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An apparatus for fast and frequent samplingof large aliquots
ANALYTICAL BIOCHEMISTRY 3 2 ,
454-459 (1969)
An Apparatus for Fast and Frequent Sampling of Large Aliquots 1 t t A I M MANOR 2 AXD DONALD CROWE Department o] Molecular Biology and Virwx Laboratory, University o[ California, Berkeley., Cali]ornia 94720
Received March 17, 1969 The analysis of some chemical and biochemical reactions requires a study of their time course over short periods. While in some eases it is possible to study the progress of such a reaction directly in a reaction vessel, e.g., by optical means, in other eases it is necessary to withdraw aliquots at short intervals and to analyze changes in their composition
(1). In this communication is described an apparatus constructed for studies of moderately fast reactions (3-5 see), the analysis of which requires withdrawal of particularly large aliquots (20-50 ml). This is necessary if a product to be analyzed is produced at a small concentration. The apparatus is operated in the following way: The reaction is initiated in a vessel by rapid mixing of its components; then the mixture is distributed into several separate chamber-, in each of which the reaction continues and is terminated independently at a different time. EXPERIMENTAL The apparatus. A detailed drawing of the apparatus is shown in Figure 1 and photographs are presented in Figures 2 and 3. The instrument consists of two parts, an upper and a lower part, which can be separated from each other as shown in Figures 1 and 2. During actual operation, the upper part fits into the lower part as shown in Figure 3. The lower part consists of a cylindrical piece of Lucite, in which a large central hole (a) and four smaller peripheral holes (b) were bored. To the central hole, a cylinder (e) built of stainless steel is fastened with screws. The cylinder is painted black on the outside for rapid temperature
1This investigation was supported in part by U. S. Public Health Service research grant CA 02129 from the National Cancer Institute. Postdoctoral Research Fellow of The Helen Hay Whitney Foundation. Present Address : Department of Biochemistry, The Weizmann Institute of Science, Rehovoth, lsrael. 454
FAST AND FREQUENT SAMPLING
455
Fro. 1. Detailed drawing of the rapid mixing and sampling apparatus. The various parts, which are labeled with small letters, are described in the rex&
equilibration with the surroundings. A piston (d), which can be moved up and down manually by pressing the lever (e), is shown in Figure 1 in its upper position. A stationary cylindrical piece (f) with a hole in its center directs the movement of a cylindrical lucite rod (g) glued to the piston; thus when the lever is pressed, an equal force is applied to the entire circumference of the piston. A neoprene O-ring seals the side of the piston and two other O-rings seal the sides of the stationary piece (f) and the rod (g). An air outlet (h) is provided for the space between the stationary piece and the piston. We shall refer to the chamber formed above the piston as the central chamber. When the piston is moved down, it opens four oblique connections (i) between the central chamber and the four peripheral chambers, which were formed by gluing Lucite pieces to the bottom of the peripheral holes
~6
:MANOR A N D C R O W E
FIG. 2. Photograph of the rapid mixing and sampling apparatus separated into its two parts.
(b). Four syringe holders (j) are constructed such that a syringe placed in each of them points into one peripheral chamber through an oblique hole (k). The upper part of the apparatus carries five 6 V DC electrical motors (Barber-Coleman BYQM-2184). A central motor (1) revolves a stainlesssteel stirrer (in) which stirs the contents of the central chamber. Each of four peripheral motors (n) revolves a Lucite or a stainless-steel stirrer (o), which stirs the contents of a peripheral chamber. The motors are operated from a 6 V automotive battery connected to a variable resistance. The central chamber is completely covered by the upper part, except for a hole (p) which is provided for aerat-on, and an opening provided for a syringe in a holder (q).
FAST AND FREQUENT SAMPLING
457
FIG. 3. Photograph of the rapid mixing and sampling apparatus ready for operation.
Operation. During operation, the top part of the apparatus is placed on the lower part, so that each of the stirrers fits into its respective chamber. This is shown in Figure 3. The piston (d) is maintained in its upper position; thus the central chamber is sealed from the peripheral chambers. For our experiments we left the apparatus overnight in a room maintained at a constant temperature before carrying out the experiment at that temperature. With modifications the apparatus may be immersed in a water bath. If high temperatures are required (>_37°), the appara£us may need to be placed in a chamber maintained at a high humidity to prevent excessive evapora£ion due to the vigorous stirring. A relatively large volume (in our experiments, 100 ml) of a mixture lacking one of the components necessary for initiation of a reaction is placed in the central chamber and all five stirrers are operated. A syringe containing the missing component of the reaction mixture is placed in the holder (q), and four syringes containing a solution that will terminate the reaction when injected into the mixture are placed in the holders (j).
458
MANOR AND CROWE
The syringes may need to be plugged with cotton to prevent premature dripping of their contents into the reaction mixture. The reaction is initiated when the contents of the syringe (q) are injected into the central chamber. About 2 see later, the handle (e) is pressed and the piston is moved down. Thus, the connections (i) are opened and the mixture flows by gravity into the peripheral chambers. Most of the liquid flows down in less than 2 see. The connections are closed again after 2 see to prevent a further slow flow of the liquid remaining in the central chamber. The reaction, which continues in the peripheral chambers, is terminated in eaeh of them separately by injecting a "terminating" solution from a syringe in (j). Employing manual operation, as we have done, it is possible to terminate the reaction in these chambers at intervals of 3-4 see. Finally, the upper part of the apparatus is removed and the samples are removed from the peripheral chambers and analyzed as required. AppLication. We have utilized the rapid sampling apparatus for a series of measurements of rates of polymerization of RNA molecules in vivo in exponentially growing bacterial cultures. The details and the results of these measurements are presented elsewhere (2). DISCUSSION The present model of the rapid mixing and sampling apparatus has been designed to withdraw four 25-50 ml a liquors. If necessary, similar machines ean be built to sample much larger volumes, or by increasing the number of peripheral chambers, to obtain more samples. Manual operation did not permit sampling at intervals of less than 3-4 see. It is, however, possible to introduce automation and thus decrease the intervals to fractions of a second. In either case, the apparatus is useful only if there exists a method for stopping the reaction in a time which is much shorter than the sampling interval. There are eertain limitations inherent in the design of the apparatus: (a) In order to analyze the sample after the reaction has been terminated, one has to remove the upper part to remove the samples; therefore this apparatus is not useful, for example, for experiments in which the samples have to be cooled down immediately following termination of a reaction. (b) Another limitation results from the fact {hat the mixture flows from the central to the peripheral chambers by gravity, and this requires 2-3 see; hence the first sample can be taken no sooner than about 3 see after the reaction is initia~ed, tIowever, this last difficulty can be overcome, if necessary, by designing an apparatus which would differ from the present one in that the mixture would flow into the peripheral chambers under pressure, and hence more rapidly.
FAST AND FREQUENT SAMPLING
459
The limitations considered above did not apply to our measurements of RNA chain propagation in bacteria. Furthermore, the present appara£us had the advantage that the bacteria could be grown in it for a long time and could be shown to undergo steady-state growth before and during rapid labeling experiments. Thus, it may be utilized in its present form for other in vivo studies of moderately fast reactions, in which these criteria must be met. It may, of course, be also utilized for studies of other kinds of chemical and biochemical reactions. SUMMARY An appara£us has been constructed to perform large-scale mixing and sampling experiments for studies of moderately fast reactions. In the form described in the present communication, the apparatus is operated manually and allows rapid mixing of 100-200 ml reaction mixtures and sampling of four 25-50 ml aliquots at 3-4 see intervals. ACKNOWLEDGMENTS We wish to thank Dr. G. S. Stent and David Goodman for helpful discussions. We also thank Alfred Stern, Tony Rago, and George Lauterbaeh for constructing the apparatus, and Avram
Maitlis and Ruben Cohen for the drawing. REFERENCES
1. CtIAI~'CE,B., EISENtIARDT, R. H., GIBSON, Q. ]=i., AND LONBERG-I-IoLM,K. K., eds., "Rapid Mixing and Sampling Techniques in Biochemistry" 1st Intern. Colloq; Philadelphia, July, 1964. Academic Press, New York, 1964. 2. MANOR, H., G00D-VlAN,D., AND STENT, G. S., ]. MoI. Biol. 39, 1 (1969).
454-459 (1969)
An Apparatus for Fast and Frequent Sampling of Large Aliquots 1 t t A I M MANOR 2 AXD DONALD CROWE Department o] Molecular Biology and Virwx Laboratory, University o[ California, Berkeley., Cali]ornia 94720
Received March 17, 1969 The analysis of some chemical and biochemical reactions requires a study of their time course over short periods. While in some eases it is possible to study the progress of such a reaction directly in a reaction vessel, e.g., by optical means, in other eases it is necessary to withdraw aliquots at short intervals and to analyze changes in their composition
(1). In this communication is described an apparatus constructed for studies of moderately fast reactions (3-5 see), the analysis of which requires withdrawal of particularly large aliquots (20-50 ml). This is necessary if a product to be analyzed is produced at a small concentration. The apparatus is operated in the following way: The reaction is initiated in a vessel by rapid mixing of its components; then the mixture is distributed into several separate chamber-, in each of which the reaction continues and is terminated independently at a different time. EXPERIMENTAL The apparatus. A detailed drawing of the apparatus is shown in Figure 1 and photographs are presented in Figures 2 and 3. The instrument consists of two parts, an upper and a lower part, which can be separated from each other as shown in Figures 1 and 2. During actual operation, the upper part fits into the lower part as shown in Figure 3. The lower part consists of a cylindrical piece of Lucite, in which a large central hole (a) and four smaller peripheral holes (b) were bored. To the central hole, a cylinder (e) built of stainless steel is fastened with screws. The cylinder is painted black on the outside for rapid temperature
1This investigation was supported in part by U. S. Public Health Service research grant CA 02129 from the National Cancer Institute. Postdoctoral Research Fellow of The Helen Hay Whitney Foundation. Present Address : Department of Biochemistry, The Weizmann Institute of Science, Rehovoth, lsrael. 454
FAST AND FREQUENT SAMPLING
455
Fro. 1. Detailed drawing of the rapid mixing and sampling apparatus. The various parts, which are labeled with small letters, are described in the rex&
equilibration with the surroundings. A piston (d), which can be moved up and down manually by pressing the lever (e), is shown in Figure 1 in its upper position. A stationary cylindrical piece (f) with a hole in its center directs the movement of a cylindrical lucite rod (g) glued to the piston; thus when the lever is pressed, an equal force is applied to the entire circumference of the piston. A neoprene O-ring seals the side of the piston and two other O-rings seal the sides of the stationary piece (f) and the rod (g). An air outlet (h) is provided for the space between the stationary piece and the piston. We shall refer to the chamber formed above the piston as the central chamber. When the piston is moved down, it opens four oblique connections (i) between the central chamber and the four peripheral chambers, which were formed by gluing Lucite pieces to the bottom of the peripheral holes
~6
:MANOR A N D C R O W E
FIG. 2. Photograph of the rapid mixing and sampling apparatus separated into its two parts.
(b). Four syringe holders (j) are constructed such that a syringe placed in each of them points into one peripheral chamber through an oblique hole (k). The upper part of the apparatus carries five 6 V DC electrical motors (Barber-Coleman BYQM-2184). A central motor (1) revolves a stainlesssteel stirrer (in) which stirs the contents of the central chamber. Each of four peripheral motors (n) revolves a Lucite or a stainless-steel stirrer (o), which stirs the contents of a peripheral chamber. The motors are operated from a 6 V automotive battery connected to a variable resistance. The central chamber is completely covered by the upper part, except for a hole (p) which is provided for aerat-on, and an opening provided for a syringe in a holder (q).
FAST AND FREQUENT SAMPLING
457
FIG. 3. Photograph of the rapid mixing and sampling apparatus ready for operation.
Operation. During operation, the top part of the apparatus is placed on the lower part, so that each of the stirrers fits into its respective chamber. This is shown in Figure 3. The piston (d) is maintained in its upper position; thus the central chamber is sealed from the peripheral chambers. For our experiments we left the apparatus overnight in a room maintained at a constant temperature before carrying out the experiment at that temperature. With modifications the apparatus may be immersed in a water bath. If high temperatures are required (>_37°), the appara£us may need to be placed in a chamber maintained at a high humidity to prevent excessive evapora£ion due to the vigorous stirring. A relatively large volume (in our experiments, 100 ml) of a mixture lacking one of the components necessary for initiation of a reaction is placed in the central chamber and all five stirrers are operated. A syringe containing the missing component of the reaction mixture is placed in the holder (q), and four syringes containing a solution that will terminate the reaction when injected into the mixture are placed in the holders (j).
458
MANOR AND CROWE
The syringes may need to be plugged with cotton to prevent premature dripping of their contents into the reaction mixture. The reaction is initiated when the contents of the syringe (q) are injected into the central chamber. About 2 see later, the handle (e) is pressed and the piston is moved down. Thus, the connections (i) are opened and the mixture flows by gravity into the peripheral chambers. Most of the liquid flows down in less than 2 see. The connections are closed again after 2 see to prevent a further slow flow of the liquid remaining in the central chamber. The reaction, which continues in the peripheral chambers, is terminated in eaeh of them separately by injecting a "terminating" solution from a syringe in (j). Employing manual operation, as we have done, it is possible to terminate the reaction in these chambers at intervals of 3-4 see. Finally, the upper part of the apparatus is removed and the samples are removed from the peripheral chambers and analyzed as required. AppLication. We have utilized the rapid sampling apparatus for a series of measurements of rates of polymerization of RNA molecules in vivo in exponentially growing bacterial cultures. The details and the results of these measurements are presented elsewhere (2). DISCUSSION The present model of the rapid mixing and sampling apparatus has been designed to withdraw four 25-50 ml a liquors. If necessary, similar machines ean be built to sample much larger volumes, or by increasing the number of peripheral chambers, to obtain more samples. Manual operation did not permit sampling at intervals of less than 3-4 see. It is, however, possible to introduce automation and thus decrease the intervals to fractions of a second. In either case, the apparatus is useful only if there exists a method for stopping the reaction in a time which is much shorter than the sampling interval. There are eertain limitations inherent in the design of the apparatus: (a) In order to analyze the sample after the reaction has been terminated, one has to remove the upper part to remove the samples; therefore this apparatus is not useful, for example, for experiments in which the samples have to be cooled down immediately following termination of a reaction. (b) Another limitation results from the fact {hat the mixture flows from the central to the peripheral chambers by gravity, and this requires 2-3 see; hence the first sample can be taken no sooner than about 3 see after the reaction is initia~ed, tIowever, this last difficulty can be overcome, if necessary, by designing an apparatus which would differ from the present one in that the mixture would flow into the peripheral chambers under pressure, and hence more rapidly.
FAST AND FREQUENT SAMPLING
459
The limitations considered above did not apply to our measurements of RNA chain propagation in bacteria. Furthermore, the present appara£us had the advantage that the bacteria could be grown in it for a long time and could be shown to undergo steady-state growth before and during rapid labeling experiments. Thus, it may be utilized in its present form for other in vivo studies of moderately fast reactions, in which these criteria must be met. It may, of course, be also utilized for studies of other kinds of chemical and biochemical reactions. SUMMARY An appara£us has been constructed to perform large-scale mixing and sampling experiments for studies of moderately fast reactions. In the form described in the present communication, the apparatus is operated manually and allows rapid mixing of 100-200 ml reaction mixtures and sampling of four 25-50 ml aliquots at 3-4 see intervals. ACKNOWLEDGMENTS We wish to thank Dr. G. S. Stent and David Goodman for helpful discussions. We also thank Alfred Stern, Tony Rago, and George Lauterbaeh for constructing the apparatus, and Avram
Maitlis and Ruben Cohen for the drawing. REFERENCES
1. CtIAI~'CE,B., EISENtIARDT, R. H., GIBSON, Q. ]=i., AND LONBERG-I-IoLM,K. K., eds., "Rapid Mixing and Sampling Techniques in Biochemistry" 1st Intern. Colloq; Philadelphia, July, 1964. Academic Press, New York, 1964. 2. MANOR, H., G00D-VlAN,D., AND STENT, G. S., ]. MoI. Biol. 39, 1 (1969).