Rearing of Germfree and Monocontaminated Chicks in RigidPlastic Isolators1

PHILLIPS, NEWEMCOMB, LACHAPELLE. ANI)

Biological Iesearch Laborator ies, Department of Bacter iology and Botany, Syractise University, Syr ac use, N\ew Y'or k

Received for publication November 27, 1961

ABSTRACT

13HILLIPS, A. W. (Syracuse University, Syracuse, N. Y.),H. R. NEWCOMB, R. LACHAPELLE, AND E. BALISH. Rearingof germfree and monocontaminated chicks in rigid plasticisolators. Appl. Microbiol. 10:224-230. 1962. A Plexiglasisolator for rearing germfree vertebrates is described.Plastic components were precut and finished by a Plexiglassupplier. This equipment is relatively inexpensive; more-over, neither special facilities nor skills are required intheir construction. Advantages of this type of isolator are:readily dismantled for cleaning, durable, excellent visi-bility of the interior, and minimal maintenance. In 30separate groups of animals, 27 of these yielded germfreechicks. Many of these chicks were reared germfree for4 weeks; and several were maintained for about 2 months,without microbial contamination. Pure cultures of Candidaalbicans, Streptococcus faecalis, and Clostridium perfringenswere established as monocontaminations in the intestinaltracts of chickens. In contrast, Alcaligenes faecalis ATCC9220 failed to grow in the gut, of chicks, following peroraladministration of this organism. All chicks described wereemployed on other experiments, and were killed for variousassays at times specified.

The applications of germfree vertebrates in experi-mental biology have recently been reviewed by lPhillipsand Smith (1959), Levenson et al. (1959), and Gordon(1960). The different types of apparatus and proceduresemployed by various workers were described in two recentsymposia: New York Academy of Sciences (1959) andNotre Dame University (1960).

T'lastic isolators have been introduced in recent. yearswhich are less costly than previous e(uipment. For in-stance, flexible film isolators were described by Trexlerand Reynolds (1957); and rigid plastic isolators weredescribed by Phillips et al. (1960). The present reportexhibits our improved I'lexiglas isolators which have beenemployed successfully in research on germfree (hicks.Some of our results with these isolators are presented.

I These studies were supported in part by research grant E-2999from the National Institute of Allergy and Infectious Diseases,U. S. Public Health Service.

2 Predoctoral fellow, U. S. Public Health Service.I Rohm and Haas Company, Philadelphia, Pa.

MIATERIALS AND 1IETHODSIsolator construction. A pair of isolators prepared for the

incubation of fertile eggs, hatching, and rearing of germ-free chicks, is shown in Fig. 1. These units are constructedof transparent acrylic plastic (type R, clear Plexiglas)3sheets, 38 in. thickness, from materials which were precutto specified dimensions by the supplier. The unit is essen-tially an inverted box (Fig. 2) inserted into a moated lid(Fig. 3). Each isolator is 2 ft in width, 3 ft in depth (frontto back), and 2 ft in height. In the construction of theunits, both a solvent cement (I-B)3 and a monomer-catalyst cement (P'S-18)3 were employed; the latter ispreferable for additional strength. The management ofexperiments in the isolator, and particularly the assemblyand removal of cages and equipment, was greatly facili-tated by the detachability of the entire top of the unitfrom the moated floor panel. A vertical shute (12 by 12 by16 in.) was cemented to the floor panel and extended intothe germicidal tank (see Fig. 1 and 3). This shute permitsthe transfer of sterile items to or from the isolator, as wellas between isolators, in which case two isolators wouldextend into the same germicidal tank. Should it be neces-sary to transfer items between isolators not linked to acommon germicidal trap, the protectve cover showni inFig. 4 (left side) is placed over the shute opening in thetrap; this protects the unit against contamination while itis removed to the appropriate germicidal tank.The chamber was ventilated by means of a small

centrifugal blower fan model no. 50748-J1184 shown inFig. 1; approximately 2 ft3 per min of air was passedthrough the isolator. The sterilization of air was achievedby the device shown in Fig. 1. This is similar to that de-scribed by Trexler and Reynolds (1957) and was purchasedfrom the American Sterilizer Company.5 These filterswere wrapped with two layers of 50-FG glass wool media.6Exhaust air was passed through the device described byTrexler (1959). The air pressure differential at the inletfilter was about 0.5 in. of water, and was negligible at theair exhaust, device.The germicidal tank under the isolators shown in

Fig. I was constructed of materials as described for theisolator; however, these tanks were roiinforced by cement-

Fasco Industries, Rochester, N. Y.American Sterilizer Company, Erie, Pa.

6 American Air Filter Company, Louisville, Ky.

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ing 1-in.2 bars of Plexiglas across the interior joints ofthe tank.

Each isolator was equipped with one pair of arm lengthrubber gloves.7 The glove ports shown in Fig. 1 were 7 in.inside diameter and were placed 18 in. apart (center tocenter); their centers were located 8 in. above the floor ofthe isolator. Gloves were mounted on Plexiglas rings ce-mented to the outside wall of the isolator. These ringswere 8% in. in outside diameter by 6 in. inside diameterby 34 in. in thickness (Fig. 4, right side). The outer edgeof the ring had a 0.25-in. groove cut in the circumferencefor accommodating the 0-rings. The latter were 61 in.inside diameter, and 0.275 in. in circular thickness (sizeno. 2S438, standard soft neoprene,8 40 to 50 durometerhardness).

All isolators were mounted on movable dollies as shownin Fig. 1. These dollies were constructed of Dexion9 with

7 Neoprene, no. 8N-2532, Charleston Rubber Company, Charles-ton, S. C.

8 Hercules Packing Company, Alden, N. Y.9 Acme Steel Co., Chicago, Ill.

rubber casters. Germicidal tanks are removable withoutdisturbing the isolators on the dolly.Animals were housed in stainless steel double utility

cages.10 This cage is 21 in. deep by 16 in. high bv 24 in.wide and has a removable partition.The peripheral moat in the bottom of the isolator was

filled with 2 % mercuric chloride solution, as was thegermicidal trap. Polyethylene film was used as a protectivedust cover for these solutions.

Testing isolators for leaks. Before each experiment, iso-Jators were tested for leaks by filling with water; all jointswere observed for leaks. In some instances, a slight positiveair pressure was established in the isolator, and a bubble-forming solution brushed on all seams and joints to testfor leaks. The Freon leak detector may be more convenientand accurate than the above method (Trexler and Rey-nolds, 1957).

Sterilization of isolator. All materials that went into theisolator, as well as all interior surfaces of the isolator, were

10 Model DUC-2, B. Feneno Company, Boston, Mass.

FIG. 1. Plexiglas isolators prepared for rearing germfree chicks, or chicks selectively contaminated with specific microorganisms. Inletair filters are the horizontal units above the isolators. Two exhaust air devices are shown as the vertical units between the isolators. Feed,water, etc., are passed into the isolators through the 2% HgCl2 solution in the tank under the isolators.

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first thoroughly cleaned with warm detergent solution,rinsed, and dried. Care was taken to insure the removal ofoil films. Items were handled only with rubber gloves.Where possible, articles were wrapped in Kraft paper andsterilized in the oven or autoclave. The air filter was pre-

sterilized at 165 C for 2 hr before being attached to theisolator; the filter was also subse(uently exposed to per-

acetic acid vapor during sterilization of the isolators (seebelow).Eaeh isolator was sterilized with 200 ml of a 2c% per-

acetic acid solution using a De Vilbiss atomizer." Thechamber was sealed and allowed to stand for 3 hr beforeventilating for 24 hr.

Sterilization of drinking water. Tap water was put inevaporated milk type cans (375 ml per can). The hole inthe can was plugged with cotton, and the can autoclaved20 min at 121 C. The hot cans were sealed with solder,and again autoclaved 20 min. Cans were put into paper

bags and given a final autoclaving for 45 min.Liquid in air exhaust device. Two per cent mercuric

chloride was placed in the air exhaust trap and a 0.25-in.layer of mineral oil placed on the surface of the solution toprevent liquid evaporation.

Sterilization of diet. The purified casein-starch diet ofForbes and 1'ark (1959) was used in this study.'2 The dietwas packaged in plastic pouches, 6 by 65x in., consisting

"No. 251, The DeVilbiss Company, Toledo, Ohio.12 Prepared by Nutritional Biochemicals Corp., Cleveland,

Ohio.

FIG. 2. Top of an isolator being removed fronm its moated floorpanel for cleaning after the termination of an experiment.

of 0.005 in. Mylar and an inner laminant of 0.002 in.polyethylene. Each package contained approximately200 g of diet. Packages were compressed in a Carver pressat 8 to 10 thousand pounds per square inch. The open endof the pouch was then heat-sealed while under pressure.The bags were tested for leaks by immersion under water,then sealed in a second bag, under vacuum; a maximalpackage thickness of 0.5) in. was maintained to complywith radiation sterilization requirements. The diet wasthen sent to High Voltage Engineering Corp., Burlington,1lass., for sterilization by electron beams at 5 megarads.Just before introducing the diet package into the sterileisolator, the outer wrap was removed; and the innerpackage was then passed through the germicidal trapwhere it was allowed to remain for 20 min before introduc-ing into the isolator. All articles entering the isolatorthrough the germicidal trap were rinsed with water toremove mercuric chloride solution.

Procedure for germfree chicks. The sterilized isolatorswere placed in a room which was controlled at 39 +4 1 Cand 50 to 60 % relative humidity. Fertile White Leghorneggs (17 day) were washed in a warm detergent'3 solutionfor 5 min at 39 C. The eggs were placed in a neoprene netand immersed in the 2 %o mercuric chloride solution of thegermicidal trap of the isolator for 10 min at 39 C. Whilethe eggs were immersed in the mercuric chloride solution,they were gently rotated to assure exposure of all sides tothe germicide. Inside of the chamber, the eggs were rinsedwith sterile water and placed on the wire mesh of thecages for incubation. From 15 to 20 eggs were placed ineach isolator. Since the eggs were placed in the isolatorafter 17 to 19 days of incubation, no further turning of theeggs was necessary.

After hatching, egg shells were removed and the chicksgiven numbered leg bands. Cloacal swabs were taken forbacteriological examinations before feeding the birds.Also, each batch of irradiated food was checked for mi-crobial contamination before feeding. The bacteriologicalprocedures were essentially those described by Wagner(1959) for germfree animals; fecal samples were takenweekly for bacteriological analysis.

Transfers. The following procedure was employed forthe removal of germfree chicks from an isolator to theexterior, or into another sterile isolator. A wide-mouth,2-quart, polyethylene jar with screw cap was thoroughlycleaned with detergent, then placed in the germicidaltrap. It was allowed to remain in the trap for 20 min, thenbrought into the chamber and rinsed with sterile water.The animal was then placed in the jar, which was thentightly sealed, and immediately removed from the isolator.Some of these jars were of high density polyethylene whichcould be autoclaved. In some instances, items were auto-claved in jars within paper bags with caps loosened. After

13 Sanegg, a product of Dare Products, Inc., Battle Creek,Mich.

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autoclaving, caps were tightened before removing jarsfrom bags. Jars were then immersed for 20 min in thegermicidal trap, then passed into the isolator.

Heat-sensitive materials, such as a pure culture of amicroorganism, were introduced into a germfree isolatoras follows. The culture was sealed in a glass ampule whichwas then immersed in potassium dichromate-sulfuric acidcleaning solution for 20 min, rinsed, placed in the germi-cidal trap for 20 min, brought into the isolator, rinsed,and opened for use.

Glove protection in isolators. To prevent glove punctures,neoprene gauntlet gloves were worn over the arm lengthisolator gloves whenever possible.

Cultures. Candida albicans ATCC 10231 was grown inSabouraud dextrose broth14 at 37 C for 48 hr. Streptococcusfaecalis, isolated from the intestinal tract of a mono-contaminated chicken, and Alcaligenes faecalis ATCC9220, were grown in nutrient broth'3 at 37 C for 48 hr.Clostridium perfringens, isolated from chickens (gut), wasgrown in thioglycolate broth'3 at 37 C for 48 hr. About

14 Difco Laboratories, Inc., Detroit, Mich.

10 ml of a broth culture were mixed with about 200g ofdiet in the feed tray of specified groups of germfree chicks.

RESULTS

A summary of our isolator operations from May 1960through April 1961 is given in Table 1. Most of the germ-free chicks obtained during May through July 1960 (ex-periments 13 to 16) manifested an illness similar to thatdescribed by Gordon et al. (1959). Hyperexcitability andtremors with unsteady gait were observed in newlyhatched chicks; symptoms progressed with loss of weightand high mortality. However, in experiments 17 to 20,which were carried out between January and April 1961,apparently healthy germfree chicks were obtained and noevidence of the previous illness was seen during thisperiod. Out of 30 attempts to rear germfree chicks, 27 ofthese were successful from a bacteriological standpoint.In two experiments, contamination was first noticed at21 to 25 days and, in another experiment, contaminationoccurred within 2 days of hatching. The sources of thesecontaminations were not determined, although the bac-teriology of the food was negative.

FIG. 3. Floor panel of an isolator showing the transfer shute projecting into a germicidal bath

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Most of the chicks shown in Table 1 were for use onexperiments of 4 weeks in duration. The abilitv of theapparatus to conduct, germfree experiments beyond thistime was proved, in a few instances, in which chicks werereared germfree up to 70 days.At the conclusion of each experiment the animals were

autopsied and bacteriological examinations performed oneach of the following segments of the gastrointestinaltract: crop, duodenum, jejunuim, ileum, ceca, and colon.No viable microorganisms were recovered from any por-tion of the intestinal tract of those animals specified asgermfree.

Table 2 summarizes the results obtained wheni purecultures of various microorganisms were introduced intogermfree chicks. No extraneous contaminations wereencountered wheii C. albicans, S. faecalis, and C. per-fringens were introduced independently into separategroups of germfree chicks. Repeated attempts to establishA. faecalis in the gut of a single group of germfree chickswere unsuccessful. No viable organisms of this specieswere obtained from fecal samples taken during the periodof feeding this organism to germfree chicks, or on bac-teriological examination of the intestinal contents at thetime of sacrifice. Broth cultures (10 ml) of A. faecalis wereadded to the diet twice a week for 4 weeks; on one occasionthe culture was added to the drinking water.

Figure 5 shows the comparative growth rates of germ-free and conventional control chicks reared under similarconditions of isolation. These data do not include birdswith the illness previously mentioned, but include therelatively healthy germfree chicks described in experi-ments 17 through 20. The mean growth rate of the germ-free birds was comparable to that of the control birds,but the weight gain in our germfree chicks was not asgreat as that described by Forbes, I'ark, and Lev (1959)on the same diet. However, these authors sterilized theirdiet by steam, whereas our diet was sterilized by radia-tion and it is not known whether this difference in growthrate is due to the difference in method of sterilization. Wemade no determinations on vitamin losses due to radiationsterilization. Data reported by Luckey et al. (1955) suggestthat radiation sterilization results in less loss of vitaminsthan steam sterilization.

DISCUSSION

With little change in the design of the apparatus, itshould be possible to employ this equipment for the rearingof germfree mammals; also this equipment may be em-ployed for performing caesarian sections. For the latter,an antechamber could be cemented to the bottom of theisolator to accomodate the pregnant animal. A Mylarwindow could be installed in the floor of the isolator above

FIG. 4. Left side: moated cover for the transfer shulte of an isolator; this is used to protect an isolator from contamination when the isolatoris to be removed to another germicidal tank. Right side: two views of a Plexiglas glove ring, which is cemented to the outer wall of an isolatorto support an arm length rubber glove.

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the antechamber, through which the caesarotomy couldbe performed. The size of the isolator may be determinedby the species and numbers of animals to be housed.Our isolators have been kept in somewhat dusty quar-

ters; however, it is preferable to maintain them in rela-tively dustfree rooms. Where large accumulations of dustare encountered it has been found necessary to use a pre-filter on the air supply unit to the isolators. Without theprefilter, it may be necessary to transfer the chicks toisolators with fresh filters after 3 months.

Mercuric chloride has been employed throughout as agermicidal liquid in the trap, but other germicidal solu-tions may be satisfactory. No study was made on theadequacy of other germicides. The walls and floors of the

TABLE 1. Germfree chick production statistics

Expt. Starting Isolator Eggs Chicks Survi- Dura- Terminal micro-no. date no. set hatched vors tion* bial statust

days

13 5/16/60 4 10 9 9 30 C at 21 days5 10 6 2 30 gf7 10 8 2 15 gf

14 6/6/60 2 16 11 11 28 gf3 16 6 6 28 gf

15 6/28/60 4 15 14 7 30 gf5 15 14 3 32 C at 25 days6 15 11 11 25 gf7 15 11 2 14 gf

16 7/18/60 2 15 12 12 20 gf3 15 14 11 31 gf

17 1/2/61 6 15 10 9 28 gf7 15 8 1 7 gf8 15 6 1 4 gf9 15 5 2 70 gf10 15 7 1 8 C at 2 days11 15 4 1 4 gf

18 1/15/61 10 15 8 5 28 gf11 15 12 5 28 gf

19 2/27/61 5 15 5 1 29 gf6 15 9 4 45 gf7 15 3 1 49 gf10 15 4 1 30 gf11 15 3 1 25 gf

20 4/10/61 2 20 7 6 30 gf3 20 8 7 30 gf8 15 3 2 30 gf9 15 3 2 30 gf10 15 4 4 30 gf11 15 7 7 30 gf

* Each group of chicks was removed from the isolators at

specified times, killed, and submitted to various analyses.t gf = Germfree; C = contaminated. Results of bacteriological

examinations were negative on fecal samples taken at weekly in-tervals during the life of the birds, and on intestinal contentsexamined after autopsy of chicks.

chambers as well as the gloves should be rinsed occasion-ally to prevent the accumulation of mercuric chloridewithin the isolator.

It would be desirable to have a more convenient methodof supplying the relatively large quantities of sterile waterrequired in experiments of the type described. Sterilewater may be provided directly to an isolator through abacteriological filter.The Plexiglas isolator may be equipped with a transfer

autoclave for either introducing steam-sterilized items orproviding a direct connection with other types of germ-free isolators. Although with a specially designed packagesuch as that described by Trexler and Reynolds (1957)which can be steam sterilized and then passed through agermicidal trap, the necessity for direct connection betweenisolator and a steam autoclave may not be necessary insome instances.

ACKNOWLEDGMENTS

The assistance of M. Pfeffer, D. Willett, and C. Dence isgratefully acknowledged.

TABLE 2. Deliberate contamination of germfree chicks with purecultures of microorganisms*

No. of Age at Duration Terminalchicks

inocula- Organism of contam- microbialchicks tion inationt statust

days days

6 45 Candida albicans 30 Pure culture2 49 Streptococcus faecalis 30 Pure culture5 10 Clostridium perfringens 30 Pure culture8 10 Clostridium perfringens 30 Pure culture

* Ten milliliters of a broth culture of the specified organismwere mixed with 200 g of diet.

t Time at which birds were autopsied and examined for micro-

bial status.t As determined by bacteriological analyses of intestinal con-

tents on killed birds.

15AGE (days)

FIG. 5. Comparative growth rates of germfree and conventional

White Leghorn chicks (Kimber strain). Diet: casein-starch (Forbesand Park, 1969); sterilized by 5 megarads of electron beams. Experi-ment no. 17.

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LITERATURE CITEDFORBES, M., AND J. T. PARK. 1959. Growth of germfree and con-

ventional chicks: effect of diet, dietary penicillin and bac-terial environment. J. Nutrition 67:69-84.

FORBES, M., J. T. PARK, AND M. LEV. 1959. Role of the intestinalflora in the growth response of chicks to dietary penicillin.Ann. N. Y. Acad. Sci. 78:321-327.

GORDON, H. A. 1960. The germfree animal. Am. J. Digest. Diseases(N.S.) 5:841-867.

GORDON, H. A., M. WAGNER, T. D. LUCKEY, AND J. A. REYNIERS.1959. An encephalomeningeal syndrome selectively affectingnewly hatched germfree and monocontaminated chickens. J.Infectious Diseases 105:31-37.

LEVENSON, S. M., R. P. MASON, T. E. HUBER, 0. J. MALM, R. E.HOROWITZ, AND A. EINHEBER. 1959. Germfree animals in sur-

gical research. Ann. Surgery 150:713-730.LUCKEY, T. D., M. WAGNER, J. A. REYNIERS, AND F. L. FOSTER,

JR. 1955. Nutritional adequacy of a semi-synthetic dietsterilized by steam or by cathode rays. Food Research 20:180-185.

New York Academy of Sciences. 1959. Germfree vertebrates: pres-ent status. Ann. N. Y. Acad. Sci. 78:1-400.

Notre Dame University. 1960. Proc. second symposium on

gnotobiotic technology. May 1959. Notre Dame Univ. Press,Notre Dame, Ind. 195 p.

PHILLIPS, A. W., AND J. E. SMITH. 1959. Germfree animal tech-niques and their applications. Advances in Appl. Microbiol.1:141-174.

PHILLIPS, A. W., F. A. Rupp, J. E. SMITH, AND H. R. NEWCOMB.1960. A Plexiglas isolator for germfree animal research. Proc.second symposium on gnotobiotic technology, p. 49-54. NotreDame Univ. Press, Notre Dame, Ind.

TREXLER, P. C. 1959. The use of plastics in the design of isolatorsystems. Ann. N. Y. Acad. Sci. 78:29-36.

TREXLER, P. C., AND L. I. REYNOLDS. 1957. Flexible film apparatusfor the rearing and use of germfree animals. Appl. Microbiol.5:406-412.

WAGNER, M. 1959. Determination of germfree status. Ann. N. Y.Acad. Sci. 78:89-102.

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