APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1992, p. 900-9040099-2240/92/030900-05$02.00/0Copyright © 1992, American Society for Microbiology

Model System Using Coliphage (X174 for TestingVirus Removal by Air Filters

MARTIN L. RAPP,1t TERESA THIEL,`* AND ROBERT J. ARROWSMITH2Department of Biology, University of Missouri-St. Louis, 8001 Natural Bnidge Road,

St. Louis, Missouri 63121,1 and Pernea Inc., St. Louis, Missouri 631462

Received 20 September 1991/Accepted 6 January 1992

Short-term (15-min-duration) and long-term (5- to 6-day-duration) test procedures have been developed fordetermining the efficiency of the removal of bacteriophage 4X174 by air-sterilizing filters. These procedureswere sensitive enough to measure a 108-fold reduction in the number of bacteriophage. A filter commonly usedin industrial air sterilizations (Domnick-Hunter Bio-X borosilicate glass) effected a 108-fold removal of viablephage in both short-term and long-term tests. A prototype low-flux, hollow-fiber membrane gave similarresults; however, a prototype high-flux, hollow-fiber membrane removed only about 99.999% of thebacteriophage in short-term tests.

High microbiological purity of process streams is requiredin applications such as clean rooms for pharmaceuticaloperations, sterile hoods for the manipulation of sensitive orhazardous biological materials, and bioreactors, includingtissue culture reactors, fermentors, and some enzyme reac-tors. In many fermentation processes, large amounts of airare required and the process times are long (15). Low-levelcontamination is accepted in some industrial processes,including yeast propagation for beverage and fuel ethanolproduction (7). However, overgrowth of a contaminatingorganism because of a high growth rate, early entry, or highconcentration may result in loss of the batch. Bacteriophagecontamination of fermentation processes is a particularproblem because of the rapid multiplication of bacterio-phages. For example, a single host-specific bacteriophagewith a burst of 150 phage per cell every 15 min theoreticallycould lyse a 1,000-liter bacterial culture (generation time of30 min) of 1010 cells per ml in less than 1.5 h. To prevent suchcontamination, bacteriophage present in ambient air must beremoved or inactivated.

Filtration is the most economical and prevalent method ofair sterilization, as it was over 30 years ago (9, 13). Standardmethods for measuring performance of air-sterilizing filtersare forward flow testing (9), mineral oil or dioctyl phthalateaerosol challenge (10), and bacteriophage challenges (4, 6,12, 14, 17, 18, 21, 22). If removal of viruses is the goal,bacteriophage may be the most realistic challenge. Mostviral challenge tests that have been developed include thegeneration of an aerosol containing viruses and measure-ment of the concentration of viruses in the air stream beforeand after it passes through the filter. There are reports on thetesting of air filters with animal viruses (8, 22); however,more reproducible assays using bacteriophages have beendeveloped (6, 16-18, 21, 23). Quantitation of downstreamradioactivity after filters are challenged with radiolabelledcoliphage T-2r provides a sensitive assay (18); however, ithas the disadvantages that hazardous aerosols are producedand the viability of the phages is not measured. Other phagesystems that have been used to measure filter performances

include coliphage S-12, actinophage Si, and coliphages T5and T7 (6, 16, 17, 21, 23).The most widely used bacteriophage for aerosol chal-

lenges of air filters is coliphage Ti. Ti is approximately 50 by150 nm in size (12). A 106_ to 107-fold reduction in Ticoncentration has been observed at a challenge of 2.8 x 1010PFU/ml in tests of HEPA filters (14). Ti aerosols at up to1011 PFU/liter have been generated inside autoclaves to testautoclave vent filters (4). Ti has not been detected inexhaust from a Pall 9-ft2 (ca. 1-M2), 0.2-,um-pore-size Emflonfilter when challenged at 1 standard cubic foot per minute(ca. 3 x 10-2 m3) with a total of 2.5 x 1010 PFU (9). Testresults published by a filter manufacturer demonstrate 104-to 106-fold reduction in viable bacteriophage after a chal-lenge with up to 1.6 x 109 PFU (3). The actual efficiencies ofthe test filters are greater than those calculated, sincepenetration was not detected. A 108-fold reduction in thenumber of Ti has been demonstrated by using borosilicateglass filters (if efficiency is calculated as described below inMaterials and Methods) (2).

If the effectiveness of a filter is determined primarily bythe size of the particle, a small phage should provide agreater challenge than a large one. The icosahedral coliphage4X174, which is 27 nm in diameter and has a total mass of6.2 x 106 Da, was selected for this study primarily becauseof its small size and roughly spherical shape (12, 19). 4X174is a single-stranded DNA phage that has no membraneenvelope. The burst of each host cell approximately 16 minafter infection yields 150 to 200 infectious virions (20).Particles of this size are in the most penetrating particle sizerange for at least one type of air-sterilizing membrane (11).The first objective of this research project was to develop

a method for rigorously challenging air filters in both short-term and long-term tests with aerosols containing highconcentrations of coliphage (X174. The second objectivewas to measure phage removal efficiencies of filters that arecommonly used in industrial air sterilization systems and ofprototype polysulfone hollow-fiber filters (provided by Per-mea, Inc.) using the 4X174 system.

MATERIALS AND METHODS* Corresponding author.t Present address: Monsanto Co., St. Louis, MO 63198.

Growth of host. Escherichia coli C was grown in Luriabroth (tryptone, 10 g/liter; yeast extract, 10 g/liter; NaCl, 5

900

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B

AIR

D

FYHAI JRT

FIG. 1. Schematic drawing of test system. (A) Rotameter measuring flow through the nebulizer. Typical flow, 5.5 standard liters/min(SLPM). (B) Devilbiss model 646 nebulizer. Five-milliliter lysate charge, 0.5 ml/min nebulization rate. (C) Three-liter aerosol evaporationtank. (D) Test filter. (E) Downstream impinger, Ace Glass model 7542. (F) Rotameter measuring flow through the test filter. Typical flow, 4.0SLPM. (G) Upstream impinger, Ace Glass model 7542. (H) Rotameter measuring flow through the upstream impinger. Typical flow, 4.0SLPM. (I) Rotameter measuring bypass flow. Typical flow rate, 15 to 25 SLPM. It was used with nebulizer bypass flow to control humidity.(J) Humidity sensor.

g/liter) with 1.0 mM MgCI2. Logarithmic-growth-phase cul-tures were prepared by inoculating 10 ml of medium with0.25 ml of a culture grown overnight to the stationary phaseof growth. Cultures were incubated with vigorous shaking at37°C for 2 to 3 h (optical density at 660 nm of 0.2 to 0.4) andkept at 4°C until used.

Production of phage lysates. Although some lysates in earlyexperiments were made by the plate lysate technique (1),most of the phage in these experiments were produced asfollows. Log-phase cells (108 cells per ml) in 33% Luriabroth-67% M9 minimal medium with glucose (M9 minimalmedium with glucose is Na2HPO4, 6 g/liter; KH2PO4, 3g/liter; NH4Cl, 3 g/liter; NaCl, 0.5 g/liter; MgSO4, 0.2 g/liter;and glucose, 0.2%) were infected with 4X174 at a multiplic-ity of infection of 0.01. The infected cells were incubated at37°C with aeration until lysis, indicated by a reduction inbroth turbidity, was observed. The lysed suspension waspurified by centrifugation (4,000 x g, 15 min) and filtrationthrough a 0.45-,um-pore-size Nylon 66 filter. The lysates(titer, 1010 to 1011 PFU/ml) were stored at 4°C. Lysatesprepared by this method were used either full strength ordiluted 1:10 with phage buffer (10 mM Tris, 10 mM NaCl, 5.0mM MgCl2, pH 7.0).

Challenge testing apparatus. A diagram of the test appara-tus, a modification of one used previously (3), is given in Fig.1. For the long-term test (5 to 6 days), an additional impingerwas installed in parallel to the one shown as item E in Fig. 1.One of the impingers was removed after 15 min for theshort-term challenge test. The second impinger was removedupon completion of the long-term test at 5 to 6 days. In sometests of disc-type filters, the upstream impinger and flow-meter (items G and H in Fig. 1) were not present. In theseexperiments, upstream phage concentration was determinedby recovering viable phage by washing the filter disc.

Filters tested. Two prototype filters consisting of hollow-fiber filter bundles enclosed in aluminum housings wereused. These units were steam sterilized at 121°C (15 lb/in2)for 30 min prior to initial use and after each four to fiveexperiments. Filter 1 was a 6,968-cm2, high-flux unit. Filter2 was a 6,968-cm2, low-flux unit. A single unit of each typewas used for this study. Particulate removal properties ofthese filters are unknown. Filter 3 was a borosilicate glassmembrane filter enclosed in a stainless-steel housing. Thisapproximately 23-cm2 filter is commercially available fromDomnick Hunter Filters Ltd. (BIO-X model MER1). Asingle-filter element was used for this study and was steril-ized as described above. This type of filter element iscommonly used in fermentation applications. The manufac-turer certifies that each element has a maximum penetrationof less than 0.001% of dioctyl phthalate droplets in the 0.1- to0.3-pum-diameter size range (2). Filter 4 was a 47-mm-diameter, 5.0-p.m-pore-size Nylon 66 disc filter (MicronSeparations, Inc., catalog no. N50SP04700) enclosed in areusable polysulfone housing (Nalgene). The housing wasautoclaved and a new membrane was inserted before eachtrial.

Short-term test procedure. The evaporation tank, im-pingers, and all sterilizable components were autoclaved at121°C for 30 min. Impingers contained 50 or 100 ml of sterilephage buffer. Early experiments used 100 ml of phage buffer;in later experiments, the volume was reduced to 50 ml.Impingers were sampled prior to the initiation of the exper-iment to confirm sterility. The nebulizer was filled with 5 mlof phage buffer, and then airflow was started and valves wereadjusted to attain the indicated flowrates. After 5 min ofbuffer nebulization, the nebulizer was emptied and refilledwith 5.0 ml of phage lysate. In early runs, the lysate wasnebulized for 15 min, and then airflow was stopped. In some

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tests, two 5-mi lysate charges were nebulized for 10 to 15min each in an attempt to increase challenge levels. Toincrease the sensitivity for detection of phage that pene-trated the filter, five 1.0-ml aliquots of the solution from thepostfilter impinger were titered.

In some tests of filter 4, the membrane disc filters wereassayed for viable phage instead of an upstream impingerbeing assayed to determine challenge levels. After the chal-lenge was completed, the filters were removed from thehousing, placed in a sterile culture plate with 4 ml of phagebuffer, and gently stirred to recover the phage. After 15 min,an aliquot was titered. In separate experiments, the percent-age of recovery of ~X174 applied to filter 4 was measured.The recovery of virus applied to the filter was 85 to 90%.

Long-term test procedure. Long-term tests were con-ducted for 5 to 6 days, during which time the nebulizer wasfilled with 5 ml of phage lysate once a day, as describedabove for short-term tests. To minimize evaporation, airflowrates between nebulizations were reduced to about 10% ofthose used during a nebulization. For long-term tests, anadditional impinger was connected in parallel with the down-stream impinger. After completing a typical short-term test,one of the downstream impingers was removed and thephage buffer was titered. During this test (the equivalent ofthe short-term test described above), the flow of filtered airwas through only one impinger. After this impinger wasremoved, flow was through the second downstream impingerfor the duration of the long-term test.

Quantitation of phage concentration. For solutions ex-pected to contain significant numbers of phage, 0.1-ml sam-ples from appropriate 10-fold serial dilutions of the samplewere mixed with 0.3 ml of host cells undergoing exponentialgrowth and 2.5 ml of 45°C soft agar (Luria broth with 0.7%Difco agar and 1.0 mM MgCl2). The contents of the tubewere mixed and quickly poured into a 100-mm-diameterculture plate containing 20 ml of solidified medium (Luriabroth with 1.5% Difco agar). For solutions expected tocontain low numbers of phage, the above procedure wasmodified so that 1.0 ml of undiluted test solution and 4.0 mlof soft agar were added to host cells and poured onto agarplates. After incubation of the plates at 37°C, plaques couldbe counted in 5 to 7 h. Phage concentration (titer) wascalculated by multiplying the number of plaques per plate bythe dilution factor, and the results were expressed as PFUper milliliter. Plaque size varied from 4 to 8 mm; the meanplaque size was about 6 mm. There was no significantdifference in plaque size or number when agar concentrationwas varied between 0.5 and 0.7%. This experiment was doneto mimic the variation in final agar concentration caused bydifferences in phage sample volume.

Calculation of filter efficiency. The phage challenge (CH)was calculated as:

CH = tUvJUlfd (1)where t,, is the titer in the upstream impinger, vu is thevolume of buffer in the upstream impinger, ft is the airflowrate through the upstream impinger, andfd is the airflow ratethrough the downstream impinger. The concentration ofphage in the air upstream of the filter (C") was calculated as:

CU = tuvul(fut) (2)where t is the test time. Phage concentration in the air streamafter the filter (Cd) was calculated as:

Cd = tdVd/(fdt) (3)where td is the titer in the downstream impinger and Vd is thevolume of the downstream impinger. If no plaques were seen

TABLE 1. Short-term tests of filter 4 (5.0-pum-pore-sizeNylon 66)

TilRelative PFU/liter in air Calne Removal rtono. humiUpdity Down- (PFU) e ficiency detected?(%) Upstream stream(%

1 38 5.0 x 103 2.2 2.8 x 105 99.96 Yes2 36 1.4x 10' 11 8.6x 102 20 Yes3 39 1.6 x 104 39 1.0 x 106 99.7 Yes4 39 1.3 x 104 240 8.2 x 105 98.2 Yes5 39 6.2 x 104 160 4.0 x 106 99.7 Yes6 33 4.8 x 104 0.3 3.0 x 106 99.999 Yes

on any of the five plates containing 1.0 ml of the undilutedsample, one plaque (per 5-ml sample) was assumed forpurposes of calculation and titer is expressed as less than (<)one plaque per 5 ml. For short-term tests, phage removalefficiency (Rs) was calculated as:

Rs = 100(1 - Cd/CU) (4)For tests in which an upstream impinger was not used but

a membrane disc was washed with buffer to recover filteredphage, the upstream phage concentration was calculated asabove, except that the impinger volume of 50 to 100 ml wasreplaced with the 4-ml buffer wash volume. In these tests,the total challenge was the sum of all recovered phage.

Calculation of removal efficiency in long-term tests by theprocedure described above was problematic because ofeffects of variation of airflow rates. Airflow rates betweenchallenges may have fluctuated with changes in air pressure,and it was difficult to set the airflow accurately at very lowlevels. For long-term tests, phage removal efficiency (R,)was calculated as:

RI = 100[1 (tdvdIt,v,)I (5)This method assumed equal airflow through both impingers,which was usually true for the duration of the experiment.

RESULTS AND DISCUSSION

Phage removal by filters. A variety of filters were subjectedto short-term tests of viral retention ability. As expected,penetration was detected in all challenges of 5.0-,um-pore-size Nylon 66 filters (filter 4) (Table 1). Removal efficienciesranged from 20 to 99.999%. Penetration was also detected infour of six short-term tests of filter 1, the high-flux, hollow-fiber membrane (Table 2). Removal efficiencies ranged from>99.98 to 99.999%. Since penetration was detected in anumber of tests, the challenge level was sufficient to assessthe performance of this filter. Results of tests of filter 2

TABLE 2. Short-term tests of filter 1 (high flux, hollow fiber)

TilRelative PF/liter in air Reoano humidity Challenge efficien Penetrationno. () Upstream Down- (PFU) Ceny detected?M ~~~stream (%

1 46 9.4 x 102 <0.16 6.1 x 104 >99.98 No2 45 5.6 x 104 0.79 3.5 x 106 99.999 Yes3 42 1.2 x 103 0.16 7.7 X 104 99.99 Yes4 43 7.9 X 104 0.16 5.2 x 105 99.998 Yes5 NDIa 3.2 x 103 <0.15 2.1 x 105 >99.995 No6 ND 1.4 x 105 1.6 1.2 x 107 99.999 Yesa ND, not determined.

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TABLE 3. Short-term tests of filter 2 (low flux, hollow fiber)

PFU,/1iter inairReoaTrial Challenge Removal Penetrationno. Upstream mDown- (PFU) etficiency detected?no Upstream stream (%q)

1 1.8 x 105 <0.12 1.6 x 107 >99.99993 No2 4.4 x 1(0 <0.12 3.8 x 1(7 >99.99997 No3 1.9 x 107 0.23 1.6 x 1(0 99.999999 Yes

(low-flux, hollow-fiber membrane) are given in Table 3(short-term tests) and Table 4 (long-term tests). In the singleshort-term test in which penetration was detected, the re-moval efficiency was 8 orders of magnitude. The penetrationdetected was two plaques on one of five replicate plates andwas probably contamination from the work area. Penetrationwas not detected in long-term tests. Since penetration wasdetected only once, it is likely that the removal efficiency ofthis membrane was greater than 8 orders of magnitude andthat this procedure was not sensitive enough to determinethe actual efficiency. Results of tests of filter 3 (borosilicateglass) are given in Table 5 (short-term tests) and Table 6(long-term tests). Removal efficiencies of up to >99.999998%were obtained in short-term tests. Penetration was detectedin one of the seven short-term tests. Penetration was notdetected in either of the two long-term tests, and removalefficiencies were greater than 8 orders of magnitude. Sincepenetration was detected only once for filter 3, this methodwas not capable of determining the true removal efficiency.

Particle size. One objective of this study was to produce astream of viable, dry, individual virion particles from ahigh-titer phage lysate. There were at least four factors thataffected the particles: initial droplet size, average number ofvirions per droplet, droplet evaporation rate, and loss ofviability on nebulization and drying. Ideally, the droplet sizewould be such that there was on average less than one virionper droplet and the droplet would evaporate prior to contactwith the test filter, with no viability loss on nebulization anddrying. The diameter of the droplets dispersed by the nebu-lizer was estimated by the manufacturer of the device to be0.3 to 5.0 l.m under the conditions of these experiments andwas not measured in these experiments. With this assump-tion of droplet sizc, the number of virions per droplet for therange of lysate titers used in this work was determined. Fora lysate titer of 109, the average number of virions perdroplet was 1.4 x 10-5 and 0.065 with droplets of 0.3- and5.0-p.m diameter, respectively. For a lysate titer of 10", theaverage number of virions per droplet was 1.4 x 10-3 and6.5 with droplets of 0.3- and 5.0-p.m diameter, respectively.Thus, aerosol droplets were likely to contain on average lessthan one virion per droplet. The residence time of a dropletin the test system ranged from 3.9 to 21 s, with the majorityof experiments having a residence time of 18 to 21 s. It wasassumed that the droplets were dry when they contacted thetest filter. Visual observation supported this assumption.

TABLE 4. Long-term tests of filter 2 (low flux, hollow fiber)

Trial No. of No. of Upstream Removal Penetrationno.days charges titer efficiency detected?no. days charges (PFU/ml) (c)

1 5 6 2.1 x 10(' >99.999990 No2 6 7 5.0 x 105 >99.99996 No3 5 6 3.5 x 1(" > 99.999994 No

TABLE 5. Short-term tests of filter 3 (borosilicate glass)

Tal Relative PFU/liter in air Removal Penetration'rilhumidity Dw-Challenge efficiency Petriono. (%) Upstream Down- (PFU) e detected?stream(%

1 73 1.0 x 106 <11 9.1 x 107 >99.999 No2 62 5.0 x 104 <13 2.1 x 105 >99.97 No3 32 6.4 x 104 <24 2.7 x 106 >99.96 No4 80 1.1 x 106 <7.8 7.0 x 107 >99.9993 No5 50 9.9 x 104 0.46 6.6 x 106 99.99995 Yes6 ND" 2.0 x 106 <0.12 1.8 x 10" >99.999994 No7 ND 1.5 x 107 <0.31 1.0 x 109 >99.999998 No

" ND, not determined.

Exhaust from the nebulizer appeared as a fine smoky mist,whereas air in the inlet to the test filter was visually clear. Ifindividual dried virions did not agglomerate, it is likely thatthe challenge particles were individual, dry phages.

Test conditions. Because the operable flow rate range ofthe test system was limited and the areas of the test filterswere diverse, the test flow rate rather than the flux was heldconstant (or nearly so). The range of fluxes and transmem-brane velocities in this work is given in Table 7. Transmem-brane velocity can have a significant effect on penetration(5), with bacteriophage penetration more likely at increasedvelocity.

Significant loss of phage viability occurred in the testsystem, typically about 4 orders of magnitude. Viruses wereprobably killed by nebulization, by drying, or by both. Up to30% loss of viable Ti upon nebulization and drying has beenreported (2). Drying experiments done in petri plates as partof this project indicated a viability loss of about 80% for4X174. Three possible reasons for significantly higher via-bility losses on drying in an airstream relative to drying on aplate are (i) the rapid evaporation rate of drying in anairstream, (ii) the vigorous aeration associated with thenebulization process, and (iii) foaming of the lysate onnebulization. Foaming is attributed to protein denaturationwhich could have inactivated viruses. Because of this loss ofviable phage, only the titers of viable phage recovered afternebulization and drying were used in the calculation ofretention efficiency.Removal efficiencies. The phage removal efficiency re-

quired for a particular process varies considerably, depend-ing on the concentration of contaminating phage, length ofthe process, rate of airflow to the process relative to processvolume, effect of phage contamination, and kinetics of phagegrowth. As an example, the following assumptions for afermentation process will be made: 100 PFU/ft3 (ca. 2.8PFU/m3); airflow, 1 volume of air per volume of liquid;process time, 7 days (13). If penetration of less than onevirion is allowable, greater than 99.9999% of the phage mustbe removed (106-fold reduction). If a safety factor of 100 isincluded, a greater than 108-fold reduction of the phageconcentration in the air supply must be effected. A 108-fold

TABLE 6. Long-term tests of filter 3 (borosilicate glass)

Trial No. of No. of Upstream Removal Penetrationno. days charges (P)titer efficiency detected?no.days charges (PFU/ml) (%

1 6 5 3.0 x 107 >99.999999 No2 5 6 3.4 x 107 >99.999999 No

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TABLE 7. Test fluxes and velocities

Filter area Flux range VelocityFilter material (CM2) (liters/min/cm2') range(cm ~~~~~~~~(cm/min)Polysulfone 6,968 4.3 x 10-4-6.6 x 10-4 0.43-0.66

(filters 1 and 2)Borosilicate 23 0.13-0.20 130-200

(filter 3)Disc membrane 17 0.17-0.26 170-260

(filter 4)

removal efficiency was the target for development of the testmethod.The first objective of this study was to develop a method

of challenging air filters with aerosols of coliphage 4)X174, asmall coliphage not previously used for filter challenge tests.By optimizing the system configuration, lysate titer, airflowrates, impinger volume, and analytical practices, this proce-dure was capable of measuring phage removal efficiencies of108-fold for 4~X174 in both short-term (15- to 20-min) andlong-term (6-day) tests.The second objective of this work was to use the proce-

dure to characterize phage removal performance of proto-type polysulfone hollow-fiber filters provided by Permea,Inc. and to compare this performance with that of a filtercommonly used in industrial air sterilization systems. Sincethe fluxes were not the same for all filters tested andpenetration is somewhat dependent on flux, a direct compar-ison of test results of different filters was not possible.As expected, penetration was detected in each trial of

membranes with large pores (5.0-p.m pore size; filter 4).Permea filter 1, the high-flux unit, delivered a reduction of upto 5 orders of magnitude in the concentration of 4X174,allowing detectable penetration of the filter in 66% of thetrials. The low-flux Permea membrane, filter 2, delivered a

reduction of up to 8 orders of magnitude in the concentrationof 4X174 in short-term tests and a reduction of up to 7 ordersof magnitude in long-term tests. Penetration was not usuallydetected (and those rare instances were probably attribut-able to contamination of the plates rather than actual pene-tration); therefore, the actual removal was probably greaterthan 108-fold for the short-term test and 107-fold for thelong-term test.The Domnick Hunter borosilicate glass filter, filter 3,

delivered a reduction up to 108-fold in the concentration of4X174 in both short-term and long-term tests. Althoughpenetration was detected in one of the seven short-term testswith 4X174, it was probably an artifact. The filter appears tobe capable of reducing phage concentrations by at least 8orders of magnitude.

Conclusions. The procedure described here using 4XX174aerosols to challenge air sterilization filters was capable ofdetermining removal efficiencies as high as previously pub-lished efficiencies for protocols using much larger phages. Inaddition, the long-term test more closely simulating indus-trial applications was capable of determining removal effi-ciencies of 8 orders of magnitude. Such high efficiencies ofremoval are necessary to prevent infection of large fermen-tors by viruses. Two filters tested by the procedure de-scribed here appeared capable of providing 108-fold removalof very small viruses from air. One was the Domnick Hunter

borosilicate glass filter; the other was a prototype, low-flux,hollow-fiber filter (Permea, Inc.).

ACKNOWLEDGMENTSWe thank Selena Rogers for excellent technical assistance.This research was supported by a grant from the Missouri

Research Assistance Act.

REFERENCES1. Adams, M. H. 1959. Bacteriophages. Interscience Publishers

Inc., New York.2. Anonymous. 1985. Compressed air sterilization. Domnick Mi-

cro-filtration, Inc., Durham, England.3. Anonymous. 1987. Validation guide for Pall EmflonRII mem-

brane cartridges for air and gas sterilization. Pall UltrafineFiltration Corporation, East Hills, N.Y.

4. Barbeito, M. S., and E. A. Brookey. 1976. Microbiologicalhazard from the exhaust of a high-vacuum sterilizer. Appl.Environ. Microbiol. 32:671-678.

5. Billiet, C. T., and J. I. T. Stenhouse. 1985. Theoretical study offilter systems used in the production of sterile air. Filtration andSeparation 22:375-377.

6. Bolton, N. E., T. A. Lincoln, J. A. Otten, and W. E. Porter. 1976.A method for biological testing of containment systems for viralagents. Am. Ind. Hyg. Assoc. J. 37:427-431.

7. Brodl, G. B. 1988. Continuous fermentation technology. Fuelethanol workshop, St. Louis, Mo.

8. Burmester, B. R., and R. L. Witter. 1972. Efficiency of com-mercial air filters against Merek's disease virus. Appl. Micro-biol. 23:505-508.

9. Conway, R. S. 1984. State of the art in fermentation air filtration.Biotechnol. Bioeng. 26:844-847.

10. Domnick, K. R. 1982. On site performance evaluation and bestinstallation arrangements for cartridge type air sterilizationfilters, abstr. 184. Meeting of American Chemical Society.

11. Eberhardt, L., C. Dowell, and R. Levy. 1989. Preparation andpurification of 4)X174 (cs7O am-3) and T, bacteriophage (ATCC11303 B) for use in aerosol challenge testing of sterilizing grademembrane filters, abstr. Q55. Abstr. 89th Annu. Meet. Am.Soc. Microbiol. 1989.

12. Frankel-Conrat, H. 1985. The viruses. Catalogue characteriza-tion and classification, p. 202 and 266. Plenum Press, NewYork.

13. Gaden, E. L., and A. E. Humphrey. 1956. Fibrous filters for airsterilization. Ind. Eng. Chem. 48:2172-2176.

14. Harstad, J. B., and M. E. Filler. 1969. Evaluation of air filterswith submicron viral aerosols and bacterial aerosols. Am. Ind.Hyg. Assoc. J. 30:280-290.

15. Humphrey, A. E., and E. L. Gaden. 1955. Air sterilization byfibrous media. Ind. Eng. Chem. 47:924-930.

16. McGarrity, G. J., and L. L. Coriell. 1973. Mass airflow cabinetfor control of airborne infection of laboratory rodents. Appl.Microbiol. 26:167-172.

17. Roelants, P., B. Boon, and W. Lhoest. 1968. Evaluation of acommercial air filter for removal of viruses from the air. Appl.Microbiol. 16:1465-1467.

18. Sadoff, H. L., and J. W. Almoff. 1956. Testing of filters for phageremoval. Ind. Eng. Chem. 48:2199-2203.

19. Sinsheimer, R. L. 1959. Purification and properties of bacterio-phage 4X174. J. Mol. Biol. 1:37-42.

20. Sinsheimer, R. L. 1970. The life cycle of a single-stranded DNAvirus (+X174). Harvey Lect. 64:69-86.

21. Songer, J. R., P. J. Matthews, D. T. Braymen, and D. E. Dennis.1974. An ultrahigh-efficiency filter for use with flexible filmgermfree animal isolators. Lab. Anim. Sci. 24:943-945.

22. Wallis, C., J. L. Melnick, V. C. Rao, and T. E. Sox. 1985.Method for detecting viruses in aerosols. Appl. Environ. Micro-biol. 50:1181-1186.

23. Washam, C. J., C. H. Black, W. E. Sandine, and P. R. Elliker.1966. Evaluation of filters for removal of bacteriophages fromair. Appl. Microbiol. 14:497-505.

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