Plate colonization of Methanococcus maripaludis and Methanosarcina thermophila in a modified canning jar

MICROBIOLOGY LETTERS FEMS Microbiology Letters 145 (1996) 131-137

Plate colonization of Methanococcus maripaludis and Methanosarcina thermophila in a modified canning jar Ethel Center of Marine

Biotechnology.

University

A. Apolinario,

Kevin

of Mrrr~~land Biotechnology Baltimore,

MD

R. Sowers *

Institute,

Columbus Center, Suite 236, 701 E. Prutt

Street,

21202, C’SA

Received 28 August 1996; revised 15 September 1996: accepted 18 September 1996

Abstract Conditions have been optimized for colonization of fastidiously anaerobic microorganisms on solidified plating medium incubated in modified canning jars. Two species of methanogenic Archaea, the mesophilic, hydrogen utilizer Methanococcus maripuludis and the moderately thermophilic methylotroph Methunosarcina thertnophila, were grown with high efficiency on agar-solidified medium in petri plates. Maximum colony size and plating efficiencies of 50-100% of total cell counts were obtained by optimizing inoculation method, H2S concentrations, and agar moisture content. The simple modifications required provide a readily available source of inexpensive anaerobic growth vessels for investigations requiring colonization of methanogens on solidified plating medium. Kqwords.

Methanogen: Archaea; Methylotroph: Hydrogen utilizer; Plating; Anaerobe jar

1. Introduction Many obligate anaerobes that tolerate brief exposure to oxygen can be plated on pre-reduced medium in the open air and subsequently incubated in anaerobic jars that have been evacuated or chemically reduced [ 11. However, colonization on solidified medium by fastidiously anaerobic microorganisms such as the methanogenic Archaea requires complete exclusion of oxygen at all times and a growth atmosphere with a redox potential below -330 mV [2]. Although this was initially accomplished in early

* Corresponding author. Tel.: + I (410) 234-8878; Fax: +l (410) 234-8899; E-mail: [email protected]

studies by the roll-tube technique, later interest in replica plating for genetic studies resulted in the development of techniques for colonization on solidified medium in petri plates [1,3]. In all these reports, methanogens are inoculated onto solidified medium by standard techniques such as spreading or molten agar overlay inside an anaerobic glove box, then incubated in anaerobe jars that contain a reducing agent such as hydrogen sulfide. Due to the long incubation periods required for growth of most methanogens, anaerobe jars partially or wholly composed of polymer must be incubated in the limited space of the anaerobic glove box due to gradual permeation of oxygen through the polymer components [3-51. To overcome this limitation, investigators have employed anaerobic culture jars composed of metal with O-ring seals that have negligible permeability

0378-1097/96/$12.00 Copyright 7 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PIISO378-1097(96)00401-6

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EA.

Apolimrio,

K. R. SowerslFEMS

to gasses. Reports include metal jars available commercially [6,7], modified jars made from pressure cookers and paint pressure canisters [S-l 11, and custom made metal jars [3,12-151. While these anaerobe jars yield high plating efficiencies, greater plating capacities required by many genetic experiments, especially slow growing microorganisms, are limited by the expense of acquiring or constructing these apparatus. A recent report describes the use of a glass canning jar for colonization of the extreme thermophile Thermotogu neupolitanu on solidified plating medium [16]. Although the inclusion of palladium in the jars catalyzed an adequately anoxic atmosphere to grow thermotoga, methanogenic Archaea require a more reduced atmosphere usually generated by the addition of H2S. Since H2S irreversibly inactivates palladium, anaerobic jars must be equipped with an outlet for gas purging before the plates can be removed within an anaerobic glove box. Herein we describe a plating protocol that utilizes an inexpensive canning jar with simple modifications for efficient colonization of Methunococcus muripuludis and Methunosurcinu thermophilu on agar-solidified media. These anaerobic culture jars are fitted with a pressure gauge to monitor gas production or depletion and a butyl rubber septum for gas exchange and outgassing of hydrogen sulfide.

Microbiology

Letters I45 (1996) 131-137

as previously described [7]. M. muripuludis was grown in McC medium as previously described [l 11. Solidified agar medium consisted of McC liquid medium without the addition of NazS9HzO. For growth of M. thermophilu, trimethylamine-HCl was added to a final concentration of 0.05 M. Media (100 ml) were transferred to 160 ml serum vials that contained 1.25% or 0.5% (w/v) purified agar for bottom or top agar, respectively. The vials were sealed under a N2-C02 headspace with a butyl rubber stopper secured by an aluminum crimp seal. Media were sterilized by autoclaving at 121°C for 20 min. To prepare plating medium, molten agar medium was transferred to a Freter-type anaerobic glove box (Coy Manufacturing Co., Ann Arbor, MI) that contained an atmosphere of N~-CO~-HZ (15:4: 1). The oxygen concentration was maintained below 3 ppm with palladium catalyst. Relative humidity was monitored with an electronic hygrometer (Fisher Scientific Co., Pittsburgh, PA) and maintained at 30 & 5% with trays of CaC12.HZ0. Bottom agar (10 ml) cooled to 55°C was dispensed into sterile polystyrene petri plates (60x 15 mm) and allowed to solidify to room temperature. Plates were dried at room temperature in the anaerobic glove box for 2 days before use except where indicated. All plasticware and glassware were equilibrated in the anaerobic glove box 24 h before use. 2.3. Colony plating procedures

2. Materials and methods 2.1. Bucterial strains The source for Methanosarcinu thermophilu TM-l (= DSM 1825) was described previously [6]. Methunococcus muripuludis JJ (= DSM 2067) was a gift from W. Whitman (University of Georgia, Athens, GA). 2.2. Medium preparation Sterile media were prepared anaerobically in an atmosphere that contained Nz-CO2 (4:l) by a modification of the Hungate technique [3]. All gasses were passed through a column of reduced copper turnings at 350°C to remove traces of 02. M. thermophilu was grown in disaggregated single-cell form

Solidified plating media were inoculated from liquid cultures of M. thermophilu harvested in midexponential growth (OD55a = 0.2-0.3; 1 ODjso = 2.6~ 10s cells/ml) and M. muripuludis in lateexponential growth (ODr”~0.6; 1 ODcoo = 7.4 x lO”cells/ml) shown previously to yield viable cells for growth [7,9]. Cultures were harvested by centrifugation at 1OOOOXg for 10 min. Methunococcus muripuludis grows with equal efficiency when inoculated in a molten agar overlay or by spreading [17], but the plating efficiency of Methanosurcinu thermophilu when inoculated by spreading is only 3 + 1% of the efficiency observed with agar overlays [7]. Therefore, plates were inoculated by adding 0.1 ml serially diluted cell suspension to 1 ml molten overlay medium (50°C) and layering the suspension on predried bottom agar.

E. A. Apolinurio, K. R. Sowers I FEMS Microbiology Letters 145 11996) 131-137

133

A

Fig. 1. Construction of modified canning jar showing assembly of components (A), and photograph of assembled jar (B). Anaerobic culture containers were constructed from I quart, wide-mouth glass canning jars (Ball Corporation. El Paso. TX, No. 14400-67000) with dome tops and ring seals. Two holes were made in the top with 12.8 mm and 9.5 mm metal punches. A butyl rubber septum stopper (Bellco Glass, Inc.. Vineland, NJ, No. 2048-l 1800) was inserted through the larger hole and secured with a 12.8 mm pushnut bolt retainer (Serv-A-Lite Products, Inc., E. Moline. IL, No. 595-F). A compound gauge (-100 kPa/lOO kPa, Master-Carr Supply Co., Dayton, NJ, No. 4004K4) attached to an adapter and O-ring (Cajon Co., Macedonia, OH, No. SS-4-SAE-7-4) was inserted into the smaller hole and secured with a flat washer (9.5 mm id.) and hex nut. Clear strapping tape was applied to the exterior of the jar to contain glass in the event of breakage. Up to 10 plates were inverted and stacked into cylindrical polypropylene bottles (Qorpak, Pittsburgh, PA, No. 73396) that had the top I cm removed and mere slotted along two sides for viewing.

2.4. Incubation

in anaerobic culture containers

A 5 ml serum vial containing 2.5% (w/v) Na+9H20 solution was placed in the bottom of the canning jar alongside the plate stack (Fig. 1). The jar was sealed with the modified top and removed from the anaerobic glove box. For jars that contained plates inoculated with M. maripaludis, the gas phase was exchanged with a gas mixture of

HZ-CO2 (4:l) by inserting into the septum a gassing cannula fitted with a 23 gauge needle and a second needle as a vent. After displacing the Nz-C02, the jar was pressurized to 35 kPa by removing the vent needle. Culture containers were repressurized to 35 kPa with HZ-C02 as the gasses were depleted. Pressures greater than 35 kPa were avoided as a safety concern due to the glass construction of the jar. Jars that contained plates inoculated with A4. thevmophila

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Microbiology Letters 145 (1996) 131-137

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Sodium sulfide (g) Fig. 2. Effect of NazS*9H20 on plating efficiencies of M. thermophila (A) and -8-L maripaludi~ (B). Indicated amounts of NaaS9Hz0 in Hz0 were added to a serum vial and incubated with plates in the modified canning jars. Plates were incubated at 35°C for 14 days. The average CFUs on six replica plates from two experiments are reported k I standard deviation.

were directly transferred from the anaerobic glove box to an incubator without purging. Stainless steel anaerobic culture containers (Torbal model AJ-3, Torsion Balance Co., Clifton. NJ) were used as previously described [7]. Prior to opening the modified canning jars in an anaerobic glove box, HPS was purged from the containers with a gas mixture of Na-CO2 in a fume hood. 2.5. Chemicals Purified agar was from Difco Laboratories, Detroit, MI. Trimethylamine-HCl was from Sigma Chemical Co., St. Louis, MO. All other chemicals were of reagent grade.

3. Results and discussion 3.1. Modification methanogens

qf canning jars for growth of

Inoculated plates were sealed in the modified canning jar and HzS was generated from NaaS9HsO as it was acidified by CO2 in the gas phase [ll]. Since H2S irreversibly inactivates palladium catalyst used to scavenge oxygen in the anaerobic glove box, the jar was modified by inserting a butyl rubber septum into the top of the lid as a means of purging the hydrogen sulfide before introducing and opening the jar inside the glove box. The septum also provided a means of replacing gas phase with Ha-CO2

E. A. Apolinurio, K. R. Sowers I FEMS Microbiology Letters 145 (I 996) 131-137

for growth of hydrogen-utilizing methanogens. The septum was secured with a retainer to prevent extrusion of the septum as a result of pressure from methane production or from pressurization with Hz-COZ. The compound gauge had a dual function. The gauge was used to monitor an increase in pressure from CH4 and CO2 production by M. thermophilu grown on trimethylamine. The gauge also measured the decrease in Hz gas pressure as it was consumed by AL voltae. Media containing resazurin remained clear after over 2 months in modified jars incubated under pressure (+35 kPa) or vacuum (-35 kPa). The modified jar tops were reusable as long as the septa were periodically changed and the rubber seals remained clean. When leaks eventually developed it was either due to multiple perforations of the rubber septa or to eventual corrosion and pitting on the inside of the metal top where the inner coating had been abraded. 3.2. E&t

of HzS on growth

M. thermophila and M. muripaludis had different tolerances to the amount of H2S released into the vessels. Maximum colony forming units (CFU) recovery for M. thermophilu occurred using 0.02 g NazS9HzO (=0.20/o v/v HzS) with a significant reduction in efficiencies using 0.01 g (= 0.1% v/v HsS) and 0.04 g (= 0.4% v/v H2S) Na2S9HzO (Fig. 2A). This optimal concentration range is lower than that previously reported for growth of M. thermophila in stainless steel jars (0.550.8% v/v) [7]. The likely explanation for the discrepancy is that the latter container has more metal surface area that will potentially sequester HsS, reducing the effective HgS concentration for growth [5]. However, the modified canning jar has minimum metal surface area, thereby increasing the effective H2S concentration available for growth. Maximum CFU recovery for M. maripaludis was observed using 0.16 g NazS*9HZ0 (= 1.6% v/v HzS) with a reduction in efficiencies at lower concentrations of NazS*9Hz0 (Fig. 2B). No growth occurred with 3.2 g NazS9H20 (~3.2% v/v H$S), the highest H$ concentration tested. In contrast to a previous report showing that M. maripaludis tolerated a narrow range of H2S (1.3% v/v) in metal anaerobe jars, A4. maripaludis growth efficiency in modified canning jars increased linearly as the

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HZ’S was increased from 0.2 to 1.6% (v/v). This observation suggests that at concentrations below 1.6% (v/v), H2.S is sequestered by the metal jar below threshold levels required for growth, whereas HzS is available for growth at all concentrations in the modified canning jar. 3.3. Eflect of media hydration on growth The extent of predrying agar medium is critical for effective colonization since anaerobe jars are closed to the outside atmosphere, thereby preventing desiccation of the medium. Solidified medium must be dried adequately to prevent condensation and confluence of colonies, but must not be too dry for efficient growth of colonies. The effect of agar Hz0 content on growth efficiencies was tested on agar medium predried for l-7 days inside an anaerobic glove box (Fig. 3). The relative humidity was maintained at 30 f 5% throughout the drying period by introducing trays of CaClz into the glove box as a desiccant. Recovery of A4. thermophila and A4. maripaludis CFUs was optimal on plates dried for 2 days with a significant reduction in efficiency on plates dried for 3-7 days. Plates dried for 1 day or used immediately after preparation could not be counted due to confluence of the colonies caused by excess moisture. Plating medium dried for 2 days could be stored in canning jars and inoculated at a later time without a reduction in plating efficiency. 3.4. Effect of growth conditions on colony size Maximum colony size generally coincided with conditions for maximum CFU recoveries. Under optimal H2S and moisture content M. thermophilu colonies were 1.78kO.25 mm after 2 weeks. Under optimal growth conditions colony sizes of M. maripaludis were more variable on individual plates (0.46 + 0.39 mm), ranging from 0.2 to 1.2 mm. The range of sizes is greater than reported previously [17]. However, plates were inoculated by spreading in the former study in contrast to inoculation with molten agar described here. A previous report also described variability of colony sizes for hydrogenutilizing methanogens grown with molten agar and attributed it to hindered diffusion of hydrogen to cells at the bottom of the soft agar overlay [12]. In

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Drying Time in Days Fig. 3. Effect of predrying solidified medium on plating efficiencies of M thermophila (A) and M. maripalrdis (B). Plates were predried for the indicated time under conditions described in Section 2. Plates were incubated at 35°C for 14 days. The average CFUs on six replica plates from two experiments are reported f 1 standard deviation

this report colonies below the top agar were smaller on average than colonies on the surface, which is consistent with differential diffusion of hydrogen into the medium. Colonies of M. maripaludis were larger and less variable in size (1 .l ? 0.4 mm) when inoculated by spreading. As reported previously with conventional anaerobe jars, M. thermophilu colonies were visible within 5 days, and M. maripaludis colonies were observed within 2 days [7,17]. Colonies of both species were readily transferred by streaking or replica plated by patching. In conclusion, a canning jar has been adapted for incubation of hydrogen-utilizing and methylotrophic methanogenic Archaea grown on solidified plating media. The simple modifications allow for purging of HzS and replenishment of gasses without opening

the jar. The addition of a compound pressureivacuum gauge allows monitoring of CH4 production or depletion of Hz. Overall recoveries of CFU for M. muripaludis and M. thermophilu grown in modified canning jars were 88 + 12% and 82 + 6%, respectively. of CFU recovered from stainless steel jars after 14 days of incubation. However, optimal HZS concentration and inoculation method for maximum growth must be determined empirically for each species. The modified jar remained anaerobic for periods required to grow methanogens, and based on previous reports with Thermotoga neapolitana, they can be used at growth temperatures required to grow hyperthermophilic methanogens without the deterioration associated with commercial polycarbonate jars [16]. Unlike metal anaerobe jars, these glass ves-

E. A. Apolinnrio. K. R. Sowers I FEMS Microbiology Letters 145 (1996) 131-137

sels allow visual monitoring of colony growth without opening and removing the plates. The ready availability of canning jars and simple modifications required provide an virtually inexhaustible source of anaerobic growth jars for investigations that require plate colonization of methanogens.

Acknowledgments

We are grateful to K. No11 and H. May for helpful discussions. This work was supported by Department of Energy Grant DE-FG02-93ER20106.

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[7] Sowers, K.R., Boone, J.E. and Gunsalus. R.P. (1993) Disaggregation of Methanosarcina spp. and growth as single cells at elevated osmolarity. Appl. Environ. Microbial. 59, 38323839. [E] Balch, W.E. and Wolfe, R.S. (1976) New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonit acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressurized atmosphere. Appl. Environ, Microbiol. 32, 781-791. [9] Tumbula, D.L., Makula. R.A. and Whitman, W.B. (1994) Transformation of Methanococcus maripaludis and identification of a PstI-like restriction system. FEMS Microbial. Lett. 121, 3099314. [lo] Bowen, T.L. and Whitman, W.B. (1987) Incorporation of exogenous purines and pyrimidines by Methcrnococcus voltae and isolation of analog-resistant mutants. Appl. Environ. Microbiol. 53, 1822-1826. [II] Tumbula, D.L., Bowen, T.L. and Whitman, W.B. (1995) Growth of methanogens on solidified medium. In: Archaea: A Laboratory Manual (Robb, F.T., Place, A.R., Sowers. K.S.. Schreier, H.J., DasSarma, S. and Fleischmann, E.M., Eds.), pp. 49-55. Cold Spring Harbor Laboratory Press, Plainview, NY. [12] Kiener, A. and Leisinger, T. (1983) Oxygen sensitivity of methanogenic bacteria. Syst. Appl. Microbial. 4, 305-312. [13] Worrell, V.E., Nagle, D.P.M., Jr.. McCarthy. D. and Eisenbraun, A. (1988) Genetic transformation system in the archaebacterium Methanobucterium thermoautotrophicum Marburg. J. Bacterial. 170, 6533656. [14] Balch, W.E., Fox, G.E., Magrum, L.J., Woese, C.R. and Wolfe. R.S. (1979) Methanogens: reevaluation of a unique biological group. Microbial. Rev. 43, 260-296. [15] Micheletti, P.A., Sment, K.A. and Konisky, J. (1991) Isolation of a coenzyme M-auxotrophic mutant and transformation by electroporation in Methanococcus volume. J. Bacterial. 173. 34143418. [16] Vargas, M. and Noll. K.M. (1994) Isolation of auxotrophic and antimetabolite-resistant mutants of the hyperthermophilic bacterium Thermotoga rteapolitana. Arch. Microbial. 162, 357-361. [17] Jones, W.J., Whitman. W.B. and Wolfe, R.S. (1983) Growth and plating efficiency of methanococci on agar media. Appl. Environ. Microbial. 46. 220-226.