Isolation of Anaerobic Bacteria from Human Gingiva. Box Procedure' and Mouse Cecum by Means of a Simplified Glove

Transcription

1 APPLIED MICROBIOLOGY, Apr. 1969, p Copyright ( 1969 American Society for Microbiology Vol. 17, No. 4 Printed in U.S.A. Isolation of Anaerobic Bacteria from Human Gingiva and Mouse Cecum by Means of a Simplified Glove Box Procedure' ALEXANDER ARANKI,2 SALAM A. SYED, ERNEST B. KENNEY, AND ROLF FRETER Department of Microbiology, The University of Michigan, Ann Arbor, Michigan Received for publication 7 February 1969 An anaerobic glove box constructed of clear flexible vinyl plastic is described. It is sufficiently inexpensive and simple in operation to be used not only in research but also in a clinical laboratory by technicians without special training. Conventional bacteriological techniques may be used inside the glove box for culturing and transferring anaerobic bacteria. The box may be heated to 37 C and thus serve as an anaerobic incubator as well, permitting inspection of cultures at any time. Media may be prepared and agar plates may be poured on the laboratory bench in the conventional manner. An overlay of trace amounts of palladium black catalyst over plated agar media reduces the medium to an oxidation-reduction (0-R) potential of -300 mv within 2 days after introduction into the glove box. In spite of its greater simplicity, the system matched or excelled the roll tube method with respect to all parameters tested, including O-R potential obtainable in the media, 02 concentration in the gas phase, and efficiency in isolating anaerobic bacteria from the mouse cecum. Comparative studies indicate that the conventional anaerobic jar method was inadequate for the isolation of strict anaerobes from human gingival specimens and from the mouse cecum. This was due to the exposure of specimens and media to air during plating on the open laboratory bench. Anaerobic jars were adequate for maintaining the proper conditions for growth of anaerobic bacteria once these had been established in the glove box. As reviewed in an earlier publication (14), anaerobic bacteria are now recognized as the predominant component of the intestinal flora of man and mammals. Human oral flora also contains a large proportion of anaerobes (8). It is also well known that the majority of these anaerobic bacteria cannot be cultured with conventional anaerobic technique (10, 14, 15). Recent studies in this laboratory (14) have shown that a significant improvement in the recovery of anaerobes from the mouse cecum may be obtained if care is taken to avoid even momentary exposure of the specimen and of the medium to atmospheric oxygen. These latter studies were carried out by use of Hungate's (4) roll tube technique. In Hungate's method, exclusion of air is achieved by simply using rubber-stoppered tubes or flasks for the preparation and tubing of media. During transfer of media or specimens, atmospheric oxygen is kept out of the tubes by im- 1 A preliminary account of this study was given earlier (Bacteriol. Proc., p. 94, 1968). 2 Present address: Escuela Dental, Laboratorio Microbiologia, Avenida Santa Maria 571, Santiago, Chile. mediately introducing a stream of sterile inert gas whenever the rubber stopper is removed. Isolated colonies are obtained from specimens with mixed flora by incorporating serial dilutions of the specimen into liquid agar medium, which is then allowed to solidify on the walls of a rubberstoppered test tube (roll tube). The roll tube technique is thus the anaerobic equivalent of the ''pour plate" method for enumerating bacteria. There can be no doubt that the Hungate method has opened up an entire field of microbiological endeavor, i.e., the ecology of the rumen flora. Nevertheless, when compared to conventional bacteriological procedures, such as those used for the isolation and identification of Enterobacteriaceae in a clinical laboratory, the Hungate method is extremely complex. The preparation and tubing of media under exclusion of oxygen are time consuming. Since this is a pour plate method, each specimen has to be serially diluted first (under exclusion of oxygen), and then replicate roll tubes have to be prepared from each dilution. An apparatus for streaking prehardened roll tubes under a stream of inert gas has been de- 568

2 VOL. 17, 1969 ISOLATION OF ANAEROBES IN SIMPLIFIED GLOVE BOX 569 scribed by Moore (6). This constitutes a considerable improvement but is still more complex and less efficient than the standard "triple streak" agar plate known to all bacteriologists. The simultaneous handling of rubber stoppers, gas outlets, and tubes during the transfer of cultures or media requires specially trained technicians. Therefore, we attempted to achieve the desired exclusion of atmospheric oxygen by some more convenient means and thus explored the possibility of using oxygen-free glove boxes. Our objective was to devise a method which would be sufficiently simple and inexpensive to be used not only in the research laboratory but also in a routine diagnostic laboratory by technicians having no special training beyond routine bacteriological methods. Anaerobic glove boxes for the cultivation of bacteria have been described by Socransky et al. (13), Rosebury and Reynolds (9), Gravens et al. (Bacteriol. Proc., 1967, p. 77), and Draser (1). Those of Rosebury and Reynolds and Gravens et al. were complex and expensive metal chambers considerably beyond the technical competence of the average bacteriological technician and beyond the budget of many laboratories. The other glove boxes described in the literature require elaborate set-up procedures before each use, and for this reason are unsuitable for prolonged or routine operation. There is no data available in the literature concerning the relative performance of glove boxes in relation to other techniques. Socransky et al. (13) and Rosebury (9) stated that spirochetes may be grown in the box as surface colonies. Drasar (1) reported that he can recover 1011 bacteria from 1 g of human feces. Described below is an inexpensive glove box which is simple to operate and which can be assembled entirely from components that are commercially available (Fig. 1). [Those interested in using a glove box may obtain a list of all necessary components, supplies, commercial sources, and drawings by writing to one of us (R. F.). The entire outfit may be purchased for less than $1,000.] Data on the efficiency of this device in recovering anaerobic bacteria is given and is related to the performance of other methods for the cultivation of anaerobic bacteria. MATERIALS AND METHODS The glove box is constructed of flexible 20-mil clear vinyl plastic with 25-mil neoprene gloves. This is the type of chamber which is now widely available in a great variety of sizes for germ-free animal work. A chamber 2 by 2 by 3 feet (60.9 by 60.9 by 91.4 cm) wide is sufficient if only the plating and transferring of cultures are to be carried out in the chamber. In this case, the cultures must be incubated in anaerobic jars outside the glove box. The work described below was carried out in a glove box 2 by 2 by 7 feet (60.9 by 60.9 by cm) wide with two pairs of gloves in the front panel. The chamber was heated to 37 C and half of it was used simply as an anaerobic incubator. This eliminates the need for anaerobic jars and has the further advantage that cultures may be observed at any time during incubation. The plastic chamber is clamped with a worm-drive steel clamp to a round steel airlock, 18 inches (45.7 cm) long by 12 inches (30.5 cm) in diameter. The lock has an airtight acrylic plastic door at either end which may be closed by tightening a single bolt. On top of the lock are two valves, one connecting to the gas tank, the other to a vacuum pump. A vacuum gauge on the top of the airlock completes the list of controls. Inside the chamber, a blower located at one end of the glove box forces the interior atmosphere through a plastic hose into a rectangular box, located at the opposite end. The top of the box is open and holds a removable tray of stainless-steel screening which is covered with catalyst pellets [palladiumcoated alumina, Engelhard; the same material was first used in a glove box by Rosebury and Reynolds (9)]. This system continuously circulates the chamber atmosphere through the catalyst. It has no connection to the outside. If one wishes to use part of the glove box as an anaerobic incubator, a cone-type heating element is placed into the box below the catalyst tray. The heater is controlled by a bimetallic thermostat located at the blower intake. This arrangement holds the temperature in the chamber to within -4l C. Closer temperature control may be achieved by placing an insulating blanket of styrene foam on top of the glove box. The atmosphere in the chamber used in the experiments described below was a mixture of 10% H2 and 5% CO2 in N2. We also used 10% H2 in 90% CO2 which, in preliminary studies, gave no better recovery of anaerobes from our specimens than the above mixture. Gases may be obtained premixed from most suppliers. Any mixture can be used as long as it contains some H2 to react with oxygen in the presence of the catalyst. During development of the glove box technique, oxygen levels were determined with a trace oxygen analyzer (model GP; Lockwood & McLorie, Horsham, Pa.). Precise oxygen analysis is not required in routine use, because the proper function of the glove box can be controlled by adding oxidationreduction (O-R) indicator dye to the media. Electric leads are introduced into the plastic chamber through a rubber stopper which fits into a plastic nipple in the rear wall. The bacteriological loops used in the glove box have metal handles (Matheson no ). The tip of the handle is attached via an alligator clip to a thin flexible electric cord leading to a 6-v transformer. The second lead from the transformer connects to a piece of sheet metal [1 by 1 inch (2.54 by 2.54 cm)] mounted on a suitable stand. When the tip of the loop touches this metal, the loop wire heats up to the proper (red-hot) temperature for sterilization. A coil of Nichrome wire is connected via a foot switch to the 6-v transformer. When the

3 570 ARANK.I ET AL. APPL. MICROBIOL. switch is depressed, the coil heats up to red heat and serves to "flame" the rim of test tubes and flasks. A maximum-minimum indicating thermometer and a hygrometer are kept in the glove box for control purposes. A tray containing 2 lb of reusable silica gel desiccant (Tel-Tale, Davison Commercial Chemical, Baltimore, Md.) serves to keep the relative humidity in the glove box between 30 and 50%. At higher humidities, the drying of agar plates is impaired and the problem of swarming bacteria becomes serious. When the glove box is set up for the first time, it is evacuated by opening the inside door of the lock and turning on the vacuum pump. The chamber then simply collapses, draping itself tightly around any items which may already be inside. (Articles too large to pass through the lock, such as shelves, may be introduced before the plastic chamber is clamped to the lock.) After evacuation, the valve leading to the pump is closed, and the chamber is filled with the gas mixture by opening the gas valve on top of the lock. After the chamber is inflated, the inner door of the chamber is closed. A tray with catalyst is then introduced into the box in the usual manner and placed on top of the catalyst box. Within 1 to 2 days, the oxygen concentration drops to below 10 ppm and the glove box is ready to be used indefinitely. In normal use, it is not necessary to add gas to the chamber atmosphere. To introduce dry materials into the glove box, these materials are placed in the lock via the outside door, keeping the inside door locked. The lock is then evacuated to 1 mm of Hg. After closing the valve to the vacuum pump, the lock is filled with gas mixture from the tank, the inside door is opened, and the materials are taken into the chamber. With a vacuum pump with a free air capacity of 75 liters/ min, the entire process takes less than 5 min. Liquid media and agar plates are introduced in a similar manner except that the lock is evacuated to only 4 cm of Hg. The chamber is then filled with gas, and the process of evacuation and filling is repeated twice. The items may then be taken into the glove box by opening the inner door of the lock. With a pump with a free air capacity of 75 liters/min, evacuation of the lock to 4 cm of Hg takes only a few seconds, and the entire process of introducing liquids can be completed in 5 min. For reasons of economy, we used less expensive nitrogen from a separate tank connected to the lock to fill the lock after the first two cycles of evacuation, with the gas mixture only for the final filling. Depending on the widely divergent prices charged for gas mixtures by the various manufacturers, one entry (i.e., one filling of the lock with gas mixture) costs between $0.10 and $0.25. To remove items from the glove box, the inside door of the lock is opened, the items are placed into the lock, the inside door is closed, and the items are removed via the outside door. No gas needs to be expended for withdrawals from the chamber when these operations are carried out alternately with introduction of materials into the chamber. During routine use of the glove box, the efficiency of the catalyst decreases in time. It can be rejuvenated by heating to 160 C for 2 to 4 hr (same conditions as for sterilizing glassware). We have two trays of catalyst for each glove box which are exchanged when the oxygen concentration exceeds 10 ppm. In chambers without oxygen analyzer, a routine schedule of exchange and rejuvenation of catalyst trays twice a week is recommended. The catalyst should be replaced completely every 4 months. This schedule gives an ample margin of safety. Probable reasons for the decrease in catalyst efficiency are moisture and poisoning by H2S elaborated by the bacteria. Both of these are removed on heating to 160 C. To reduce the release of H2S into the glove box, broth cultures were kept in screw-cap tubes and flasks. Agar plates were kept in 0.5 gallon (1.9 liter) size freezer-type plastic containers (Sears Roebuck & Co., no. 9704). These containers were fitted with a suspended bottom of galvanized-steel wire mesh. The space under the wire bottom was filled with a slurry of PbCO3 in water which absorbed any H2S formed. Media. For the cultivation of bacteria from tooth scrapings, a slight modification of Huntoon's medium was used. This is essentially an enriched veal infusion agar and is referred to below as EVIA. It was composed of Veal Infusion Agar (Difco), 40 g/liter;.g/ml; cysteine hydrochloride, 0.05%O; menadione, 5 Na2CO3, 0.056%; and normal rabbit serum, 10%. The ingredients were sterilized and plated as described below for enriched Trypticase Soy Agar (ETSA). ETSA was used for the cultivation of bacteria from the mouse cecum. It was composed of trypticase Soy Broth (TSB; BBL), 30 g/liter; hemin, 1 /Ag/ml; menadione, 0.5,ug/ml; cysteine hydrochloride, 0.05%; Na2CO3, 0.042%; and agar (Difco), 2%. The base medium was autoclaved in a cotton-stoppered Erlenmeyer flask at 120 C for 20 min and cooled to 56 C in a water bath. Menadione was made up in alcoholic stock solution, sterilized by filtration, and added to the autoclaved base medium. Cysteine hydrochloride and Na2CO3 were autoclaved separately in aqueous solution and added to the base medium after autoclaving. Hemin was kept in alkaline alcoholic stock solution and was autoclaved with the base medium. Rabbit serum was sterilized by filtration and added to the sterile EVIA medium at 56 C. The concentration of Na2CO3 had been adjusted in preliminary experiments to give a neutral medium after equilibration in the glove box atmosphere containing 5% CO2. Agar media were poured into glass petri dishes covered with metal covers carrying an absorbent disc (BBL no A). A small amount (0.05 ml of a 0.1% solution) of indigo carmine or phenosafranine indicator dye was placed into the bottom of two petri dishes and mixed with the agar as it was poured. These served as control plates. The media were placed into the glove box as soon as possible after hardening. They were used as soon as the dye in the control plates had noticeably faded or had become entirely colorless. The control plate was then removed from the glove box, in which case the original color returned immediately. Reduction of indigo carmine was usually complete within 2 days. Media to be plated with a palladium black overlay

4 VOL. 17, 1969 ISOLATION OF ANAEROBES IN SIMPLIFIED GLOVE BOX 571 were autoclaved in two portions. The first portion was plated as usual in a shallow layer (15 to 20 ml in a dish with an inner diameter of 9 cm). When this layer had solidified, the second portion of medium was mixed with palladium black (Fisher no. P-3) which had been dry sterilized in a test tube for 2 hr at 160 C. Sufficient palladium black to make a 1 mg/ml suspension was used, and 10 ml of this was placed on top of the primary layer of medium. Control plates with phenosafranine were prepared as described above, by mixing the dye with the lower layer of medium. Two days after introduction into the glove box, all agar plates were placed into closed plastic containers, as described above, in order to minimize drying and to trap H2S from those plates which had already been inoculated. Diluting fluid was composed of TSB (BBL), 30 g/liter; Na2CO3, 0.042%; and cysteine hydrochloride, 0.05%. The three ingredients were autoclaved separately and mixed afterwards as described for agar media above. The fluid was then dispensed in 9-ml amounts into sterile screw-cap test tubes (for making serial dilutions before quantitative culture) or in 100- ml amounts in Erlenmeyer flasks (for use in the Waring Blendor). The broth was introduced into the glove box immediately after dispensing and was used only after it had remained there for at least 2 days. O-R potential. Measurements were taken with a Beckman model G ph meter located inside the glove box. In liquid media, the reading of three freshly burnished platinum electrodes (Beckman no ) against a Calomel electrode (Beckman no ) were taken. The average of the three readings was corrected for the voltage of the Calomel electrode to give the Eh. The potentials of the three electrodes agreed closely. The system was tested against media containing various O-R indicator dyes and gave the theoretical potentials during the progressive reduction of these indicators. O-R potentials on the surface of agar plates were measured by pressing the platinum electrodes against the surface and reading potentials against the reference part of a surface combination electrode (Beckman no ). In all instances, the ph was measured with a Beckman glass electrode. It ranged between ph 6.9 and 7.1, and no corrections for ph were made in the Eh values presented in this paper. Gingival flora. Specimens were taken from a healthy volunteer, who had his 32 teeth, no open cavities, and no periodontal pockets. His mouth had been cleaned in our School of Dentistry 6 months before this study. The samples were taken according to the criteria of Socransky et al. (12), before breakfast and before brushing of the teeth. The teeth were isolated with cotton rolls and all 32 teeth were scraped with a bacteriological loop. The material on the loop was placed into a rubber-stoppered tube of diluting fluid which had been removed from the glove box just before the experiment. While the stopper was opened, a stream of nitrogen was directed into the tube through a sterile 18-gauge hypodermic needle which was attached to a short piece of rubber tubing containing sterile cotton. The specimen was then placed in the glove box and blended for 30 sec in 100 ml of sterile diluting fluid in a Waring Blendor at low speed (with a semimicro jar, A. H. Thomas no F). From this homogenate, serial 10-fold dilutions were made in diluting fluid, with a Fisher no pipette filler to operate the pipettes. Portions (0.1 ml) of the appropriate dilution were finally transferred to agar plates and spread evenly over the surface with a sterile glass rod. Ten replicate plates were inoculated from each dilution. Microscopic counts were made from the original homogenate in a Petroff-Hausser chamber as described earlier (14). Inoculated media were incubated inside the glove box, and the number of colonies appearing were counted daily. When incubation was to be in anaerobic jars, these were filled inside the glove box. Heated palladium asbestos catalyst (5 g per jar) was included in each jar. We used glass jars (desiccators kept closed by a slight vacuum), stainless-steel jars (Torbal jar), and inexpensive airtight jars made in our shop from acrylic plastic tubing [wall, V8 inch (0.96 cm)] and acrylic plastic sheet [0.5 inch (1.27cm)]. (Anaerobic jars used in conjunction with the glove box do not require valves or gauges because no evacuation and gas filling is required.) There was no difference in the performance of these various jars. For incubation of cultures, the jars were removed from the glove box to a conventional incubator at 37 C. When plating of the specimen was to be done outside the glove box (to simulate the conventional anaerobic jar technique), a small amount of the homogenate was taken from the blendor jar and removed from the chamber. Serial dilutions were then made in ordinary TSB (BBL) which was freshly prepared but had never been inside the glove box. Plating was carried out as described above, except that it was done outside the glove box. The agar plates used for this type of culture were of the same batch as used for plating inside the glove box. They were removed from the glove box 30 min before plating. The inoculated plates were incubated in anaerobic jars. Mouse cecum. The animals used were strain CD-1 from Charles River Laboratories and strain BALB/ Wm maintained in this Department by William Murphy. The animals were killed by cervical dislocation and immediately thereafter introduced into the glove box. The cecum was then removed under aseptic conditions and its contents were washed into a sterile Waring Blendor jar containing 100 ml of diluting fluid. Subsequent steps were as described above for tooth scrapings. Statistical analysis. The significance of the difference between mean plate counts was analyzed by means of the t test with Table 3 of Fisher and Yates (2). RESULTS Oxygen tension in the glove box. During the experiments described below, the 02 tension in the glove box was maintained between 5 and 10 ppm. In a chamber 2 by 2 by 7 feet (60.9 by 60.9 by

5 572 ARANKI ET AL. APPL. MICROBIOL cm) wide and made of 20-mil vinyl plastic; this required 200 g of catalyst spread in a single layer over 5.5 by 10.5 inch (13.9 by 26.6 cm) wire mesh tray. With twice this amount of catalyst in a 21 by 5.5 inch (53.3 by 13.9 cm) tray, the 02 tension was maintained between 2 and 5 ppm. We have not as yet determined whether this lower 02 concentration results in a higher rate of recovery of anaerobes from our specimens. Vinyl chambers of thicker material (40 mil) were also used, but with these we did not obtain significantly lower 02 concentrations. It seems probable that both the plastic and the rubber gloves are permeable to oxygen. When the catalyst was removed from an established glove box [2 by 2 by 7 feet wide (60.9 by 60.9 by cm), 20-mil vinyl] having an initial 02 concentration of 5 to 10 ppm and an internal temperature of 37 C, the O2 concentration rose at a rate of 1 ppm per min. In the same box kept at room temperature (29 C), the rise was 0.6 ppm per min. The 02 concentration in an established glove box is thus in a steady state determined by the rate of inward diffusion of 02 and the rate of its removal by the catalyst. Theoretically this should result in a lowering of the H2 concentration in the chamber over a period of several weeks. This is, however, not a practical consideration because with every entry of materials into the chamber fresh gas (approximately 4% of the total chamber volume) is mixed in with the gas already in the glove box. O-R potential of media. To evaluate various methods for preparing media, TSB buffered with 0.056%7 Na2CO3 was autoclaved for 20 min in 500-ml amounts in cotton-stoppered Erlenmeyer flasks, cooled rapidly, and introduced into the glove box. A second batch of the same medium was prepared in a similar manner, except that the flasks were closed with a check valve which permitted escape of air during autoclaving but not its reintroduction after removal from the autoclave. These flasks were cooled rapidly after autoclaving and were introduced into the glove box immediately afterward. The medium was thus kept entirely free from contact with atmospheric oxygen. At periodic intervals of time beginning immediately after introduction (time-zero), the O-R potential was measured in samples taken from the flasks. Broth autoclaved with a check valve showed a potential of -280 mv immediately after introduction into the glove box and this remained constant thereafter. Broth autoclaved in cotton-stoppered flasks was at first quite oxidized (+65 mv), but after 1 day had reached the same O-R potential as that autoclaved with a check valve. This was a constant observation in numerous repetitions of this experiment with this and other media. In all instances, media autoclaved in cotton-stoppered flasks reached, after 1 to 2 days in the glove box, the same O-R potential as similar media autoclaved with a check valve. The above experiment was repeated with TSB to which Na2CO3 and cysteine hydrochloride had been added (i.e., our "diluting fluid"). When this medium was autoclaved in cotton-stoppered flasks and introduced into the glove box, its initial O-R potential was already quite low (-200 mv). Surprisingly, however, the final potential (-280 mv) was not lower than that of TSB without added cysteine. The reducing activity of the cysteine could only be demonstrated when the medium was poised by the addition of % indigo carmine. In this case, reduction of the dye occurred several hours earlier in broth containing cysteine as compared to broth without this reducing agent. O-R potentials were also measured on the surface of standard ETSA plates with and without paladium black overlay. The O-R potential in the presence of paladium catalyst was significantly more negative (-290 mv) than that found on plain ETSA plates (-250 mv). Phenosafranine added to broth or regular agar media never became reduced. In contrast, agar plates with palladium black overlay consistently reduced phenosafranine, and this reaction is now used routinely by us to control the proper preparation of agar media. Microscopic bacterial counts. To relate the present results to those of others, our standard method of making Petroff-Hausser chamber counts was compared with a method involving quantitation of a Gram-stained smear. In this method, the specimen was mixed with a suspension of polystyrene particles of known concentration as described by Socransky et al. (12). A conventional Gram-stained slide was then prepared from this mixture. The total count of bacteria in the original specimen was calculated from the proportion of bacteria to polystyrene particles in the average microscopic field. Since human feces have been the standard of reference for many authors, we used this type of specimen first. The counts obtained (Table 1) ranged from 1.37 X 101" to 6.16 X 1011 bacteria per g of fresh feces. These values are of the same order of magnitude as those reported by Van Houte and Gibbons (15). Table 2 shows comparative counts of bacteria in the mouse cecum obtained by the two methods mentioned above. As may be seen, the counts of Gram-stained smears were consistently lower. We interpreted this as indicating that some bacteria (and perhaps some polystyrene particles)

6 VOL. 17, 1969 ISOLATION OF ANAEROBES IN SIMPLIFIED GLOVE BOX 573 TABLE 1. Microscopic counts, in a Petroff-Hausser chamber, of normal formed human stools from five individuals Individual No. of bacteria per g of fresh stool A 1.37 X 1011 A 2.40 X 1011 C 2.82 X 10'1 D 3.34 X 101 B 3.62 X 10'1 E 4.40 X 1011 A 6.16 X 1011 TABLE 2. Comparison of two methods determining total microscopic counts bacteria in the mouse cecum" Specimen no. No. of bacteria per cecum for of Chamber -(Gram Counted in Counted on smear) chamber Gram smear X X X X X X X X X X X X X X X X X X X X X X a Method 1, Petroff-Hauser Chamber; method 2, Counts On a Gram-stained slide. had been washed off the slides during the staining procedure. If more polystyrene particles than bacteria had been washed off, one would expect the counts to be too high. These results obviously do not invalidate all methods of counting Gramstained smears, because factors such as the nature of the bacteria and of the suspending medium, the method of fixing, etc., may influence the results. The data in Table 2 do indicate, however, that a counting procedure based on stained smears should be tested by some independent method before the results are accepted. On the basis of the above data, we decided to use Petroff-Hausser chamber counts as the basis for the experiments described below. The main drawback of this method lies in the necessity of having to distinguish bacteria from other particulate matter that may be observed in the counting chamber. This was facilitated considerably by adding dye to the suspending medium as detailed in the original description of the method (14). In a specimen such as human stool or mouse cecum which consisted mainly of bacteria, there were relatively few particles that were difficult to identify. In case of doubt, a particle was counted as a bacterium, and the total counts shown in the table below may thus be slightly too high (and, consequently, the percentages of recovery slightly too low). In the case of tooth scrapings, the proportion of undefinable particles was relatively high, and the counts from these specimens (Table 3) must be regarded as approximations only. Bacteria from gingival flora. These bacteria were cultured in three different ways: (i) by carrying out the entire procedure (serial dilutions, plating, incubation of plates) in the glove box with EVIA medium; (ii) as above but with EVIA medium with palladium black overlay; and (iii) by carrying out the serial diluting and plating procedures on the laboratory bench and incubating the plates in anaerobic jars. The results are shown in Table 3. The recovery on agar with palladium overlay was higher than that on regular medium. Although this difference was not statistically significant for any single experiment, it was a consistent finding in all instances. The conventional anaerobic jar procedure gave consistently lower recoveries than cultures carried out in the glove box, and these differences were statistically significant in every experiment. In experiments 2 to 5, all of the colonies which grew on one or two of the original plates (total of 20 to 40 colonies per experiment) were transferred to two agar plates each, one of which was incubated aerobically, the other in the glove box. In this manner, the fraction of anaerobes among the total cultivated flora was determined. The per cent recovery shown in Table 3 was then multiplied by this fraction to TABLE 3. Total counts of bacteria cultured from human gingival flora under various conditions of cultures (II) (I) Per cent re- (I Per cent ) re- (V Experi- Per cent re- covery in Factor of ment covery in glove box on covery in difference no. glove box on EVIA with anaeron l columns plain EVIA palladium EVIA (11)/(III) overlay b aaverage counts are based on 10 replicate plates. bfor all values in this column, difference is significant at the <0.01 level.

7 574 ARANKI ET AL. APPL. MICROBIOL. FIG. 1. Plastic glove box and entry lock. The outer door of the lock is open, and the yoke-type closure, which allows the door to be closed by means of tightening a single bolt, can be seen. give the recovery of anaerobes under the various conditions tested (Table 4). The relative ability of the standard anaerobe jar method to recover anaerobes (Table 4) was still lower than would have been suspected from the difference in total counts (Table 3). In other words, plates processed and incubated in the chamber had not only a higher total count, but the proportion of anaerobes among the organisms cultured was also higher. An experiment was carried out to determine the critical step in the total procedure of culturing. A specimen of gingival flora was processed inside the glove box in the usual manner, except that 20 replicate plates were inoculated. The tubes containing the serial dilutions of the specimen were then taken from the chamber and plated on 20 replicate plates as described above for the anaerobic jar procedure. Ten of the plates inoculated by each method were incubated inside the chamber, and the other 10 were incubated in anaerobic jars in the usual manner. Incubation inside the chamber appeared to be equivalent to incubation in anaerobic jars. The critical step was the method of plating. With incubation in the glove box, there was 50% recovery of bacteria plated in the glove box, but only 23.8% recovery of bacteria plated on the bench. With incubation in jars, there was 51.8% recovery of bacteria plated in the glove box, and 26.1 % recovery of bacteria plated on the bench. Differences in recovery between plating in the glove box versus plating on the bench were significant at the < 0.01 level. Apparently, contact of the specimen with air and plating on oxidized media (as were the conditions in plating on the laboratory bench) were the factors responsible for the low recovery rates obtained with the standard anaerobic jar method in the present study. Bacteria from the mouse cecum. Table 5 gives the results obtained with ETSA medium with and without palladium black overlay. As with the specimens from dental scrapings, higher recoveries were obtained in every experiment on the medium with palladium. This difference was statistically significant only in experiment 4. The mice used in experiments 1 to 3 were Charles River strain CD1, that in experiment 4 was BALB/Wm. The former mice had a high proportion of microaerophilic gram-positive, rodshaped bacteria in their cecal flora. The BALB/ Wm mouse yielded only gram-negative anaerobic rods. This presumably accounts for the difference in results, because the lower O-R potential achieved with the palladium overlay would be advantageous only in the culture of strict anaerobes. It should be noted that the per cent recovery obtained in the glove box (Table 5) equalled or exceeded that obtained TABLE 4. Recovery of anaerobic bacteria from gingivalflora under various conditions of culturea (II) I) Per cent* re- (I ) PereI (IV) ment covery in glove box on covery oi difference no. glove box on EVIA with jars on plain columns plivapalladium EVIA (II/III) Experi- Per cent re- covery in Factor of pliva overlay > a Same experiments as shown in Table 3. The counts in this table are reduced according to the percentage of anaerobes found under the various conditions tested. TABLE 5. Total recovery ofbacteria from the mouse cecum Experi- Medium Per cent ment no. recovery'~ 1 ETSA, plain 21.6 ETSA with Pd overlay ETSA, plain 26.6 ETSA with Pd overlay ETSA, plain 42.9 ETSA with Pd overlay ETSA, plain 10.2b ETSA with Pd overlay 20. 2b a Based on average counts from 10 replicate plates. b Difference is significant at the <0.01 level.

8 VOL. 17, 1969 ISOLATION OF ANAEROBES IN SIMPLIFIED GLOVE BOX with the same type of specimen in earlier studies with the roll tube method (14). DISCUSSION The long established standard of anaerobic bacteriology is Hungate's roll tube method. To be acceptable, newly developed simplified methods for the culture of strict anaerobes must be shown to at least equal the conditions obtainable with this standard. The roll tube method has two (and only these two) critical advantages. (i) Bacteria may be transferred and cultured without significant exposure to atmospheric oxygen. (ii) The media used may be kept at a low O-R potential at all times, even at the moment of inoculation. There can be no doubt that an anaerobic glove box is superior to any other method in allowing manipulation of cultures and transfer of bacteria without contact with air. In fact, one of the principal advantages of a glove box is that these manipulations can be accomplished easily and in a "foolproof" manner. The data given in this paper show that the glove box technique also equals or even exceeds the roll tube method in achieving a low O-R potential in the media. As reviewed recently by Hungate et al. (5), a medium able to reduce indigo carmine has about the "right" Eh (-150 mv or lower), whereas reduction of phenosafranin indicates an Eh (-300 mv or lower) which is in excess of that required to cultivate such fastidious anaerobes as the methanogenic bacteria. In our experience with the roll tube technique, it was impossible to obtain reduction of phenosafranin in media unless potentially toxic reducing agents, such as sodium dithionite, were employed. All media used in the glove box reduced indigo carmine even in the absence of cysteine. With the addition of a palladium overlay, plated agar media regularly reduced phenosafranin and thus had a lower O-R potential than the roll tube media used by most workers. One must realize, of course, that the O-R potential as measured in a complex bacteriological medium is only of limited significance (3). Nevertheless, the data shown in Table 5 show that the present method gave results which were equal to or better than those obtained in our earlier work with the roll tubes (14). Very few data are available in the literature concerning the effect of the oxygen concentration in the gas phase on the growth of strict anaerobes. Hungate (11) noted that the growth of a methanogenic bacterium was inhibited in the presence of 300 ppm of oxygen and that a slight reduction in growth rate could be noted at levels down to 40 ppm. In view of these data, 575 the 02 concentration routinely obtainable in the glove box (2 to 5 ppm or 5 to 10 ppm, depending on the amount of catalyst used) appears to be sufficiently low to cultivate strict anaerobes. The principal advantages of the glove box system are as follows. (i) Anyone who has sufficient technical ability to evacuate and fill an anaerobic jar can also operate the glove box. (ii) Only standard bacteriological techniques are required for the isolation and cultivation of anaerobes, except that the manipulations have to be done through gloves, which are not a major hindrance. "Triple streaking" of an agar plate is the most convenient and efficient means of isolating strains from a mixture, and this is possible only in a glove box. Moreover, the agar plates may be dried sufficiently to prevent spreading of bacteria, a complication which, with our specimens, occurred frequently in roll tubes even when very lean media were used. Also, some diagnostic reactions (e.g., hemolysis and the disc method of antibiotic sensitivity testing) can only be carried out on agar plates. (iii) Media may be prepared in the conventional manner on the laboratory bench. This has been possible because of the efficiency of the palladium catalyst in reducing plated media. Although palladium has been employed before in roll tubes (7), its use has not been common, most likely because of the dangers inherent in working with hydrogen on the open laboratory bench. The anaerobic glove box permits the use of this catalyst in a convenient and safe manner. (iv) Viewing ports may be attached to the plastic chamber to allow operation of a microscope in the glove box for observation of motility, etc. (v) The cost of a complete glove box is not excessive. A price of less than $1,000 includes items such as a vacuum pump, which may already be available in many laboratories. All parts and accessories can be obtained commercially. (vi) Plastic chambers can easily be fabricated in any desired size or shape. Oversize airlocks can also be obtained commercially. Another use of this type of glove box may therefore be to provide an anaerobic environment for various types of apparatus, such as continuous culture devices, chromatographic columns, etc. Without experience in operating a flexible plastic glove box one may suspect several disadvantages, such as the possibility of puncturing the material. We have operated four glove boxes for as long as 18 months with only one puncture in one of the chambers. Because of the positive inside pressure, small punctures do not affect

9 . 576 ARANK I ET AL. the oxygen tension in the box. They may be repaired simply with plastic tape. We have never had a puncture in a 25-mil neoprene glove. The thinner plastic gloves available from some manufacturers are more susceptible to puncture but have the advantage that they can be replaced without significantly disturbing the anaerobic conditions in the glove box. A small glove box takes up about as much bench space as is normally provided for a bacteriological technician. A large chamber also functions as an incubator and requires as much additional space as would be taken up by a separate incubator. The atmosphere in the glove box contains 10% H2; thus, the largest chamber we have used [2 by 2 by 7 feet (60.9 by 60.9 by cm)] contains 2.8 ft5 of pure H2. Even if by some accident the contents of the entire glove box were to be emptied into the laboratory, the concentration of flammable gas in the air would probably be lower than that developing when some illuminating gas is inadvertently released from an incompletely closed Bunsen burner. One may therefore conclude that, in terms of the various criteria tested, the glove-box system described in the present paper matched or exceeded the roll tube technique in providing suitable conditions for the culture of strictly anaerobic bacteria. Its various advantages should simplify the task of isolating fastidious anaerobic bacteria, especially in institutions (such as diagnostic hospital laboratories) where anaerobic culture is not the only or even the major activity. As has been emphasized before (14), the present results show again that the conventional anaerobic jar method is inadequate for the culture of fastidious anaerobes. The data presented suggest that this was not due to the conditions in the jar itself. Therefore, this inadequacy must be attributed to the fact that the bacteria and media were exposed to atmospheric oxygen during the process of plating. Earlier studies (14) have shown that only 1 to 5% of the total bacterial population from the mouse cecum may be isolated with the anaerobic jar technique. Thus, the rate of 20 to 45% recovery achieved in the present study (Table 5) is a significant improvement. Nevertheless, one must realize that 55 to 80% of the total cecal flora is still not cultivated. It is possible that some of these bacteria may be APPL. MICROBIOL. dead. On the other hand, it is obvious that anaerobic methods, roll tube or glove box, can accomplish no more than the exclusion of oxygen, which is only one of the requirements for growth of fastidious anaerobes. It is quite possible, then, that better recoveries of intestinal flora may be achieved by providing various additional growth factors and nutrients in the medium or by varying the composition of the gas phase. Relevant studies are now in progress. ACKNOWLEDGMENT This investigation was supported by Public Health Service grants AI and AI from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Drasar, B. S Cultivation of anaerobic intestinal bacteria. J. Pathol. Bacteriol. 94: Fisher, R. A., and F. Yates Statistical tables for biological, agricultural and medical research. Hafner Publishing Co., New York. 3. Hewitt, L. F Oxidation-reduction potentials in bacteriology and biochemistry. E. S. Livingstone, Edinburgh. 4. Hungate, R. E The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Rev. 14: Hungate, R. E., M. P. Bryant, and R. A. Mah The rumen bacteria and protozoa. Ann. Rev. Microbiol. 18: Moore, W. E. C Techniques for routine culture of fastidious anaerobes. Intern. J. System. Bacteriol. 16: Mylorie, R. L., and R. E. Hungate Experiments on the methane bacteria in sludge. Can. J. Microbiol. 1: Rosebury, T Microorganisms indigenous to man. McGraw-Hill Book Co., Inc., New York. 9. Rosebury, T., and J. B. Reynolds Continuous anaerobiosis for cultivation of spirochetes. Proc. Soc. Exptl. Biol. Med. 117: Savage, D. C., and R. Dubos Alterations in the mouse cecum and its flora produced by antibacterial drugs. J. Exptl. Med. 128: Smith, P. H., and R. E. Hungate Isolation and characterization of Methanobacterium ruminantium n. sp. J. Bacteriol. 75: Socransky, S. S., R. J. Gibbons, A. C. Dale, L. Bortnick, E. Rosenthal, and J. B. MacDonald The microbiota of the gingival crevice area of man. Arch. Oral Biol. 8: Socransky, S. S., J. B. MacDonald, and S. Sawyer The cultivation of Treponema microdentium as surface colonies. Arch. Oral Biol. 1: Spears, R. W., and R. Freter Improved isolation of anaerobic bacteria from the mouse cecum by maintaining continuous strict anaerobiosis. Proc. Soc. Exptl. Biol. Med. 124: Van Houte, J., and R. J. Gibbons Studies of the cultivable flora of normal human feces. Antonie van Leuwenhook J. Microbiol. Serol. 32: