General Description

The Graz Organ Bath consists of a Plexiglass baseplate with 4 vertical columns, each of which carries organ vessel, tissue carrier and transducer for measuring the contraction forces. Organ vessels are available in different sizes of 2, 5, 10 and 20 ml. The organ vessels carry a drain cock for draining the solution. Fresh solution which must be pre-warmed and aerated is introduced into the vessel with a syringe of appropriate size.

A frit is fused into the base of each organ vessel for aerating the solution. A needle valve is provided for separately adjusting the aeration rate of each vessel. The standard tissue carrier is suitable for mounting vascular rings. Other tissue carriers can be supplied in different forms to special order, depending on tissue type and whether electrical stimulation is required. 

Application

The Graz organ bath is intended for experiments on isolated tissue preparations in fundamental research in physiological and pharmacological laboratories. The standard version is particularly suitable for experiments on vascular tissues. 

Getting Started

The following items and equipment are required for work with the organ bath:

• Thermocirculator with approximately 3 liter capacity, distilled water, and algicidal agents such as Thermoklar.
• Carbogen supply for aeration, through a tank or central supply. 
• Reservoir for perfusion solution with frit for equilibrating the solution supply. The amount of solution required depends on the needs of the intended experiment. The solution reservoir should be placed inside the thermostat bath so that the solution is kept at the correct temperature and tissue contractions through temperature changes during washing are prevented. 
• Beakers (4) to collect the waste solution during washing.
• Transducers (4) to measure the muscle contractions (isometric or isotonic as required) with micrometer height control for fine adjustment of the preload setting. For isometric contraction measurement the use of the HSE force transducer FT20 or FT50 is recommended.
• A bridge amplifier for each transducer, for signal amplification and adaptation to a recorder or to a computer data capture system. 

Set-Up

Careful packing at the factory largely excludes transport damage. The organ bath is normally supplied completely assembled. The tissue vessels as ordered are mounted in position and the tubing is pre-affixed. The water circuit for your thermocirculator must be connected to the connection on the circulation thermostat. In addition the carbogen supply must be connected up, the transducers have to be fitted in position and connected to the measuring apparatus (bridge amplifier, recorder and/or data acquisition system). 

When deciding on the apparatus location the following should be observed. Vibrations are oft overlooked or their impact to the experiment underestimated. In particular thermostats, equipment with fans or amplifiers should not be located on the bench on which the apparatus is placed. This applies also to vessels containing sample dilutions. Equipment driven by electric motors can be picked up by the transducer.

In a recording these disturbances can not be distinguished from electrical hum interference. Care should be taken for other laboratory equipment that produce vibrations (e.g. centrifuges). The apparatus should not be placed near a window or door. Draughts or heating through vents or direct sunlight can interfere with the proper function of the thermostating or the transducers. With very sensitive measurements any draughts may even be transmitted through the connecting thread from tissue to the transducer and simulate faulty contractions.

In the measurement of tissue contraction forces (F) where rapidly varying forces have to be determined accurately, especially when the differential coefficient (dF/dt) has to be evaluated, exact experiments are only possibly by placing the system on an anti-vibration table. 

It is often necessary to handle liquids close to an organ bath. Be sure that the amplifiers and recording system are close but far enough away to be protected from accidental spills and splashes.

In order to prevent electrical noise it is important that the apparatus is not set up close to a mains power supply. Consider also that there may be electrical control panels, lifts or elevators that are not immediately seen. Please be sure that the equipment is properly powered through a grounded mains line. 

Once the apparatus location has been determined, the organ bath is set up on the table or bench or on the HSE subframe and aligned as accurately horizontal as possible.

1. Basic Unit (Plexiglass)
2. Stand Column
3. Tissue Vessel (pictured 20ml)
4. Tissue Holder
5. Force Transducer FT50 (not pictured)
6. Vernier Control
7. Manifold for Water Circulation
8. Gas Distribution Block with Needle Valves
9. Cables of the FT50/20 to the necessary Bridge Amplifiers

Detailed Views and Description

Legend for the illustrations above:

1. Stand column
2. Tissue vessel 20 ml (left and center illustrations) and 2 ml (right illustration)
3. Holder for tissue vessel
4. Glass frit, fused into the bottom of the tissue vessel
5. Shut-off stopcock for draining the tissue vessel
6. Gas supply tubing for the frit
7. Bosshead for securing the tissue by the holder (3)
8. Tubing connections for thermostating the tissue vessel. Note the connections of the individual tissue vessels: on the 2ml vessel the glass connections are fused in at the same level at the top edge of the vessel; on the 20 ml vessel one connection is fused in at the bottom and the other at the top of the vessel. on the 20 ml vessel the connections have to be made so that the thermostating liquid flows through the tissue vessel jacket from the bottom upwards. 
9. Stop ring for the bosshead (7). The stop ring has to be so adjusted that the tissue holder (13) just does not touch the Bottom (=frit) of the vessel when the tissue vessel is in its working position (center illustration).
10. Bosshead for the tissue holder (13)
11. Control knob for the micrometer vernier control for adjusting the tissue preload.
12. Isometric force transducer FT50/FT20
13. Tissue holder
14. Tissue hook with transmission wire to the force transducer
15. Fixed hook for mounting the tissue

Connecting the thermostatic circuit

The Plexiglass baseplate carries at the center of the back edge the manifold for the thermostatic circuit. The two connections at the side are intended to be connected to the thermostat (The TC100/TC120 manual may be useful here.) The tubing connections pointing upwards (two group of four each) are connected to the thermostating connections of the tissue vessels. It is important to ensure that for each vessel one connection is made to the left groups and the other to the right groups. When using larger tissues vessels (>5ml) it is important that the flow direction through them is from the bottom upwards. Otherwise air bubbles are not being flushed out of the thermostating jacket by the liquid. 

Connection of the thermostating circuit

1. Manifold
2. Connection for the inflow from the thermostat pump
3. Connection for the return to the thermostat
4. Supply connection to the tissue vessel (bottom), connection for the return to the manifold (top)

Tubing Connections for Thermostating

The tubing of the thermostating circuit is completely installed when the equipment is supplied. After the apparatus has been in use, despite regular and proper cleaning to inhibit the growth of algae, the tubing will need to be changed. We recommend using Silicone tubing, and if not available we recommend PVC tubing. 

When you replace the tubing, please ensure that the direction of flow through the larger tissue vessels (5 - 10 ml) is from the bottom upwards. On the smaller tissue vessels (2ml) the flow direction is not important. Here the connections are at the same level so that it is impossible for air pockets to form.

Connection for Aeration

A glass frit is fused into the bottom of the tissue vessels to ensure equilibration of the perfusion solution (carbogen is normally used for aeration). The gas is fed to the tissue vessels at the glass nipple which protrudes from the side below the frit (diameter 4mm approximately). A needle valve is provided for each tissue vessel for adjusting the degree of aeration. An additional needle valve serves for adjusting the aeration in the reservoir; a loose glass frit with tubing is supplied for this purpose. The 5 needle valves are combined in the Plexiglass valve block which is mounted with a clamping screw at the top end of the extra-long vertical column. This ensures that the needle valves do not fill with solution when the gas pressure is switched off, thereby preventing corrosion. At the front Plexiglass valve block the gas is connected to the gas supply using suitable tubing (Silicone tubing 3mm ID, 5mm OD).

The gas supply can be a central supply system or a gas cylinder. The supply must be provided with a reducing valve which should have an output pressure of about 0.5 bar but must not exceed 1 bar. 

WARNING: If you are using a high-pressure gas cylinder as your gas supply it is essential that you observe the regulation for securing and handling it.

1. Plexiglass needle valve block
2. Connection for gas supply
3. Needle valves for adjusting the aeration of the tissue vessels
4. Connecting tubing from the side outlet of the needle valves to the gas connections of the tissue vessels
5. Tissue vessels (2, 5, 10 or 20 ml)
6. Glass connection 
7. Glass frit fused into the bottom of the tissue vessel
8. Needle valve for adjusting the aeration in the solution reservoir
9. Glass frit with glass connection for aerating the solution reservoir
10. Connecting tubing

Filling the Thermocirculator

After all the tubing connections have been made correctly and the thermocirculator is assembled according to its own user manual, the next step is to fill the system. Use only distilled water; you can add one of the following to suppress algae growth:

• Sorbic Acid 0.1%
• Octanol or higher alcohol
• Thermoklar
• RBS 50
• Mucasol

1. First fill the thermocirculator.
2. Switch on the thermocirculator
3. If the tubing has been properly installed the water jackets for the tissue vessels should fill from the bottom up. If this does not happen check for leaks or kinks in the tubing.
4. As the system fills you can top off the thermocriculator tank.
5. If you find air pockets in the tissue vessels, try flushing them out by briefly lifting one side of the organ bath. Air bubbles in the tubing should be flushed out by tapping and squeezing the tubing while the thermocirculator is running.
6. Make sure that the thermostatic circuit is switched on early enough so that all parts of the system have reached the correct temperature at the beginning of the experiment. During the first few days of operation there is possibility for air bubbles to form in the thermocirculator through the outgassing of the liquid. Please be sure to remove them as described.

NOTE: If the thermocirculator is not running after it has been switched on please check the user manual. 

Spares

Tubing for the thermocirculator

Tubing for Aeration

Maintenance 

The daily effort involved in cleaning the apparatus depends largely on the substances being tested. If the substances do not adhere strongly to glass surfaces, cleaning can be limited to simply flushing the tissue vessel with distilled water. If this is not sufficient, a cleaning solution such as RBS 50 or Mucasol should be employed. When working with strongly adhering substances it is recommended to remove the glass vessels from the apparatus and to clean them conventionally with chrome-sulphuric acid. The stopcock keys must first be dismantled. The plastic parts of the stopcock (nut and pressure ring) should be kept away from concentrated cleaning agents. On re-assembly the stopcock keys have to be lubricated (for assembly and lubrication see below).

You should make it a regular rule to clean the apparatus thoroughly with the aid of a cleaning solution before each weekend. This involves filling and draining the vessels several times (with tissue holder immersed). Finally the cleaning solution should be left in the vessels stand over the weekend. Before starting experimental work again the solution has to be drained out of the vessels and the apparatus flushed thoroughly with distilled water several times.

Maintenance of the thermostatic circuit is limited to replacing the liquid immediately when algae are seen to form in the circuit. In any case the circulating liquid should be replaced regularly at least every 6 months. Any leakage in the thermostatic circuit should be dealt with immediately. The liquid level may fall in the course of time through evaporation; you should therefore check from time to time whether the distilled water has to be topped up.

In addition the correct aeration should be monitored. If the gas bubbles are too large it has to be suspected that the particular aeration frit has become partly blocked. It is important to ensure free movement and sealing of the stopcocks. When necessary the stopcock key has to be removed, cleaned, and re-lubricated with stopcock grease. To remove the key the plastic nut at the back has to be unscrewed. Watch out for the O-ring between the plastic pressure ring and the nut. Apply only a very thin layer of grease to the stopcock key. The bores must of course be kept free from grease. Reassemble the stopcock in the reverse order. Remember that the plastic nut has only a safety function against the key slipping out; tighten up the nut only very slightly. When the stopcock has been fully assembled the key must turn with a "sucking" action without any "scratching."

In principle the stopcock keys are interchangeable. After several hundred operations there is however a possibility that the stopcock become ground in so that they no longer seal perfectly if a key has been placed in a different stopcock body. It is therefore advisable to mark the stopcock keys before cleaning and to fit them always in the same stopcock. If you have to replace a stopcock keys, e.g. if it is broken, you have for the reason explained above to grind in the key in the stopcock body with grinding powder.

It goes without saying that external contamination through blood or saline solution has to be removed immediately. Use a moist cloth for that purpose. Any contamination difficult to remove can be attacked with a cleaning agent. In order to avoid damage to the apparatus you must use only the cleaning agents recommended by the manufacturer. If for any special reasons you require a different cleaning agent you must before using it carry out a compatibility test with the components of the apparatus. Note here the compatibility list for Plexiglass as shown in the appendix. In case of doubt contact the manufacturer of the apparatus.

Cleaning and Maintenance Agents

As cleaning solution we recommend RBS (supplied by Roth) or Mucasol (available from Merz). These solutions are supplied in concentrated form and have to be diluted with water in accordance with the manufacturer's instructions.

USE ONLY THE RECOMMENDED CLEANING AGENTS!

IMPORTANT: not all cleaning agents used in the laboratory are suitable for cleaning components made from Plexiglass. For example, Mucocit F produced by Merz (Frankfurt) attacks Plexiglass.

If for any special reasons you require a different cleaning agent you must before using it carry out a compatibility test with the components of the apparatus. Note here the compatibility list for Plexiglass as shown in Section 13. In case of doubt contact the manufacturer of the apparatus.

Alternative Cleaning Agents:

3% H₂O₂ Solution (3-percent hydrogen peroxide solution).

10% acetic acid. Do not use stronger solutions.

NOTE: NEVER USE ALCOHOL FOR CLEANING THE SYSTEM.