Journal ofAppliedBacteriology 1978, 45,411415
SHORT COMMUNICATION An Anaerobic Glove Box for the Isolation and CuItiva ti on of Metha nogen ic Bacteria D. J. COX
AND
S . D. HERBERT
Department of Biology, University of York, York YO 1 500 Received 31 January 1 9 7 8 and accepted 2 5 A p r i l 1 9 7 8 1. An anaerobic glove box and growth chamber capable of isolating and culturing methanogenic
bacteria is described.
EVENTHE SIMPLEST manipulations of methanogenic bacteria, which are among the most rigorous of obligate anaerobes (Morris 1975), necessitate the application of techniques designed to prevent any exposure of the culture to oxygen during processing and growth. The Hungate (1969) or roll-tube technique precludes the use of Petri dishes, and thus imposes difficulties in single-colony purification of isolates. Several workers have resorted to the use of glove boxes, as reviewed, for example, by Aranki & Freter (1972). These are generally satisfactory for the isolation of some of the less fastidious anaerobes, but in many cases gaseous oxygen concentrations have exceeded the 10 pl/l reported to be inhibitory to the growth and survival of methanogens by Edwards & McBride (1975). These authors successfully grew methanogens using a combination of a vinyl glove box and an inner chamber, termed an Ultra Low Oxygen Chamber (ULOC), in which the culturing took place, and where gaseous oxygen was present at ca. 1 pl/l. Media were prepared aerobically and then reduced in the ULOC. This necessitated long periods of equilibrium of medium in Petri dishes before spreading, with the danger of interim growth of contaminating colonies. Because such equipment is complex and very expensive we describe in this communication an inexpensive rigid anaerobic glove box, incorporating the better features of a number of other such boxes, in which we have isolated and cultured methanogens.
Materials and Methods Media The methanogenic medium (MM) used for growth was similar to the phosphate buffered synthetic acetate broth used by Pate1 & Roth (1977) but also included (g/l): sodium acetate (instead of acetic acid), 2.5; sodium formate, 3 .O; DL-2-methylbutyric acid, 2.5; sodium chloride, 0.2. The medium was solidified with agar (20 gh). All chemicals were obtained from BDH Ltd. with the exception of the agar (Oxoid Ltd.), vitamins (Sigma Ltd.) and methylbutyric acid (Cambrian Chemicals Ltd.). They were of analytical grade where possible. 0021-8847/78/030411+05%01.00/0
[411]
0 1978 The Society for Applied Bacteriology
D. J. COX A N D S. D. HERBERT
412
Digesting sludge All isolates were obtained from a laboratory digester (1-1) operating at 35 "C with a retention period of 12.5 days and using domestic sewage sludge as feed. Anaerobic cabinet The anaerobic cabinet was adapted from an obsolete glove box originally supplied by SLEE Ltd. (Hither Green Lane, London). Two access ports (21 cm diam.) were cut in the top of the box, closed by steel discs retained by screw clamps and sealed with butyl rubber 0 rings. Inlet and outlet gas and water bulkhead connectors were brazed into the wall of the cabinet, and all electrical switchgear was mounted on the outside of the cabinet, the overall features of which are shown in Fig. 1. Pyrotenic cables supplied Electrical
External out
Circulating water for growth chamber
\Front window
I
76 cm
I
Furnace and copper reducing tubes
I
Glove port and clomp
Fig. 1. Frontal view of the anaerobic glove box (schematic).
apparatus within the cabinet: a 13 W Sylvania U.V. germicidal lamp (Thorn Electric Ltd.) and a 70 V purpose built 'warm box' used for keeping media molten. The completely sealed warm box, of stainless steel, was fitted with ports for sparging with 0, free gas (Fig. 2). The front and top windows of the cabinet were removed and resealed with neoprene sealant (Greens Aqua Division, Leeds) as a precautionary measure against 0, diffusion, and then the seals were covered inside and out with 5 cm PVC tape. The cabinet was fitted with neoprene rubber gloves (SLEE) retained with neoprene sealant and PVC tape, and, when not in use, the glove ports were closed off with aluminium discs retained by screw clamps. The glove box contained 100 g (equivalent to approx. 1 g/2 1 of cabinet volume) palladianized asbestos (Engelhard, Cinderford, Glos.), distributed into four gauze sachets hung around the walls. The catalyst was regenerated every two or three complete runs by removing the sachets from the cabinet and heating (16OoC/2 h). All tubing used for the conveying of gases into and out of the cabinet was of butyl rubber (Gallenkamp Ltd).
ISOLATION OF METHANOGENIC BACTERIA
4 13
General operating of the cabinet All the equipment necessary for experimentation was placed in the cabinet in an anaerobic jar (Gallenkamp Ltd.) under H, 18-24 h before operation. The equipment included plastic Petri dishes (Sterilin), spreaders, Finn pipettes and sterile tips (Jencons Scientific Ltd., Heme1 Hempstead, Herts.), sharp sterile sticks (for picking colonies) and sterile pre-reduced anaerobic dilution fluid (Toerien & Siebert 1967) dispensed in screw capped bottles in volumes of 49.5 and 9.0 ml. The dilution fluid was generally prepared and reduced on the bench and dispensed using the cabinet in a separate run. The cabinet was equilibrated, in a manner similar to that described by Leach et al. (1971), by flushing CO, (Distillers Co. Ltd.) into it at 6.0 l/min. A Gulfstream CO, heater (F. Myers Welding Supplies Ltd., Leeds) prevented the icing of the cylinder
Screw boss
I
70 V heater element
,Sparging
valve
Supporiing legs
Fig. 2. Cross section of the anaerobic warm box.
reduction valve at this high flow rate. To remove traces of 0, from the gas it was passed through a glass tube (25 mm diam.) containing 500 g copper turnings at 350 "C before delivery to the cabinet. After initial equilibration for 3 h (or much less if the cabinet already contained CO, from a previous run) samples of sludge and the molten media were put into the cabinet. After a further 30 min of CO, flushing, hydrogen was added to the gas stream at a flow rate of 1.0 l/min. This served to keep the copper reduced and, together with the Pd catalyst, to scavenge residual 0, from the cabinet. After 1-2 h of H, flow, the cabinet was ready for use.
Growth chamber The growth chamber, similar to the ULOC of Edwards & McBride (1975), was a polished copper anaerobic jar (Baird and Tatlock, Ltd.), around which was coiled 2 m of butyl rubber tubing (8.5 mm i.d.). Water at 39OC circulating through this tubing served to maintain an internal temperature of 35 "C. The chamber contained 10 g Pd catalyst and 50 g silica gel.
414
D. J. C O X AND S. D. HERBERT
Preparation of media Medium MM was prepared outside the cabinet in a stout Pyrex bottle with a neoprene bung. The bottle was placed in a boiling water bath and oxygen was displaced with a continual stream of N, (0, free) passed into the medium through a 23 cm syringe needle. Reducing fluid was then added and sparging was continued until five minutes after the resazurin indicator in the medium had decolourized. The medium was then autoclaved (121 OC/lS min), holding the bottle in a screw clamp t o prevent the bung being ejected. The sterile medium was transferred while still hot to the cabinet and placed in the warm box previously equilibrated to 50 "C. To reduce to a minimum the number of times the cabinet was opened, the digesting sludge sample was usually put in at this time. The warm box was flushed with CO, for 30 min using an additional sparging line plumbed into the cabinet, and then sealed off. Hydrogen was then added to the main CO, stream to the cabinet. After 2 h the medium was removed from the warm box, poured, allowed to set and dried for 1 h under U.V. light. The sludge sample was serially diluted in the anaerobic dilution fluid and spread on to the plates in volumes of 0.1 ml. Plates were immediately transferred to the growth chamber which was flushed with H,/CO, (80:20, BOC Special Gases). Flushing was repeated every 2-5 days according to the number of plates in the chamber and the degree of growth of the colonies. Throughout incubation, 0, in the (outer) cabinet was maintained at a low partial pressure by continual flushing with CO, at 0.5 l/min. Slopes Slopes in thick Pyrex tubes were prepared similarly except that 5 ml portions of the reduced unsterilized medium were dispensed inside the equilibrated cabinet. The tubes, sealed with butyl rubber bungs (C. W. Meadows & Co. Ltd., Torquay), were removed for autoclaving and then immediately returned to set inside the cabinet. After inoculation, the slopes were individually sparged with sterile (filtered) H,/CO, mixture. They were removed from the cabinet inside an anaerobic jar (without catalyst) and incubated at 37 OC. The jar was sparged every other day with H,/CO, mixture and the slopes were individually sparged every 4 d. Analysis of gases Oxygen The preliminary equilibration time of the cabinet was estimated from analysis of the exhaust gases using a Radiometer R M 1302 blood gas analyser (V. A. Howe Ltd., Peterborough Road, London). The limit of sensitivity of the instrument was 400 ,ul O,/l. Methane Methane was measured in a Pye 104 GLC fitted with an activated alumina column.
Results Equilibration of the cabinet The time taken for the cabinet gas phase to reach 0, concentrations below the limit of sensitivity of the analyser was approx. 3 h. This was chosen as the suitable time to add H, to the CO, gas stream.
ISOLATION O F METHANOGENIC BACTERIA
415
Growth of methanogens After 14 days incubation a strong negative pressure had developed within the vessel and, even after this had been equalized with H,/CO, mixture, the gas phase showed a methane content of approx. 10% (v/v). The jar was opened in the fully equilibrated cabinet. Total colony counts averaged 2.5 x 108/ml digesting sludge. Fifty colonies were picked at random and subcultured to slopes of MM. Only five of these cultures showed obvious growth and methane production after 14 days incubation, although trace methane was evident in 30 others. After 2 1 days a total of 45 cultures showed growth, 40 of which were methane positive. All positive isolates were further purified by single colony streaking on plates and, on the basis of colonial and cell morphology, some were presumptively identified with Methanobacterium, Methanosarcina and Methanospirillum.
Discussion Single colonies of methanogenic bacteria have been isolated on Petri dishes using an anaerobic glove box without the constraints of the roll tube method. Pre-reduced media were preferred, because although aerobically prepared media could be reduced in our growth chamber, it was considered that media prepared, poured, dried and spread within a matter of hours were less likely to become contaminated. A rigid metal cabinet was considered more suitable than a flexible vinyl one as it enabled the various services to be easily conveyed through and attached to the walls. Operation of the cabinet was like that of a large anaerobic jar, i.e., a combination of hydrogen gas and palladium catalyst to scavenge traces of oxygen. The apparatus was relatively inexpensive to construct and operate, and requires less technical skill in its use than does the roll tube method. Complete identification of the methanogenic isolates was not attempted. The cabinet was primarily intended for a study of fastidious non-methanogenic bacteria in digesting sludge; the growth of methanogens serves, however, to assess biologically the efficiency of the apparatus. The authors acknowledge the assistance provided by the Biology Department workshops. Financial support was provided by the Wolfson Foundation.
References ARANKI,A. & FRETER,R. 1972 Use of anaerobic glove boxes for the cultivation of strictly anaerobic bacteria. American Journal of Clinical Nutrition 25, 1329-1 334. EDWARDS, T. & MCBRIDE,B. C. 1975 New method for the isolation and identification of methanogenic bacteria. Applied Microbiology 29,504-545. HUNGATE, R. E. 1969 A roll tube method for cultivation of strict anaerobes. In Methods in Microbiology ed. Norris, J. R. & Ribbons, D. W. Vol. 3B, London: Academic Press. LEACH,P. A., BULLEN, J. J. & GRANT,I. D. 1971 Anaerobic CO, cabinet for the cultivation of strict anaerobes. Applied Microbiology 22,824-827. MORRIS,J. G. 1975 The physiology of obligate anaerobiosis. Advances in Microbial Physiology 12,169-246. PATEL,G. B. & ROTH,L. A. 1977 Effect of sodium chloride on growth and methane production of methanogens. Canadian Journal of Microbiology 23,893-897. TOERIEN,D. F. & SIEBERT,M. L. 1967 A method for the enumeration and cultivation of anaerobic ‘acid-forming’ bacteria present in digesting sludge. Water Research 1,397-404.
SHORT COMMUNICATION An Anaerobic Glove Box for the Isolation and CuItiva ti on of Metha nogen ic Bacteria D. J. COX
AND
S . D. HERBERT
Department of Biology, University of York, York YO 1 500 Received 31 January 1 9 7 8 and accepted 2 5 A p r i l 1 9 7 8 1. An anaerobic glove box and growth chamber capable of isolating and culturing methanogenic
bacteria is described.
EVENTHE SIMPLEST manipulations of methanogenic bacteria, which are among the most rigorous of obligate anaerobes (Morris 1975), necessitate the application of techniques designed to prevent any exposure of the culture to oxygen during processing and growth. The Hungate (1969) or roll-tube technique precludes the use of Petri dishes, and thus imposes difficulties in single-colony purification of isolates. Several workers have resorted to the use of glove boxes, as reviewed, for example, by Aranki & Freter (1972). These are generally satisfactory for the isolation of some of the less fastidious anaerobes, but in many cases gaseous oxygen concentrations have exceeded the 10 pl/l reported to be inhibitory to the growth and survival of methanogens by Edwards & McBride (1975). These authors successfully grew methanogens using a combination of a vinyl glove box and an inner chamber, termed an Ultra Low Oxygen Chamber (ULOC), in which the culturing took place, and where gaseous oxygen was present at ca. 1 pl/l. Media were prepared aerobically and then reduced in the ULOC. This necessitated long periods of equilibrium of medium in Petri dishes before spreading, with the danger of interim growth of contaminating colonies. Because such equipment is complex and very expensive we describe in this communication an inexpensive rigid anaerobic glove box, incorporating the better features of a number of other such boxes, in which we have isolated and cultured methanogens.
Materials and Methods Media The methanogenic medium (MM) used for growth was similar to the phosphate buffered synthetic acetate broth used by Pate1 & Roth (1977) but also included (g/l): sodium acetate (instead of acetic acid), 2.5; sodium formate, 3 .O; DL-2-methylbutyric acid, 2.5; sodium chloride, 0.2. The medium was solidified with agar (20 gh). All chemicals were obtained from BDH Ltd. with the exception of the agar (Oxoid Ltd.), vitamins (Sigma Ltd.) and methylbutyric acid (Cambrian Chemicals Ltd.). They were of analytical grade where possible. 0021-8847/78/030411+05%01.00/0
[411]
0 1978 The Society for Applied Bacteriology
D. J. COX A N D S. D. HERBERT
412
Digesting sludge All isolates were obtained from a laboratory digester (1-1) operating at 35 "C with a retention period of 12.5 days and using domestic sewage sludge as feed. Anaerobic cabinet The anaerobic cabinet was adapted from an obsolete glove box originally supplied by SLEE Ltd. (Hither Green Lane, London). Two access ports (21 cm diam.) were cut in the top of the box, closed by steel discs retained by screw clamps and sealed with butyl rubber 0 rings. Inlet and outlet gas and water bulkhead connectors were brazed into the wall of the cabinet, and all electrical switchgear was mounted on the outside of the cabinet, the overall features of which are shown in Fig. 1. Pyrotenic cables supplied Electrical
External out
Circulating water for growth chamber
\Front window
I
76 cm
I
Furnace and copper reducing tubes
I
Glove port and clomp
Fig. 1. Frontal view of the anaerobic glove box (schematic).
apparatus within the cabinet: a 13 W Sylvania U.V. germicidal lamp (Thorn Electric Ltd.) and a 70 V purpose built 'warm box' used for keeping media molten. The completely sealed warm box, of stainless steel, was fitted with ports for sparging with 0, free gas (Fig. 2). The front and top windows of the cabinet were removed and resealed with neoprene sealant (Greens Aqua Division, Leeds) as a precautionary measure against 0, diffusion, and then the seals were covered inside and out with 5 cm PVC tape. The cabinet was fitted with neoprene rubber gloves (SLEE) retained with neoprene sealant and PVC tape, and, when not in use, the glove ports were closed off with aluminium discs retained by screw clamps. The glove box contained 100 g (equivalent to approx. 1 g/2 1 of cabinet volume) palladianized asbestos (Engelhard, Cinderford, Glos.), distributed into four gauze sachets hung around the walls. The catalyst was regenerated every two or three complete runs by removing the sachets from the cabinet and heating (16OoC/2 h). All tubing used for the conveying of gases into and out of the cabinet was of butyl rubber (Gallenkamp Ltd).
ISOLATION OF METHANOGENIC BACTERIA
4 13
General operating of the cabinet All the equipment necessary for experimentation was placed in the cabinet in an anaerobic jar (Gallenkamp Ltd.) under H, 18-24 h before operation. The equipment included plastic Petri dishes (Sterilin), spreaders, Finn pipettes and sterile tips (Jencons Scientific Ltd., Heme1 Hempstead, Herts.), sharp sterile sticks (for picking colonies) and sterile pre-reduced anaerobic dilution fluid (Toerien & Siebert 1967) dispensed in screw capped bottles in volumes of 49.5 and 9.0 ml. The dilution fluid was generally prepared and reduced on the bench and dispensed using the cabinet in a separate run. The cabinet was equilibrated, in a manner similar to that described by Leach et al. (1971), by flushing CO, (Distillers Co. Ltd.) into it at 6.0 l/min. A Gulfstream CO, heater (F. Myers Welding Supplies Ltd., Leeds) prevented the icing of the cylinder
Screw boss
I
70 V heater element
,Sparging
valve
Supporiing legs
Fig. 2. Cross section of the anaerobic warm box.
reduction valve at this high flow rate. To remove traces of 0, from the gas it was passed through a glass tube (25 mm diam.) containing 500 g copper turnings at 350 "C before delivery to the cabinet. After initial equilibration for 3 h (or much less if the cabinet already contained CO, from a previous run) samples of sludge and the molten media were put into the cabinet. After a further 30 min of CO, flushing, hydrogen was added to the gas stream at a flow rate of 1.0 l/min. This served to keep the copper reduced and, together with the Pd catalyst, to scavenge residual 0, from the cabinet. After 1-2 h of H, flow, the cabinet was ready for use.
Growth chamber The growth chamber, similar to the ULOC of Edwards & McBride (1975), was a polished copper anaerobic jar (Baird and Tatlock, Ltd.), around which was coiled 2 m of butyl rubber tubing (8.5 mm i.d.). Water at 39OC circulating through this tubing served to maintain an internal temperature of 35 "C. The chamber contained 10 g Pd catalyst and 50 g silica gel.
414
D. J. C O X AND S. D. HERBERT
Preparation of media Medium MM was prepared outside the cabinet in a stout Pyrex bottle with a neoprene bung. The bottle was placed in a boiling water bath and oxygen was displaced with a continual stream of N, (0, free) passed into the medium through a 23 cm syringe needle. Reducing fluid was then added and sparging was continued until five minutes after the resazurin indicator in the medium had decolourized. The medium was then autoclaved (121 OC/lS min), holding the bottle in a screw clamp t o prevent the bung being ejected. The sterile medium was transferred while still hot to the cabinet and placed in the warm box previously equilibrated to 50 "C. To reduce to a minimum the number of times the cabinet was opened, the digesting sludge sample was usually put in at this time. The warm box was flushed with CO, for 30 min using an additional sparging line plumbed into the cabinet, and then sealed off. Hydrogen was then added to the main CO, stream to the cabinet. After 2 h the medium was removed from the warm box, poured, allowed to set and dried for 1 h under U.V. light. The sludge sample was serially diluted in the anaerobic dilution fluid and spread on to the plates in volumes of 0.1 ml. Plates were immediately transferred to the growth chamber which was flushed with H,/CO, (80:20, BOC Special Gases). Flushing was repeated every 2-5 days according to the number of plates in the chamber and the degree of growth of the colonies. Throughout incubation, 0, in the (outer) cabinet was maintained at a low partial pressure by continual flushing with CO, at 0.5 l/min. Slopes Slopes in thick Pyrex tubes were prepared similarly except that 5 ml portions of the reduced unsterilized medium were dispensed inside the equilibrated cabinet. The tubes, sealed with butyl rubber bungs (C. W. Meadows & Co. Ltd., Torquay), were removed for autoclaving and then immediately returned to set inside the cabinet. After inoculation, the slopes were individually sparged with sterile (filtered) H,/CO, mixture. They were removed from the cabinet inside an anaerobic jar (without catalyst) and incubated at 37 OC. The jar was sparged every other day with H,/CO, mixture and the slopes were individually sparged every 4 d. Analysis of gases Oxygen The preliminary equilibration time of the cabinet was estimated from analysis of the exhaust gases using a Radiometer R M 1302 blood gas analyser (V. A. Howe Ltd., Peterborough Road, London). The limit of sensitivity of the instrument was 400 ,ul O,/l. Methane Methane was measured in a Pye 104 GLC fitted with an activated alumina column.
Results Equilibration of the cabinet The time taken for the cabinet gas phase to reach 0, concentrations below the limit of sensitivity of the analyser was approx. 3 h. This was chosen as the suitable time to add H, to the CO, gas stream.
ISOLATION O F METHANOGENIC BACTERIA
415
Growth of methanogens After 14 days incubation a strong negative pressure had developed within the vessel and, even after this had been equalized with H,/CO, mixture, the gas phase showed a methane content of approx. 10% (v/v). The jar was opened in the fully equilibrated cabinet. Total colony counts averaged 2.5 x 108/ml digesting sludge. Fifty colonies were picked at random and subcultured to slopes of MM. Only five of these cultures showed obvious growth and methane production after 14 days incubation, although trace methane was evident in 30 others. After 2 1 days a total of 45 cultures showed growth, 40 of which were methane positive. All positive isolates were further purified by single colony streaking on plates and, on the basis of colonial and cell morphology, some were presumptively identified with Methanobacterium, Methanosarcina and Methanospirillum.
Discussion Single colonies of methanogenic bacteria have been isolated on Petri dishes using an anaerobic glove box without the constraints of the roll tube method. Pre-reduced media were preferred, because although aerobically prepared media could be reduced in our growth chamber, it was considered that media prepared, poured, dried and spread within a matter of hours were less likely to become contaminated. A rigid metal cabinet was considered more suitable than a flexible vinyl one as it enabled the various services to be easily conveyed through and attached to the walls. Operation of the cabinet was like that of a large anaerobic jar, i.e., a combination of hydrogen gas and palladium catalyst to scavenge traces of oxygen. The apparatus was relatively inexpensive to construct and operate, and requires less technical skill in its use than does the roll tube method. Complete identification of the methanogenic isolates was not attempted. The cabinet was primarily intended for a study of fastidious non-methanogenic bacteria in digesting sludge; the growth of methanogens serves, however, to assess biologically the efficiency of the apparatus. The authors acknowledge the assistance provided by the Biology Department workshops. Financial support was provided by the Wolfson Foundation.
References ARANKI,A. & FRETER,R. 1972 Use of anaerobic glove boxes for the cultivation of strictly anaerobic bacteria. American Journal of Clinical Nutrition 25, 1329-1 334. EDWARDS, T. & MCBRIDE,B. C. 1975 New method for the isolation and identification of methanogenic bacteria. Applied Microbiology 29,504-545. HUNGATE, R. E. 1969 A roll tube method for cultivation of strict anaerobes. In Methods in Microbiology ed. Norris, J. R. & Ribbons, D. W. Vol. 3B, London: Academic Press. LEACH,P. A., BULLEN, J. J. & GRANT,I. D. 1971 Anaerobic CO, cabinet for the cultivation of strict anaerobes. Applied Microbiology 22,824-827. MORRIS,J. G. 1975 The physiology of obligate anaerobiosis. Advances in Microbial Physiology 12,169-246. PATEL,G. B. & ROTH,L. A. 1977 Effect of sodium chloride on growth and methane production of methanogens. Canadian Journal of Microbiology 23,893-897. TOERIEN,D. F. & SIEBERT,M. L. 1967 A method for the enumeration and cultivation of anaerobic ‘acid-forming’ bacteria present in digesting sludge. Water Research 1,397-404.
