APPLIED MICROBIOLOGY, July 1968, p. 1086-1092Copyright @ 1968 American Society for Microbiology

Vol. 16, No. 7Printed in U.S.A.

Microbiological Studies on the Performance of a

Laminar Airflow Biological CabinetJOSEPH J. McDADE, FRED L. SABEL, RONALD L. AKERS, AND ROBERT J. WALKER

Biohazards Department, Pitman-Moore Division, The Dow Chemical Company, Zionsville, Indiana 46077, andEngineering Department, Behrent Engineering Company, Denver, Colorado 80211

Received for publication 9 May 1968

Engineering and microbiological tests indicated that a typical, commercial laminarairflow cabinet was not effective in providing either product protection or agentcontainment. The cabinet was modified and tested through a series of alternateconfigurations to establish a set of design criteria. A mock-up cabinet was de-veloped from these design criteria. The mock-up unit was evaluated for efficiencyin providing both product protection and agent containment. In these evaluations,challenge methods were developed to simulate normal, in-use laboratory opera-tions. Controlled bacterial or viral aerosol challenges were used at higher thannormal levels to provide stringent test conditions. Test results indicated that themock-up unit was considerably better in preventing agent penetration (0.1 to 0.2particles per 100 ft3 of air) than the commercial cabinet (5 to 6 particles per 100ft' of air) during product protection tests. Similarly, agent containment was con-siderably better in the new cabinet (particle escape of 2 to 3 per 100 ft3 of air at onlyone of the five test sites) than in the commercial cabinet (particle escape of 2 to 14per 100 ft8 of air at three of the five test sites).

The laminar airflow principle developed byWhitfield (15) has been widely and successfullyapplied in industry for the control of particulatecontamination during clean assembly work.More recently, laminar airflow clean rooms andclean work benches have been used with successfor control of microbial contamination in theaerospace (4, 8-10) and medical fields (11, 14).Use of laminar airflow cabinets is also increasingin the pharmaceutical field and in microbiologi-cal laboratories (5).A variety of horizontal (crossflow) and vertical

(downflow) laminar airflow cabinets are com-mercially available. The general areas of applica-tion of such units comprise (i) product protection,i.e., use of laminar airflow cabinets for operationsinvolving manipulations of materials that must bekept sterile or free from unwanted ecologicalagents, and (ii) agent containment, i.e., use oflaminar airflow cabinets for manipulations involv-ing etiological agents, materials, or proceduresrequiring personnel protection. The item(s) in usemust be confined within the working area andmust not be allowed to escape from the cabinet.The horizontal laminar airflow cabinet is best

suited for maximal product protection. However,when agent containment is required, the vertical

laminar airflow cabinet is more applicable. Air-handling-system modifications have led to thedevelopment of downflow units that have beenreported to provide product protection and agentcontainment. In such units, a larger volume ofmoving air is exhausted from the cabinet thanthe volume of air supplied through the filter.This creates a negative pressure at the face of thecabinet, thereby drawing room air into the faceor front opening of the cabinet. Theoretically, aprotective air curtain is established at the cabinetface to provide both product protection and agentcontainment.

In this study, a commercial vertical laminarairflow cabinet was tested for its ability to es-tablish and maintain laminar flow of air. Thecabinet was also tested to establish a quantita-tive measure of the degree of product protectionand agent containment. In general, the unit wasdeficient in establishing laminar airflow withinthe cabinet and was not as good as desired forproduct protection or agent containment.

Since vertical laminar airflow clean roomswere effective in controlling particulate andmicrobial contamination, it was decided todevelop a downflow cabinet that was more nearlylaminar in airflow. Laminar airflow is defined inFederal Standard 209a (6) as "Air flow in which

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LAMINAR AIRFLOW BIOLOGICAL CABINET

the entire body of air within a confined areamoves with uniform velocity along parallelflow lines, with a minimum of eddies." Thus, inour study, the design goal for the cabinet wasdevelopment of uniform, parallel, and nonturbu-lent airflow throughout any given cross sectionof the cabinet. This report describes the engi-neering approach, tests and analysis, and themicrobiological studies conducted to developand to evaluate the performance of the newcabinet.

MATERIALS AND METHODS

Test cultures. Stock cultures of Serratia marcescens(ATCC 14756) were maintained at 4 to 6 C on Tryp-tose Phosphate Agar (TPA) slants. Routine cultureswere propagated at 30 C on TPA slants and weretransferred at 24-hr intervals during tests. Test cul-tures were prepared as suspensions for aerosolizationby heavily streaking a plate (100 mm in diameter)of TPA with a 3-mm loopful of inoculum from aroutine culture. The freshly streaked plate was in-cubated at 30 C for 16 hr. Then, the resultant growthwas harvested from the plate and resuspended in 200ml of Tryptose Phosphate Broth (TPB). This sus-pension contained from 1.0 to 6.0 X 109 cells per ml.

Test bacteriophage was prepared by inoculating1,000 ml of a 5-hr TPB Escherichia coli culture (ATCC11303-B) in a Fernbach flask with 200 ml of a sus-pension of TI coliphage (ATCC 11303-BI) contain-ing 7.0 to 9.0 X 109 plaque-forming units (PFU)per ml. The mixture was placed on a magnetic stirrerand slowly agitated at room temperature until themedium cleared. Upon clearing, 10 ml of chloroformwas added, and the lysate was centrifuged for 30min at 680 X g. The number of PFU in the lysatewas determined according to the method describedby Adams (1). The phage suspension was stored at4 to 6 C for periods up to 2 months.

Product protection tests. An aerosol generator(model 200 A; Schoeffel Instruments, Westwood,N.J.) was used to create the challenge aerosols forthe product protection tests. This self-containedgenerator dispenses 0.2 ml of fluid per minute.Particle distribution in the resultant aerosol rangesfrom 1 to 4 ,u in diameter (3, 7). In these tests (Fig. 1),a Schoeffel generator was placed 12 inches (30.5cm) from the face of the cabinet and 6 inches (15.2 cm)above the level of the perforated work surface. Posi-tioned in this manner, the aerosol produced wouldclosely approximate the location of an aerosolgenerated by a laboratory technician working at thecabinet. To measure the extent of aerosol penetra-tion into the cabinet, 12 rows of five agar settlingplates (100 by 15 mm) each were placed across theperforated work surface. These plates covered ap-proximately one-third of the total area of the worksurface. An aerosol was generated for 2 or 3 min pertest. A Reyniers slit sampler was placed under theperforated work surface of the hood to sample airfrom the exhaust portion of the cabinet in order toindicate the presence of viable particles. Controlsamples were obtained in the challenge aerosol prior

A1ROOM EXHAUST GRIIILLS

A A A A

FiG. 1. Setup for product protection tests.

to each trial. Aerosols of S. marcescens were col-lected on TPA plates and were incubated at 30 C for24 hr prior to counting. The T1 coliphage aerosolswere collected on TPA plates which had been over-layered previously with a thin layer of 1.0% TPAcontaining 2.5 to 9.5 X 107 viable E. coli cells/ml.The settling plates (100 by 15 mm) were overlayeredwith 3 ml of the inoculated agar per plate and theReyniers plates (150 by 25 mm) with 8 ml per plate.After sample collection, test plates were incubatedat 37 C, and the number of PFU was counted after 6and 24 hr of incubation.

Agent containment tests. A model 1001 WaringBlendor was used to create the challenge aerosolduring agent containment tests. The blendor wasplaced in the center of the cabinet on the perforatedwork surface and 24 inches (61 cm) from the rightinside edge. Test aerosols were produced by blending200 ml of the bacterial or viral suspension within thecabinet for a 1-min period with the top on the blendor.Then, the blendor was turned off, and the top wasremoved for a 1-min period, after which the top wasreplaced. The particle size distribution of the resultantaerosol ranged from 1 to 3.5 , (3). Challenge aerosolswere collected with volumetric air samplers (Rey-niers) on appropriate medium described above. Theair samplers were equipped with a 60-min timingmechanism and were calibrated to sample air at a

rate of 1 ft3 of air per minute. Six air samplers were

used per test, and they were located in the followingpositions (Fig. 2): sampler 1 (R-1) was placed underthe perforated work surface of the cabinet, beneath

and adjacent to the blendor; sampler 2 (R-2) was

placed outside of the cabinet, 12 inches (30.5 cm)from the left comer and 6 inches (15.2 cm) from the

face opening, level with the work surface; sampler

TEST CABINET

SCHOEFFEL XAEROSOL 3 S HIGHGENERATOR

SUPPLY DIFFUSERS

r---------- -I

; _ _ ____. I__________

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I

MCDADE ET AL.

3 (R-3) was located outside of the cabinet at the repiratory level of an operator seated at the cabinei.e., center front of the unit, 6 inches from the facopening and 18 inches (45.7 cm) above the worsurface; sampler 4 (R-4) was located outside of tkhood, 12 inches from the right corner and 6 inchfrom the face opening and level with the work surfaclsampler 5 (R-5) was located 12 inches from the leicorner, level with the work surface and 6 ft (1.97 nfrom the face opening; sampler 6 (R-6) was locate12 inches from the right corner, level with the worsurface and 6 ft from the face opening. In generatrials were conducted as follows. The samplers weioperated for a period of 10 min to obtain a bacdground count. At the completion of the aerosol cycithe sampler continued to run for 8 min, makingtotal for each trial of 10 min or 10 ft3 of air sample(Five trials were conducted during each series of plate

Cabinet airflow studies. A complete description (the engineering phase of this study has been presenteelsewhere (in press). For continuity, a brief descriptioof the engineering is included. A theoretical analysindicated that airflow in the commercial cabinet wanonuniform along the bottom edge of the front wiIdow panel, at the face of the cabinet, and across thperforated work surface. This analysis also identifleareas of extreme eddy or turbulence at the upptedge of the face opening. Actual airflow velocitimeasured in this cabinet proved the theoretical calcilations to be correct. All velocity profiles were mea;ured with an Alnor Thermo-Anemometer employina special 24-inch (61 cm) probe. Measurements weitaken (Fig. 3) at 2-inch (5.1 cm) intervals 2.5 (6.4 cninches below the high efficiency particulate air (HEPAfilter bank (filter traverse), at 1-inch (2.54 cm) intei

ROOM EXHAUST GRILLS

A-A A I.A

FIG. 2. Setup for agent containment tests.

5-

-k

e,e;Ift1i)

1I1 WINDOW

reC-

,e,a fd. Ll MID-LEVEL TRAVERSE (26

Df FACE OPENING TRAVERSE (7M2")FLOOR TRAVERSE (26")

n f-iskPRRAEPERFORATED WORK SURFACEas

Ax CABINET FRONT PANEL

er FIG. 3. Measurement locations for velocity pro-es files.a-

s- vals at the bottom edge of the front window panel1g (mid-level traverse), at 1-inch intervals 1 inch abovere the perforated work surface (work surface traverse),) and at 1-inch intervals up the face opening (face open-

ing traverse). All traverse planes were 19 inchesr- (49.3 cm) from the left end of the cabinet. The air-

handling system of the commercially available cabinetwas modified to allow recirculation, and an auxiliaryexhaust fan, pitot tube, and manometer were attachedto measure the portion of the exhaust air discarded.Velocities at each traverse line were measured. The airquantity for each representative plane was calculatedand totaled against the fan-flow totals to confirm

> velocity readings. A proffle of the cabinet face openingwas calculated to determine airflow distortion, areas ofprobable turbulence, and effect of the inner air mass.Airflow patterns within the cabinet were convertedthrough a series of configurations to evaluate theflow conditions typical of a number of commerciallyavailable cabinets. After these evaluations, the fol-lowing set of design criteria were established: (i)airflow paths should be as nearly a straight line aspossible; (ii) highly different velocity air massesshould not be mixed; (iii) the airflow in the faceopening should be turned to more nearly parallel

> the inner air stream; and (iv) the quantity of air ex-hausted should be minimal to reduce face turbulence,

> exhaust filter size, and building conditioned airlosses.

RESULTS

Engineering studies. It was found that theentire airflow of the commercial cabinet wasshifted toward the back of the unit. This direction

1088 APPL. MICROBIOL.

LAMINAR AIRFLOW BIOLOGICAL CABINET

of flow was due to two factors: the 40% free area

perforated work surface, and the fact that thecabinet air was exhausted below the perforatedwork surface at the back of the cabinet. The 40%free area perforated work surface did not createsufficient back pressure to equalize effects causedby the location of the return air exhaust. Thevelocity profile of the mid-level traverse was

shown to drop near the front of the cabinetdue to the expanding air stream formed by theoutward sloped window. None of the otherconfigurations evaluated appeared to be betterin laminar airflow characteristics nor to haveparticular advantage over the original test cabi-net.

Positioning the window parallel to the innerairstream and the back of the cabinet correctedthe variation in velocity caused by the expandingairstream. The free area in the perforated worksurface was reduced to correct the backwardsweep of the inner mass of cabinet air. Reducingthis free area to 10% caused the air that was

drawn into the cabinet face to be "pulled" intothe work area of the cabinet. However, when theportion of the work surface extending beyond thewindow was increased to 20% free area (keepingthe portion of the perforated work surface behindthe window at 10% free area), airflow conditionswithin the cabinet improved (Fig. 4). Finally, theattachment of an angled front lip further im-proved flow conditions at the face opening byturning the face flow to more nearly parallel the

HEPA FILTERED AIR

LIP ANGLE °h300 FREE 10% FREE

FIG. 4. Schematic cross section of the design mock-up cabinet.

TABLE 1. Product protection tests in the commercialcabineta

Test no. nRoom air- Total virus pen- Viral penetra-hading system etain(F)tion per 100 f-of cabinetft

1 On 659 27.82 On 1,428 60.23 Off 24 1.04 Off 230 9.7

a The cabinet air supplied during challenge was2,372 fts. The titer of Ti coliphage was 5 X 107PFU/ml. The 2-min aerosol challenge was 2 X107; the aerosol challenge per 100 ft' was 8.4 X105.

inner air stream (Fig. 4). At this point, it appearedthat all of the established design criteria had beenfulfilled, and the design mock-up (Fig. 4) wasdeveloped for further microbiological testing.

Product protection tests. A typical set of re-sults obtained in the commercial cabinet arepresented in Table 1. More than 10 tests wereconducted with bacteriophage and 8 tests with S.marcescens. The data shown for tests 1 and 2represent the greatest viral penetrations recorded.Results similar to those shown in tests 3 and 4were observed repeatedly with both the bacterialand the viral challenge aerosols. At present, dataare not available to resolve the differences inagent penetration noted when the room air-han-dling system was on or off. A possible explanationlies in the "induction type" diffuser used for theroom air supply. The air velocity produced bysuch a diffuser was turbulent and at a muchgreater value than any in the cabinet. Thus, theroom turbulence tended to distribute the challengeaerosol more uniformly across the face openingof the cabinet and at velocities greatly in excessof the low-velocity areas at the top of the faceopening. When the room air was off, the aerosolwas more readily controlled by the higher velocityair flowing into the exhaust grille at the cabinetface.

Table 2 contains a typical set of results ob-tained during product protection tests in thedesign mock-up of the new cabinet. Resultssimilar to those given in Table 2 have been ob-tained repeatedly in some 15 more tests witheither S. marcescens or T-1 coliphage. In all ofthese tests with the new cabinet, it should benoted that the room air-handling system wason, the aerosol challenge was three to four ordersof magnitude larger, and the aerosol challengeperiod was an additional 60 sec. All of the aboveitems provided a more stringent test, yet productprotection was vastly improved in the new cabi-net. The commercial cabinet tests showed an

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MCDADE ET AL.

TABLE 2. Product protection tests in a design mock-up of the new cabineta

Aerosol challenge Aerosol challenge Toa irba irbial penetrationTest no. Agent titer (3-min) per 100 ft3 penetration ofbiarl p fVcabinetpe10ft

1 3 X lOsb 1.8 X 108 6.9 X 105 0 02 1.5 X 109b 9 X 108 3.5 X 107 2 0.083 4 X 109b 2.4 X 109 9.2 X 107 5 0.191 5 X 108c 3 X 108 1.2 X 107 5 0.192 5 X 1O8c 3 X 108 1.2 X 107 3 0.12

a The room air-handling system was on; the cabinet air supplied during challenge was 2,598 ft3.b S. marcescens expressed as bacteria per ml.c Ti coliphage expressed as PFU per ml.

TABLE 3. Comparison of viral containment testresults in both cabinetsa

Total no. of particles Particle recovery per ft3Reyniers recoveredb of room air sampledsampler

no. Commercial Design Commercial Designcabinet mock-up cabinet mock-up

I TNTO TNTC TNTC TNTC2 8 3 0.14 0.033 1 0 0.02 04 0 0 0 05 7 0 0.12 06 0 0 0 0

a Based on 10 trials.b Aerosol: 5.9 X 109 PFU of TI coliphage/ml.c Too numerous to count.

average penetration of five to six microbial par-ticles per 100 ft3, whereas the new cabinet indi-cated an average penetration of 0.1 to 0.2 micro-bial particles per 100 ft3.Agent containment tests. The results of a typical

set of agent containment tests in both cabinets arecompared in Table 3. The test agent was TIbacteriophage. As presented in Table 3, contain-ment was considerably better in the design mock-up (particle escape 0.03 per ft3 at one site, 0 atthe other four) than in the commercial cabinet(particle escape 0.02 to 0.14 per ft3 at three sites,0 at the other two). Results similar to thosegiven in Table 3 have been obtained in more than30 trials in the commercial cabinet and in morethan 60 trials in the design mock-up cabinet.

Table 4 contains a second set of typical resultscomparing bacterial containment tests in bothcabinets. The bacterial containment efficiency ofthe design mock-up cabinet was far better thanthat of the commercial cabinet (Table 4). In thisseries of tests, the challenge aerosol used with thedesign mock-up cabinet was two orders of mag-nitude larger than that used in the commercial

TABLE 4. Comparison of bacterial containment testresults in both cabinetsa

Total no. of particles Particle recovery per ft3Reyniers recoveredb of room air sampledsampler

no. Commercial Design Commercial Designcabinet mock-up cabinet mock-up

I TNTCc TNTC TNTC TNTC2 0 0 0 03 6 0 0.12 04 2 1 0.04 0.025 1 0 0.02 06 0 0 0 0

a Based on five trials.I Aerosol: commercial cabinet, 5.6 X 107/ml;

design mock-up, 4.9 X 109/ml.c Too numerous to count.

cabinet, yet agent containment was superior inthe design mock-up cabinet.

DIsCUSSION

In the present study, a number of differentconfigurations and airflow patterns were eval-uated in an attempt to compare the productprotection or agent containment features or bothof several commercially available laminar flowcabinets. It was possible to apply a theoreticalengineering analysis to all phases of the develop-ment of a newly designed laminar downflowcabinet. Assumptions and conclusions made inthis theoretical analysis were supported by actualtests and airflow measurements. It was essentialto use a long probe (24 inches, 61 cm) to measureairflow. Smaller, hand-held thermo-anemometerswould create a cone of turbulence due to thepresence of the operator's hand in the airstream.Thus, it was felt that the measurements taken inour study represented realistic, repeatable values.Early in the study, design criteria were estab-

1090 APPL. MICROBIOL.

LAMINAR AIRFLOW BIOLOGICAL CABINET

lished and, through a series of modifications, itwas possible to meet the design criteria. Whenthis was accomplished, the design mock-up cabi-net functioned very satisfactorily during bothproduct protection and agent containment tests.The degree of agent containment or product

protection is dependent on the airflow patternand the degree of turbulence within the hood.Smoke has been used successfully by Turner (13)and Schulte et al. (12) to evaluate fume hoodswith respect to operating conditions and hooddesign. Unfortunately, there is a paucity of pub-lished information concerning the biologicalevaluation of safety cabinets and particularly oflaminar flow hoods. Williams and Lidwell (16)used Bacillus subtilis spores and Chromobacteriumprodigiosum cells to evaluate the containmentefficiency of an open-face cabinet. Barbeito (2)presented a detailed evaluation of a laboratoryhandling infectious microorganisms. His testsincluded evaluation of safety cabinets, filters,and exhaust systems. In each case, the spread ofthe challenge aerosol was limited by the pressuredifferential of the units being evaluated and notby a laminar airflow movement. The containmentefficiency and the protection afforded a biologicalproduct by a laminar flow unit has not, until thistime, been described in detail. It is felt that theproduct protection or the agent containmenttests or both represent a very high challenge intypical in-use situations. In the product protectiontests, the aerosol generator was positioned tosimulate the location of an operator. In thesetests, the units were exposed to a microbial aerosolconsiderably in excess of that normally encoun-tered, yet the newly designed cabinet providedexcellent product protection. Similarly, in theagent containment tests, the use of a blendor wasconsidered to be a realistic aerosol generator.The repeated sequence of hand insertion andremoval during the creation of each challengeaerosol was felt to simulate the normal operationsof a technician using the cabinet.The engineering phase of this study centered

around development of a more nearly laminarairflow cabinet. The microbiological phase cen-tered around the development of tests and pro-cedures to evaluate the performance of the newcabinet. Taken collectively, the results obtainedin this study represent a joint effort betweenengineering and microbiology to produce a

laminar airflow cabinet that functioned veryefficiently in providing agent containment andproduct protection.

Design drawings of the new cabinet may be

obtained from William E. Barkley, NationalCancer Institute, Wiscon Building, Room 600.7550 Wisconsin Ave., Bethesda, Md. 20014.

ACKNOWLEDGMENTS

We thank C. B. Henke for helpful suggestions dur-ing the engineering phase of this study. Appreciationis also extended to M. T. Kenny for assistance duringthe microbiological testing.

This investigation was supported by Public HealthService contract 43-65-1045 from the National CancerInstitute.

LITERATURE CITED

1. Adams, M. H. 1950. Methods of study of bac-terial viruses. Methods Med. Res. 2:1-73.

2. Barbeito, M. S. 1965. Biological safety evaluationof a commercial vaccine production laboratory.U.S. Army Biol. Labs., Ft. Detrick, Frederick,Md. Tech. Rept. No. 65.

3. Dow Biohazards Department. 1967. Evaluationof safety cabinets and test laboratory. The DowChemical Co., Pitman-Moore Div. Tech. Rept.No. BH 67-01-014.

4. Favero, M. S., J. R. Puleo, J. H. Marshall, andG. S. Oxborrow. 1966. Comparative levels andtypes of microbial contamination detected inindustrial clean rooms. Appl. Microbiol.14:539-551.

5. Favero, M. S., and K. R. Berquist. 1968. Use oflaminar air-flow equipment in microbiology.Appl. Microbiol. 16:182-183.

6. Federal Standard No. 209a. 1966. Clean room andwork station requirements, controlled environ-ment. General Services Administration, Wash-ington, D.C.

7. Jensen, M. M. 1963. Inactivation of virus aerosolsby ultraviolet light in a helical baffle chamber.Proc. 1st Intern. Symp. Aerobiol., p. 219-226.

8. McDade, J. J., W. Paik, M. W. Christensen,D. Drummond, and V. S. Magistrale. 1965.Microbiological studies conducted in the ex-perimental assembly and sterilization labora-tory. Jet Propulsion Laboratory Space Pro-grams Summary 4:30-39.

9. McDade, J. J., M. S. Favero, and G. S. Michael-sen. 1966. Environmental microbiology and thecontrol of microbial contamination. NASA(Natl. Aeron. Space Admin.), Publ., NASASP 108, p. 51-86.

10. McDade, J. J., M. S. Favero, and L. B. Hall.1967. Sterilization requirements for spaceexploration. J. Milk Food Technol. 30:179-195.

11. McDade, J. J., J. G. Whitcomb, E. W. Rypke,W. J. Whitfield, and C. M. Franklin. 1968.

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Microbiological studies conducted in a ver-tical laminar airflow surgery. J. Am. Med.Assoc. 203:125-130.

12. Schulte, H. F., E. C. Hyatt, H. S. Jordan, andR. N. Mitchell. 1954. Evaluation of laboratoryfume hoods. Am. Ind. Hyg. Assoc. Quart.15:195-202.

13. Turner, J. F. 1951. New laboratory fume hoodscut air conditioning load. Heating, Piping AirConditioning 23:113-116.

APPL. MICROBIOL.

14. Whitcomb, J. G., and W. E. Clapper. 1966.Ultraclean operating room. Am. J. Surg.112:681-685.

15. Whitfield, W. J. 1962. A new approach to cleanroom design. Sandia Corporation (Albuquer-que, N.M.) Tech. Rept. No. SC4673(RR).

16. Williams, R. E. O., and 0. M. Lidwell. 1957.A protective cabinet for handling infectivematerials in the laboratory. J. Clin. Pathol.10:400-402.