- Email: [email protected]
[7] An anaerobic laboratory
[7]
AN ANAEROBIC LABORATORY
49
tained, permitting the use of solute-detecting apparatus distal to the concentration device. Such systems do not interfere with further flow into a fraction collector along with concomitant registration of the collected fractions on the recorded pattern. Comments
A large number of UF systems for direct application to enzyme chemistry have been reviewed. In selecting among these techniques, a number of considerations exist. Choice of membrane should not be entirely dictated by flux, but rather lack of adsorption; systems should be chosen to provide the maximum depolarization, i.e., to achieve maximum flux, commensurate with the need to ensure minimal denaturization. When in doubt for labile proteins, the single-pass system is probably best suited; if the enzyme can withstand the shear, graduation to recirculating systems with the attendant higher flux may be warranted.
[7] An Anaerobic Laboratory ByJ. MICHAEL POSTON, THRESSA C. STADTMAN, a n d EARL R. STADTMAN The investigation of many aspects of microbiology and biochemistry is hampered by the oxygen lability of the material under study; enzymes, electron carriers, and metabolic intermediates may be rapidly lost in the presence of air. Many bacteria are severely inhibited or may be killed by exposure to air. These properties have made the study of anaerobic mutants particularly difficult since many of the techniques that are well established for use with aerobes cannot be adapted to anaerobic use. Although any given manipulation can be carried out in conventional anaerobic glove boxes, suitably equipped with remote control devices, experimental operations become extremely difficult, if not impossible, when they involve multistep procedures employing filtration, chromatography, electrophoresis techniques or the use of massive instrumentation such as spectrophotometry, centrifugation, and refrigeration. To facilitate anaerobic experimentation under conditions that allow great versatility in the utilization of standard laboratory techniques, an anaerobic laboratory chamber was constructed? The chamber is a ~The anaerobic facility at the National Institutes of Health, Bethesda, Maryland, was designed by and built under the supervision of the Linde Division of the Union Carbide Corporation.
50
GENERAL METHODS
[7]
space of approximately 1400 cubic feet. (40 cubic meters) enclosed by a gas-tight partition. By displacing most of the air with N2 gas and then removing the remaining oxygen by combining it with hydrogen on a catalyst bed, oxygen tensions of less than 100 ppm can be maintained. The laboratory contains a fume hood, incubators, and a cold room. Most portable laboratory equipment may be used in this chamber. A fairly wide spectrum of experiments have been conducted in its anaerobic environment.
Description The anaerobic chamber was constructed in a laboratory module and its dimensions were, in part, dependent upon the existing structure. The gas-tight partition is made of 3/~ inch carbon steel plates supported on a framework of I-beams. All the floor plates are riveted to the concrete floor and all joints and seams are welded. Doors to the gas locks and the emergency doors were fabricated at the site from the same steel as the shell of the chamber. Observation windows are of double-strength safety glass. The cold room is a commercial, walk-in refrigerator box of sufficient size to accommodate a fraction collector or similar equipment used in cold-room operations. Refrigeration is supplied by liquid nitrogen which sprays as saturated gas at --195 ° from a perforated pipe (the spray header) mounted at ceiling level (Polar Stream System, Linde Division, Union Carbide Corp.). The cold room is designed to hold any temperature between - 1 0 ° and +4 °. Its use is optional. The temperature can be brought from ambient (ca. 25 °) to operating levels in 30-60 minutes. Nitrogen for the cold room is conveyed directly from a large storage vessel outside the building through insulated copper tubing. T h e same storage vessel is the source of the nitrogen atmosphere in the chamber itself. It is passed first through a vaporizer assembly that is bathed in water at 21 ° and then through a volume meter before entering the anaerobic chamber.
Operation of the Chamber Placing the chamber in service once it is stocked with supplies and equipment is a relatively simple matter. Prior to closing the doors, the drain in the waste sink is filled with water. After checking the operation of the emergency system, the chamber is sealed and nitrogen is added through the purge valve. The chamber is vented at the other end of the N2 circulation loop (Fig. 1). After about an hour, the 02 level is less than 0.5% oxygen-in-nitrogen (Beckman Oxygen Analyzer,
[7]
AN ANAEROBIC LABORATORY
"~ EMERGENCYEXHAUST PURGING _. r...~._ ~] N2"MAKEUP
HUMIDFIER~
N, 7,VALV~
"-<"" , H2-ADDITION 0'T'O"."2"4 m~'~ ~ ~ L,
'~--~
h
I
~ ~
,OR~--:--I IH
.~
PRESSURE
I*I EMERGENCY .RELIEF I'1 EX.AUST /DEVICE
f-COZY/.. '~ -~,¢)1. , ~ E . ~
/
Lo~K ~ \
i
.AIN
LABORATORY ~
LOUVRES
It -
~.~ ~ v ' ~ ' T "~ ~ I L_. ._J
~
CRITICAl
CRITICAL
PURGEVENTVALVE IIT ][ JI
I..~c.".( ~ "~"
PORT
I~
H2ipS~MT PLE '
~,~.
'IO2SAMPLE
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51
,
,
I F-'/~VENT
D~N
l~l
[
I
,
VALVE
I
IHOO1
Fie. 1. Schematic illustrating the circulation and purification of gases within the chamher. Model D2). When this level is reached, the purge is stopped, recirculation is begun, and the addition of hydrogen is started at a rate of about 20 standard cubic feet (scf) per hour. The hydrogen content is monitored with a thermal conductivity analyzer (Model 204A, Analytic Systems Co.) and automatically maintained at 0.5-0.6%. After 4-5 hours of recirculation over a catalyst bed (Deoxo Gas Purifier, Model D-10,000-100, Engelhard Industries), the oxygen level of the hydrogen enriched atmosphere is less than 100 ppm. After 6-7 hours of recirculation, the atmosphere is less than 20 ppm oxygen. The tension continues to drop as recirculation continues and reaches a minimum of 2-3 ppm after about 48 hours. Oxygen tension is measured electrometrically by an oxygen trace analyzer (ASC Model 306 W, Analytic Systems Co.) in which a continuous stream of the gas to be monitored is passed through a cell containing a lead plate covered by a KOH solution in which a silver screen is suspended. The generation of current is governed by the following reactions: O ~ + 2 A g ~ 2AgO 2AgO+2H20+4e~2Ag+4(OH)2Pb+4(OH)-~2PbO+2H~O+4e A pressure-sensing make-up valve keeps the environment within the chamber at about 1 inch (water column) above the atmospheric pressure. Nitrogen lost through leakage or through operation of the fume hood is replaced by this system. The maximum pressure within the chamber is controlled by the pressure release device (Fig. 1). This
52
GENERAL METHODS
[7]
is a vented water tank containing a weir so a r r a n g e d that gas on the internal side will escape u n d e r it if the pressure rises above 4 inches o f water.
Life Support and Emergency System T h e life-support system consists o f face masks (two individuals may work within the c h a m b e r at once) to which are attached a b r e a t h i n g air line, a v a c u u m line to r e m o v e exhaled air, an o x y g e n m o n i t o r for the b r e a t h i n g air, and an alarm buzzer. B r e a t h i n g air is supplied to the mask by a c o m p r e s s o r h o u s e d nearby. I f this should fail, a large tank o f breathing air (4 hours' supply) is automatically c o n n e c t e d to the air line. I f this back-up source o f air should fail or be e x h a u s t e d , a pressure activated alarm notifies the p e r s o n in the c h a m b e r to o p e n the valve on the small bottle o f b r e a t h i n g air that is carried o n the harness o f the life-support system. This second back-up source has a m i n i m u m o f 7 minutes o f air and will p e r m i t the p e r s o n to make an o r d e r l y exit f r o m the c h a m b e r . D u r i n g the time w h e n a n y o n e is inside the c h a m b e r , a second p e r s o n is outside observing the operations and well-being o f the o n e inside and m o n i t o r i n g the o x y g e n level o f the b r e a t h i n g air and o f the c h a m b e r a t m o s p h e r e . T h e r e are observation windows in the wall and each o f the doors, as well as strategically placed mirrors so that at no time is the person inside the c h a m b e r out o f sight or h e a r i n g o f the monitor. In the event o f a situation in which it is vital to aid the p e r s o n within the c h a m b e r , a b u t t o n on the control panel activates the e m e r g e n c y system. T h e e m e r g e n c y doors and the butterfly drain valve on the pressure release device o p e n pneumatically. Large exhaust blowers (Fig. 1) begin to draw fresh air into the c h a m b e r t h r o u g h the e m e r g e n c y doors. Within about 7 seconds, the a t m o s p h e r e inside the c h a m b e r will s u p p o r t h u m a n life. Even in the event o f an electric power failure, the e m e r g e n c y doors can be o p e n e d individually by means o f the p n e u m a t i c system or by m a n u a l release o f the latches.
Entrance to the Laboratory Access to the c h a m b e r is p r o v i d e d t h r o u g h the p e r s o n n e l lock (Fig. 2) in which the life-support mask is d o n n e d . Once the mask is tightly fitted to the face, the w o r k e r closes the outside d o o r and the lock is p u r g e d . D u r i n g this time the pressure within the c h a m b e r is raised close to its m a x i m u m in o r d e r to p r e v e n t the p u r g e f r o m driving o x y g e n t h r o u g h the d o o r seals into the anaerobic room. After 13 minutes, the p u r g e is e n d e d and the main laboratory is e n t e r e d . T h e i n n e r laboratory may be e n t e r e d t h r o u g h the critical lock. In practice, it has been f o u n d that the inner critical laboratory and the main laboratory are sufficiently
[7]
AN ANAEROBIC LABORATORY
53
I
FA~
J 0 N2 ENTRANCE EMERGENCY[T~ CRITICALLABORATORY CRITICAL
i CONTROLPANEL HooDFUME I
O~ERVAT,ON.
W,NOOW "-...
L
~'xI'-'-IL-~y //"
>,,FAN
COLDROOM
EQUIPMENT
LOCal{ I
H
...... ~ . ~
~:.kl i
i...~
'\11 L'FEII/ "~~~F
i~FAN
N,ExITO .-/\ n~
~el
EMERGENCYDOOR Fro. 2. Floor plan of the anaerobic chamber. low in oxygen so that this passive-flow lock may be o p e n thereby permitting u n h i n d e r e d passage f r o m one laboratory to the other. T h e lock is kept in operation d u r i n g the time when workers are not inside the chamber. T h e r e is also a small e q u i p m e n t lock t h r o u g h which items may be sent into or out o f the c h a m b e r with only a 5 minute purge. Exit f r o m the c h a m b e r is uncomplicated. T h e doors to the lock between the two laboratories are closed, portable e q u i p m e n t is placed in the personnel lock, the life-support umbilical cord is g a t h e r e d up, and the d o o r f r o m the main laboratory into the personnel lock is closed. Once the main laboratory is closed off from the personnel lock, the o u t e r lock d o o r may be o p e n e d and the mask and its assorted p a r a p h e r n a l i a removed. Consumption of Gases Typical c o n s u m p t i o n o f nitrogen for bringing the r o o m f r o m 21% to 100 p p m o x y g e n is about 6000 scf. Recirculation for 24 hours will consume about 4500 scf N2. An entry p u r g e uses about 1800 scf above the
54
GENERAL METHODS
[7]
normal recirculation value. Hydrogen consumption is not excessive; a week of operation of the chamber may exhaust one to two cylinders (200-400 scf). If the chamber is not in use for several days, e.g., over a week end or longer, the various gases and the recirculator are turned off. This quiescent stage maintains the chamber at a low level of 02 since diffusion into the room is slow. This action reduces consumption of N2 and H2 and saves time in bringing the room to operational levels when the room is reactivated. Uses of the Chamber The anaerobic chamber has been used for purification of oxygen labile enzymes by means of anaerobic chromatographic procedures; preparation and purification of reduced cobalamin derivatives; study of enzymatic reduction and oxidation of various substrates; study of the formation of an enzyme-substrate complex; plating and isolation of anaerobes from enrichment cultures; isolation of mutants of clostridia following mutagenic treatment and replica plating; study of anaerobic mutants of facultative strains; study of the formation of methane by enzyme preparations of methane bacteria.
AN ANAEROBIC LABORATORY
49
tained, permitting the use of solute-detecting apparatus distal to the concentration device. Such systems do not interfere with further flow into a fraction collector along with concomitant registration of the collected fractions on the recorded pattern. Comments
A large number of UF systems for direct application to enzyme chemistry have been reviewed. In selecting among these techniques, a number of considerations exist. Choice of membrane should not be entirely dictated by flux, but rather lack of adsorption; systems should be chosen to provide the maximum depolarization, i.e., to achieve maximum flux, commensurate with the need to ensure minimal denaturization. When in doubt for labile proteins, the single-pass system is probably best suited; if the enzyme can withstand the shear, graduation to recirculating systems with the attendant higher flux may be warranted.
[7] An Anaerobic Laboratory ByJ. MICHAEL POSTON, THRESSA C. STADTMAN, a n d EARL R. STADTMAN The investigation of many aspects of microbiology and biochemistry is hampered by the oxygen lability of the material under study; enzymes, electron carriers, and metabolic intermediates may be rapidly lost in the presence of air. Many bacteria are severely inhibited or may be killed by exposure to air. These properties have made the study of anaerobic mutants particularly difficult since many of the techniques that are well established for use with aerobes cannot be adapted to anaerobic use. Although any given manipulation can be carried out in conventional anaerobic glove boxes, suitably equipped with remote control devices, experimental operations become extremely difficult, if not impossible, when they involve multistep procedures employing filtration, chromatography, electrophoresis techniques or the use of massive instrumentation such as spectrophotometry, centrifugation, and refrigeration. To facilitate anaerobic experimentation under conditions that allow great versatility in the utilization of standard laboratory techniques, an anaerobic laboratory chamber was constructed? The chamber is a ~The anaerobic facility at the National Institutes of Health, Bethesda, Maryland, was designed by and built under the supervision of the Linde Division of the Union Carbide Corporation.
50
GENERAL METHODS
[7]
space of approximately 1400 cubic feet. (40 cubic meters) enclosed by a gas-tight partition. By displacing most of the air with N2 gas and then removing the remaining oxygen by combining it with hydrogen on a catalyst bed, oxygen tensions of less than 100 ppm can be maintained. The laboratory contains a fume hood, incubators, and a cold room. Most portable laboratory equipment may be used in this chamber. A fairly wide spectrum of experiments have been conducted in its anaerobic environment.
Description The anaerobic chamber was constructed in a laboratory module and its dimensions were, in part, dependent upon the existing structure. The gas-tight partition is made of 3/~ inch carbon steel plates supported on a framework of I-beams. All the floor plates are riveted to the concrete floor and all joints and seams are welded. Doors to the gas locks and the emergency doors were fabricated at the site from the same steel as the shell of the chamber. Observation windows are of double-strength safety glass. The cold room is a commercial, walk-in refrigerator box of sufficient size to accommodate a fraction collector or similar equipment used in cold-room operations. Refrigeration is supplied by liquid nitrogen which sprays as saturated gas at --195 ° from a perforated pipe (the spray header) mounted at ceiling level (Polar Stream System, Linde Division, Union Carbide Corp.). The cold room is designed to hold any temperature between - 1 0 ° and +4 °. Its use is optional. The temperature can be brought from ambient (ca. 25 °) to operating levels in 30-60 minutes. Nitrogen for the cold room is conveyed directly from a large storage vessel outside the building through insulated copper tubing. T h e same storage vessel is the source of the nitrogen atmosphere in the chamber itself. It is passed first through a vaporizer assembly that is bathed in water at 21 ° and then through a volume meter before entering the anaerobic chamber.
Operation of the Chamber Placing the chamber in service once it is stocked with supplies and equipment is a relatively simple matter. Prior to closing the doors, the drain in the waste sink is filled with water. After checking the operation of the emergency system, the chamber is sealed and nitrogen is added through the purge valve. The chamber is vented at the other end of the N2 circulation loop (Fig. 1). After about an hour, the 02 level is less than 0.5% oxygen-in-nitrogen (Beckman Oxygen Analyzer,
[7]
AN ANAEROBIC LABORATORY
"~ EMERGENCYEXHAUST PURGING _. r...~._ ~] N2"MAKEUP
HUMIDFIER~
N, 7,VALV~
"-<"" , H2-ADDITION 0'T'O"."2"4 m~'~ ~ ~ L,
'~--~
h
I
~ ~
,OR~--:--I IH
.~
PRESSURE
I*I EMERGENCY .RELIEF I'1 EX.AUST /DEVICE
f-COZY/.. '~ -~,¢)1. , ~ E . ~
/
Lo~K ~ \
i
.AIN
LABORATORY ~
LOUVRES
It -
~.~ ~ v ' ~ ' T "~ ~ I L_. ._J
~
CRITICAl
CRITICAL
PURGEVENTVALVE IIT ][ JI
I..~c.".( ~ "~"
PORT
I~
H2ipS~MT PLE '
~,~.
'IO2SAMPLE
I . I~'1~ I o ~ I o L.~
I~I I ~ ~- I I'~1
. I ".~%'" J ~ I-_l I
51
,
,
I F-'/~VENT
D~N
l~l
[
I
,
VALVE
I
IHOO1
Fie. 1. Schematic illustrating the circulation and purification of gases within the chamher. Model D2). When this level is reached, the purge is stopped, recirculation is begun, and the addition of hydrogen is started at a rate of about 20 standard cubic feet (scf) per hour. The hydrogen content is monitored with a thermal conductivity analyzer (Model 204A, Analytic Systems Co.) and automatically maintained at 0.5-0.6%. After 4-5 hours of recirculation over a catalyst bed (Deoxo Gas Purifier, Model D-10,000-100, Engelhard Industries), the oxygen level of the hydrogen enriched atmosphere is less than 100 ppm. After 6-7 hours of recirculation, the atmosphere is less than 20 ppm oxygen. The tension continues to drop as recirculation continues and reaches a minimum of 2-3 ppm after about 48 hours. Oxygen tension is measured electrometrically by an oxygen trace analyzer (ASC Model 306 W, Analytic Systems Co.) in which a continuous stream of the gas to be monitored is passed through a cell containing a lead plate covered by a KOH solution in which a silver screen is suspended. The generation of current is governed by the following reactions: O ~ + 2 A g ~ 2AgO 2AgO+2H20+4e~2Ag+4(OH)2Pb+4(OH)-~2PbO+2H~O+4e A pressure-sensing make-up valve keeps the environment within the chamber at about 1 inch (water column) above the atmospheric pressure. Nitrogen lost through leakage or through operation of the fume hood is replaced by this system. The maximum pressure within the chamber is controlled by the pressure release device (Fig. 1). This
52
GENERAL METHODS
[7]
is a vented water tank containing a weir so a r r a n g e d that gas on the internal side will escape u n d e r it if the pressure rises above 4 inches o f water.
Life Support and Emergency System T h e life-support system consists o f face masks (two individuals may work within the c h a m b e r at once) to which are attached a b r e a t h i n g air line, a v a c u u m line to r e m o v e exhaled air, an o x y g e n m o n i t o r for the b r e a t h i n g air, and an alarm buzzer. B r e a t h i n g air is supplied to the mask by a c o m p r e s s o r h o u s e d nearby. I f this should fail, a large tank o f breathing air (4 hours' supply) is automatically c o n n e c t e d to the air line. I f this back-up source o f air should fail or be e x h a u s t e d , a pressure activated alarm notifies the p e r s o n in the c h a m b e r to o p e n the valve on the small bottle o f b r e a t h i n g air that is carried o n the harness o f the life-support system. This second back-up source has a m i n i m u m o f 7 minutes o f air and will p e r m i t the p e r s o n to make an o r d e r l y exit f r o m the c h a m b e r . D u r i n g the time w h e n a n y o n e is inside the c h a m b e r , a second p e r s o n is outside observing the operations and well-being o f the o n e inside and m o n i t o r i n g the o x y g e n level o f the b r e a t h i n g air and o f the c h a m b e r a t m o s p h e r e . T h e r e are observation windows in the wall and each o f the doors, as well as strategically placed mirrors so that at no time is the person inside the c h a m b e r out o f sight or h e a r i n g o f the monitor. In the event o f a situation in which it is vital to aid the p e r s o n within the c h a m b e r , a b u t t o n on the control panel activates the e m e r g e n c y system. T h e e m e r g e n c y doors and the butterfly drain valve on the pressure release device o p e n pneumatically. Large exhaust blowers (Fig. 1) begin to draw fresh air into the c h a m b e r t h r o u g h the e m e r g e n c y doors. Within about 7 seconds, the a t m o s p h e r e inside the c h a m b e r will s u p p o r t h u m a n life. Even in the event o f an electric power failure, the e m e r g e n c y doors can be o p e n e d individually by means o f the p n e u m a t i c system or by m a n u a l release o f the latches.
Entrance to the Laboratory Access to the c h a m b e r is p r o v i d e d t h r o u g h the p e r s o n n e l lock (Fig. 2) in which the life-support mask is d o n n e d . Once the mask is tightly fitted to the face, the w o r k e r closes the outside d o o r and the lock is p u r g e d . D u r i n g this time the pressure within the c h a m b e r is raised close to its m a x i m u m in o r d e r to p r e v e n t the p u r g e f r o m driving o x y g e n t h r o u g h the d o o r seals into the anaerobic room. After 13 minutes, the p u r g e is e n d e d and the main laboratory is e n t e r e d . T h e i n n e r laboratory may be e n t e r e d t h r o u g h the critical lock. In practice, it has been f o u n d that the inner critical laboratory and the main laboratory are sufficiently
[7]
AN ANAEROBIC LABORATORY
53
I
FA~
J 0 N2 ENTRANCE EMERGENCY[T~ CRITICALLABORATORY CRITICAL
i CONTROLPANEL HooDFUME I
O~ERVAT,ON.
W,NOOW "-...
L
~'xI'-'-IL-~y //"
>,,FAN
COLDROOM
EQUIPMENT
LOCal{ I
H
...... ~ . ~
~:.kl i
i...~
'\11 L'FEII/ "~~~F
i~FAN
N,ExITO .-/\ n~
~el
EMERGENCYDOOR Fro. 2. Floor plan of the anaerobic chamber. low in oxygen so that this passive-flow lock may be o p e n thereby permitting u n h i n d e r e d passage f r o m one laboratory to the other. T h e lock is kept in operation d u r i n g the time when workers are not inside the chamber. T h e r e is also a small e q u i p m e n t lock t h r o u g h which items may be sent into or out o f the c h a m b e r with only a 5 minute purge. Exit f r o m the c h a m b e r is uncomplicated. T h e doors to the lock between the two laboratories are closed, portable e q u i p m e n t is placed in the personnel lock, the life-support umbilical cord is g a t h e r e d up, and the d o o r f r o m the main laboratory into the personnel lock is closed. Once the main laboratory is closed off from the personnel lock, the o u t e r lock d o o r may be o p e n e d and the mask and its assorted p a r a p h e r n a l i a removed. Consumption of Gases Typical c o n s u m p t i o n o f nitrogen for bringing the r o o m f r o m 21% to 100 p p m o x y g e n is about 6000 scf. Recirculation for 24 hours will consume about 4500 scf N2. An entry p u r g e uses about 1800 scf above the
54
GENERAL METHODS
[7]
normal recirculation value. Hydrogen consumption is not excessive; a week of operation of the chamber may exhaust one to two cylinders (200-400 scf). If the chamber is not in use for several days, e.g., over a week end or longer, the various gases and the recirculator are turned off. This quiescent stage maintains the chamber at a low level of 02 since diffusion into the room is slow. This action reduces consumption of N2 and H2 and saves time in bringing the room to operational levels when the room is reactivated. Uses of the Chamber The anaerobic chamber has been used for purification of oxygen labile enzymes by means of anaerobic chromatographic procedures; preparation and purification of reduced cobalamin derivatives; study of enzymatic reduction and oxidation of various substrates; study of the formation of an enzyme-substrate complex; plating and isolation of anaerobes from enrichment cultures; isolation of mutants of clostridia following mutagenic treatment and replica plating; study of anaerobic mutants of facultative strains; study of the formation of methane by enzyme preparations of methane bacteria.