Axon Axoporator 800A Theory And Operation Download

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Axoporator 800A

SINGLE-CELL ELECTROPORATOR

Theory and Operation

Part Number 2500-0158 Rev C March 2005 Printed in USA

No part of this manual may be reproduced, stored in a retrieval system, or
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Summary of Contents for Axoporator 800A

Page 1: ... stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying microfilming recording or otherwise without written permission from Molecular Devices Corp QUESTIONS See Axon s Knowledge Base http support axon com 800 998 MATE www autom8 com 650 University Ave 5 Berkeley CA 94710 USA t e l 5 1 0 8 4 5 6 2 8 3 f a x 5 1 0 6 6 5 3 9 7 5 e m a i l i n f o a ...

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Page 3: ...vertheless researchers should independently verify the basic accuracy of the controls using resistor models of their micropipettes WARNING If this equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired DISCLAIMER This equipment is not intended to be used and should not be used in human experimentation or applied to humans in any way...

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Page 5: ...ns 10 Optional Configurations 11 Oscilloscope 11 Chapter 4 Reference Section 13 Audio Monitor 13 Headstage 14 High Voltage Precautions 14 Static Precautions 14 Hot Plugging 14 Types 14 Voltage Clamp Circuit 14 Noise 16 Mounting 16 Bath Connection 16 Electroporating while Recording 16 Cleaning 16 Front Panel Controls 17 Front Panel Display 18 Rear Panel Inputs Outputs 22 Table of Contents ...

Page 6: ...Loading Dissociated Cells in Culture 40 Testing the Setup 43 Chapter 6 Troubleshooting Guide 45 General Rule 45 Symptom High or infinite tip resistance 45 Symptom Decrease in tip resistance 45 Symptom Positional drift 46 Symptom Contents leaking from micropipette tip 46 Symptom Unable to load cells 46 Symptom Current or power reading is out of range 47 Symptom Grey out of pulse frequency or width ...

Page 7: ...e mode internal 52 LCD display 53 Rear panel 54 Power requirements using the provided power supply 56 Model Cell 56 Accessories Provided 56 References 57 Technical Assistance 61 Warranty and Repair Service 63 Declaration of Conformity 67 Important Safety Information 69 Index 71 Table of Contents ...

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Page 9: ...ne potential reaches approximately 0 25 V to 1 V the electrostatic forces holding the lipid bilayer together break down causing a reconfiguration of the membrane phospholipids and creation of minute pores in the small regions of the membrane at each pole Neumann et al 1999 Ho et al 1996 Once formed these pores 20 nm to 120 nm in diameter remain open even after the external electric field is remove...

Page 10: ...livery of molecules A number of technical approaches have been devised to target individual cells including microelectrodes Lindqvist 1998 Olofsson 2003 electrolyte filled capillary tubes Nolkrantz 2001 and electronic chips Huang 2000 A most efficient method for single cell electroporation employs a glass micropipette that restricts the electric field to a single cell Haas 2001 Rae 2002 This is ac...

Page 11: ...Haas 2001 Sin 2002 The ability of single cell electroporation to co deliver multiple molecules including multiple genes to any cell type makes single cell electroporation a powerful and versatile method With Axon Instruments Axoporator 800A it is possible to electroporate individually targeted cells Axoporator 800A provides precise control of the voltage pulse and train parameters The result is op...

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Page 13: ... sure that the Axoporator 800A disconnected from its power supply Connect the AP 1A headstage to the Headstage input connector at the rear of the Axoporator 800A Connect the SCE 1U model cell as shown in Figure 1 The 1 mm pin of the SCE 1U must make a firm connection to the 1 mm input socket at the front of the AP 1A headstage Make sure that the toggle switch on the SCE 1U model cell is set so tha...

Page 14: ...nd adjust the Offset Voltage knob beneath the Pulse Voltage knob so that the DC offset reads 0 0 V Adjust the Pulse Width far right row of panel controls until the display reads 100 ms Press and hold the Trigger button bottom right of front panel controls The current reading upper left of the display should be 100 1 nA and the power reading near the center of the display should be 1 0 01 µW Termin...

Page 15: ...Functional Checkout 7 Figure 1 Connections between the AP 1A headstage to the SCE 1U model cell Functional Checkout ...

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Page 17: ...le cfm ArticleID 433 Before connecting your Axoporator 800A to an electrical outlet read Important Safety Information starting on page 69 Micromanipulator The micromanipulator must provide appropriate access to the biological specimen when viewed through the microscope For cells in dissociated cultures or slice preparations manipulators with fine and course adjustments are required Coarse manipula...

Page 18: ...to a region of tissue thought to contain the cell bodies of the target cells Successful electroporation then depends on the probability that the micropipette tip makes contact with the target cells While blind targeting using low magnification objectives will reduce electroporation efficiency the long working distances of these objectives allow rapid movement between sites in the preparation There...

Page 19: ...h 2 mm plugs to fashion a connector to run from the ground electrode to the back of the headstage Optional Configurations Additional inputs and outputs that extend the flexibility of the Axoporator are available as well To follow the voltage or current signals at the micropipette connect to the Voutput and Ioutput respectively To use the audio monitor plug in a headset or external speaker To trigg...

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Page 21: ...has entered the preparation or detecting the increases in tip resistance as it comes into proximity to the cell membrane The output is monitored via the Headphone output on the rear panel The audio monitor settings are user selectable See Figure 6 The audio output determines the volume Menu 1 The starting pitch level sets the VCO offset Menu 2 The sensitivity setting adjusts the rate at which the ...

Page 22: ...a grounded metal object immediately before touching the AP 1A headstage Hot Plugging Never connect the headstage when the Axoporator 800A is switched on Types Axoporator 800A has two types of headstages AP 1A 1MU with a feedback resistor Rf of 1 MΩ AP 1A 0 1MU with a feedback resistor Rf of 0 1 MΩ Never attempt to connect any other headstage or device to the headstage input of the Axoporator 800A ...

Page 23: ...action of the simple resistive divider formed by Re and Rc the voltage at the membrane in the vicinity of the pipette tip is Vin Rc Re Rc In a typical example Vin 6 V Rc 6 MΩ and Re 30 MΩ resulting in a voltage at the outer surface of the membrane Vm of about 1V Clearly increasing Rc will result in a higher fraction of Vin being delivered to the cell surface This is accomplished by pressing the mi...

Page 24: ... recording from it has not been attempted It is possible that large voltage pulses would damage the recording headstage It is also possible that the recording noise would increase during when an electroporating pulse is in progress Cleaning Switch off the Axoporator 800A and disconnect the headstage from the Axoporator 800A Wipe the headstage connector with a damp cloth to clean salt spills Avoid ...

Page 25: ...igger activation such as the number of cells electroporated E PULSE TYPE Selects pulse type rectangular bipolar and bi level Rectangular and bi level pulses can be selected as either positive or negative F TRAIN DURATION Sets the duration of trains when pulse trains are selected Train durations can last between 10 ms and 100 s G PULSE FREQUENCY Sets the frequency of the pulses H SINGLE TRAIN Toggl...

Page 26: ...y screen provides a measure of the current and the power of the pulse or pulse train For example if a 10 V step is applied to a 10 MΩ micropipette then one would expect the resulting current to be 1 µA and power to be 10 µW However this does not account for the duty cycle If the pulse width is 300 ms and the pulse frequency is 1 Hz then the duty cycle is 0 3 or 30 The Axoporator automatically acco...

Page 27: ...orts Active when stimuli are being delivered and Single Train selection at other times H Rf Indicates the nominal value of the feedback resistor for the headstage in use I Total Power output of a single pulse or train is presented in µW When power cannot be computed four arrows appear Blank when an external waveform generator is used J Micropipette Resistance MΩ Provides measure of the sum of the ...

Page 28: ... E Pulse Type Displays a representation of the selected pulse type F Train When trains are selected this display shows twin representation of the selected pulse types The individual pulses within a train are defined by type amplitude and duration in the same way that single pulses are defined In addition when trains are selected the train duration and pulse frequency must be set Pulse frequency pu...

Page 29: ...level pulse External Command Disables the internal waveform generator of the Axoporator when Ext Cmd is set to On Menu 2 VCO Offset Indicates starting pitch level of audio tone Sensitivity Indicates the rate at which the pitch of the audio tone changes Inverse Indicates 0 when black on white is selected and 1 when white on black selected Backlight Indicates duration in seconds that backlight is to...

Page 30: ...witch accessory Sync Output Primarily designed for synchronizing the input trigger of a monitoring system such as an oscilloscope There is trigger pulse that coincides with that of the resistance measurement frequency 100 Hz and that of the pulse frequency The Sync Output puts out a pulse of 100 Hz even when an external command is employed For automated triggering of the Axoporator the Ext Trigger...

Page 31: ...ommand always sums with the internal command even if the external command mode is set to Off Thus no external signal generator should be connected to the Axoporator unless it is to be used as the only source of a command The resistance current and power readings on the display screen will not be valid when the external command is active Successful single cell electroporation is critically dependen...

Page 32: ... Connector for AP 1A headstage For Factory Test Only For factory test procedures 12 14 VDC Connector for external power supply On Off switch Axoporator 800A Theory and Operation Copyright 2005 Axon Instruments Molecular Devices Corp ...

Page 33: ...lar and bi level Pulse amplitude and timing are set via the intuitive control interface discussed in the Front Panels Controls portion of the Reference Section Train duration Pulse width 1 frequency b a Positive rectangular pulse Bipolar pulse Positive Bi level pulse Sync Figure 9 Axoporator pulse types Reference Section ...

Page 34: ... order of presentation within the pulse is component a followed by component b or vice versa The polarity of the two components is the same and either negative or positive polarity can be selected The voltage amplitude of component a is selected first The amplitude of component b is determined by changing the b a V ratio The total bi level pulse duration is selectable the duration of component a i...

Page 35: ...n Using Axoporator s Pulse Generator The maximum duty cycle is 50 Stated differently the period cannot be less than double the pulse width With an external pulse generator you can increase the duty cycle Train duration must be at least equal to the pulse width If the train duration is to remain fixed then the pulse duration must decrease as pulse frequency increases Sample problems 1 Calculate the...

Page 36: ...ring Pulses are triggered by pressing the front panel TRIGGER button by pressing the footswitch or by an external input Micropipette Holder The HL U series holder provides a universal fit for a very wide range of glass pipette diameters and will fit any of the U type headstages from Axon Holder Design The barrel of the holder is made of polycarbonate There are two different barrel lengths 16 mm an...

Page 37: ...ette OD Color coding aids identification of the four sizes of cone washers 1 0 mm orange 1 3 mm cl pipette OD falls between two sizes of cone washers the larger size cone washer should be used For instance if the pipette OD is 1 6 mm then use a cone washer with an ID of 1 7 mm An Ag AgCl pellet offe Moreover the diameter of the pellet 1 mm restricts its use to pipettes with a large ID 1 1 mm There...

Page 38: ...ck end of the micropipette ds of the the pipette should be filled with solution The chlorided tip of the wire should be inserted into this solution Avoid wetting the holder since this will increase the noise Make sur relieved but do not rem through the cap and cone washer until it presses against the end of the bore Gently tighten the cap so that the micropipette is gripped firmly To minimize cutt...

Page 39: ...ments are suggestive of the need for rechloriding The chloride region should be long enough so that the micropipette solution does not come in contact with the bare silver wire Heat smoothing the back end of the electrode extends the life of the chloride coating by minimizing the amount of scratch damage Another way to protect the AgCl coating is to slip a perforated Teflon tube over the chlorided...

Page 40: ... the AP 1A input An unlabeled the rear Two resistance settings 10 MΩ and 15 MΩ are available input of the AP 1A headstage Model Cell SCE 1U model cell is a stand The model cell h 2 mm gold socket on the model cell connects to the 2 mm grounding socket on of the AP 1A headstage The circuit is shown in Figure 12 Figure 12 SCE 1U circuit Axoporator 800A Theory and Operation Copyright 2005 Axon Instru...

Page 41: ...ropipette tip shank may also be an important consideration Micropipettes of the type described here will work for almost any cell type tried to date Use capillary glass whose internal diameter is 1 15 to 1 2 mm and outside diameter is 1 6 to 1 7 mm with a glass filament fused to its interior to facilitate tip filling Inexpensive glass like Corning Pyrex or Kimble Kimax is available from a variety ...

Page 42: ...icropipette either by immersing it in a small volume of the solution or by injecting the solution from a syringe A less desirable filling method would be to immerse the microelectrode tip into the solution containing the molecules to be electroporated and apply suction to the back of the micropipette to fill the tip This would be an extremely slow process for viscous solutions Typically the molecu...

Page 43: ...nerally done in two stages 1 Use a hand held pipetter with a 10 µl disposable tip Add 2 to 3 µl of the solution to be electroporated to the back of the micropipette The solution moves along the internal glass filament by capillary forces and fills the tip It may take a few taps of the glass to get the flow started After 30 seconds or so apply a few gentle taps near the shank to dislodge bubbles 2 ...

Page 44: ...place Attach the HL U holder to the AP 1A headstage that is mounted on a micromanipulator Positioning the Micropipette Tip While the current is measured the micropipette tip is slowly advanced against the cell membrane identical to a standard patch clamp measurement at the time of seal formation As the pipette pushes against the cell and increases the cleft resistance the current falls as the sum ...

Page 45: ...gree angle common for a patch clamp setup The thickness of the cell is drawn to scale It is important to note that the site of electroporation need not be restricted to the cell soma and can be directed at any portion of the cell This can be useful for those studies using polarized cells such as photoreceptors or those cases where it is desirable to induce an initial localized increase of the mole...

Page 46: ... For most applications simple rectangular pulses suffice The pulses must result in total channel open time sufficient for an effective concentration of molecules to enter the cell Not surprisingly several different combinations of pulse widths frequencies and train durations may work equally well For further flexibility an external waveform generator can be used Pulse amplitude and duration influe...

Page 47: ...sion The attachment is sufficiently strong that a tissue cultured cell can be torn from the bottom of the culture dish as the micropipette is withdrawn Therefore for α TN4 cells an additional constraint can be placed on micropipettes average dissipated power should be 0 6 µW or less Therefore extreme caution is required when using low resistance e g 1 MΩ to 7 MΩ micropipettes The 0 6 µW limit for ...

Page 48: ... a general rule to maximize cell loading and optimize cell health use high concentrations of the molecules to be delivered and minimize the voltage amplitude and stimulus duration Overview of Protocols for Loading Dissociated Cells in Culture Since the details for single cell electroporation of various molecules are documented in the scientific literature we present only an abbreviated list of sug...

Page 49: ...large DNA molecules For genes ranging from 4 to 5 kb 5 5 V to 6 V is sufficient Larger voltages are required for larger genes To date 14 kb is the largest gene that has been successfully electroporated In general expression of the larger genes is problematic As always the polarity of the voltage pulse must be the same as the net charge on the gene Start with a pulse voltage of 6 V and width of 1 m...

Page 50: ...has worked for the electroporation of small molecules e g dyes oligos salts is one in which level a precedes level b The a component of the bi level pulse opens the channels and b component drives the dye molecules into the open channels of the cell The initial settings for this type of bi level pulse are a 20 V b a 5 that is the amplitude of b is 1 V Pulse duration 100 ms The result is a 20 V pul...

Page 51: ...ion of fluorescent dye like rhodamine dextran 3000 Place the saline solution in a small chamber such as a 35 mm culture dish on the stage of an epifluorescence microscope with the ground electrode immersed in the saline Position the micropipette in the saline and focus on the tip Look for dye leaking from the tip under epifluorescent illumination Molecules may leak out of the micropipette when it ...

Page 52: ...mine which pulse parameters provide best control over dye movement Optimal parameters should be able to deliver concise boluses of dye with no leakage Axoporator 800A Theory and Operation Copyright 2005 Axon Instruments Molecular Devices Corp ...

Page 53: ... ground electrode is in contact with bath or preparation Test all wires and connections for disconnections or breaks Possible cause Clogged micropipette Suggestions Place micropipette tip in bath and apply pulses of inverted polarity Replace micropipette tip Spin down all pipette solutions to remove undissolved particles Symptom Decrease in tip resistance Possible cause Broken tip Troubleshooting ...

Page 54: ...omponents service manipulator or replace it Symptom Contents leaking from micropipette tip Possible cause Offset potential not set optimally Suggestions Chloride silver wire in micropipette holder and use chloride internal solutions Adjust offset voltage Symptom Unable to load cells Possible cause Incorrect tip geometry Suggestions Try smaller tip diameter Try fluorescent dextran dye to find appro...

Page 55: ...ompute correct current I A Can t compute correct power reading µW displays four arrows Suggestions Decrease amplitude frequency and or width of pulse Symptom Grey out of pulse frequency or width reading Possible cause Duty cycle requested is 50 Suggestion Reduce pulse frequency or width Symptom No Rf value is reported Possible cause Headstage is not properly connected Suggestion Switch off the Axo...

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Page 57: ...pulses AP 1A Headstage voltage clamp Main unit connection via DB 15 connector Connect to or disconnect from the main unit only when the power is off Size 2 25 x 1 14 x 0 87 57 2 mm x 29 0 mm x 22 1 mm Mounting rod length 102 mm 4 Mounting rod diameters 1 4 5 16 standard or 3 8 6 3 mm 7 0 mm or 9 5 mm Specify non standard mounting rod diameter with order Mounting plate Acrylic Ground socket 0 08 2 ...

Page 58: ...Current 10 0 µA Power 100 µW Maximum deliverable to micropipette correct pulse measuring subsystem overloaded Current 30 0 µA AP 1A 0 1MU optional Gain Rf 100 kΩ Maximum deliverable to micropipette with measurement subsystem system functional Current 100 0 µA Power 100 µW Maximum deliverable to micropipette correct pulse measuring subsystem overloaded Current 300 0 µA Axoporator 800A Theory and Op...

Page 59: ... x 10 3 wide x 7 3 deep 105 mm x 262 mm x 185 mm Net weight 3 2 lbs 1 45 kg Operating modes Active mode A train or single pulse is generated in response to a trigger Approach mode In this mode the system generates a low level approach waveform for resistance measurement Approach mode is activated when in Active mode is in operation Type Square wave Frequency 100 Hz Amplitude 0 1 to 1 0 V in steps ...

Page 60: ...9 20 22 24 46 48 50 55 60 65 90 95 100 Pulse Voltage 1 to 100 V in steps following the 10 pattern Sum of DC offset and pulse voltage does not exceed 100V Bi level pulse Component b to component a voltage ratio is 5 to 95 in 5 steps Pulse Width Mono polar pulse 200 µs to 1 s Bi polar pulse 400 µs to 1 s Bi level pulse 10 ms to 20 s Component a width to whole pulse width a b ratio is fixed at 2 1 50...

Page 61: ...Pulse and train parameters DC offset and menu options bi level pulse ratio screen appearance and audio monitor Readouts All modes Items cleared on power up or clear button Trigger count 0 9999 Manual count 0 9999 Elapsed time hh mm ss Active mode Micropipette tip resistance MΩ measurement AP 1A headstage feedback resistance Rf Items held for 2 seconds following a return to approach mode Delivered ...

Page 62: ...ation AP 1A Headstage DB 15F connector Used only for AP 1A 1MU and AP 0 1A 0 1MU headstages Connect to or disconnect from the main unit only when the power is off Outputs Ioutput micropipette current Sensitivity 1 V µA for AP 1A 1MU headstage Sensitivity 0 1 V µA for AP 1A 0 1MU headstage Output impedance 500Ω Voutput voltage applied to the micropipette Sensitivity 0 1 V V Output impedance 475Ω Sy...

Page 63: ... db The lower of 40 db or last setting is power up default Offset frequency at 10 x Rf 50 100 200 400 800 Hz 200 Hz is factory default Inputs Ext Trigger external trigger Positive going pulse starts internal pulse generation by switching to active mode Minimum width 100 ms Above 2 4 V accepted as logic HIGH below 0 5 V is accepted as logic LOW Protected to 15 V Foot Switch external trigger Normall...

Page 64: ... 0 9A Line frequency 50 60 Hz DC out to main unit 12 14 VDC Model Cell SCE 1U 10 MΩ 15 MΩ switchable Accessories Provided Theory and Operation Manual AP 1A 1MU headstage AP 1A 0 1MU headstage optional may be substituted on request with order SCE 1U 10 MΩ 15 MΩ switchable One HL U electrode holder Two 2 mm plugs for use with headstages Two ground wires One mounting rod Foot Switch Axoporator 800A T...

Page 65: ...S and Orwar O Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes Proc Natl Acad Sci USA 95 10356 10360 1998 Nolkrantz K Farre C Brederlau A Karlsson R I Brennan C Eriksson P S Weber S G Sandberg M and Orwar O Electroporation of single cells and tissues with an electrolyte filled capillary Anal Chem 73 4469 4477 2001 Rae J L and Levis R A Single cel...

Page 66: ...onductance in human erythrocyte membranes Biochim Biophys Acta 554 479 497 1979 Koshiba Takeuchi K Takeuchi J K Matsumoto K Momose T Uno K Hoepker V Ogura K Takahashi N Nakamura H Yasuda K and Ogura T Tbx5 and the retinotectum projection Science 287 134 7 2000 Marszalek P E Farrell B Verdugo P and Fernandez J M Kinetics of release of serotonin from isolated secretory granules II Ion exchange deter...

Page 67: ...Toensing K Fundamentals of electroporative delivery of drugs and genes Bioelectrochem Bioenerg 48 3 16 1999 Olofsson J Nolkrantz K Ryttsen F Lambie B A Weber S G and Orwar O Single cell electroporation Curr Opin Biotechnol 14 29 34 2003 Sakamoto K Nakamura H Takagi M Takeda S and Katsube K Ectopic expression of lunatic Fringe leads to downregulation of Serrate 1 in the developing chick neural tube...

Page 68: ...rowth Differ 42 203 206 2000 Yasugi S and Nakamura H Gene transfer into chicken embryos as an effective system of analysis in developmental biology Dev Growth Differ 42 195 197 2000 Zheng Q A and Chang D C High efficiency gene transfection by in situ electroporation of cultured cells Biochim Biophys Acta 1088 104 110 1191 Axoporator 800A Theory and Operation Copyright 2005 Axon Instruments Molecul...

Page 69: ... to resolve a problem there are several ways to contact Axon Instruments Molecular Devices World Wide Web www axon com Phone 1 800 635 5577 Fax 1 510 675 6300 E mail axontech axon com Questions See Axon s Knowledge Base http support axon com Technical Assistance ...

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Page 71: ... product to the customer If the shipment is to a location outside the United States the customer will be responsible for paying all duties taxes and freight clearance charges if applicable The warranty is valid when the product is used for its intended purpose and does not cover products which have been modified without approval from Axon Instrument Molecular Devices or which have been damaged by ...

Page 72: ...solidly built instrument designed to survive shipping around the world However in order to avoid damage during shipping the Axoporator 800A must be properly packaged In general the best way to package the Axoporator 800A is in the original factory carton If this is no longer available we recommend that you carefully wrap the Axoporator 800A in at least three inches 75 mm of foam or bubble pack she...

Page 73: ... it is inexpensive compared to the cost of repairing an instrument that has sustained shipping damage It is your responsibility to package the instrument properly before shipping If the packaging is inadequate and the instrument is damaged during shipping the shipper will not honor your claim for compensation Warranty and Repair Service ...

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Page 75: ...ication of Council Directives EC EMC Directive 89 336 EEC as amended EC Low Voltage Directive 73 23 EEC as amended Harmonized Standards to which Conformity is Declared EMC EN 61326 1998 A2 2001 EN 55011 AS NZSCISPR11 2002 Group 1 Class B Safety EN 61010 1 2001 I the undersigned hereby declare that the equipment specified above conforms to the above Directives and Standards Authorized Signature and...

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Page 77: ... to the outlet and the power supply to the Axoporator Basic Equipment Setup and Safety 1 Connections Use the included IEC power cord to connect the instrument to a GROUNDED power receptacle That is the mains power cord must be plugged into an earthed mains outlet Routinely inspect the mains power cord and replace it if it s damaged 2 Assembly The headstage connects to the instrument through the re...

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Page 79: ...h Connection 16 Cleaning 16 69 Mounting 16 Noise 16 Precautions High voltage 14 Hot plugging 14 Static 14 Types 14 Voltage Clamp Circuit 14 Inputs Outputs 22 EXT COMMAND 23 EXT TRIGGER 22 FOOT SWITCH 22 Headphone 22 HEADSTAGE 24 IOUTPUT 23 SYNC OUTPUT 22 VOUTPUT 23 Micromanipulator 9 Micropipette Holder 28 Adapters 31 Design 28 Maintenance 31 Use 30 Micropipettes Fabricating 33 Filling 34 Mounting...

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