Flexible Film Apparatus for the Rearing and Use ofGermfree Animals1'2

P. C. TREXLER AND LOUISE I. REYNOLDS

Technology Division, Lobund Institute, University of Notre Dame, Notre Dame, Indiana

Received for publication June 10, 1957

Apparatus and methods for the rearing of germfreevertebrates have been reviewed by Gustafsson (1948).With the exception of rubber gloves, the closed systemisolators containing the sterile environment have beenconstructed of rigid materials. Isolators available com-mercially for use with hazardous agents are of the sameconstruction.

Flexible film isolators have been described by G. B.Phillips et al. (1955) for use with pathogens and byWebb and Softky (1953) for the handling of deliques-cent materials. Undoubtedly, similar apparatus havebeen made by many others but apparently the generalutility of plastic films has not been recognized. Flexibleplastic films are inexpensive and made readily intobacteria-tight structures. Their flexibility permits thetransmission of limited movement through any portionof the wall without disturbing the contamination bar-rier. They can be sterilized either with a liquid germi-cide applied as a spray or gaseous germicides (Green-span et al., 1955).

Flexible films have been used to construct bacteria-tight protective garments. A steam sterilized tank 8ft in diameter and 15 ft long, serviced by an attendantin such a garment has been described by Reyniers(1956) for the rearing of germfree animals. This opera-tion depends upon a rapid and effective method forthe cold sterilization of plastic or rubber surfaces.Peracetic acid3 appears to be satisfactory for thispurpose and can be used with an entire room linedwith a plastic coating as well as small isolators (Green-span et al., 1955; Trexler, 1956).The investigations herein reported describe methods

for sterile manipulations without the large investmentpreviously required (Reyniers, 1946). These studies areconcerned with animals in the germfree or gnotobioticstate, that is, isolated, or in association with knownorganisms (Dougherty, 1953). While the apparatusdescribed has not been in use long enough to provide a

' Presented at the Spring Meeting of the Maryland Branchof the Society of American Bacteriologists, Fort Detrick,Frederick, Md., April 6, 1957.

2 Support by contract NR 131-067 from the Office of NavalResearch, Department of the Navy, and Research GrantD-631 from the U.S. Public Health Service.

3Becco Chemical Division, Food Machinery and ChemicalCorp., Buffalo, N. Y.

long term comparison with steam sterilized rigid-walledisolators, the results to date warrant reporting becauseplastic isolators may soon be on the market.4 Germfreechickens (Reyniers et at., 1949) guinea pigs (B. P.Phillips et al., 1955) and monkeys (Reyniers et al.,1946) have been obtained germfree without muchdifficulty. Germfree rats and mice can be shipped fromcolonies established at Lobund.

MATERIALS AND METHODS

Sterilization. Some flexible films and sheets may besterilized by steam under pressure or dry heat (Mylar,5Kel-F6). However, the most useful materials, vinyl andpolyethylene, may be damaged by heat sterilizationtemperatures. Peracetic acid in a 2 per cent aqueoussolution with the addition of 0.1 per cent sodium alkyl-arylsulfonate inactivates resistant spores dried on anetched glass bead carrier within 30 sec in the liquidphase and within 10 min in the gas phase (Greenspanet al., 1955). This solution, dispersed by a glass orstainless steel atomizer, has been used to sterilize boththe isolator and protective garment. A sterilization timeof 10 min was used and the residue removed by flushingwith sterile air.

Peracetic acid has several limitations. It is very cor-rosive and many substances such as heavy metalscatalyze decomposition. Plastics, stainless steel, andglass are the construction materials of choice. Animalcages made of stainless steel wire mesh were difficultto sterilize with a peracetic acid spray, apparently dueto an accumulation of dirt in the sheet metal bindingstrips. They were treated satisfactorily when wrappedin paper and oven sterilized before spraying with thegermicide.

Isolators. The plastic isolator shown in figure 1 is aversatile design because it can be operated with positiveor negative pressure. The dimensions can be alteredeasily and either one or two operators accomodated.Transparent vinyl film (12 to 20 mils thick) was usedbecause it is very flexible and is sealed with heat orsolvent cements. The familiar plastic Scotch tapes canbe used for patching or sealing openings. Wrist-length

4 American Sterilizer Co., Erie, Pa.5 E. I. du Pont de Nemours and Co., Inc., Wilmington, Del.6 The Visking Corp., Terre Haute, Ind.

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rubber gloves are attached to sleeves by the aluminumring clamp7 shown in figure 3A. The sleeves are madeof the same material as the walls. This isolator hasbeen used for the work reported herein.The isolator shown in figure 2 is also made of vinyl7 Reyniers and Son, Chicago, Ill.

film8; however, it can be used only with positive pres-sure. All corners are rounded to reduce the build-upof stress and tearing of the plastic. Shoulder lengthrubber gloves are attached to the plastic walls by meansof a clamp (figure 3B) so arranged that only rubber and

8 Snyder Mfg. Co., New Philadelphia, Ohio.

Figure 1. (left) Plastic isolator. Air inlet filter and sterile lock is located on the left end of the isolator.Figure 2. (right) Plastic isolator. Air inlet filter and sterile lock entry door on the right end of the isolator.

Interior Isolator

Interior |Glovet

c " Interior,,,,,] , I so lator

I InteriorGlove

A BFigure S. Glove attachment clamps. A. Wrist attachment clamp. The spun aluminum ring a, is slipped into the cuff of surgical

glove b, which is then inserted in the cuff of inverted sleeve c. "O" ring d, is snapped around the sleeve and is held in place byno. 850 Scotch tape e.

B. Shoulder attachment clamp. The inner cast aluminum ring a, is placed within a 30 in. dry box glove* with the roll resting inthe annular groove. The glove and inner ring are placed within the isolator and the matching outer ring attached so as to bind theplastic film wall c, of the isolator.

* Charleston Rubber Co., Charleston, S. C.

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plastic are exposed to the interior of the isolator. Mylaror Kel-F may be used in place of vinyl film to permitsteam sterilization.

Sterile locks. The small (9 in. diameter and 12 in.length) jacketed double-door autoclave used with theReyniers Germfree System7 was attached to the plasticfilm wall by means of an 18 in. O.D. stainless steelflange to limit heat transfer to the plastic. Materialswere steam sterilized directly into the isolator or re-moved from the isolator without breaking the sterilebarrier. The weight and cost of the autoclave detractsfrom the inherent advantages of the flexible film sothis combination has limited usefulness. The flange

I I Cut I

ii+IB_ I , o

g_ I "1 1 I-_e B

Figure 4. Heat sealable bag transfer system. Diagram Ashows the introduction of an object a. The object is placed inbag b, which is then attached to collar c, fastened to the wall d,of the isolator. The germicide is introduced and removedthrough openings e. After sterilization the inner bag stub iscut as indicated. For removal of an object (diagram B) bag b,is attached and sterilized through e. The inner bag stub is cutas above, the object introduced, the neck of the bag pinched off

as indicated by the dotted lines, heat sealed, and cut at theseal as shown.

Sterile Lock

Interior Isolator

Figure 5. Germicidal spray sterile lock. A plastic bag or

isolator (not shown) is attached to collar a. A rubber sleeve b,with roll is attached to collar a, and to a cast aluminum clampring similar to figure 3B. The inner surface of the clamp ring ispadded with hemispherical sponge rubber weather strip c.

The rigid plastic door d, is attached by means of a flexibleconnector f, to hinged crossbar e. The hinge and closing mem-ber are attached to the clamp ring and are sealed to the wall ofthe isolator by sleeves h. The door is opened and closed fromthe inside of the isolator, but the sealing force is obtained fromoutside the isolator by means of the knurled nut g.

and attached inner door of the autoclave permits theattachment of the plastic isolator to the Reyniers units.A heat sealable bag method for transfer of materials

is shown in figure 4. A rigid cylindrical sleeve attachedto the wall of the isolator serves as an entrance port.The port is closed by a polyethylene bag slipped overthe external opening and fastened with a ring or ad-hesive tape against a taper. An object to be introducedis placed in a second bag which is slipped over the firstand sealed by the ring or tape. Some practice is neces-sary to produce the second seal without disturbingthe first. The first bag is cut from the inside and theobject introduced. An object to be removed is placedin the distal end of the second bag which is then closedby an externally applied heat-seal. A seal X to Y4 in.wide is used and cut so the object can be removedwithout breaking the isolation barrier. The object it-self remains sealed in the protective bag. This methodis particularly effective to confine a hazardous agenit.For sterile operation, connections must be sealed to thebag in order to admit and remove the germicide.A sectional view of a sterile lock for use with a germi-

cidal spray or gas is shown in figure 5. The lock may bein the form of a bag with inlet and outlet tubes foradmitting and removing the germicide or in the formof another isolator with attached gloves to manipulatethe objects passed. The door connecting the sterilelock to the isolator is open and closed from within theunit but the force necessary to obtain a tight seal isapplied externally. Sterile transfer from 1 unit to an-other is accomplished by connecting the sterile locksof 2 units by means of a plastic sleeve or collar havingtwo openings for admitting and removing germicides.

Objects that are damaged by germicide must beprotected in the above sterile lock. Towels, dry feed,and similar materials are sealed in a plastic bag (figure,6) equipped with a vent, plugged with cotton, anidsterilized in the autoclave. These bags made either ofMylar, Marlex,9 or Scotchpac'0 film are sealed by meansof a Vertrod Impulse Sealer.11 The material thus pack-aged is steam sterilized. After cooling the vent is sealedbelow the cotton plug and a hermetic package with asmooth exterior is produced by cutting through theseal lengthwise to remove the cotton plug.The sterilization of the packages was checked by

means of filter paper strips containing resistant Bacil-lus stearothermophilus spores placed in the center of thecontents. A vacuum must be pulled to remove the airbefore steam is admitted. Sterilization was obtaiinedrepeatedly using a vacuum of 25 in. of mercury followedby 15 lb steam pressure and a holding time dependingupon the load.

9 Phillips Chemical Co., Bartlesville, Okla.10 Minnesota Mining and Mfg. Co., St. Paul, Minn.11 Vertrod Corp., Brooklyn, N. Y.

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1957]APPARATUS FOR REARING AND USE OF GERMFREE ANIMALS

Ventilation. When contaminants are confined withinan isolator, a negative pressure is maintained and whenexcluded, a positive pressure is used to reduce theirmigration through a leak in the wall. Ordinarily, pres-sures of 1 in. of water or less are effective in an isolator.Animals require a flow of air not only for respirationbut to prevent high humidities.

Glass wool filters have been found to be most con-venient for sterilizing air supplied to the germfreeanimals (Reyniers and Trexler, 1943). The filters whichare permanently attached to the Reyniers type unitwere made by packing fine glass wool in copper orstainless tubing 1Y in. O.D. A }6 in. tube connectedto a steam line passes through the center of the tubelengthwise in order to supply the necessary heat todry the glass wool after steam sterilization. Thesefilters in the form used at the Lobund Institute requireabout 5 lb air pressure to pass 2 to 3 cfm air.A more compact and lighter filter is shown in figure

7. It consists of a cylinder of %/4 in. mesh 18 gauge stain-less steel screen 2 in. diametel and 12 in. long, sup-ported at both ends by a 2 in. length of stainless steeltubing to which the wire is welded. One end is pluggedand the other bears an adapter for Tygon tubing toconnect with the isolator. The screen is covered withfour layers of glass wool filter mat Type PF-10512 andsecured in place by wire. The filter may be wrapped inpaper and sterilized in the autoclave or it may be at-tached to the isolator and sterilized in place with agaseous germicide. A plastic or metal shroud carryinga hose adaptor is placed around the filter. Air under 4in. water pressure has been found adequate to pass 2

12 Owens-Corning Fiberglas Corp., Toledo, Ohio.

...:§.o.,;. .:

to 3 cfm air through the isolator. Pressure within theisolator can be maintained at 12 in. water pressure orless by means of a "surgical glove outlet trap." One ortwo fingers of the glove are removed and a 1 in. Tygonoutlet tube is inserted through the finger opening to thepalm. The cuff floats freely on the surface of the germi-cide and offers little resistance to the outward move-ment of the air. If the flow stops, or a negative pressureis created in the isolator by the movements of theoperator, the cuff sinks, effectively checking any back-flow. We have had no contamination traceable to thisdevice.Leak detection. Leaks can be detected by swabbing

the suspect area with a soap or detergent solutionwhile the vessel is under air pressure. The air pressuremust overcome the capillary force of the solution in theleak in order to form a bubble. The pressure that canbe used in the plastic isolators and protective garmentsis not great enough to detect small leaks.The Freon Leak Detector Type H-113 is much more

sensitive than the bubble test and is used to check theplastic isolators. Freon is available in containers ofdifferent sizes at any store carrying refrigeration sup-plies.

Preliminary tests have indicated that plastic isolatorscan be subject to an electrical leakage test similar tothat used for lineman's gloves. Essentially, the in-tegrity of the isolator is determined by the applicationof a potential across the plastic and rubber walls usingan electrolyte solution as the conductor. The leakagemay be determined with a microammeter or the po-tential at which a destructive arc is formed. Eithermethod gives a rapid complete check for tightness.This test can be used only with isolators made of non-conductors.

Protective garment. The protective garment used is3 General Electric, Schenectady, N. Y.

' I

I.. =,.,.

Figure 6. Plastic package for steam sterilization. The pack-age as removed from the steam sterilizer is shown on the left.and with the cotton plug and vent removed, on the right.

Figure 7. Air filter. A plastic shroud is shown at the top ofthe photograph with the stainless steel frame in the center,and the completely assembled unit at the bottom.

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made in two parts. It is heat sealed at the waist topresent a continuous surface to the germicide (Trexler,1955, 1957). The lower portion made of 20 mil vinylfilm is attached to a rigid metal waist band. Vinylboots, slush molded in our laboratory are heat sealedto the legs. The metal waist band carries shoulderstraps, air hose, communication lines, and is coveredwith a layer of asbestos to restrict the transfer of heatduring the sealing process. The upper portion of thesuit is made of transparent vinyl film.

Gloves are made by dipping a porcelain form inplastisol followed by baking. They are sealed to thearms of the jacket. Air is supplied to the headpiecethrough a 14 in. rubber air hose. This hose togetherwith wires for a lip microphone and ear set are carriedin a 1-1i in. industrial vacuum hose which in turn passesthrough a Tygon hose that is sealed to the trousersDuring operation, a negative pressure is maintained inthe suit by means of a domestic vacuum cleaner .t.-tached to the vacuum hose.

RESULTS

Sterilization of vinyl surfaces by peracetic acidsprays has been demonstrated by 227 successful entriesof an attendant into the large sterile tank (table 1). Onlyone contamination occurred that was traceable to theprotective garment. This was caused by a pinhole inthe armpit that escaped detection before entry ordeveloped while the garment was in use. No contamii-nant found to date has been resistant to the germicide.A plastic film isolator provided with a stainless steel

door and flange and attached to the steam sterilizedReyniers Germfree System was used for 15 separateexperiments without contamination. Animals werepassed into the plastic isolator for examination andsurgical procedures such as the canulation of bloodvessels. The optical disturbance created by the flexiblefilm has not caused difficulties so far.On two occasions, a litter of guinea pigs was reared

TABLE 1Operation of the sterile tank, serviced by an attendant in a

sterile protective garment*

Da te Entries Operated PRotuced Source of Contamination

3/5/51 13 57 - None25/10/51 30 227 End tank4/6/52 9 26 - End tank17/9/52 24 105 - Attendant error2/1/53 8 47 - None19/12/53 55 221 23 Rearing unit31/12/54 4 20 - End tank9/2/55 5 8 - Rearing unit1/4/55 56 251 223 Air compressor8/2/56 11 60 21 Corrosion entry door22/5/56 12 56 19 Protective garment

* Data obtained with the assistance of B. A. Teah.

for 3 months in isolators sterilized with peracetic acid.The procedures were similar to those of B. P. Phillipset al. (1955) as used with the steam sterilized rigid-wallunits. The young were obtained by a caesarian opera-tion performed in a small plastic isolator attached tothe unit shown in figure 1. No contamination wasfound. The units were operated by professional per-sonnel with considerable experience in this field.The same isolators (figure 1) were used to hatch and

rear chicks using the method described by Reyniers etal. (1949) for steam sterilized rigid wall apparatus. Theisolators were operated by a nonprofessional crew withno previous experience in sterile technique. Only 2 of18 runs remained sterile. The apparatus and procedurewas checked periodically to determine the source of thecontaminants. Plastic sleeves to which the gloves werefastened and the bags used in packaging diet frequentlydeveloped leaks. Invariably leakage at the inner doorof the sterile lock was found with the sensitive FreonLeak Detector.The isolator shown in figure 2 was designed to elimi-

nate the above leakage and simplify the procedures toreduce the training and experience needed for successfuloperation. The units built so far have been Freon-tight. They have not been in use for a sufficient lengthof time to determine their performance.

DISCUSSIONThe data presented shows that flexible plastic films

can contain a sterile environment suitable for therearing and study of germfree animals or gnotobiotes.3l'astic isolators have two advantages over the stainlesssteel equipment now in use. First of all, many delicatemanipulations such as the canulation of blood vesselsin experimental animals can be performed in theflexible film apparatus. Secondly, potential manufac-turers4 estimate the cost of the plastic isolator to beless than one-tenth that of the stainless steel units. Thecost of installation and operation is reduced becauseno special facilities or personnel are required. The firstfeature suggests a more extensive use of the germfreeanimal and the second should encourage the investiga-tion of germfree animals in other laboratories.The relative security provided by flexible and rigid-

walled isolators can only be determined by a compari-son of the operation of a series of finished units. Theresults apply only to the models under test and mayrequire a considerable length of time if the contamina-tion rate is low. For this reason, the factors involved incontamination control with gnotobiotes should beconsidered to provide an additional estimate of thepotential value of the two systems.

Sterility itself cannot be demonstrated experimen-tally but only the absence of certain organisms asdetermined by accepted procedures. The same limita-tion applies to the concept of gnotobiota. While the

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sterilization of inanimate objects may be monitored bya resistant organism (Bryce, 1956), this procedure can-not be applied to a living form. The determination ofthe absence of contaminants, in vertebrates at least, iscomplicated by new environments produced by theabsence of microbiota and some defense mechanisms(Wagner, 1955). While it may be possible to examine agnotobiote for the presence of all known organisms, thenew environment may produce adaptations or modifi-cations which obscure the interpretation of such exami-nations. The acquisition of new knowledge requiresthat these examinations be a continuing process ratherthan a static set of tests.The gnotobiotic state of an organism cannot be

determined in the absolute sense, but it can be deter-mined within the limitations of existing knowledgeprovided two conditions are met. First, following isola-tion, the gnotobiotes must be maintained so as to ex-clude the entrance of contaminants. Second, the gnoto-biote must reproduce to supply specimens for a seriesof examinations. There are considerable practicallimitations to these examinations particularly if theyare confined to one laboratory. With the developmentof transportation facilities these examinations need notbe confined to one laboratory but can be made by thosewho have specialized in the different groups of organ-isms or have discovered new testing procedures. Theresults of such examinations can accumulate to form a''pedigree" and in this way become available to usersof these strains.The contamination of an isolator involves not only

the loss of its contents but shows a defect in the sterilebarrier. Gnotobiotes subjected to a defective sterilebarrier must be reexamined completely and the "pedi-gree" mentioned above started anew. As any operationis subjected to accidents, physical or other means mustbe used to detect breaks in the sterile barrier whetherstructural or operational and those defective unitsremoved from the series. It is important to subject allisolators to the same testing procedures to avoid theselection of inapparent contaminants. These procedurescomplicate the operation and are probably not war-ranted except for long term investigations.

Three grades of gnotobiotes can be obtained withuseful combinations of sterile barrier checks and thediligence used to search for contaminants. Because thecategories formed are of practical importance thefollowing terms have been used:Alpha gnotobiotes are gnotobiotes reared through

generations in a sterile environment and subject toexamination for the presence of all living forms thatare suspect as contaminants. The integrity of the sterilebarriers is checked by regular physical and other tests.Routine examinations for the common contaminantsserve primarily to indicate the performance of theprocess (Davies and Fishburn, 1948). The successful

avoidance of contamination may be termed alphaoperation.

Beta gnotobiotes are gnotobiotes that are likewisemaintained with alpha operation of the environment.Because the animals do not reproduce, hence differfrom the alpha grade, their gnotobiotic status is deter-mined by a statistically significant examination ofsimilar organisms.Gamma gnotobiotes are gnotobiotes maintained under

circumstances in which accidental contamination mayoccur. The gnotobiotic status is based primarily uponthe results of an examination of the individual animalor the population of an isolator. The hazard of an ac-cumulation of inapparent contaminations must berecognized with gamma operations. Observations withour present equipment which is old and has a highcontamination rate indicate this hazard is not greatwith runs of 6 months or less. Bacteriological testinghas been more difficult with longer runs, particularlywith the colonies of rats and mice. The gamma grade ofoperating has been the type used so far. It apparentlywill continue to be the practical method used for mostinvestigations.The following situations probably will justify the

more elaborate alpha operation: (1) Animals such asrats and mice are difficult to rear germfree from con-ventional stock. They should be maintained in a sterileenvironment for purely economic reasons. Alpha opera-tions are necessary to keep the strain pure and establishpedigree lines, but the bulk of the breeding can becarried on with less rigorous control. At intervals thebreeding stock can be replaced by fresh stock from thealpha colony just as commercial seed growers obtainfresh starts periodically from sources that are geneti-cally controlled.

(2) The complete examination for some contaminantssuch as masked viruses is difficult and a considerableeffort is required. If the strains so examined can beused by other investigators, the results of these exami-nations have more than academic value. Any maskedviruses or other contaminants found may limit theusefulness of the animal but not destroy its value. Thedevelopment of techniques for unmasking virus andother parasites may indicate that it is not possible toobtain an animal completely free of other forms oflife. If the problem becomes this complex, the needfor alpha operation and pedigree stock will increaserather than decrease.

(3) The rearing of animals in the absence of micro-biota relieves the animal of the necessity for defenseagainst these agents. Under the circumstances it seemslikely that genetic strains of animals can be producedwhich have lost the ability to generate some defensemechanisms such as gamma globulin or in which genesfor susceptibility to different infective agents may ac-cumulate. These animals would be extremely valuable

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in a number of fields and should be maintained underalpha conditions.The control of contamination required for alpha

operation depends primarily upon the detection ofmechanical breaks in the sterile barrier or failure of asterilization procedure. The break down rate is ofeconomic importance and determines the number ofduplicate series necessary to reduce the probability ofcomplete loss to some predetermined level. Plasticisolators have two advantages over stainless steel unitsas far as detection of the sterile barrier failure is con-cerned. They are less complicated and the entire unitcan be subject to an electrical leakage test as used forlineman's gloves.

Plastic film isolators have been operated in a sterileroom lined with a sprayed-on vinyl coating. The at-tendant worked in a protective garment similar to thatused with the colony tank. If the atmosphere of theroom is made germicidal or the entire room is sprayedwith germicide periodically, the sterile barrier becomesa germicidal barrier. The isolator protects the animalsfrom the germicide and the garment protects the at-tendant. The room serves to confine the germicidalatmosphere.

SUMMARY

Flexible plastic film has been used to construct ap-paratus for the maintenance of a sterile environmentsuitable for the rearing and use of germfree laboratoryanimals. Peracetic acid is used for sterilization.The apparatus is used with steam sterilized supplies

either by a direct connection to a steam sterilizer or byimpervious packaging of steam sterilized supplies.A completely sealed plastic garment was sterilized

with peracetic acid and used to tend germfree animalsin a sterile room. One contamination resulting fromfailure of the garment occurred in 227 entries.The flexible film apparatus can be used either alone

or as an adjunct to steam sterilized rigid-walled equip-ment.

REFERENCES

BRYCE, M. 1956 Tests for the sterility of pharmaceuticalpreparations. The design and interpretation of sterilitytests. J. Pharm. Pharmacol., 8, 561-572.

DAVIES, 0. L. AND FISHBURN, A. G. 1948 Sampling forsterility tests. Pharm. J., 106, 184-185.

DOUGHERTY, E. C. 1953 Problems of nomenclature for thegrowth of organisms of one species with and without asso-ciated organisms of other species. Parasitology, 42, 259-261.

GREENSPAN, F. P., JOHNSEN, M. A., AND TREXLER, P. C. 1955Peracetic serosols. Chem. Specialties Mfrs. Assoc., Proc.42nd Ann. Meeting.

GuSTAFSSON, B. 1948 Germfree rearing of rats. Acta Pa-thol. Microbiol. Scand. Suppl. LXXIII, 1-130.

PHILLIPs, B. P., WOLFE, P. A., REES, C. W., GORDON, H. A.,WRIGHT, W. H., AND REYNIERS, J. A. 1955 Studies onthe ameba-bacteria relationship in amebiasis. Am. J.Trop. Med. Hyg., 4, 675-692.

PHILLIPs, G. B., NOVAK, F. E., AND ALG, R. L. 1955 Portableinexpensive plastic safety hood for bacteriologists. Appl.Microbiol., 3, 216-217.

REYNIERS, J. A. 1946 Germfree life applied to nutritionstudies. Lobund Reports, p. 101. University Press.Notre Dame, Ind.

REYNIERS, J. A. 1956 Germfree life methodology (gnoto-biotics) and experimental nutrition. Proc. Third Intern.Congr. Biochem., Brussels 458-466.

REYNIERS, J. A. AND TREXLER, P. C. 1943 The germfreetechnique and its application to rearing animals free fromcontamination. In Micrurgical and germfree techniques,114-143. Charles C Thomas, Springfield, Ill.

REYNIERS, J. A., TREXLER, P. C., AND ERVIN, R. F. 1946Rearing germfree albino rats. Lobund Reports, 1-84.

REYNIERS, J. A., TREXLER, P. C., ERVIN, R. F., WAGNER, M.,LUCKEY, T. D., AND GORDON, H. A. 1949 Rearing germ-free chickens. Lobund Reports, 1-116. University Press,Notre Dame, Ind.

TREXLER, P. C. 1955 U. S. Patent 2,705,489.TREXLER, P. C. 1956 Germicides as a step in the elimination

of microbic contamination. Chem. Specialties Mfrs.Assoc., Proc. 42nd Mid-year Meeting. 100-103.

TREXLER, P. C. 1955 U. S. Patents 2,786,464 and 2,779,331.WAGNER, M. 1955 Serologic observations in germfree ani-

mals. Bull. N. Y. Acad. Med., 31, 236-239.WEBB, L. A. AND SOFTKY, S. D. 1953 Inexpensive dry box

for working with deliquescent materials. Rev. Sci. Instr.,24, 472.

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