Appun MICROBIOLOGY, Aug. 1975, p. 229-237Copyright 0 1975 American Society for Microbiology

Vol. 30, No. 2Printed in U.S.A.

Microbiological Hazards of Household Toilets: DropletProduction and the Fate of Residual Organisms

CHARLES P. GERBA, CRAIG WALLIS, AND JOSEPH L. MELNICK*Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas 77025

Received for publication 12 March 1975

Large numbers of bacteria and viruses when seeded into household toilets wereshown to remain in the bowl after flushing, and even continual flushing could notremove a persistent fraction. This was found to be due to the adsorption of theorganisms to the porcelain surfaces of the bowl, with gradual elution occurringafter each flush. Droplets produced by flushing toilets were found to harbor bothbacteria and viruses which had been seeded. The detection of bacteria andviruses falling out onto surfaces in bathrooms after flushing indicated that theyremain airborne long enough to settle on surfaces throughout the bathroom.Thus, there is a possibility that a person may acquire an infection from an aerosolproduced by a toilet.

The transmission of disease by aerosols fromtoilets has received only limited study. It hasbeen suggested that, aside from coughing andsneezing, this must be the most common pro-cess involved in the generation of infectiousaerosols (6). Darlow and Bale (6) demonstratedthe production of bacterial aerosols, with theaid of both a liquid impinger and a slit sampler,from flushed bowls containing Serratiamarcescens. These aerosols were found to per-sist for at least 12 min after the flush. Thegeneration of aerosols by toilets seeded withcoliform bacteria has been demonstrated byBound and Atkinson (3) and more recently byNewsom (14). The size of particles produced bythe flushing toilet was found to be in the rangethat was capable of reaching the lower respira-tory tract (6). In addition, pathogenic fecalcontaminants, such as Escherichia and Sal-monella, have been isolated from the respira-tory tract of infected humans (6).The fallout of droplets containing pathogens

on bathroom surfaces is also of concern, sincehand contact with contaminated surfaces canresult in self-inoculation by touching of the noseor mouth (11). Hutchinson (12) traced thespread of Shigella sonnei in a nursery school tocontaminated toilet surfaces. Contact with con-taminated surfaces has also been shown to beimportant in the spread of animal viruses (11).To date no information exists on the genera-

tion of viral aerosols by household toilets. Thisstudy was carried out to gather more informa-tion on the fate of both bacteria and viruses influshed toilets.

MATERIALS AND METHODS

Viruses and virus assays. E. coli bacteriophageMS-2 and a plaque-purified line of type 1 poliovirus(strain LSc) were used in this study. MS-2, likepoliovirus, is a small (25-nm diameter) icosahedralribonucleic acid virus. All bacteriophage assays weredone by a modification of the agar overlay method asdescribed by Adams (1). Overlay agar and broth usedfor bacteriophage samples were prepared according toDavis and Sinsheimer (8). Stock poliovirus was grownin baboon kidney cells, concentrated 10-fold, andpartially purified by membrane chromatography (22).Poliovirus samples were diluted in tris(hydroxy-methyl)aminomethane-buffered saline containingpenicillin (100 U/ml) and streptomycin (100 gg/ml).Poliovirus assays were made with BSC-1 cells by theplaque-forming unit method as used in this laboratory(20).Bacteria and bacterial assays. A strain of E. coli

isolated from domestic sewage was used (identifica-tion based on ImVic test). All coliform assays wereperformed on Levine eosin methylene blue (EMB)agar. Total aerobic bacterial counts were performedon Standard Methods agar (BBL, Cockeysville, Md.).Cultures of E. coli used in seeding experiments weregrown overnight in Trypticase soy broth (TSB) (BBL,Cockeysville, Md.). All bacterial samples were dilutedin tris(hydroxymethyl)aminomethane-buffered sa-line.

Toilets. Standard household tank or valve toiletswere used. The tank toilets had a reservoir containingapproximately 20 liters, of which 13.7 liters wasreleased during a flush, unless otherwise noted. Thebowl of the tank-type toilet contained a volume of 3.5liters unless indicated otherwise. The bowl volume ofthe valve toilet was approximately 4.2 liters, with astandard but undetermined amount of water re-leased during a flush. In the valve toilet used, the

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230 GERBA, WALLIS, AND MELNICK

amount of water released was dependent on the waterline pressure in the building, which was usuallyconstant. Before being seeded with bacteria or virusesthe toilets were cleaned with commercially availablechlorine-containing cleanser and flushed repeatedlyto eliminate any bacteria or viruses naturally present.A solution of 5 g of sodium thiosulfate per liter wasthen added to inactivate any chlorine present in thewater, at a ratio of 1 ml of solution to 1 liter oftapwater. A tank-type toilet was used in all experi-ments unless indicated otherwise.

RESULTSResidual infectious material in toilet

bowls. The first group of experiments wasconducted to determine the fate of infectiousagents in toilet waters after flushing of a typicaldomestic toilet. Toilet bowls were first cleanedas described in Materials and Methods andthen seeded with either an overnight culture ofE. coli or MS-2 phage. In both cases theorganisms were suspended in 100 ml of TSB tosimulate the presence of organic matter foundin actual fecal material. After the bowl waterwas mixed, a baseline sample was obtained.The toilet was then flushed, and the toilet waterwas sampled for residual organisms. This proce-dure was repeated several times, and the resultsof a typical experiment for both bacteria andviruses can be seen in Fig. 1. As anticipated, theinitial flush eliminated the major proportion ofexogenously added organisms. However, afterrepeated flushes, instead of diminishing, therewas often an increase in the number of residualorganisms detected in the bowl. In the case ofboth bacteria and viruses, the number of orga-nisms in the bowl reached a plateau belowwhich their number could not be reduced, evenafter repeated flushing. From this evidence, itappeared that significant numbers of bacteriaand viruses were being adsorbed to the toiletporcelain and then eluted during the flushingaction.To test this speculation, the previous experi-

ment was repeated, and, after the third flush,Tween 80, a nonionic detergent, was added toyield a final concentration of 0.1% in the bowlwater. The sides of the bowl were then scrubbedwith a brush, and a sample was obtained forassay. The results of this experiment for E. coliare shown in Fig. 2. A 10,000-fold increase in thebacterial count occurred after the Tween 80treatment, indicating that bacteria were ad-sorbing to the porcelain surfaces and could beeluted by scrubbing in the presence of an eluentsuch as Tween 80. When the toilet was flushedafter the Tween 80 treatment, almost all of thebacteria were removed from the bowl water

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NUMBER OF FLUSHESFIG. 1. Effect of flushing on removal of exoge-

nously added bacteria and viruses to the toilet bowl.The log,, number of organisms indicated above wasadded to the toilet bowl water, and water sampleswere removed from the bowl for assay after each flush.Log,1 values of organisms represent the numberpresent in the entire volume contained in the toiletbowl. Symbols: 0, MS-2 phage; 0, E. coli.

(Fig. 2). In other experiments it was found thatsimple mixing of Tween 80 into the bowl wateror scrubbing the bowl with a brush withoutaddition of Tween 80 were equally efficient inthe removal of bacteria from the sides of thetoilet bowl.When the same experiment was performed

with poliovirus or MS-2 phage (Fig. 3), anincrease in the number of viruses was also notedafter Tween 80 treatment, but subsequentflushing resulted in only a gradual loss of virusfrom the bowl water. Thus, it did not appearthat all of the viruses were eluted from the bowlsurfaces by the Tween 80 treatment. Perhapsviruses are more difficult to desorb from theporous surface of the porcelain than bacteria.To determine the number of bacteria usually

present in toilets, toilet bowls were monitored inrestaurants, service stations, etc. Toilets weretreated with Tween 80 and agitated to elutebacteria from the bowl surfaces. A sample ofbowl water was then removed and plated onboth EMB agar and Trypticase soy agar. Theresults of these experiments are shown in Table1. Aerobic bacteria were present at levels from

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MICROBIOLOGICAL HAZARDS OF HOUSEHOLD TOILETS

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NUMBER OF FLUSHESFIG. 2. Elution of E. coli from toilet bowl surfaces

by addition of Tween 80. Same methods as in Fig. 1,except Tween 80 was added to the bowl water afterthe flush indicated and the bowl was scrubbed with a

brush.

101 to over 109 in the bowls tested.Droplet production. Dye studies were per-

formed to determine if water droplets ejectedinto the air during flushing reach the seat levelof the bowl. Crystal violet dye was added toboth the bowl and the tank water, and aftercovering the bowl with a sheet of white absorb-ent paper the toilet was flushed. By examina-tion of the paper the number of visible dropletsproduced during flushing was determined.Tank-type toilets produced a random pattern ofdroplets on the paper, ranging in number from27 to 104 during a given flush. Valve-type toiletsproduced fewer visible droplets (between 7 and10), which were always found towards the rearof the bowl. It appeared that the high pressureof the water coming into the bowl in this type oftoilet caused the droplets to be ejected to therear.

If the bowl was first seeded with E. coli andEMB agar plates were exposed at the seat level(taped to a support and facing the bowl water),coliform colonies appeared on the agar plates in

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NUMBER OF FLUSHESFIG. 3. Elution of MS-2 phage from toilet bowl

surfaces by addition of Tween 80. Same methods as inFig. 2.

TABLE 1. Recovery of bacteria from toilet bowlsa

Total no./vol intoilet bowl

(log,.)Sam- Type of TypeofToaple establishment toilet Coli- Totalno. forms bac-

teria

1 Hamburger stand Tank 2.50 6.902 Service station Tank 5.50 8.053 Hospital Valve 5.40 7.504 Restaurant Tank 3.10 6.655 Motel Valve 3.90 9.256 Service station Tank 2.50 7.507 Home Tank 3.30 8.258 Research institute Valve 3.70 6.809 Restaurant Valve 8.10 9.3010 Research institute Valve 6.10 6.6511 Research institute Valve 2.28 7.1012 Research institute Valve 3.10 6.8013 Service station Valve 4.50 9.4014 Hospital Valve 2.25 6.00

a Toilets were treated with 220 ml of 1% Tween 80 toremove adsorbed bacteria before sampling.

the same pattern as the droplets.Collection of ejected organisms with gauze.

To determine more quantitatively the numberof organisms which reach the seat level during

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232 GERBA, WALLIS, AND MELNICK

flushing, cotton gauze was utilized to collectejected organisms. A series of experiments wasfirst performed to determine the efficiency withwhich bacteria and virus entrapped by thegauze could be recovered. Suspensions of eitherbacteria or viruses were added to cut squares ofgauze (6 by 6 inches [ca. 15 by 15 cm], twothicknesses, 12 actual layers of gauze), andvarious solutions were evaluated for their abilityto elute the organisms. Gauze prewetted withTSB was found to give the best recoveries of E.coli and MS-2, and the average percentage ofrecoveries for several experiments is shown inTable 2. It has been shown that enteric virusesadsorbed to a variety of surfaces can be elutedat high pH (17). Thus, glycine buffer adjustedto pH 11.5 was used to elute poliovirus fromgauze prewetted with glycine. The average re-covery using this method to elute poliovirusfrom the gauze was 84% (Table 3). With polio-virus it was necessary to concentrate the virusfrom the gauze eluate before assay. This wasaccomplished with adsorption onto membranes(Millipore Corp.) as described by Wallis et al.(21).The following procedure was used to deter-

mine the number of organisms reaching the seatlevel in droplets. The toilet bowl and rim were

TABLE 2. Elution of E. coli and MS-2 phage fromgauze with TSBa

Pretreatment No. of No. of or-of gauze orga- ganisms

Organism prior to ad- nlsms eluted % Re-dition of test placed on from gauze coveryorganism (x 104) (x 10')

E. coli TSB 19.0 19.5 10419.0 20.0 1065.0 4.76 955.0 5.56 1135.0 4.90 98

E. coli None, dry 5.0 7.76 155.0 5.95 12

19.0 7.00 36MS-2 TSB 117 106 90

117 89 76117 92 79117 80 68117 91 78

a Gauze pads (6 by 6 inches, 2 thicknesses, 12 actuallayers of gauze) were used dry or wetted with 5 ml ofTSB. Excess fluids were expressed, and then 0.1 ml ofthe indicated organisms suspended in tapwater wasplaced onto the gauze. After 5 min the gauze wassoaked in 20 ml of broth, and approximately 7 ml ofeluate was expressed from the gauze.

first sterilized by igniting alcohol placed on therim, and the bowl was seeded with the testorganism. The bowl was then covered with apiece of gauze (14 by 17 inches [ca. 35.6 by 43.2cm ], 12 thicknesses, double layer) presoakedwith 50 ml of TSB or glycine buffer, and thetoilet was flushed. The gauze was then soakedfor 5 min in 150 ml of eluent with occasionalsqueezing of the gauze to obtain a maximalamount of eluate. Approximately 100 ml ofeluate was usually obtained. The number ofbacteria or viruses in the eluate was thenquantified. EMB agar placed on top of thegauze held over the bowl indicated that E. colibacteria did not penetrate the gauze afterflushing. Table 4 shows the number of E. coliejected from two tank-type toilets with differentvolumes and the amount of variation in thenumber of organisms recovered from replicateexperiments.The number of bacteria and phage ejected

from the toilet during a flush was found to bedirectly proportional to the amount present atthe time of the flush (Fig. 4). Studies withpoliovirus (Table 5) indicated that similarquantities of this virus were ejected from thebowl as found for MS-2 phage when the bowlwas seeded with 106 plaque-forming units.When the number of seed organisms ap-proached numbers found in human stool (about1012 for bacteria [18] and 101 for virus [16D, ashigh as 6.6 x 101 coliforms and 2.8 x 103plaque-forming units of virus were recoveredfrom the gauze.The number of coliforms and the total num-

ber of aerobic bacteria recovered from the gauze

TABiz 3. Elution ofpoliovirus from gauze pads withpH 11.5 glycine buffer

- PFUGauze pad PFU added detected

no.-a to1) in eluate Recovery(x 105) (xlO'1)

1 4.00 3.40 85.02 4.00 3.90 97.53 4.00 3.00 75.04 4.00 3.20 80.0

a Gauze pads (4 by 7 inches, 2 thicknesses, 12 actuallayers of gauze) were wetted with 5 ml of pH 11.5glycine buffer, and 0.1 ml of LSc poliovirus type 1suspended in tapwater was placed onto the gauze.After 5 min the gauze pads were soaked in 20 ml ofpH11.5 glycine buffer for another 5 min, after which 20ml of eluate was squeezed from the gauze, and the pHwas immediately adjusted to 7.5. PFU, Plaque-form-ing units.

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MICROBIOLOGICAL HAZARDS OF HOUSEHOLD TOILETS 233

TABLE 4. Number of E. coli ejected from toiletsduring flushinga

No. of bacteria added to No. of bacteriatoilet bowl (x 10') ejected"

Experimental toilet 1(bowl volume, 3,500 ml)9.97 1,2609.97 1,095

15.70 1,340105.0 5,500157.0 3,040245.0 66,500

Experimental toilet 2(bowl volume, 5,900 ml)

44.8 1,17021.8 1,92537.7 2,440

318.0 1,700171.0 9,000283.0 2,630

aBoth toilets had tank volumes of 21,000 ml.b Represents number of bacteria collected from

gauze pad held at seat level over the bowl duringflushing.

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FIG. 4. Concentration of bacteria and virus intoilet bowl and numbers ejected during flushing. Thelog,0 number of E. coli or MS-2phage indicated abovewas placed in the bowl water, a gauze wetted withTSB was placed over the bowl, and the toilet was

flushed. The log,. of the number of organisms re-covered from the gauze is indicated in the ordinateof the above figure. Symbols: 0, MS-2 phage; 0, E.coli.

when actual fecal material was present areshown in Table 6. These data indicated thatnumbers of bacteria reaching the seat level ofthe bowl were not appreciably different whenthe bowl was seeded with similar numbers ofbacteria as cultures, homogenized stool, or as a

solid fecal pellet. The apparent increase in theratio of total bacteria to coliforms may be theresult of the use of selective media in the assayof the coliforms; i.e., bacteria may be damagedduring aerosolization and fail to grow on theselective media.

TABLE 5. Number of polioviruses ejected from toiletduring flushing

No. of PFU No. of PFU Estimated no. ofadded to toilet detected in PFU present inbowl (x 10') eluate eluatea

2.88 1,570 2,8022.67 675 1,2053.78 300 5362.94 893 1,594

aTo determine efficiency of concentration, a por-tion of the baseline sample was added to a volume ofpH 11.5 glycine buffer and concentrated by the samemethod as the eluate. Estimated number of plaque-forming units (PFU) was calculated as follows: actualnumber of viruses collected by gauze = (100/%efficiency of elution from gauze) (100/% efficiency ofconcentration from eluate) (number of viruses de-tected in eluate). Efficiency of elution from gauze,84%; average efficiency of concentration using thismethod, 67%.

TABLE 6. Number of bacteria ejected from toiletsduring flushing using human stool

No. of bacteria present in No. of bacteriatoilet water at time of flush e acted'

(x 10')

Coliforms Total Coliforms Total

Homogenized stool0.182 2.8 10 4,5001.19 1.54 35 2,0006.47 7.0 35 4,0007.35 12.9 60 18,000

18.20 28.2 38 8,000Solid stool

0.0007 0.0035 2 6,0001.01 9.10 85 137,0000.168 2.30 145 10,0000.178 1.68 30 21,0001.53 3.06 280 7,000

Controlb0 0 0 2,5000 0 0 4,5000 0 0 7100 0 0 1,090

aRepresents number of bacteria collected fromgauze pad held at seat level over the bowl duringflushing.

b Gauze was placed over the bowl, but the toilet wasnot flushed. Organisms detected in the controls prob-ably represent those naturally present in the bowlwater or from contamination during collection of theeluates.

Fallout of airborne E. coli onto bathroomsurfaces. To determine if bacteria ejected intoair from the bowl during flushing were fallingout onto surfaces in the bathroom, EMB agar

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234 GERBA, WALLIS, AND MELNICK

plates were exposed at various times after atoilet seeded with E. coli had been flushed. Thedesign of the experimental bathroom can beseen in Fig. 5. Agar plates were placed at 6-inch(ca. 15-cm) intervals throughout the room for atotal number of 50 plates exposed at one time.Four sets of plates were usually exposed 2, 4,and 6 h after a flush, plus a control set exposed 2h before the toilet was flushed. The results ofthese experiments are summarized in Table 7.Within the first 2 h, bacteria were usuallydetected only in a limited area around thetoilet, whereas bacterial colonies detected atlater intervals were more randomly distributedthroughout the room.To detect the fallout of airborne particles

containing viruses from flushed toilets, 10- by8.75-inch (ca. 25.4 by 20.9 cm) squares of gauzemounted on metal screens and wetted with TSBwere placed at the locations shown in Fig. 5. Atthe end of the exposure time (each set of gauzewas exposed at 2-h intervals), as much fluid aspossible was expressed from the gauze andassayed for virus. Since it was desirable to keepthe amount of elution fluid to as small a volumeas possible, a series of experiments was con-ducted to determine the effect of eluate volumeon the efficiency of virus recovery. The results ofthese experiments are shown in Table 8. Whenthe eluate volume was less than 10 ml, theefficiency of virus recovery was appreciablyreduced. Thus, enough TSB was added to thegauze so that the eluate volume did not fallbelow this amount.The results of fallout experiments using MS-2

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location of gauze pads for viral fallout experiments.

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TABLE 7. Number of bacteria detected after fallingout on bathroom surfaces after flushing

EstimatedAvg no. Avg no. no. of

Time of plates of bacteria Venti-after on which colonies on floor lationc

flush (h) bacteria detected surfacegrewa per flush area of

bathrooms

0-2 7.0 9.0 737 Closed2-4 2.8 3.0 246 Closed4-6 0 0 0 Closed0-2 7.8 9.2 639 Open2-4 2.0 2.0 164 Open4-6 0 0 0 Open

a Represents the average of six experiments for each2-h period. In each experiment 1011 E. coli were addedto the bowl before flushing. Fifty EMB agar plates,mainly on the bathroom floor, were exposed duringeach 2-h period and replaced by another set of plates.

Calculated as follows: (total floor surface area ofbathroom/total surface area of all agar platesexposed) (total number of colonies on agar plates).

c The ventilation of the room was considered closedwhen all air vents in the room were sealed and thebathroom door remained closed.

TABLE 8. Effect of eluate volume on recovery ofMS-2phage from gauze padsa

Vol of wetting Avg vol of Avg % recoveryfluid (ml) eluate (ml) of virus

35 11.3 7230 10.1 7825 5.0 5922 4.2 46

a Gauze pads (10 by 8.75 inch) were wetted with theamount of TSB indicated above. Virus was added astwo drops of 0.05 ml each. After 15 min as much fluidas possible was expressed from each of the gauze pads.This eluate was then assayed for virus. 1.2 x 103to 6.0 x 10" viral plaque-forming units were added toeach gauze pad.

phage are shown in Table 9. As was observed forthe bacteria, most of the virus appeared to fall-out within 2 h after the flush. However, smallnumbers of virus were often detected on controlsets of gauze exposed before the toilet flush,indicating that virus from experiments per-formed several days previously was still presentin the room. These background counts weresubtracted from those obtained after each toiletflush.Natural contamination of bathroom sur-

faces by coliforms. Using 2.5-inch (6.4-cm)diameter Rodac plates of Levine EMB agar,various surfaces in a number of household and

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MICROBIOLOGICAL HAZARDS OF HOUSEHOLD TOILETS 235

TABLE 9. Number of MS-2 phage falling out onbathroom surfaces after toilet flushing

TotalTotal no. of PFU Estimated

PFU after no. ofPFUTime eluted correction on floor Venti-after from all for ef- surface lationc

gauze ficiency area ofpadsa of bathroom

elution'

0-2 4,107 5,175 35,468 Closed2-4 244 307 2,532 Closed4-6 0 0 0 Closed0-2 209 263 1,540 Open2-4 0 0 0 Open4-6 0 0 0 Open

'Represents the average of five to six experimentsfor each 2-h period. In each, approximately 4 x 10$plaque-forming units (PFU) of MS-2 phage wereadded to the bowl before flushing. Eluate from eightgauze pads was pooled before assay. The location ofthe gauze pads in the bathroom can be seen in Fig. 5.

The average efficiency of elution was approxi-mately 79%. e

c The ventilation of the room was considered closedwhen all air vents in the room were sealed and thebathroom door remained closed.

TABLE 10. Detection of naturally occurring coliformson bathroom surfaces"

No. ofagar Maxi-plates mum

Nof on %Sam- no. ofSurface sam- which plea coliforms

pcoli- positive detectedplea om positive oforms on awere singlede- plate

tected

Walls 54 11 20 5Floor 120 31 26 > 100Seat, toilet 70 27 38 15Rim, toilet 35 10 28 >60Flush handle 9 1 11 2Bathtubs, sinks, 125 6 5 > 100

cabinets, etc.

a Rodac plates of EMB agar were used.

public bathrooms were sampled for the presenceof coliforms. The results are summarized inTable 10. Over 20 bathrooms were tested andcoliforms were detected in all, indicating thatsurface contamination in the bathroom is com-mon.

DISCUSSIONConsiderable numbers of bacteria and viruses

were shown to remain in the bowl water after

flushing, and even continual flushing could notremove a persistent fraction. This was attrib-uted to the adsorption of the organisms to theporcelain surfaces of the bowl, with gradualelution occurring after each flush. This ad-sorbed fraction could explain the heavy bacte-rial aerosols detected by Darlow and Bale (6)after the second flush of toilets seeded withbacteria. In toilet bowl waters tested by us,chlorine was usually absent or was present invery small amounts (< 1 mg/liter), probably dueto its rapid loss to the atmosphere. In addition,organic matter in the stool would combine withany free chlorine that might be present. Thus, agradual build-up of both bacteria and viruses intoilets could occur during regular use.

Droplets produced by flushing toilets werefound to harbor both bacteria and virusesplaced in the toilet before flushing. The averagehuman stool weighs approximately 100 g andcontains a total of about 10l2 bacteria (18), ofwhich 10i0 or more are coliforms (19). In in-fected persons, up to 10"1 Salmonella (19) and108 to 10"1 Shigella (14) have been detected inthe stool. Concentrations as high as 10i0 Salmo-nella paratyphi B per g of feces have beendetected in carriers (19). The number of polio-viruses present in the stool of infected personscan be as high as 106 per g of feces, giving a totalof 10. in the stool (16). For these values, thenumber of infectious organisms ejected from thebowl would range from 1,000 to over 10,000based on data obtained in this study.Organisms collected on the gauze probably

represent those contained in the larger-sizeddroplets that quickly settle out on surfaces inthe bathroom and do not take into account theorganisms present in smaller droplets whichmay be airborne for considerable lengths oftime. The detection of coliform bacteria andviruses falling out onto surfaces in the bathroomafter flushing indicated that these organismsremain airborne long enough to settle on sur-faces throughout the bathroom. The number ofE. coli detected on the agar plates is probably aminimal value since airborne bacteria are dam-aged during aerosolization and by environmen-tal stresses, making growth on selective mediamore difficult (13). These data also do not takeinto account the accumulation of organisms onthe walls and other surfaces of the bathroom.The number of viruses calculated to be fallingout onto the floor surface of the bathroom wasfound to be over a log greater than that detectedwhen the gauze was held over the bowl. Thismay be due to large numbers of virus beingpresent in smaller-sized drops which do not

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236 GERBA, WALLIS, AND MELNICK

impact onto the gauze during flushing. Whereasthe number of both bacteria and viruses deter-mined to be impacting on surfaces was lesswhen the bathroom door and vents were leftopen, it should be pointed out that the orga-nisms not settling out in the room under theseconditions are probably being carried to otherlocations by air currents.The presence of fecal organisms on bathroom

surfaces is undoubtedly widespread, as evi-denced by the isolation of coliform bacteria onsurfaces in all of the bathrooms sampled.Hutchinson (12) detected the presence of Shi-gella sonnei on bathroom floors and toilet seats.He also found that this organism could persistfor as long as 17 days on wooden water closetseats. Newsom (14) reported that Salmonellasurvived for 11 days after desiccation whensuspended in either tapwater or feces. In thispresent study, large numbers of coliform bacte-ria were found on several occasions under sham-poo bottles in bathrooms, which might indicatethat regrowth or prolonged persistence of theseorganisms may occur where organic materialhas a chance to accumulate. In addition, en-teroviruses, as well as members of the adenovi-rus and reovirus groups, have been found tosurvive desiccation on surfaces (4).Doses of less than 102 Shigella flexneri have

been found to be capable of initiating infectionin man (9). Also, Darlow et al. (7) have shownthat the lethal dose of Salmonella typhimuriumin mice was lower when they were infected byinhalation rather than ingestion. With virus,however, the minimal infectious dose for hu-mans may be as low as one tissue cultureinfectious dose (15). Thus, it would appear thatthe numbers of bacteria and viruses ejectedfrom the toilet are sufficiently large to initiateinfection, especially in the case of viruses.

In a study of airborne transmission of coxsack-ievirus type 21, Couch et al. (5) detected only 150% tissue culture infectious dose per 3.5 ft3(0.10 mi) in a barracks in which aerosol trans-mission of the virus between humans occurred.In an army barracks in which transmission ofadenovirus was occurring, air sampling revealedthe presence of only 1 50% tissue cultureinfectious dose in 920 ft3 (25.76 min) of air (2).The results of our study would seem to indicatethat greater concentrations of virus would existas aerosols in bathrooms in which infectiousmaterial has been flushed. The spread of viraldisease by aerosols from toilets may take onadded importance in those viral diseases inwhich low numbers of viruses are excreted innasopharyngeal secretions as compared to theamount excreted in the feces. In addition, the

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overall importance of enteric virus transmissionby respiratory secretions is still in dispute. Forexample, the spread of poliovirus within fami-lies when pharyngeal excretion occurred dif-fered little from that following purely fecalexcretion (10). Also, recent studies with adeno-virus type 4 indicate that fecal sources may bemore important than respiratory sources in thespread of this virus (10).The significance of enteric virus disease

transmission by contact with surfaces harboringinfectious material should not be overlooked, asevidenced by the recent findings of Hendley etal. (11) on the transmission of rhinovirus fromfomites to the hands and self-inoculation of theeyes and nose. Additional evidence would benecessary to substantiate the role of aerosols inthe epidemiology of disease transmission bytoilets, but the results of this study indicatethat it appears to be a distinct possibility.

LITERATURE CITED

1. Adams, M. H. 1959. Bacteriophages. Interscience Pub-lishe's, New York.

2. Artenstein, M. S., and W. S. Miller. 1966. Air samplingfor respiratory disease agents in army recruits. Bacte-riol. Rev. 30:571-572.

3. Bound, W. H., and R. I. Atkinson. 1966. Bacterial aerosolfrom water closets. A comparison of two types of panand two types of cover. Lancet i:1369-1370.

4. Buckland, F. E., and D. A. J. Tyrrell. 1962. Loss ofinfectivity on drying various viruses. Nature (London)195:1063-1064.

5. Couch, R. B., R. G. Douglas, Jr., K. M. Lindgren, P. J.Gerone, and V. Knight. 1970. Airborne transmission ofrespiratory infection with coxsackievirus A type 21.Am. J. Epidemiol. 91:78-86.

6. Darlow, H. M., and W. R. Bale. 1959. Infective hazards ofwater-closets. Lancet i:1196-1200.

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8. Davis, J. E., and R. L. Sinsheimer. 1963. The replicationof bacteriophage MS-2. I. Transfer of parental nucleicacid to progeny phage. J. Mol. Biol. 6:203-207.

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14. Newsom, S. W. B. 1972. Microbiology of hospital toilets.Lancet ii:700-703.

15. Plotkin, S. A., and M. Katz. 1967. Minimal infectivedoses of viruses for man by the oral route, p. 151-156. InG. Berg (ed.), Transmission of viruses by the water

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poliomyelitis viruses in experimentally infected humanvolunteers. Am. J. Med. Sci. 230:1-8.

17. Sobsey, M. D., C. Wallis, M. Henderson, and J. L.Melnick. 1973. Concentration of enteroviruses fromlarge volumes of water. Appl. Microbiol. 26:529-534.

18. Stanier, R. Y., M. Doudoroff, and E. A. Adelberg. 1970.The microbial world. Prentice-Hall, Englewood Cliffs,N.J.

19. Thomson, S. 1954. The number of bacilli harboured by

enteric carriers. J. Hyg. 52:67-70.20. Wallis, C., S. Grinstein, J. L Melnick, and J. E. Fields.

1969. Concentration of viruses from sewage and excretaon insoluble polyelectrolytes. Appl. Microbiol.18:1007-1014.

21. Wallis, C., M. Henderson, and J. L. Melnick. 1972.Enterovirus concentration on cellulose membranes.Appl. Microbiol. 23:476-480.

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VOL. 30, 1975

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