LEV, M. (1962). J . appl. Bact. 25 ( 1 ) . 30-34.

AN AUTOCLAVABLE PLASTIC UNIT FOR REARING ANIMALS UNDER GERM FREE CONDITIONS

BY M. LEV National Irtstitute for Research i n Dairying, University of Reuding

SUMMARY: A plastic unit for rearing germ free animals is described. This is sterilized by steam under pressure and has been successfully used to rear germ free chicks.

ALTHOUGH QERM FREE animals are now recognized tools in biological research, their use is restricted by the expense of the apparatus involved. For the apparatus previously described, steam under pressure is the sterilizing medium of choice and this consideration has influenced the design. The two main types of apparatus in use are those which are autoclaves and those which are autoclavable. Reyniers, Trexler & Ervin (1946) and Reyniers (1959) described an apparatus which is essentially a stainless steel autoclave modified to take air sterilized by filtration through glass wool. This apparatus has attached to it a small subsidiary autoclave for entry and exit of materials. Most of the experimental work reported has been done with this apparatus modified when necessary for special purposes.

Gustafsson (1948, 1959) has used an elegantly designed light gauge stainless steel apparatus consisting of a box fitted with a plate glass top, entries into which are made via a germicidal trap. Air is sterilized by incineration at 300".

A plastic unit described by Trexler & Reynolds (1957) has the disadvantage of not withstanding heat sterilization but requiring the use of chemical disinfectants. Gustafsson (1959) has stressed the advantages to be gained by using an autoclavable plastic for the construction of germ free rearing units.

It was felt that the design of a cheap reliable autoclavable plastic unit would be an asset to germ free animal research. Such a unit is described and has been used for the rearing of germ free chicks.

MATDIC~ALS AND METHODS

Comtruction of the unit. The unit consists essentially of a nylon tube 8 f t long, 78 in. in circumference and 0.002 in. thick, made from two pieces of 20 in. wide lay-fiat Portex (Portland Plastics Ltd., Hythe, Kent) nylon tube cemented together with nylon adhesive. The joint is covered with tape for extra protection. The tube is drawn over an angle-alloy frame 40 in. in length, 21 in. wide at the base, 114 in. high at the front and 19 in. high at the back (Fig. 1, a and b). One end of the tube is constricted around three stainless steel rings of 8 in. diameter which fit into a germicidal trap consisting of a stainless steel trough 10 in. x 10 in. x 20 in. fitted with

Germ free apparatus 3'

a lid to cover the part not occupied by the nylon tube (Fig. lb, Plate 1). The free end of the nylon tube is used for loading the apparatus with cages, water, etc. and is then sealed by twisting and folding. The fold is tightly wrapped and tied with rubber tubing (Plate 2).

The gloves are of rolled neoprene (28 in. x 6 in. and 0.015-0.022 in. thick, Veedip Ltd., Slough) and are supported on stainless steel rings of 6 in. diameter. For assembly two holes of 5 in. diameter are cut in the nylon tube 19 in. apart, and stretched to a diameter of 6 in. (by means of a conical flask). The collar so formed fits over the glove supports. The gloves have thickened ends which fit over the nylon collars and are secured in place by Jubilee clips (Plate 3).

(b) Fig. 1. Longitudinal (a) and lateral (b) sections of an autoclavable plastic

germ free unit.

Air is sterilized by passage through a glove box filter (Vokes L a . , Guildford, Surrey) and fed into the centre of the apparatus by an air pipe which passes through the germicidal trap (Fig. 1). Air is collected a t opposite corners through two nylon pipes which pass out via the germicidal trap into a small bottle of germicide (1 in 200 Chloramine T). The apparatus is maintained under positive air pressure which is determined partly by the depth of the outlet tube in the Chloramine T.

Sterilimtion of the unit. The sterilization procedure is based on that described by Gustafsson (1948). The apparatus is assembled, loaded and placed in the autoclave. The autoclave is fitted with a tap so that germicide can be run into the trap of the

32 M . Lev

unit while the apparatus is still inside, and is also fitted with a small-bore tube enabling sterilized air to pass into the closed autoclave after sterilization.

The autoclave is evacuated to -20 mm Hg, steam is passed in until the pressure reaches 2-3 lb/in2 and steaming at this pressure is continued for 10 min. The pressure is raised to 15 lb/in2, held for 30 min, and allowed to fall gradually. The germicide (an aqueous solution containing 1,000 p/m of benzalkonium chloride contained in a tank above the autoclave) is then run into the germicidal trap. A sterile glove box filter is then connected to the air pipe leading into the autoclave and sterile air is blown through the filter of the apparatus to dry it. The next day the apparatus is removed from the autoclave and air is blown through the filter to continue the drying. When dry the apparatus is ready for use.

The experimental procedure was as follows. A nylon mat was placed on the floor of the unit after assembly. On to it were placed 2 chick cages (17 in.x 10 in. x 11 in.) modified so that one half of the top could be slid open. Each cage contained a stainless steel feeding trough, a stainless steel water trough, and a stainless steel or glass drinking fountain. A wet and dry bulb thermometer and a tray containing hook and chain for introducing eggs, scissors, tin opener, artery forceps, gauze, and a pair of cloth over-gloves were placed on top of the cages. Forty tins of sterilized canned water (Smedleys Ltd., Whyteleafe, Surrey) or Kilner jars of distilled water were also included.

Operation for rearing germ free chicles. The diet used was the practical chick meal used in previous experiments (Lev & Briggs, 1956). This was packed between two layers of plastic and sterilized by irradiation with y-rays at 5 Mrads. This dose of irradiation was found to be adequate for sterilization without causing too much destruction of the components of the diet (Ford, Gregory & Thompson, 1959).

Fertile eggs which had been incubated for 18 days were used for the experiments. The procedure used for sterilizing the shells was based on those of Reyniers et al. (1949) and Forbes & Park (1959). Six eggs were placed in a net bag and immersed successively in 2% (v/v) Lissapol for 2 min and 2% HgC1, for 8 min, then rinsed in an aqueous solution of 1,000 p/m benzalkonium chloride and passed into tbe apparatus through the germicidal trap also containing benzalkonium chloride, by a hook and chain. All solutions except that in the trap were maintained at 37.5" and the eggs were gently manipulated during immersion. Eighteen eggs were intro- duced for each experiment.

The germicidal trap is not intended to sterilize materials: therefore all materials passed through the trap are previously surface-sterilized. They are wrapped in two layers of nylon before sterilization and the outer layer is removed just prior to immersion, and the inner layer is removed in the germicidal trap. The materials are then allowed to remain in the trap for 30 min before being passed into the chamber. Operators passing material through the trap wear sterile rubber gauntlets or a sterile armlength nylon tube.

After autoclaving and drying, the unit was transferred to a small room a t 37" in which a relative humidity of 65% was maintained. When the chicks had hatched eight were selected, if available, and excess chicks were killed and removed. The diet was introduced into the unit and sterility tests begun. The temperature in the

Germ free apparatus 3 3

room was gradually reduced to 20" over a period of two weeks. Faeces were removed from the cages at weekly intervals.

Assessment of sterility. Sterility checks of the chicks were at weekly intervals. A special stainless steel can containing 4 tubes of trypticase-soy broth, 4 tubes of fluid thioglycollate medium and 2 tubes containing swabs, was introduced into the unit. Vent swabs and samples of the faeces were taken and inoculated into the liquid media, inside the unit. Other swabs were streaked outside the unit on to trypticase- soy-blood plates which were incubated aerobically and anaerobically. Potato-dextrose agar and Sabouraud's agar were also inoculated and incubated aerobically for moulds and in addition smears from some of the swabs were examined microscopically after staining by Gram's method.

RESULTS To date three experiments have been done with this apparatus, two of which were successful in that no bacterial or fungal contamination occurred. In the first experi- ment, 2 chicks were kept in a germ free condition for a period of 5 weeks, after which they became too large for the cages. In the second, 7 chicks were maintained in a germ free condition for an experimental period of 4 weeks, but in the third, the chicks became contaminated with a Gram-positive sporeforming organism which, it was believed, gained entry into the unit when the eggs were being introduced. The eggs used in this experiment were unavoidably dirty. The germ free chicks reared in the apparatus appeared healthy and of average weight.

DISCUSSION Nylon is cheap, transparent, withstands autoclaving, and is well suited for use in germ free apparatus. Although the apparatus described has had only a limited number of runs, it has proved to be workable. There are several advantages of a unit made of flexible material compared with rigid apparatus: a positive pressure is always maintained in the apparatus (even when manipulating the gloves), which results in less chance of contamination occurring through a possible leak; lack of rigidity also means that when entry is made into the apparatus the germicide is not displaced from the trap as in the Gustafsson unit; and the layout is designed so that one person, by putting a left hand in the right hand glove, can place materials in the apparatus and remove them with ease.

The apparatus was designed as a prototype, easy modification of which can be made to suit special purposes such as those involving mammals as described in the experiments of Reyniers, Trexler & Ervin (1946), Gustafsson (1948) and Phillips, Wolfe & Gordon (1959).

The nylon tube can be used for at least two experiments and is easily replaced. Experiments are in progress to eliminate the use of a heated room for hatching eggs by fitting the apparatus with a thermostat and employing direct heat. Also, the availability of lay-flat tubing of 40 in. width will greatly facilitate the construction of the apparatus.

The author wishes to thank Mr. W. A. Cuthbert for constant help and encourage- ment, Dr. M. E. Coates for advice and for supplying fertile eggs and sterile diet,

34 M . Lev

and Mr. G. F. Harrison, Mr. S. Suffolk and Mr. B. Seed for help and advice. The diet was sterilized by the Wantage Radiation Laboratories, to whom the author’s thanks are due.

REFERENCES FORBES, M. & PARK, J. T. (1959). Growth of germ-free and conventional chicks. Effect of diet,

dietary penicillin and bacterial environment. J . Nutr. 67, 69. FORD, J. E., GREGORY, M. E. & THOMPSON, S. Y. (1959). Rep. nat. Inst. Res. Daivy., Reading,

p. 104. GUSTAFSSON, B. E. (1948). Germ-free rearing of rats. Acta path. nhicrobiol. scand. Supplement

no. 73. GTJSTAFSSON, B. E. (1959). Lightweight stainless steel systems for rearing germ free animals.

Ann. N.Y. Acad. Sci. 78, 17. LEV, M. & BRIGQS, C. A. E . (1956). The gut flora of the chick. 11. The establishment of the

flora. J. cappl. Bmt. 19, 224. PHILLIPS, R . P., WOLFE, P. A. & GORDON, H. A. (1959). Studies on rearing the guinea pig

germ-free. Ann. N . Y . Acad. Sci. 78, 183. - REYNIERS, J. A. (1959). Design and operation of apparatus for rearing germ free animals. Ann.

N . Y . Acad. Sci. 78, 47. REYNIERS, J. A., TREXLER, P. C. & ERVIN, R. F. (1946). Rearing germ-free albino rats. Lobund

REYNIERS, J. A., TREXLER, P. C., ERVIN, R. F., WAQNER, M., LUCKEY, T. D. & GORDON, H. A.

TREXLER, P. C. & REYNOLDS, L. I. (1957). Flexible film apparatus for the rearing and use of

Rep. no. 1.

(1949). Rearing germ-free chickens. Lobund Rep. no. 2 .

germ-free animals. Appl. Microbiol. 6, 146.

EXPLANATION OF PLATES Plate 1. Autoclavable plastic unit showing the germicidal trap, air filter and pump. Plate 2. Autoclavable plastic unit. End view. Plate 3. Autoclavable plastic unit. General view.

(Received 1 August, 1961)

Journal of Applied Bactclriology, 5 0 1 . 25, Part 1

Journal of dpplied Bacteriology, Vol. 25, Part 1

M. JIEV - AN AUTO(’LAVABLE PLASTIC UNIT Bolt REAKING ANIMaLS TiNDER GERM FRW; CONDITIONS. PLATE 2

Journal of Applied Bacteriology, Vol. 25, Part 1

&I. LEV - AN AVTO( LAVABLE P L A S T I C I T N I T FOlt ItEARlNCr ANIMALS U N D E R GERM FREM CONI)ITIONS. P L A T E 3