1607268
Enzyme 1991;45:67-70
Ultrasonic Baths as Substitutes for Shaking Incubator Baths Norman S. Radin, Girja S. Shukla Mental Health Research Institute, University of Michigan, Ann Arbor, Mich., USA
Key Words. Incubation bath, ultrasonic • Baths, constant-temperature ultrasonic Abstract. An easily assembled incubation bath for enzyme work is described. The bath is made from commercially available units: an ultrasonic bath of the type used for cleaning and a temperature-controlling device. The device is not only much cheaper, quieter, and more compact than a commercially built shaking-type bath, but it also gives superior mixing of heterogeneous enzyme incubation samples, particularly those containing tissue homogenates or subcellular particles.
For a great many years, biochemists have used shaking water baths of the type pio neered by Dubnoff for carrying out enzy matic and nonenzymatic reactions. The same effects can be obtained much more economically and quietly by using a ther mostatically controlled ultrasonic water bath. While it is a common belief that ultra sonic baths can damage enzymes, the fact is that ultrasonic probes and high-intensity ul trasonic baths enjoyed much usage for solu bilizing enzymes from cells and membranes for many years [1]. This usage decreased when modem detergents were found to pro duce the same effect in many systems. The
acoustic intensity in a bath of the type used for cleaning is far less than the intensity underneath the tip of a probe. This differ ence is obvious from the price and sound intensity difference and from simple exami nation of the vibrating metal: the tip of a probe becomes rough from loss of metal par ticles but the metal in a cleaning bath lasts indefinitely. Ultrasonic baths occupy less bench space than shakers since the motor-driven recipro cating mechanism is not needed and the thermostatic control can be placed on a shelf above the bath. Our tests with tissue homog enates have shown that ultrasonic baths pro duce a more uniform, better agitated suspen sion of membranes [2], This feature is partic Downloaded by: University of Exeter 144.173.6.94 - 5/4/2020 12:18:36 AM
Introduction
Radin/Shukla
68
Materials Currently available ultrasonic baths do not seem to be furnished with accurate temperature controls, but it is easy to build a system. The vibrator at the bottom of the bath produces heat, so a cooling coil is needed for a typical enzymic incubation. We use a 3/4-gallon bath (Cole-Parmer ‘cleaning bath’ No. N08851-00, 9.5 X 5.5 inches) and a 4-foot length of 1/8-inch OD copper tubing, bent into a flat set of loops to fit low down inside the bath without touching the walls. The vertical sections of the loop inlet and outlet that are immersed in water are insulated with short lengths of Tygon or rubber tubing. The exit end of the tube leads to a sink drain via flexible tubing. The entrance end is connected by a compression fit ting to 1/8-inch OD polyethylene tubing which, in turn, leads to the outlet of a solenoid valve. The inlet to the solenoid valve is connected, as closely as possi ble, to a cold water tap via the same plastic tubing. The solenoid valve, made for use with water, is connected to the 110-volt outlet of a temperature con troller (Cole-Parmer No. N-02158-00). This con troller is an on/off relay; it is installed on a shelf, together with the solenoid valve, to save table space. The sensor for the controller is a thermistor sheathed in a stainless steel tube (Cole-Parmer No. N-0843400, YSI 403), immersed in a comer of the water bath. With the solenoid activated by outlet No. 2 of the controller, it lets cold water enter the coil when the bath temperature exceeds the set point. To prevent fluctuations in the flow rate of the cooling water or overloading of the solenoid valve due to pressure surges, we attach the tubing to a pressure regulator, made for use with water (pressure range about 0-100 PSI). To minimize temperature fluctua tions, the operating pressure is set so that the cooling water flows most of the time. The cycle can be observed by watching the controller’s on /offlights. A higher water pressure would be needed in the sum mer, when the water is not as cold.
Since the tubing in the cold water line can easily become clogged by sediment-contaminated water, it may be wise to install a filter in the water line. We have found such filters to be very useful in protecting glass-washing equipment too. The bath temperature is monitored with a long mercury thermometer, with 0.1 “C divisions, from -1 to 101 °C. Bimetallic or short mercury thermometers cannot be used in an ultrasonic bath. The thermistor sensor, thermometer, and cooling coil are held in place by clamps attached to a support stand. It is important to avoid contact with the bath walls. Test tubes are held in the bath by means of a plas tic test tube support, which can be loaded outside the bath, then lowered into the water and held in place by two stainless steel sheets, bent in the shape of an ell. The vertical surface of the ell is attached to the nar row ends of the support rack (21 X 10.8 cm maxi mum dimensions for this bath size) and the horizon tal surface simply rests on the upper edge of the bath. We also use a 40-tube plastic-coated steel wire rack (Baxter Scientific Products No. S9222), in which case the support ells are attached to the ends with short pieces of stainless steel wire. A polypropylene support for drying test tubes (Fisher Scientific No. 14-781) can be used after attaching the two ells. For test tubes larger than 16 mm OD, a larger bath would be prefer able. A test tube rack with holes below each test tube is probably better than one with a solid bottom, to help the ultrasonic vibrations enter the tube. We have also made a test tube support from two 1/4-inch thick plastic sheets, with holes drilled for tubes and the upper sheet long enough to fit over the top of the bath. The two sheets are connected by four stainless steel machine screws and nuts.
Results and Discussion
The bath temperature, measured over time or at any point in the bath, fluctuated ± 0.2 °C or less. This variability is better than the specified variability of some com mercially available shaker baths. As a further test of bath uniformity, assays were carried out using a crude preparation of splenic acid phosphatase and p-nitrophenyl phosphate, in buffer at pH 4.8. Sixteen incubation tubes, Downloaded by: University of Exeter 144.173.6.94 - 5/4/2020 12:18:36 AM
ularly important for reactions involving two or three phases - lipids (liposomes), mem branes, and water. A similar improvement can be expected for incubations involving enzymes or antibodies immobilized on beads.
each containing 0.3 ml of the same premixed enzyme-substrate solution, were loaded into a 4 X 8 position rack, in alternating loca tions. After cooling in ice and addition of 1 ml of 0.1 M NaOH, the A400 values were measured. The observed values varied ran domly between 0.440 and 0.460 A (4.4% of the mean), with an SD of 0.006. The enzyme velocity was constant under these conditions for >45 min, showing the stability of the enzyme under the ultrasonic irradiation. A similar location-comparison test using a kidney homogenate and UDP-[3H]glucose with a liposomal lipoidal glucose acceptor was also used [2], This is a particularly diffi cult assay because of the tendency of the membranes to settle during pipetting and incubating and because several handling steps after incubation (solvent partitioning, evaporation, and counting) are required. The homogenate was portioned out with a plastic-tipped transfer pipettor into each reaction tube while the homogenate con tainer was suspended in a second, ice-cooled ultrasonic bath to help produce uniform ali quots. The plastic tip was cut off near the end to widen the opening. The range be tween observed maximal and minimal val ues was 7.3% and 5.3% of the means for two runs of six tubes each. These data rule out the possibility of significant fluctuations in temperature or intensity of agitation with test tube location. When a standard shakingtype bath is used with microsomes under these conditions, the membranes form a ring around the inner walls of the tubes and the variability in results is significantly greater. It is possible that incubating a tissue ho mogenate will cause an increase in observed enzyme activity due to mechanical disinte gration of subcellular particles. This would be expected to result in higher activity dur
69
ing the latter part of the incubation. How ever, in our time studies with the above homogenates no such effect was observed. To our knowledge, no one has recommended the use of ultrasonic cleaning baths for the extraction of enzymes from membranes, and it is unlikely that this kind of bath can dis rupt subcellular membranes. If the reader fears that his favorite enzyme is unusually sensitive to ultrasonic disruption, he can rea dily check this - as we did - with an avail able ultrasonic cleaning bath by controlling its temperature manually (with small addi tions of ice). The fear has been expressed that enzymes immobilized on beads could somehow be damaged, perhaps by break-up of the beads. We sonicated alkaline phosphatase bound to agarose beads (Sigma Chemical) for 1 h and compared the mixture with beads shaken in a standard shaker at 37 °C. The particles set tled at the same rate in both tubes, producing a clear supernatant liquid, showing that there was no mechanical disintegration with either incubation method. A similar test with Sephadex G100 yielded the same find ing. In normal use of enzyme-linked beads, one hopes for good access of the enzyme to the substance being attacked and some disin tegration of the bead - if it occurred - would be helpful since this would expose a larger surface. Red blood cells can be incubated in the ultrasonic bath but some lysis occurs. While we have not yet tested the idea, it should be possible to lower the ultrasonic energy inten sity inside the incubation tubes by using test tube racks with solid bottoms. Ultrasonic baths can get quite warm from spontaneous action so it is possible to run incubations or chemical reactions at ele vated temperatures, as with a shaking type or Downloaded by: University of Exeter 144.173.6.94 - 5/4/2020 12:18:36 AM
Ultrasonic Incubation Baths
70
readily emptied (partially) and refilled with a beaker, using warm tap water. Temperature equilibration to 37 °C takes only a few min utes so the bath need not be operated appre ciably longer than the actual incubation time. Older ultrasonic baths may be noisier than the new ones, but they could easily be placed inside a sound-absorbing box lined with acoustic ceiling tiles, open at the front.
Acknowledgements This work was supported by NIH grant NS03192.
References 1 2
Alliger H: Ultrasonic disruption. Am Lab, Octo ber 1975. Shukla GS, Radin NS: Glucosylceramide synthase of mouse kidney: Further characterization with improved assay method. Arch Biochem Biophys 1990;283:372-378.
Received: October 20, 1990 Accepted: November 30, 1990 Dr. Norman Radin Neuroscience Bldg. 1103 E. Huron Ann Arbor, MI 48104-1687 (USA)
Downloaded by: University of Exeter 144.173.6.94 - 5/4/2020 12:18:36 AM
magnetic stirrer bath. If necessary, hot water could be passed through the copper coil in stead of cold water. In the case of two-phase nonenzymatic chemical reactions, or extrac tion of lipids from a cell suspension or tissue homogenates, a magnetic stirrer is unneces sary. It is possible that the lifespan of an ultra sonic bath is shorter than that of a shaker bath but it would be a simple task to replace the bath, without replacing the temperature control system. Shaking baths require maintainance (lubrication can be readily over looked) but no maintenance is needed for the ultrasonic incubator. An important posi tive feature of the ultrasonic incubator, be sides its low noise level, is its relatively low cost. A shaker bath lists at roughly US$ 2,000 while the ultrasonic bath, con troller, and sensor list at about US $ 345, 225, and 60, respectively (US$630 total). With the solenoid valve, water filter, and pressure regulator the total cost comes to about one third that of a shaker bath. No mechanical aptitude or machining equip ment is necessary, only the ability to connect tubing and plug wires into sockets. Fabrica tion of test tube racks can be done with min imal equipment. While a shaking bath could also be fabricated at a relatively low cost (by a skilled mechanic), the required compo nents are not readily available. Some ultrasonic baths do not come with a drain for replacing the water but they are
Radin/Shukla
Enzyme 1991;45:67-70
Ultrasonic Baths as Substitutes for Shaking Incubator Baths Norman S. Radin, Girja S. Shukla Mental Health Research Institute, University of Michigan, Ann Arbor, Mich., USA
Key Words. Incubation bath, ultrasonic • Baths, constant-temperature ultrasonic Abstract. An easily assembled incubation bath for enzyme work is described. The bath is made from commercially available units: an ultrasonic bath of the type used for cleaning and a temperature-controlling device. The device is not only much cheaper, quieter, and more compact than a commercially built shaking-type bath, but it also gives superior mixing of heterogeneous enzyme incubation samples, particularly those containing tissue homogenates or subcellular particles.
For a great many years, biochemists have used shaking water baths of the type pio neered by Dubnoff for carrying out enzy matic and nonenzymatic reactions. The same effects can be obtained much more economically and quietly by using a ther mostatically controlled ultrasonic water bath. While it is a common belief that ultra sonic baths can damage enzymes, the fact is that ultrasonic probes and high-intensity ul trasonic baths enjoyed much usage for solu bilizing enzymes from cells and membranes for many years [1]. This usage decreased when modem detergents were found to pro duce the same effect in many systems. The
acoustic intensity in a bath of the type used for cleaning is far less than the intensity underneath the tip of a probe. This differ ence is obvious from the price and sound intensity difference and from simple exami nation of the vibrating metal: the tip of a probe becomes rough from loss of metal par ticles but the metal in a cleaning bath lasts indefinitely. Ultrasonic baths occupy less bench space than shakers since the motor-driven recipro cating mechanism is not needed and the thermostatic control can be placed on a shelf above the bath. Our tests with tissue homog enates have shown that ultrasonic baths pro duce a more uniform, better agitated suspen sion of membranes [2], This feature is partic Downloaded by: University of Exeter 144.173.6.94 - 5/4/2020 12:18:36 AM
Introduction
Radin/Shukla
68
Materials Currently available ultrasonic baths do not seem to be furnished with accurate temperature controls, but it is easy to build a system. The vibrator at the bottom of the bath produces heat, so a cooling coil is needed for a typical enzymic incubation. We use a 3/4-gallon bath (Cole-Parmer ‘cleaning bath’ No. N08851-00, 9.5 X 5.5 inches) and a 4-foot length of 1/8-inch OD copper tubing, bent into a flat set of loops to fit low down inside the bath without touching the walls. The vertical sections of the loop inlet and outlet that are immersed in water are insulated with short lengths of Tygon or rubber tubing. The exit end of the tube leads to a sink drain via flexible tubing. The entrance end is connected by a compression fit ting to 1/8-inch OD polyethylene tubing which, in turn, leads to the outlet of a solenoid valve. The inlet to the solenoid valve is connected, as closely as possi ble, to a cold water tap via the same plastic tubing. The solenoid valve, made for use with water, is connected to the 110-volt outlet of a temperature con troller (Cole-Parmer No. N-02158-00). This con troller is an on/off relay; it is installed on a shelf, together with the solenoid valve, to save table space. The sensor for the controller is a thermistor sheathed in a stainless steel tube (Cole-Parmer No. N-0843400, YSI 403), immersed in a comer of the water bath. With the solenoid activated by outlet No. 2 of the controller, it lets cold water enter the coil when the bath temperature exceeds the set point. To prevent fluctuations in the flow rate of the cooling water or overloading of the solenoid valve due to pressure surges, we attach the tubing to a pressure regulator, made for use with water (pressure range about 0-100 PSI). To minimize temperature fluctua tions, the operating pressure is set so that the cooling water flows most of the time. The cycle can be observed by watching the controller’s on /offlights. A higher water pressure would be needed in the sum mer, when the water is not as cold.
Since the tubing in the cold water line can easily become clogged by sediment-contaminated water, it may be wise to install a filter in the water line. We have found such filters to be very useful in protecting glass-washing equipment too. The bath temperature is monitored with a long mercury thermometer, with 0.1 “C divisions, from -1 to 101 °C. Bimetallic or short mercury thermometers cannot be used in an ultrasonic bath. The thermistor sensor, thermometer, and cooling coil are held in place by clamps attached to a support stand. It is important to avoid contact with the bath walls. Test tubes are held in the bath by means of a plas tic test tube support, which can be loaded outside the bath, then lowered into the water and held in place by two stainless steel sheets, bent in the shape of an ell. The vertical surface of the ell is attached to the nar row ends of the support rack (21 X 10.8 cm maxi mum dimensions for this bath size) and the horizon tal surface simply rests on the upper edge of the bath. We also use a 40-tube plastic-coated steel wire rack (Baxter Scientific Products No. S9222), in which case the support ells are attached to the ends with short pieces of stainless steel wire. A polypropylene support for drying test tubes (Fisher Scientific No. 14-781) can be used after attaching the two ells. For test tubes larger than 16 mm OD, a larger bath would be prefer able. A test tube rack with holes below each test tube is probably better than one with a solid bottom, to help the ultrasonic vibrations enter the tube. We have also made a test tube support from two 1/4-inch thick plastic sheets, with holes drilled for tubes and the upper sheet long enough to fit over the top of the bath. The two sheets are connected by four stainless steel machine screws and nuts.
Results and Discussion
The bath temperature, measured over time or at any point in the bath, fluctuated ± 0.2 °C or less. This variability is better than the specified variability of some com mercially available shaker baths. As a further test of bath uniformity, assays were carried out using a crude preparation of splenic acid phosphatase and p-nitrophenyl phosphate, in buffer at pH 4.8. Sixteen incubation tubes, Downloaded by: University of Exeter 144.173.6.94 - 5/4/2020 12:18:36 AM
ularly important for reactions involving two or three phases - lipids (liposomes), mem branes, and water. A similar improvement can be expected for incubations involving enzymes or antibodies immobilized on beads.
each containing 0.3 ml of the same premixed enzyme-substrate solution, were loaded into a 4 X 8 position rack, in alternating loca tions. After cooling in ice and addition of 1 ml of 0.1 M NaOH, the A400 values were measured. The observed values varied ran domly between 0.440 and 0.460 A (4.4% of the mean), with an SD of 0.006. The enzyme velocity was constant under these conditions for >45 min, showing the stability of the enzyme under the ultrasonic irradiation. A similar location-comparison test using a kidney homogenate and UDP-[3H]glucose with a liposomal lipoidal glucose acceptor was also used [2], This is a particularly diffi cult assay because of the tendency of the membranes to settle during pipetting and incubating and because several handling steps after incubation (solvent partitioning, evaporation, and counting) are required. The homogenate was portioned out with a plastic-tipped transfer pipettor into each reaction tube while the homogenate con tainer was suspended in a second, ice-cooled ultrasonic bath to help produce uniform ali quots. The plastic tip was cut off near the end to widen the opening. The range be tween observed maximal and minimal val ues was 7.3% and 5.3% of the means for two runs of six tubes each. These data rule out the possibility of significant fluctuations in temperature or intensity of agitation with test tube location. When a standard shakingtype bath is used with microsomes under these conditions, the membranes form a ring around the inner walls of the tubes and the variability in results is significantly greater. It is possible that incubating a tissue ho mogenate will cause an increase in observed enzyme activity due to mechanical disinte gration of subcellular particles. This would be expected to result in higher activity dur
69
ing the latter part of the incubation. How ever, in our time studies with the above homogenates no such effect was observed. To our knowledge, no one has recommended the use of ultrasonic cleaning baths for the extraction of enzymes from membranes, and it is unlikely that this kind of bath can dis rupt subcellular membranes. If the reader fears that his favorite enzyme is unusually sensitive to ultrasonic disruption, he can rea dily check this - as we did - with an avail able ultrasonic cleaning bath by controlling its temperature manually (with small addi tions of ice). The fear has been expressed that enzymes immobilized on beads could somehow be damaged, perhaps by break-up of the beads. We sonicated alkaline phosphatase bound to agarose beads (Sigma Chemical) for 1 h and compared the mixture with beads shaken in a standard shaker at 37 °C. The particles set tled at the same rate in both tubes, producing a clear supernatant liquid, showing that there was no mechanical disintegration with either incubation method. A similar test with Sephadex G100 yielded the same find ing. In normal use of enzyme-linked beads, one hopes for good access of the enzyme to the substance being attacked and some disin tegration of the bead - if it occurred - would be helpful since this would expose a larger surface. Red blood cells can be incubated in the ultrasonic bath but some lysis occurs. While we have not yet tested the idea, it should be possible to lower the ultrasonic energy inten sity inside the incubation tubes by using test tube racks with solid bottoms. Ultrasonic baths can get quite warm from spontaneous action so it is possible to run incubations or chemical reactions at ele vated temperatures, as with a shaking type or Downloaded by: University of Exeter 144.173.6.94 - 5/4/2020 12:18:36 AM
Ultrasonic Incubation Baths
70
readily emptied (partially) and refilled with a beaker, using warm tap water. Temperature equilibration to 37 °C takes only a few min utes so the bath need not be operated appre ciably longer than the actual incubation time. Older ultrasonic baths may be noisier than the new ones, but they could easily be placed inside a sound-absorbing box lined with acoustic ceiling tiles, open at the front.
Acknowledgements This work was supported by NIH grant NS03192.
References 1 2
Alliger H: Ultrasonic disruption. Am Lab, Octo ber 1975. Shukla GS, Radin NS: Glucosylceramide synthase of mouse kidney: Further characterization with improved assay method. Arch Biochem Biophys 1990;283:372-378.
Received: October 20, 1990 Accepted: November 30, 1990 Dr. Norman Radin Neuroscience Bldg. 1103 E. Huron Ann Arbor, MI 48104-1687 (USA)
Downloaded by: University of Exeter 144.173.6.94 - 5/4/2020 12:18:36 AM
magnetic stirrer bath. If necessary, hot water could be passed through the copper coil in stead of cold water. In the case of two-phase nonenzymatic chemical reactions, or extrac tion of lipids from a cell suspension or tissue homogenates, a magnetic stirrer is unneces sary. It is possible that the lifespan of an ultra sonic bath is shorter than that of a shaker bath but it would be a simple task to replace the bath, without replacing the temperature control system. Shaking baths require maintainance (lubrication can be readily over looked) but no maintenance is needed for the ultrasonic incubator. An important posi tive feature of the ultrasonic incubator, be sides its low noise level, is its relatively low cost. A shaker bath lists at roughly US$ 2,000 while the ultrasonic bath, con troller, and sensor list at about US $ 345, 225, and 60, respectively (US$630 total). With the solenoid valve, water filter, and pressure regulator the total cost comes to about one third that of a shaker bath. No mechanical aptitude or machining equip ment is necessary, only the ability to connect tubing and plug wires into sockets. Fabrica tion of test tube racks can be done with min imal equipment. While a shaking bath could also be fabricated at a relatively low cost (by a skilled mechanic), the required compo nents are not readily available. Some ultrasonic baths do not come with a drain for replacing the water but they are
Radin/Shukla
