some of thebacteria that had been suspended in chlorhexi-dine for 18 months appeared to be normal, theircytoplasm (Fig. 8A) was never seen to consist ofdiscrete ribosomes but, rather, to consist of uni-form-ly electron-dense masses of various dimen-sions. Contrast the appearance of these bacterialcells with those of an isolate of Serratiamarcesc-T1lcens (from the catheterized urinary tract of a M_patient) that had never been exposed to chlor- FIG. 6. Transmission electron micrograph of ahexidine (Figure 8B). The ribosomes and nuclear section of a ruthenium red-stained preparation of amaterial are well seen. A fibrous capsule is also fragment of a plastic chlorhexidine stock bottle (OV;evident. The cytoplasmic structure of cells of Table 1) contaminated with Serratia marcescens.

Serratia marcescens recovered from the con- This gram-negative bacterial cell is surrounded bytaminated chlorhexidine and grown on an agar

an electron-dense condensed, anionic, ruthenium red-taminatednchlorexidin and grow onuan ar positive material (P). This same electron-dense con-surfacetpismseen in Fig. 9, and this structure isdensed material (M) is seen to cover much of theentirely typical of the ultrastructure of bacterial inner surface of the plastic container. The bar idi-cels. cates 0.1ctm.


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FIG. 7. Transmission electron micrograph of sediment of contaminated chlorhexidine which on culturegrew Serratia marcescens (IOV; Table 1). At the time this sample was collected, the chlorhexidine had beencontaminated for 18 months. Many of the bacteria in this micrograph are disrupted, and others have largecytoplasmic vacuoles. Even in apparently intact bacteria, the cytoplasm appears to be abnormal. The barindicates 1.0 tm.

tamination. Pseudomonas maltophilia has beenisolated from contaminated savlon (17). Pseu-domonas cepacia was adapted to a 1:30 dilutionof savlon; however, it did not survive in thisconcentration when the pH was adjusted to 7.2with hard tap water (2). The Pseudomonas ce-pacia organisms remained viable in tap waterfor 1 year. We found that the Serratia marces-cens organisms survived in chlorhexidine formore than 27 months.The highest concentration of chlorhexidine

required to inhibit the strains of Serratia iso-lated from this disinfectant was 1,024 ,ug/ml;however, the organism could be grown fromsolutions containing 20,000 ,ug/ml without usinginhibitors of chlorhexidine in the medium. Thescanning electron micrographs showed thatmany of the organisms were embedded in a veryextensive fibrous matrix, and transmission elec-tron micrographs (Fig. 6 and 10) showed thatthis matrix was ruthenium red positive. Thismaterial is probably polysaccharide, since ruthe-nium red, a cationic dye, has an affinity for

strongly anionic polymers, most of which arepolysaccharides (9). It stains extracellular ma-terial associated with the surface of cell wallsthat is otherwise difficult or impossible to dem-onstrate. Whether this ruthenium red-positivematerial interferes with penetration of the chlor-hexidine, a cationic agent, to the surface of thecell is speculation only. Indeed, the cells shownin Fig. 10 (scraped from the surface of a chlor-hexidine storage jug and surrounded by exten-sive fibrous matrix) had abnormal cytoplasmand were similar in appearance to some of thecells free in the liquid (Figure 8A), suggestingthat the fibrous matrix did not completely pre-vent the chlorhexidine from interacting with thecell. Chlorhexidine interacts with bacterial cellsin- several ways (7). It is adsorbed onto thesurface of the bacterial cell and damages perme-ability barriers, resulting in leakage of cell con-tents (12). At higher concentrations, it precipi-tates or coagulates cytoplasm.

Stickler and Thomas (15) studied 802 bacte-rial isolates from patients with urinary tract

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infections and found that none ofthe 369 isolatesof Escherichia coli were resistant to 500 ,ug ofchlorhexidine per ml, whereas 83 and 45.7% oftheir Providencia stuartii and Pseudomonasaeruginosa isolates, respectively, were so resist-ant. In a subsequent study (16), they were unableto explain the resistance of Providencia stuartii(chlorhexidine MIC, 1,600 ytg/ml) in terms ofdifferences in lipid content between sensitiveand resistant strains. Furthermore, the resist-ance could not be attributed to reduced adsorp-tion of the antiseptic or its enzymatic degrada-tion. They speculated that there might be a

w + * layer in the cell wall of Providencia stuartiiwhich absorbs the agent and prevents furtherpenetration through to the cytoplasmic mem-brane. Our observations would suggest that thisis not so for Serratia marcescens (Fig. 7, 8A,and 10).

Cytoplasmic vacuoles (Fig. 7) were present inorganisms free in the chlorhexidine. These vac-uoles stained with Sudan black B and were notpresent in cells (from the chlorhexidine) thathad been grown on a solid medium.We have described the morphology of Serra-

tia marcescens that has survived in chlorhexi-dine for a prolonged period. Additional studiesare needed to elucidate the mechanism of thissurvival.

F4!___ We were unable to determine the clinical rel-evance of the contaminated chlorhexidine. TheSerratia isolates from contaminated chlorhexi-dine could not be 0 serotyped. The antibioticsusceptibility of such isolates (generally genta-micin resistant and amikacin sensitive) resem-bled the pattetn of isolates from the urine ofinfected patients. With the institution of an ap-propriate infection control program, many mea-sures, such as cohorting infected or colonizedpatients, obtaining separate urine measuringcontainers for each patient, etc., were introducedat about the time the contaminated chlorhexi-dine was discovered. The Serratia infection/col-onization rate before our infection control pro-

_ gram began was 132/10,500 discharges and, in1980, when the program had been operationalfor 3 years, the rate was 62.1/10,000 discharges

FIG. 8. (A) Transmission electron micrograph ofthe same sediment of contaminated chlorhexidine asthat shown in Fig. 7. The cell wall of this bacterium(Serratia marcescens on culture) is normal. The cy-toplasm is abnormal, however, in that it consists ofamorphous, electron-dense masses. In contrast, cellsof Serratia marcescens grown in BHI broth whichwas never exposed to chlorhexidine (B) show easilydiscernible ribosomes and nuclear material. (Notethe fibrous capsule surrounding these bacteria.) Thebar indicates 0.1 ,um.



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FIG. 9. Transmission electron micrograph of Serratia marcescens from contaminated chlorhexidine aftersubculture to an agar medium. The bacteria in this micrograph have no cytoplasmic vacuoles; their cell wallsare intact, and their cytoplasm appears to be normal. The bar indicates 0.1 ,um.

(P < 0.001) (Marrie et al., submitted for publi-cation).

ACKNOWLEDGMENTSThis study was supported in part by grants from the

University Internal Medicine Research Foundation and Ay-erst Laboratories Ltd., Montreal, Quebec, Canada.We wish to thank Carol Swantee, Carol Kwan, and Joyce

Nelligan for technical support and Wilma Mays for typing themanuscript. We also thank Sheila Costerton for photographicassistance.

LITERATURE CITED1. Barry, A. L., and C. Thornsberry. 1980. Susceptibility

testing: diffusion test procedures, p. 469. In E. H. Len-nette, A. Ballows, W. J. Hausler, Jr., and J. P. Truant(ed.), Manual of clinical microbiology, 3rd ed. AmericanSociety for Microbiology, Washington, D.C.

2. Bassett, D. C. J. 1971. The effect of pH on the multipli-cation of a pseudomonad in chlorhexidine and cetrim-ide. J. Clin. Pathol. 24:708-711.

3. Burdon, D. W., and J. L. Whitby. 1967. Contaminationof hospital disinfectants with Pseudomonas species. Br.Med. J. 1:153-155.

4. Burdon, K. L. 1946. Fatty material in bacteria and fungirevealed by staining dried, fixed slide preparations. J.Bacteriol. 52:665-678.

5. Cohen, A. L, D. P. Marlow, and G. E. Garner. 1968. Arapid critical point using Flurocarbon ("Freons") asintermediate and transitional fluids. J. Microsc. (Ox-ford) 7:331-342.

6. Edwards, P. R., and W. H. Ewing. 1977. Identificationof Enterobacteriaceae, 3rd ed. Burgess Publishing Co.,Minneapolis, Minn.

7. Hennessey, T. D. 1977. Antibacterial properties of hibi-tane. J. Clin. Periodontol. 4:36-48.

8. Lovell, D. J., and D. J. Bibel. 1977. Tween 80 mediumfor differentiating nonpigmented Serratia from otherEnterobacteriaceae. J. Clin. Microbiol. 5:245-247.

9. Luft, J. H. 1971. Ruthenium red and ruthenium violet. II.Fine structural purification, methods for use for electronmicroscopy and localization in animal tissues. Anat.Rec. 171:369-416.

10. Malick, L E., and B. W. Wilson. 1975. Modified thio-carbohydrazide procedure for scanning electron micros-copy: routine use for normal, pathological or experimen-tal tissues. Stain Technol. 50:265-269.

11. Reynolds, E. S. 1963. The use of lead citrate at high pHas electron-opaque stain in electron microscopy. J. CellBiol. 17:298-342.

12. Richards, R. M. E., and R. H. Cavill. 1979. Electron-microscope study on the effect of chlorhexidine onPseudomonas aeruginosa. Microbios 26:85-93.

13. Rosenberg, A., S. D. Alatary, and A. F. Peterson.1976. Safety and efficacy of the antiseptic chlorhexidine

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1102 MARRIE AND COSTERTON APPL. ENVIRON. MICROB10L.., }. . .. *_. E_ X <

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FIG. 10. Transmission electron micrograph of material scraped from the side of a chlorhexidine storagejug (7BU; Table 1) contaminated with Serratia marcescens. The cell wall of these bacteria is well resolved;however, the cytoplasm is amorphous with no discernible ribosomes seen. (Note the extensive fibrous matrix.)The bar indicates 0.1 um.

gluconate. Surg. Gynecol. Obstet. 143:789-792.14. Steers, E., E. L. Foltz, and B. S. Graves. 1959. An

inocula replicating apparatus for routine testing of bac-terial susceptibility to antibiotics. Antibiot. Chemother.9:307-311.

15. Stickler, D. J., and B. Thomas. 1980. Antiseptic andantibiotic resistance in Gram negative bacteria causing

urinary tract infection. J. Clin. Pathol. 33:288-296.16. Thomas, B., and D. J. Stickler. 1978. Chlorhexidine

resistance and the lipids of Providencia stuartii. Micro-bios 24:141-150.

17. Wishhart, M. M., and T. V. Riley. 1976. Infection withPseudomonas maltophilia. Hospital outbreak due tocontaminated disinfectant. Med. J. Aust. 2:710-712.

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