techniques for cultivating

C H A P T E R O N E

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Techniques for Cultivating

Methanogens

Ralph S. Wolfe

Contents

1. In

in

076

en

troduction

Enzymology, Volume 494 # 2011

-6879, DOI: 10.1016/B978-0-12-385112-3.00001-9 All rig

t of Microbiology, University of Illinois, Urbana, Illinois, USA

Else

hts

1

2. M
ethanogens on a Budget 2
2
.1. P reparation of oxygen scrubbers 2
2
.2. P reparation of 200 mL of anoxic medium by boiling 5
2
.3. T he vacuum–vortex method 8
2
.4. A noxic, aseptic use of a syringe 9
2
.5. G rowth of methanogens on agar medium, the bottle plate 10
2
.6. G rowth of methanogens in a Hungate roll tube 11
2
.7. P reparation of a 3-L amount of anoxic medium 12
2
.8. C ultivation of marine methanogens 16
2
.9. A septic transfer of sterile medium 16
3. U
se of an Anoxic Chamber 18
Ackn
owledgment 21
Refe
rences 21
Abstract

Basic techniques for the cultivation of methanogenic archaea in anoxic media,

where the O/R potential is maintained below (�) 330 mV under a pressurized

atmosphere of 20% carbon dioxide, are described.

1. Introduction

Although techniques for growing methanogens vary in detail amonglaboratories, these procedures follow the legacy of Hungate who perfectedthe preparation of prereduced media (Bryant, 1972; Hungate, 1950, 1969).He developed methods for the exclusion of oxygen in the preparation andsterilization of anoxic media as well as methods for the aseptic inoculationand transfer of anaerobic microbes in media where an O/R potential below

vier Inc.

reserved.

1

2 Ralph S. Wolfe

(�) 330 mV was maintained. Three essential steps persist today: (a) use ofnature’s buffer of carbon dioxide–bicarbonate–carbonate to maintain a pHnear neutrality, (b) use of cysteine and sodium sulfide as reducing agents,and (c) use of resazurin as an O/R indicator that is reddish, when oxidized,and colorless, when reduced at (�) 330 mV. To aseptically remove a stopperfrom a culture tube in which a methanogen had generated a negativepressure required exceptional skill for the operator to prevent contamina-tion or the entrance of oxygen into the tube. The demands for perfection inuse of these techniques were so high that only a few methanogens had beenisolated in pure culture prior to 1974. Development of procedures wheremethanogens could be cultured in a pressurized atmosphere produced aparadigm shift in the isolation and culture of these organisms; the chancesfor contamination or loss of reducing potential were essentially eliminated(Balch and Wolfe, 1976; Balch et al., 1979). For additional details on thecultivation of methanogens, the article by Sowers and Noll (1995) should beconsulted. The following presentation is designed to encourage the study ofmethanogens in laboratories where funds for equipment may be limiting orfor the cultivation of methanogens in teaching laboratories. Use of ananoxic chamber is then discussed.

2. Methanogens on a Budget

2.1. Preparation of oxygen scrubbers

1. Essential to the study of methanogens is a mechanism for scrubbingtraces of oxygen from commercially available compressed gases.Figure 1.1 presents a system for routing N2, or mixtures of 80% N2

and 20% CO2 (N2:CO2) or 80% H2 and 20% CO2 (H2:CO2) through aheated cylinder (Fig. 1.1(1)) that contains reduced-copper filings orpacked copper turnings, an inexpensive oxygen scrubber. Gas pressuresup to 2 atm may be used, so it is essential that a sturdy gas-tight systemwith copper or stainless-steel (ss) tubing and connecters (Swagelok-type,Whitey Co.), be constructed. Figure 1.2A shows a simple scrubber thatemploys a section of copper tubing 23 � 3 cm that contains copperfilings. Solid copper caps have been soldered to each end with hightemperature (essential!) silver solder. The top cap is penetrated by twopieces of copper tubing, 3 mm (dia) and of desired length, soldered inplace; one piece, the intake, extends to within 2 cm of the bottom, andthe exit tube extends through the cap about 1 cm.

2. Figure 1.2B shows a method of heating the cylinder with electric heatingtape connected to a variable transformer. Only heating tape specificallydesigned for use on metal can be used safely! (Fisher Scientific). Forinsulation, the cylinder should be wrapped in a thick layer of glass wool.

33

GAS

To gassingProbe station

OFF

VAC

N2

N2:CO2

H2:CO2

VAC

2

1

3

3

6

7

4

95

8

Figure 1.1 Gassing station for dispensing anoxic gases (not drawn to scale). (1)Welded stainless-steel (ss) column 3 � 25 cm containing reduced-copper filings; (2)ceramic furnace at 340 �C; (3) ss 3 mm tubing; (4) ss three-way valve with a center-offposition and Swagelok fittings Whitey Co.; (5) moisture trap (5 � 40 cm) containingCaCl2 pellets (not necessary for oil-less vacuum pumps); (6) vacuum pump pressuregauge, range (�) 100 kPa (�30 in.Hg) to 200 kPa (30 psi); (7) flexible 3 mm PVAperfluoroalkyloxy tubing (Swagelok Co.); (8) gas-dispensing attachment; (9) vacuumline on/off vent valve. Reproduced with permission, from Wolfe and Metcalf (2010).

Techniques for Cultivating Methanogens 3

Figure 1.2C shows awelded 316 ss-cylinder that is filledwith copper filingsand inserted in an electrically heated ceramic furnace (formerly distributedby a company named Sargent Welch, but is now difficult to find). Suchequipment has functioned flawlessly for decades in many laboratories.

3. The copper filings are initially reduced by passing H2 or H2:CO2 slowlythrough the hot filings. This reduction releases significant amounts of heatand should be performed carefully so as not to cause the copper filings toclump. After reduction, cool the filings by passingN2 through the cylinder;then place a high temperature thermometer in contact with the cylinder.Turn on the transformer and gradually increase the setting until the columntemperature approaches 340 �C.At higher temperatures, significant reduc-tion of CO2 to CO may occur. In active laboratories, the transformerremains on continuously. After continued use, oxidized copper is regen-erated by passing H2:CO2 through the column for a few minutes.

4. From the scrubber (Fig. 1.1(1)) the gas line branches at a T-connecter,one line going to a gassing station with three probes (Fig. 1.3) and one toa gassing attachment (Fig. 1.1(8)). A probe is easily constructed(Fig. 1.3D) from a 3-cc glass Leur Lok syringe filled with cotton.

A B C

D E

Figure 1.2 Heated reduced-copper oxygen scrubber. (A) Copper tube filled withcopper filings; (B) simple method for heating cylinder with a variable transformer andmetal-safe heating tape (Fisher); (C) welded ss-cylinder filled with copper filings andinserted in a ceramic furnace; (D) two ss-cylinders connected in series for scrubbing gasesrouted to an anoxic chamber (Section 3); (E) top of a larger ceramic furnace fitted with acustom-made ss-cylinder and a temperature probe, for use with an anoxic chamber.

A

B

CD

Figure 1.3 Gassing station for dispensing anoxic gases. (A) 3 mm copper tubing froman oxygen scrubber; (B) on/off valve; (C) adjustable metering valve; (D) gassing probe,a 3-mL glass Leur Lock syringe filled with cotton, fitted with a long, bent 16G needle(blunt end), and held in place for easy removal.


To the syringe add a bent 16-gauge (G) needle 12 cm long from whichthe point has been removed. The complete probe can be placed in a testtube holder, as shown. Two other gassing probes each with a 12-cm 18Gneedle are also constructed. Each probe is attached to the gas source withblack-rubber tubing (OD 9 mm with a 3 mm-thick wall) by insertingthe tubing firmly into the syringe. To initiate gas flow to a probe, turnon the gas flow valve (Fig. 1.3B); to regulate the amount of gas flowingto a probe, turn the metering valve (Fig. 1.3C).

2.2. Preparation of 200 mL of anoxic medium by boiling

Many methanogens grow in a simple medium where 4 mol of H2 areoxidized and 1 mol of CO2 is reduced to methane, resulting in a negativepressure within the culture tube or vial. The atmosphere of H2:CO2 isrepressurized during growth of the methanogen.

1. Here is an example of such a medium (g/L): NH4Cl, 1; NaCl, 0.6;NaHCO3, 5; KH2PO4, 0.3; K2HPO4, 0.3; MgCl2�6H2O, 0.16;CaCl2�2H2O, 0.009; resazurin 0.1% solution, 1 mL; cysteine�HCl andNa2S�9H2O are added separately (see item 4 below).

2. Prepare a solution of the following vitamins (10 mg of each/L) and add10 mL of the solution to a liter of medium: p-aminobenzoic acid,nicotinic acid, calcium pantothenate, pyridoxine, riboflavin, thiamine,and 5 mg each of biotin, folic acid, a-lipoic acid, and B12.

3. Prepare a solution of trace minerals (g/L) and add 10 mL of the solutionto 1 L of medium: trisodium nitrilotriacetic acid, 1.5; Fe(NH4)2(SO4)2,0.8; NaSeO3, 0.2; CoCl2�6H2O, 0.1; MnSO4�H2O, 0.1;Na2MoO4�2H2O, 0.1; NaWO4�2H2O, 0.1; ZnSO4�7H2O, 0.l;NiCl2�6H2O, 0.1; H3BO3, 0.01; CuSO4�5H2O, 0.01.

4. Reducing agents are most conveniently added as anoxic solutions. Preparea 0.2-M solution of cysteine HCl by adding the powder to a volume ofhot distilled water that is about one-third the volume of the container vial.For example, 50 mL in a 158-mL vial (listed as 125 mL, Wheaton). Flushout the atmosphere above the liquid with N2 from a gassing probe;Hungate seal and crimp a Balch stopper in place (Fig. 1.4E–H). Likewise,prepare 0.2 M solution of Na2S�9H2O. (Sulfite on the surface of a crystalof sulfide is toxic to methanogens; so briefly rinse the surface of the crystal,held in tweezers, under flowing water and dry the crystal with a papertowel before weighing.) Unfortunately, the strong reducing agent,sodium dithionite, is toxic to methanogens and cannot be used.

5. Open the plastic stopcock on the gassing attachment (Figs. 1.1(8) and 1.5)so that N2 is flowing through the 21G needle. Insert the needle throughthe Balch stopper on the vial of reducing solution and adjust the pressureof N2 in each vial to about 35 kPa (5 psi) so that, when desired, a sample of

A B C

D E F G H

Figure 1.4 Procedure for preparing and dispensing anoxic media. (A) Gassing probe;(B) a boiling medium being flushed with anoxic N2:CO2 gas from a probe; (C) 10 mLplastic syringe with long, blunt 16G needle being flushed out with anoxic N2:CO2 andfilled with anoxic medium; (D) medium being dispensed into the anoxic atmosphere ofa tube; (E, F) Hungate sealing a tube with a Balch stopper; (G, H) crimping analuminum seal in place.

6 Ralph S. Wolfe

liquid can be removed easily through a 21G needle on a syringe that hasbeen flushed with anoxic gas. Reducing agents are added last, whenpreparing a medium, to avoid excessive loss of volatile sulfide.

6. To prepare the anoxic medium add 200 mL of medium to a 500-mLflask and place the flask over a Bunsen burner (Fig. 1.4B). As themedium approaches boiling, insert a gassing probe with an N2:CO2

flow rate of at least 1 L per min and inject 3 mL of the 0.2 M cysteineHCl solution. Allow the medium to boil for about 1 min; then turn offthe burner. When boiling has stopped, inject 0.8 mL of the 0.2 MNa2S�9H2O solution and mix by swirling the flask.

7. Assemble 20 aluminum seal anaerobic culture tubes or 20, 40 mL serumvials with stoppers (Balch aluminum-crimp stoppers), aluminum seals, anda crimper (Bellco, Vineland, NJ). Place a gassing probe in each of twoempty culture tubes and start the flow of N2:CO2. Place a rubber bulb-type safety pipet-filler or a Scienceware type pipet pump filler/dispenser

7

8

9

10

11

12

13

Figure 1.5 Anoxic gas-dispensing attachment. (7) PVA tubing; (8) Swagelok 3 mmbrass union; (9) solder; (10) Leur Lok fitting with male tubing adapter, Beckton-Dickenson 3083; (11) plastic stopcock with Leur Lok inlet–outlet fittings; (12)0.2 mm sterile membrane filter, preferably with Leur Lok inlet–outlet fittings; (13)21G needle. Reproduced with permission, from Wolfe and Metcalf (2010).


on the end of a 10-mL pipet, or use a 15-cm long 16G needle (blunt end)on a 10cc plastic syringe. Place the pipet or 16G needle into the neck ofthe flask (Fig. 1.4C). Remove a syringe or pipet full of gas and expel itoutside the flask; repeat twice, then lower the needle or pipet into themedium and remove 10 mL. Place the needle or pipet into a tube that isbeing flushed out and inject the medium into the tube (Fig. 1.4D). Placethe syringe or pipet back into the flask. Perform a Hungate seal (Fig. 1.4Eand F) by pressing a Balch stopper into the mouth of the culture tube asthe gassing probe is withdrawn in one smooth motion. Place the gassingprobe into an empty tube. Likewise add the anoxic medium to each tube.Crimp an aluminum seal over each stopper (Fig. 1.4G and H).

8. Now the gas atmosphere in each tube can be adjusted to the needs of theexperiment. The medium is designed to have a neutral pH underpressure (173 kPa, 25 psi) of a gas mixture containing 20% CO2. Toexchange gas in a tube, use the gassing attachment (Fig. 1.5). Mostmethanogens can use hydrogen as substrate and reduce CO2 to methane;so set the regulator of the gas mixture of H2:CO2 (80:20) at 173 kPa(25 psi). When using gas atmospheres at negative or positive pressures,wear plastic goggles and work with the tubes or bottles behind aPlexiglas barrier. Close the N2:CO2 tank (Fig. 1.1) open the H2:CO2

tank, and turn on the vacuum pump. Close vacuum-release valve (9).

8 Ralph S. Wolfe

(Before turning off the vacuum pump, when finished, open valve (9) torelease the negative pressure in the line.)

9. To evacuate the N2:CO2 in a tube turn the three-way valve (Fig. 1.1(4))to VAC, insert the 21G needle (Fig. 1.5(13)) of the gassing attachmentthrough the Balch stopper, and open the plastic valve (Fig. 1.5(11)).After 10 s, turn the three-way valve to GAS and after 10 s turn the plasticvalve off and insert the needle in the stopper of another tube. Repeat thisprocedure to add H2:CO2 to each tube. Sterilize the medium at 121 �Cfor 20 min, then fast exhaust the autoclave.

2.3. The vacuum–vortex method

Recently, Wolfe and Metcalf (2010) described a quick method for the prepa-ration of anoxic solutions of nonvolatile compounds in culture tubes or in smallvials. This method also was used to prepare small volumes of an anoxicmedium in which a methanogen grew as well as in media prepared by boiling.This procedure avoids a boiling step and involves the use of the equipmentdescribed in Sections 2.1 and 2.2 with the addition of a common laboratoryvortex mixer such as a Fisher Vortex/Genie 2. In this procedure, 10 mL of asolution in a culture tube or small vial can be rendered anoxic in 90 s by threealternate cycles of strong vortexing under high negative pressure followed byaddition ofN2, (or withN2:CO2 for culturemedia). The gassing attachment isused as described in Section 2.2 (5) above, with the precaution that after eachvacuum-vortexing period a positive gas pressure is passed through the attach-ment to clear any liquid from the needle prior the next vacuum–vortex cycle;any liquid that enters the 0.2 mmmembrane may seal the filter, preventing gasfrom passing through the filter. If this happens, replace the filter.

1. Turn the three-way valve (Fig. 1.1(4)) to the OFF position. Turn on thevacuum pump and close valve 9 (Fig. 1.1). Adjust the gas pressure of N2

in the line to 100 kPa (15 psi). Set the speed of the vortex mixer to nearmaximum and to vibrate on contact.

2. Turn the three-way valve to the GAS position. Open the plastic stop-cock on the gassing attachment, and with gas flowing from the 21Gneedle, insert the needle through the Balch stopper of a tube thatcontains the aerobic solution that is to be made anoxic. Now, turn thethree-way valve to the VAC position for 10 s. Turn the plastic stopcockon the gassing attachment off and turn the three-way valve to OFF.

3. Hold the tube firmly (Fig. 1.6A) so that it does not rotate as the tube istouched to the vortex pad for 10 s (Fig. 1.6B).

4. Turn the three-way valve to GAS and open the plastic stopcock on thegassing attachment. This procedure ensures that any liquid in the needleis removed and adds gas to mix with any oxygenic gas that has beenremoved from the solution.

A B C D

Figure 1.6 The vacuum–vortex procedure for preparing anoxic solutions. (A) Tube isheld firmly with gassing attachment in place; (B) with its atmosphere under negativepressure tube contacts vortex mixer top; (C) 50 mL of liquid in a serum bottle; (D)serum bottle with its atmosphere under negative pressure contacts top of vortex mixer.


5. Turn the three-way valve to VAC and repeat the procedure (2–4) abovetwo more times. After the final step, pressurize the atmosphere in thetube to 173 kPa, 25 psi. Sterilize the anoxic liquid at 121 �C for 20 min;then fast exhaust the autoclave.

6. When this procedure is used for 50 mL amounts of liquid (Fig. 1.6C andD) the evacuation time of the atmosphere in the bottle should beincreased to 40 s and the vortex step to 30 s.

7. For culture media, prepare a medium aerobically without reducingagents, add it to tubes or vials, and by use of N2:CO2 instead of N2,render the medium anoxic as described in Section 2.3 (1–5) above. If H2

is to be the substrate, evacuate the N2:CO2 and replace it with H2:CO2.Now inject the appropriate amount of cysteine and sodium sulfide fromanoxic solutions of these reducing agents to complete the medium;sterilize the medium and fast exhaust the autoclave. For more details,consult Wolfe and Metcalf (2010).

2.4. Anoxic, aseptic use of a syringe

1. Prepare a gassing probe (Fig. 1.3D) for aseptic use by passing the 16Gneedle through the flame of a gas burner. Heat exchange with the metalis so efficient that only a brief exposure to the flame is necessary. Replacethe probe in its holder.

10 Ralph S. Wolfe

2. Add a sterile needle to a sterile 1 mL plastic syringe, and with anoxic gasflowing through the probe, insert the 21G needle into the 16G needleand pump the plunger a few times; then fill the syringe with sterileanoxic gas. If the gas is H2:CO2, hold the syringe upright to retain H2 inthe syringe.

3. Insert the needle into the flamed, rubber stopper of a tube or bottle as theplunger of the syringe is held firmly in place to avoid its being dislodgedby the pressurized gas atmosphere. Invert the vessel, expel the gas fromthe syringe, and fill it with sterile fluid while holding the piston firmly.When removing the syringe needle from a rubber stopper, hold theplastic base of the syringe needle firmly to prevent separation of theneedle from the syringe body. Transfer the liquid through the flamed,sterile stopper of the receiving vessel.

4. To economize, plastic syringes with needles may be reused many times.Simplywash out the syringe by pumping the plunger as the needle is held inrunning demineralized water. Then separate the plunger from the syringebody to prevent its rubber tip from being compressed during sterilization,and hang the syringe body and plunger in an 18 mm (dia) test tube. Add a21-mm (inside dia) plastic cap to the tube, or cover the end of the tubewithaluminum foil, and sterilize; then fast exhaust the autoclave.

2.5. Growth of methanogens on agar medium, the bottle plate

To isolate a methanogen in pure culture use of the Petri plate in an anoxicchamber is the method of choice (Section 3). Methods for obtaining isolatedcolonies on agar at the lab bench are described in this section and inSection 2.6. Use of an improved bottle plate, Bellco, (Olson, 1992) toobtain isolated colonies of a methanogen is the most simple method foruse of agar (Herman et al., 1986).

1. To the desired amount of medium (Section 2.2) in a 500-mL flask, addagar to yield a final concentration of 1.5%. Add about 300 mL of waterto a 1-L metal beaker that has been placed on a ring stand above a gasburner. Clamp the flask of medium so that its bottom is immersed in theboiling water, and add a gassing probe from which N2:CO2 is flowing.Heat the medium with occasional swirling of the flask until the agar iscompletely melted, then add solutions of cysteine and sodium sulfide.Turn off the burner. Follow the procedures in Section 2.2 to dispensethe anoxic agar medium, 10 mL per bottle. Hungate seal each bottlewith a Balch stopper, add a screw cap, and pressurize the atmospherewith N2:CO2 to 105 kPa (15 psi).

2. After sterilization, cool the bottle of medium in a water bath to about50 �C; then manipulate the agar into the plate area of the bottle, placethe bottle in a horizontal position so that the bottom reservoir is devoid


of agar, and allow the agar to solidify. Store the bottle in an uprightposition (Fig. 1.8B) so that water of condensation moves to the bottom,allowing the agar surface to remain free of water.

3. To inoculate the agar, insert the sterile needle of a gassing attachmentwith N2:CO2 flowing through the bottom Balch stopper (Fig. 1.8C).Aseptically, remove the top Balch stopper, add a drop of inoculum to thelower region of the agar, and streak the inoculum over the agar surfaceby using a streaking probe made by melting the tip of a Pasteur pipet intoa small glass ball. Dip the pipet into alcohol and touch it briefly to a flamebefore use. Hungate seal the stopper in place, replace the screw cap, andchange the atmosphere in the bottle to H2:CO2. Incubate the bottle inan upright position.

4. After incubation, repeat the procedures to add N2:CO2 to the bottompart and open the top stopper. An isolated colony may be picked with aplatinum or ss-inoculation loop, transferred quickly to the anoxic agarsurface of another bottle, and streaked. After incubation, if all “secondplate” colonies are identical, one colony may be considered to representa pure culture.

2.6. Growth of methanogens in a Hungate roll tube

The Hungate roll tube (Bryant, 1972; Hungate 1950, 1969; Sowers andNoll, 1995) is the most economical method to obtain isolated colonies onagar. The inoculated agar medium in a culture tube is rotated in flowingcold water to solidify the medium on the inside surfaces of the tube, creatinga cavity within the tube which can be pressurized with H2:CO2. Colonieswhich grow in the agar can easily be picked by use of aseptic, anoxicprocedures. There are many variations of these procedures, includingmethods for streaking a roll tube. Holdeman et al. (1977) popularized anadaptation of the roll tube method for use in clinical diagnostic laboratories.Balch-stoppered roll tubes are readily adapted to these procedures forgrowth of methanogens.

The procedures presented below are intended as a simple introductionto the use of a roll tube.

1. Prepare 200 mL of anoxic medium as described in Section 2.2 and addagar as described in Section 2.5. Add 9 mL to each culture tube; use ananoxic gassing probes with N2:CO2. After sterilization, equilibrate thetubes in a water bath at 45 �C.

2. By aseptic, anoxic use of a syringe (Section 2.4) transfer l mL of a liquidculture or enrichment of a methanogen to a tube of melted agar mediumin the water bath and invert the tube three times to mix the inoculum.With a warm sterile, anoxic syringe quickly transfer 1 mL of agarmedium from the first tube to the second tube. Invert this tube and

12 Ralph S. Wolfe

likewise carry out a serial dilution through six tubes of agar medium.Rotate each tube under cold, flowing water to form a roll tube. Evacuatethe atmosphere in each tube and add H2:CO2 to 173 kPa (25 psi).Incubate the tubes in an upright position.

3. After incubation, release some of the gas pressure in the tube thatcontains isolated colonies by penetrating the stopper with a sterile 21Gneedle. Place the tube in a clamp on a ring stand and remove the Balchstopper as a sterile gassing probe with N2:CO2 flowing is inserted intothe tube. Place the probe in a clamp to hold it firmly in position. In asimilar manner, position a tube that contains a few milliliter of sterilebroth medium and insert a sterile gassing probe into the tube.

4. Pick an isolated colony and inoculate the broth. Hungate seal this tubeand suspend the cells well; then use 1 mL of this suspension for a serialdilution through melted agar medium. If, after incubation, all “secondplate” colonies in a tube of high dilution appear to be of the same type,it may be assumed that cells in an isolated colony represent a pure culture.

2.7. Preparation of a 3-L amount of anoxic medium

The apparatus described in this section for the preparation of large amountsof medium is designed to be fabricated by laboratory personnel from readilyavailable materials for the convenient delivery of media (up to 3 L) from ananoxic reservoir where the pressure is 35 kPa (5 psi), not higher!

1. Obtain a standard, 5 L round-bottom Pyrex flask and a #11 solid black-rubber stopper, which fits snuggly into the neck. Drill two holes throughthe stopper that will accommodate 5 mm (dia) ss-tubing and one hole for3mm(dia) ss-tubing. Space the holes evenly on the stopper (Fig. 1.7A–C).To construct a gas-exit port, insert a piece of ss-tubing about 5 cm longand 5mm(dia) so that one end is flush with the bottom of the stopper. Tothe upper end of this tube attach a piece of rubber tubing and a standardplastic stopcock (Fig. 1.7A(1)). The hole through the rotator plasticcylinder of the stopcock should have a diameter of about 3 mm.

2. To prepare a dispensing tube, insert an ss-tube 10 cm long and 5 mm(dia) so that about 5 cmprojects below the bottomof the stopper. To thislower end, attach a piece of Nalgene 180 pvc nontoxic-grade plastictubing about 15 cm long and add a piece of ss-tubing about 10 cm longto serve as a weight, the total length of tubing inside the flask being about27 cm long, extending to just above the bottom of the flask.

3. To the top end of the ss-tube, which projects above the stopper(Fig. 1.7A(4)) attach a piece of Nalgene tubing about 65 cm long(Fig. 1.7B). To the end of this tubing, add a standard plastic stopcockwhich has attached a piece of Nalgene tubing about 6 cm long(Fig. 1.7B(8)).

1

87

6

5

4

3

2

1

D E F

A B C

9

2 3 4

Figure 1.7 Method for preparing 3 L of anoxic medium in a 5-L flask. (A) stopper andattachments: (1) plastic stopcock; (2) stainless-steel connector, upper ferrules replacedwith a rubber septum; (3, 4) 65 cm long Nalgene tubing; (5) 5 cm long strong stainless-steel spring, also see (c(9)); (6) #11 black-rubber stopper; (7) stainless-steel hose clampwith cut and bent threads; (B) view of complete apparatus with dispensing stopcock (8);(C) anoxic medium being cooled and gased in an ice bath: (9) 5-cm stainless-steelspring with iron wire attachments; (D) anoxic medium being dispensed into a smallbottle; (E) into a 500-mL bottle; and (F) anoxic medium sealed in a (1) 158-mL bottle,(2) 500-mL bottle, and (3), (4) in 1 L bottles.


4. Prepare a port for gas injection by inserting a 3-mm ss-tube about20 cm long through the rubber stopper so that about 8 cm projectsabove the stopper. To this end, affix a Swagelok-type ss-union(Fig. 1.7A(2)). Unscrew the top nut of the union, and remove the

14 Ralph S. Wolfe

front and back ferrules. Insert into the nut a rubber plug 5 mm longprepared from a Balch stopper by use of a No. 4 cork borer. Screw thenut firmly in place. In operation of the system, the rubber plug serves asa septum through which anoxic gas is provided from a 21G needle onthe gassing attachment (Fig. 1.7B).

5. To hold the stopper in place, obtain an ss-hose clamp that fits around theneck of the flask, which is about 6 cm (dia). To provide attachments forthe spring (Fig. 1.7A(5) and C(9)), cut and bend (Fig. 1.7A(7)), three ofthe threads on the hose clamp band which are about 4 cm from the screwhousing. On the opposite side of the neck, cut and bend a prong about4 mmwide from the solid ss-band of the clamp. These cuts of the ss-bandare easily performed with a small motorized hand tool (Dremel, Racine,WI) equipped with a reinforced, metal cutting, 426 fiber disc.

6. To each end of a strong ss-spring (Fig. 1.7C(9)), which is about 5 cmlong, add a loop of iron wire to serve as attachments to the prongs ofthe hose clamp, when the spring is stretched over the rubber stopper(Fig. 1.7A–C).

7. Clamp the 5 L flask on a ring stand so that the bottom of the flask issupported by a wire gauze on a ring above a Bunsen burner. Add 3 L ofmedium (without reducing agents) to the flask. The advantage of a 5-Lflask is that 1–3 L amounts of medium are easily accommodated.

8. Open gas-exit stopcock (Fig. 1.7A(1)), close medium dispensing stop-cock (Fig. 1.7B(8)), seat the rubber stopper firmly in the mouth of theflask, and stretch the ss-spring in place (Fig. 1.7A). Ignite the Bunsenburner gas.

9. Set the N2:CO2 tank line pressure so that the pressure gauge at thegassing station reads 35 kPa (5 psi), not higher. This low pressureprovides sufficient anoxic gas flow over the contents of the 5 L flaskduring medium preparation and later provides a sufficient pressure toforce the flow of medium, when it is being dispensed.

10. When the medium approaches boiling, insert the 21G needle on thegassing attachment, through the rubber septum in the ss-union(Fig. 1.7A(2)) and start the flow of N2:CO2.

11. Previously, prepare a solution of cysteine�HCl by dissolving 1.5 g in20 mL of hot distilled water in a beaker. Bring the solution into a 20-mLplastic syringe equipped with a 15-cm long, blunt 18G needle. Placethe needle through the gas-exit stopcock (Fig. 1.7A(1)), and inject thesolution into the 3 L of medium.

12. Bring the medium to a low boil for about 1 min as the resazurin in themedium is reduced to its colorless form. Turn off the Bunsen burnerand continue the N2:CO2 gassing for about 20 min as the mediumcools somewhat; then lower the flask carefully into an ice bath(Fig. 1.7C) as gassing continues.


13. Swirl the medium in the flask and, when it has cooled to the pointwhere it is no longer too-hot-to-touch, place the flask back on the ringstand, and inject 6 mL of 0.2 M sodium sulfide through the gas-exitstopcock (Fig. 1.7A(1)). Mix the medium by swirling the flask. (Thebasal medium is now anoxic without substrate. If H2 is to be thesubstrate, the N2:CO2 atmosphere can be replaced with H2:CO2

after the medium has been dispensed. (If the substrate is to be methanol,acetate, formate, or methylamines, the desired substrate can be injectedat this time or can be injected later to the desired bottles of sterilemedium from a sterile, anoxic stock solution of substrate.)

14. Prepare to dispense the anoxic medium and Hungate seal each con-tainer by assembling the desired bottles, stoppers, and caps. (Tubing ofsterile medium into culture tubes is described in Section 2.9) For 50 mLamounts of medium the “125” mL size serum bottles (Wheaton) areuseful (Fig. 1.7D). Each bottle has a volume of 158 mL with a Balchstopper in place. For a 250-mL amount of medium, a 500-mL heavy-walled, graduated serum bottle (Wheaton) with a #1 black-rubberstopper that can be crimped in place with a large crimper, is ideal(Fig. 1.7E). For a 500 mL amount of medium, a 1-L graduated medialab bottle (Wheaton) can be modified for use by drilling a 2-cm (dia)hole in the plastic screw cap. Cut off an aluminum-crimp culture tubeabout 6 cm from the top and insert the cut end through a #9 bored holein a black-rubber #4 stopper (Fig. 1.7F(3)). These modified bottleshave been used successfully for decades (Balch and Wolfe, 1976; Balchet al., 1979). An alternative for a 500-mL amount of medium is a 1-Lbottle with a wide mouth, rubber septum, and aluminum screw cap(#191000 Transfusions flaschen, 1L, Glasgeratebau OCHS, 37120Bovenden, Germany).

15. For anoxic dispensing of the anoxic medium, place a gassing probe(Fig. 1.7D), in each of two receiving bottles with a flow of N2:CO2

from each probe. Close stopcock 1 (Fig. 1.7A), open stopcock8 (Fig.1.7B) and allow some medium to flow into a beaker, replacingair in the dispensing tube. Discard this medium.

16. Insert the plastic tubing at the end of the dispensing tube into the neckof a receiving bottle (Fig. 1.7D), which now has an anoxic atmospherefrom the gassing probe, and open the stopcock (Fig. 1.7D) and allowthe desired amount of medium to flow into the bottle. (From 3 L ofmedium, 300 test tubes may be filled, sealed, and crimped. By use of aneight-port gas-dispensing station, the atmosphere in each of eight tubescan be simultaneously exchanged with H2:CO2 and pressurized priorto sterilization.)

17. Transfer the dispensing tube to an empty bottle that is being gassed by aprobe, and Hungate seal a stopper in the bottle to which the mediumhas just been added.

16 Ralph S. Wolfe

Move the gassing probe into an empty bottle. Likewise, dispense theanoxic medium into the desired number of bottles.

18. Prior to sterilization increase the pressure of N2:CO2 in each bottle to70 kPa (10 psi) by use of the gassing attachment (Fig. 1.5). For the 3 Lof medium to be steam-sterilized in an autoclave, the time exposure at121 �C should be increased to 1 h. For safety, the 1-L bottles areisolated in metal containers, should breakage occur. An ss-SterilizerBox (Fisher) for glass Petri dishes is ideal for use with a 1-L bottle.

19. After sterilization, the gas atmosphere in each bottle can be asepticallyadjusted to meet the needs of the experiment.

2.8. Cultivation of marine methanogens

To cultivate methanogens from a marine environment, the medium(Section 2.2 (1–6)) is modified by increasing the amount of NaCl to400 mM, MgCl2�6H2O to 54 mM, and CaCl2�2H2O to 2 mM. Use ofthe vacuum–vortex system (Section 2.3) is the most simple way to prepare amarine medium. For methods that require boiling of the medium, it isessential to prepare a separate solution that contains the components, MgCl2and CaCl2, to avoid formation of an insoluble precipitate during the initialboiling step; this separate solution is treated exactly in the manner for thepreparation of anoxic media as previously outlined. After cooling theanoxic, divalent-metal solution, it is combined with the cool, anoxic,marine-medium solution and swirled to mix. Add an anoxic substratesuch as methanol at this time. Dispense the complete anoxic medium intotubes or bottles. A precipitate will form during heat sterilization, but it willdissolve on standing at room temperature.

2.9. Aseptic transfer of sterile medium

The storage of sterile medium in bottles (Section 2.7) has the advantage thatthe investigator has flexibility in designing experiments. A bottle of sterilemedium can be inoculated directly for culture of a methanogen in largerquantities, or the sterile medium can be modified as desired and asepticallydispensed into tubes.

1. To dispense sterile medium from a bottle use of a male–male Leur Lok,in line, ss-stopcock (Sigma-Aldrich) (Fig. 1.8A) is most efficient. Such astopcock is expensive but lasts forever. A less expensive alternativeconsists of a chrome-plated brass, male to male Leur Lok needle connec-tor attached to a chrome-plated, one way, spring-clip stopcock, femaleLeur to male needle Leur Lok PS-6021 (Sigma-Aldrich), but chrome-plated brass stopcocks must be disassembled and washed thoroughly after

A

C

B

1

1

1

2 2

2

3

3

45

Figure 1.8 Aseptic techniques. (A) Sterile medium being dispensed into a culturetube: (1) bottle with sterile medium under a positive gas pressure, (2) sterile male–malestopcock with attached needles, (3) sterile, empty culture tube with negative gaspressure; (B) bottle plate: (1) agar medium, (2) reservoir for collecting moisture; (C)bottle plate held in position for streaking: (1) 0.2 mm filter, (2) bottom gassing port, (3)agar surface, (4) threads for cap, (5) stopper held in removal tool.


each use to prevent corrosion of brass by sulfide in the medium. Wrapthe metal ss-stopcock or the chrome-plated stopcock and connector inaluminum foil and sterilize in an autoclave; fast-exhaust the autoclave.

2. To each test tube, add a drop of distilled water from a 21G needle on asyringe and crimp a Balch stopper in place. This drop of water willprovide enough moisture so that the interior of the tube is subjected tomoist heat and can be sterilized at 121 �C for 20 min.

3. By use of the gassing attachment (Fig. 1.5), evacuate the air from a tubefor 10 s, add anoxic gas for 10 s, and then evacuate the gas for 10 s.Likewise, treat each tube. Sterilize the tubes in an autoclave; then

18 Ralph S. Wolfe

fast-exhaust the autoclave. Do not store tubes with a negative pressure,but dispense medium into them fairly soon.

4. The atmosphere in the bottle of medium should have a pressure of atleast 70 kPa (10 psi), so that the medium flows readily from the bottle.Invert the bottle and flame the rubber stopper. Place the inverted bottlein a clamp on a ring stand (Fig. 1.8A). Remove the aluminum foil from asterile ss-male–male Leur lok stopcock and add a sterile 21G needle toeach end. With the stopcock open, insert one needle through the rubberstopper of the bottle, and immediately close the stopcock after a smallamount of medium has passed into a beaker.

5. Insert the other needle of the stopcock through the flamed Balch stopperof a tube which has an internal negative pressure. Open the stopcock andallow 10 mL of medium to flow into the tube. Likewise add sterilemedium to each tube.

6. For culture of a methanogen on H2:CO2, flame the Balch stopper of thetube. Add a sterile needle to the gassing probe (Fig. 1.5) and asepticallypressurize the atmosphere in the tube to 173 kPa (25 psi). Likewiseprepare each tube prior to inoculation.

3. Use of an Anoxic Chamber

The laboratory of W. W. Metcalf at the University of Illinois haspioneered investigations into the genetics of Methanosarcina. These studieshave reached a level of sophistication such that almost any genetic or mole-cular procedure that can be performed with Escherichia coli can now beperformedwithMethanosarcina. An essential component of such investigationsis the Rolf Freter anoxic chamber (Fig. 1.9A), manufactured by Coy Labora-tory Products. Although this double chamber model is a significant financialinvestment, it is an indispensable tool for serious genetic and molecularstudies. A floor space of about 3 � 7 m is required for the chambers,compressed gases and ceramic O2 scrubbers (Fig. 1.9A). One of the chambersis used for dispensing anoxic liquid or agar medium, and for genetic ormolecular studies. The adjacent chamber is used to house a Coy intrachamberincubator (Metcalf et al., 1998) and plastic, anoxic jars (such as GASPAK) forincubation of inoculated Petri plates. Erratic growth of methanogens in suchjars occurs unless the jars are continuously stored and used in the chamberwhere O2 has had time to diffuse out of the plastic. Use of individual jarsprovides flexibility for incubation of plates under various conditions, and theCoy intrachamber incubator has space for more than 100 plates.

1. Gases for the chambers are passed through ceramic heated scrubbers(Fig. 1.2D and E). The gas atmosphere in the chambers consists of N2,

A

C D E

B

Figure 1.9 Working anoxic chambers in the laboratory of W. W. Metcalf. (A) dualchambers made by Coy, (B) intrachamber dry-heated bath for storing melted agarmedium prior to pouring of Petri plates, (C) automatic air lock located betweenchambers, (D) mechanism for holding a #10 stopper in a 3-L flask that contains anoxicmedium as it is passed through the negative pressure of the air lock, (E) intrachamberincubator with inoculated Petri plates under an N2:CO2:H2S atmosphere.


and the presence of 20% CO2 is essential for handling media with a CO2–carbonate buffering system. Because O2 slowly diffuses through the plasticwalls, an H2 component of the atmosphere is maintained below 5% so thattwo catalytic O2 scrubbers efficiently remove O2 from the atmosphere.The pelleted catalyst may be removed and regenerated in an oven at250 �C. (When using H2 in an anoxic chamber, there is no substitutefor eternal safety vigilance! To ensure that the H2 concentration in achamber does not exceed 5%, the Metcalf laboratory uses only a tankmixture of N2:CO2 with 5% H2 to fill the chambers. The most seriouspotential danger lies in careless operation of the air lock. For example, if ahigh level of H2 accumulates in a chamber, and the chamber door of theair lock is opened to an air lock full of air, the catalyst may turn red-hot,igniting an explosion.) A separate electric fan adds to the circulation of the

20 Ralph S. Wolfe

atmosphere. To avoid poisoning the catalyst by fumes of H2S from theculture media, a bed of charcoal is placed on a tray above the catalyst toscrub H2S form the atmosphere. It is important to replace the charcoaloften, depending on the use of the chamber.

2. An essential component of the internal equipment is a particle scrubberdesigned to supply germ-free air to a germ-free mouse cage. Thisscrubber runs continuously so that aseptic procedures can be performedin the chamber.

3. Operation of the air lock involves programmed cycles of negativepressure followed by addition of anoxic gas. Any vessel that containsliquid must have a solid rubber stopper that is securely held in place,when placed in the air lock. Figure 1.9D shows one type of mechanismused in the Metcalf laboratory for clamping a stopper in a 3-L flask.

4. To transfer plastic Petri dishes through the air lock, prick a hole in theplastic cylindrical cover so that gas exchange may take place. (Oneperson, MEM, prefers to place a Band-Aid to serve as a gas filter overthe opening.) Prior to use, it is essential to store the dishes in thechamber for days prior to use, so that O2 may diffuse out of the plastic.

5. One method for the preparation of anoxic agar for Petri plates is to storepreweighed amounts of agar folded in aluminum foil in the chamber sothat O2 has time to diffuse out of the dried agar. Then, for example, mostof the gas pressure in a stored, 1-L bottle that contains 250 mL of sterilemedium is released by inserting a 21G needle through the stopper priorto transfer into the chamber. Inside the chamber remove the stopper andpour the anoxic agar through a dry plastic funnel into the liquidmedium. Recrimp the stopper and transfer the bottle out through theair lock. Pressurize the N2:CO2 atmosphere, place the bottle into a metalcontainer, and sterilize it; then fast exhaust the autoclave. Carefully usegloves and swirl the hot medium in the container to very gently mix theagar; allow it to cool to about 60 �C.

6. Aseptically release most of the gas pressure in the bottle with a sterile21G needle. Place the bottle in a metal container and transfer it into thechamber. Aseptically inject any additional components of the medium,such as an antibiotic, at this time. To avoid premature cooling of theagar, the bottle is placed in a heated cavity of a modified dry bath (Fisher)(Fig. 1.9B), until the medium is poured into Petri dishes.

7. For growth of Methanosarcina, the Petri plate must be incubated in thepresence of 0.1% H2S in the atmosphere, which if present in thechamber will poison the catalytic O2 scrubber. Separate gas lines ofN2:CO2:0.1% H2S or H2:CO2:0.1% H2S as well as a vacuum line areconnected to the intrachamber incubator and to a port with rubbertubing for use with the GASPAK jars. To incubate inoculated Petriplates, the atmosphere in the intrachamber incubator or GASPAK jar isevacuated and replaced with a pressurized, appropriate gas mixture that


also contains 0.1% H2S. For culture of methanogens that are usingsubstrates other than hydrogen, the N2:CO2 atmosphere in the incuba-tion vessel is not pressurized; when H2:CO2 is used, the atmosphere ispressurized. (For extra safety precautions in the laboratory of John Leigh,to each inlet and outlet port on the top of an incubation jar for Petriplates is attached a quick-connect flexible metal hose (Swagelok ss-FL4-TA4-24); the male parts (ss-QC4-D-400) allow gas to flow only whenthe connection is made and shut off when uncoupled. Each metal hose isattached to a four-way cross-over valve with Swagelok fittings, allowingthe lines and the jar to be flushed and the jar filled without the possibilityof a line disconnection.) Seals on the incubator and jars must be perfect!To examine the plates after incubation, evacuate the atmosphere abovethe Petri plates and allow the chamber atmosphere to enter the incubatorvessel. There is no substitute for visiting a working laboratory andobtaining first-hand training and experience with the operation of ananoxic chamber.

ACKNOWLEDGMENT

I thank W. W. Metcalf for helpful suggestions and for use of his laboratory equipment.

REFERENCES

Balch, W. E., and Wolfe, R. S. (1976). New approach to the cultivation of methanogenicbacteria: 2-Mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobac-terium ruminantium in a pressurized atmosphere. Appl. Environ. Microbiol. 32, 781–791.

Balch, W. E., Fox, G. E., Magrum, L. J., Woese, C. R., and Wolfe, R. S. (1979).Methanogens: Reevaluation of a unique biological group. Microbiol. Rev. 43, 260–296.

Bryant, M. P. (1972). Commentary on the Hungate technique for culture of anaerobicbacteria. Am. J. Clin. Nutr. 25, 1856–1859.

Herman, M., Noll, K. M., and Wolfe, R. S. (1986). Improved agar bottle plate for isolationof methanogens or other anaerobes in a defined gas atmosphere. Appl. Environ. Micrbiol.51, 1124–1126.

Holdeman, L. V., Cato, E. P., and Moore, W. E. C. (1977). Anaerobe Laboratory Manual.4th edn. V. P. I. Anaerobe Laboratory, Blacksburg, VA.

Hungate, R. E. (1950). The anerobic mesophilic cellulolytic bacteria. Bacteriol. Rev. 14,1–49.

Hungate, R. E. (1969). A roll-tube method for cultivation of strict anaerobes. MethodsMicrobiol. 3B, 117–132.

Metcalf, W. W., Zhang, J. K., and Wolfe, R. S. (1998). An anerobic, intrachamberincubator for growth of Methanosarcina spp. on methanol-containing solid media. Appl.Environ. Microbiol. 64, 768–770.

Olson, K. D. (1992). Modified bottle plate for the cultivation of strict anaerobes. J. Microbiol.Methods 14, 267–269.

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Sowers, K. R., and Noll, K. M. (1995). Techniques for anaerobic growth, methanogenicarcheae. In “Archaea, a Laboratory Manual,” (F. T. Robb, A. R. Place, K. R. Soweres,H. J. Schreier, S. DarSarma, and E. M. Fleischmann, eds.), pp. 17–47. Cold SpringHarbor Laboratory Press, NY.

Wolfe, R. S., and Metcalf, W. W. (2010). A vacuum-vortex method for preparation ofanoxic solutions or liquid culture media in small amounts for cultivation of methanogensor other strict anaerobes. Anaerobe 16, 216–219.

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