GB2065912A

GB2065912A - Improvements in or relating to a method of and device for embedding dehydrated tissue specimens in paraffin

Info

Publication number: GB2065912A
Application number: GB8025480A
Authority: GB
Original assignee: Individual
Current assignee: Individual
Priority date: 1979-08-08
Filing date: 1980-08-05
Publication date: 1981-07-01
Also published as: GB2065912B; DE2932112A1; JPS5633527A

Abstract

Dehydrated tissue specimens are immersed in liquid paraffin wax in container 10 which is evacuated by vacuum pump. The maximum vacuum is only achieved after a predetermined period of time, determined by a controller which can control the speed of the motor of the pump. Thus any dehydration medium, e.g. acetone, in the specimen will be sucked off at a low pressure minimising the risk of bubbles forming in the sample or in the paraffin. The paraffin wax is heated by water bath 16. <IMAGE>

Description

SPECIFICATION Improvements in or relating to a method of and device for embedding dehydrated tissue specimens in paraffin The invention relates to a method of, and a device for, embedding dehydrated tissue specimens in paraffin.

Tissue specimens for histological, histochemical or histoautoradiographic examination or for examination under the electron microscope, etc., must first be dehydrated and then must be embedded in a material such that histological sections can be prepared as required for examination. Paraffin, in the form of paraffin wax, together with certain plastics, are the preferred embedding materials. Hitherto, when paraffin is used the dehydrated tissue specimens are immersed in the paraffin, which has been heated until liquid, and are kept there until sufficiently impregnated. The liquid is then cooled to the solid state.

Recently methods and devices have been developed (German Auslegeschrift 27 53 710) for dehydrating tissue samples by means of hygroscopic liquids, more particularly acetone, which can considerably shorten the time required for dehydration. This has greatly increased the existing need to shorten the duration of the subsequent process of embedding in paraffin.

An attempt has already been made to speed up the process of embedding dehydrated tissue specimens in paraffin, by using a process in which the paraffin is subjected to a vacuum during the process of embedding (U.S. Patent Specification 2,324,122). It has been found, however, that if the specimen has been dehydrated with a hygroscopic liquid, and if the container which contains the paraffin is evacuated quickly, the hygroscopic dehydration medium, e.g. acetone, remaining in the tissue evaporates abruptly and produces bubbles which tear the tissue of the specimen, more particularly fatty tissue. The bubbles cause the paraffin to foam and increase in volume, with the risk that paraffin foam will be sucked into the pipe leading from the container to the vacuum pump, and the liquid paraffin will cool and harden and stop up the pipe or stop up the pump.

The present invention, seeks to provide a process of embedding dehydrated tissue specimens which still containing liquid dehydration medium in paraffin which is faster than the above described known method, and in which the risk of excessive foaming of the paraffin may be minimised.

According to one aspect of this invention there is provided a method of embedding dehydrated tissue specimens in paraffin, which comprises the steps of immersing the specimens in heated liquid paraffin within a container, sealing the interior of the container from the atmosphere and at least partially evacuating the sealed interior of the container, the evacuation being performed so that the maximum evacuation is not achieved until a predetermined period of time has elapsed, said maximum evacuation resulting in a pressure within the container of 0.05 to 0.2 Torr.

Preferably the evacuation is brought about so that the pressure within the container gradually and continuously decreases during the predetermined period of time, although alternatively the evacuation is brought about stepwise, the interior of the container first being evacuated to a first pressure comprising a partial vacuum and the resulting partial vacuum being maintained for a period of time, after which one or more further partial evacuation steps are carried out, the resulting partial vacuums of lower pressure being maintained on each occasion, until the maximum evacuation is achieved.The bubbleforming volatile substances are sucked away at the low partial vacuum that is present at the beginning of the evacuation process, without bubbles forming and damaging the tissue, whereas the final vacuum, which is important for efficiently impregnating the tissue with paraffin, is not brought to bear until the proportion of volatile constituents remaining is negligible.

The embedding process can be further speeded up if the container is exposed to ultrasonic vibration during evacuation.

The formation of large bubbles in the paraffin, resulting in excessive foaming at the surface, can also be prevented by an air stream, directed towards the paraffin surface during the evacuation process.

Preferably said predetermined period of time is at least 10 minutes. However, depending on the amount of warm liquid paraffin in the container, i.e. on the size and number of the tissue specimens to be embedded, the predetermined period of time may be longer than this. Thus, if, as a result of the size or number of tissue specimens to be embedded, a relatively large paraffin container holding a relatively large amount of paraffin is used, the predetermined time can be correspondingly lengthened up to a few hours.

According to another aspect of this invention there is provided a device for performing a method in accordance with the first aspect of the invention said device comprising a hermetically sealable and heatable container for containing paraffin, said container being connected by a pipe to a vacuum pump, there being a control unit for controlling the evacuation power of the pump in dependence on time.

If the vacuum pump is driven by a variablespeed electric motor, the control unit can be associated with the electric motor and adapted to vary the speed of the electric motor in dependence on time.

Preferably at least one electrically controllable aeration valve is disposed in the pipe between the paraffin container and the vacuum pump the control unit being connected to control said aeration valve to affect control of the evacuation power of the pump. With such an arranyement the vacuum in the container can be increased stepwise. At the beginning of the evacuation process, the valve is first opened to atmosphere, so that some of the suction power of the vacuum pump is used up in sucking in the ambient air.

After a certain time, the electrically actuated aeration valve can be electrically closed by the timed control unit, whereupon the full evacuating power of the pump will act exclusively on the container, and the vacuum will become correspondingly higher.

In another embodiment, where the vacuum can be controlled stepwise in a number of stages two or more electrically controllable aeration valves are disposed in the pipe between the paraffin container and the vacuum pump and the control unit is associated with the aeration valves and constructed so that it opens the valves at the beginning of the evacuation process and then shuts them after successive intervals of time.

Preferably the control unit comprises an adjustable timer whereby the duration of the process of evacuating from ambient pressure to the maximum evacuation can be optionally varied from 10 minutes to several hours.

The invention also relates to a tissue specimen whenever embedded by the process described above.

In order that the invention may be more readily understood and so that further features thereof may be appreciated the invention will now be described by way of example with reference to the accompanying drawings, in which: Figure 1 is a diagrammatic sectional view through a sealable container used for embedding dehydrated tissue specimens in paraffin; Figure 2 is a schematic circuit diagram of a first embodiment of a device for embedding tissue specimens in paraffin in which the speed of the evacuation process is controlled by a means for regulating the speed of the motor driving the vacuum pump; and Figure 3 is a schematic circuit diagram of a second embodiment of a device for embedding tissue specimens in paraffin, in which the vacuum in the paraffin container can be controlled stepwise by aerating valves.

Figure 1 shows a paraffin container 10 which can be closed in airtight manner by a removable cover 12 and evacuated via a suction nozzle 14 by a vacuum pump or the like. The entire container 10 is placed in a bath 1 8 filled with water 1 6 and heatable (in a manner not shown) so that the water therein can be heated to a temperature above the liquefaction temperature of paraffin. The paraffin in container 10 can thus be liquefied by the water bath.

Dehydrated tissue specimens are inserted into container 10 in a receiver 22, which advantageously has a perforated bottom and walls so that the liquid paraffin has free access to the tissue specimen. Thus the specimens may be immersed in the liquid paraffin.

Figure 2 diagrammatically shows the construction of a device for embedding dehydrated tissue specimens in paraffin. The connecting nozzle 14 of the paraffin container 10, which contains dehydrated tissue specimens immersed in liquid paraffin is connected by a suction pipe 24 to a vacuum pump 26 driven by an electric motor 28 which can be connected to the d.c. mains by a switch 30. The speed of motor 28 can be electrically controlled, with respect to time, by a control unit 32, by which the motor speed can be varied, e.g. by means which adjust the electric power supplied to the motor, which means are controlled by timing elements. Since the evacuating power of pump 26 depends on the speed of motor 28, the control unit 32 can be designed so that the paraffin container can be gradually and continuously evacuated to a pressure of between 0.05 and 0.2 Torr.

Figure 3 shows a device for embedding specimens in paraffin which differs from the previously-described device in that the process of evacuation in the paraffin container 10 is controlled not by varying the speed of the motor 28 driving pump 26 but by using one or more electrically actuable aerating valves 34, 34' disposed in the connecting pipe 24 between container 10 and pump 26.

The aerating valves 34, 34' are controlled by unit 32 as follows: Valves 34, 34' are switched on at the same time as the motor driving pump 26, so that the pump sucks not only the air in the paraffin container but also sucks air from the atmosphere through the constricted venting apertures of the aerating valves. This additional air, sucked through the aerating valves, limits the degree of vacuum which can be reached in the paraffin container.

After a preadjusted time, the control unit 32 closes the first valve 34, thus reducing the facility for sucking additional air from the atmosphere, leading to a higher vacuum in the container. After another preset time, valve 34' is also closed, whereupon the full suction power of pump 26 is applied to evacuating container 10, which thus reaches the required final vacuum, which in this case is of the order of 0.1 Torr.

Of course, variations and developments can be made in the inventive idea. For example, the device described in connection with Figure 3 can be modified by adding further aerating valves, thus increasing the number of partial evacuation stages and reducing the changes in pressure between the individual stages. If, on the other hand, the aerating valve 34 is a steplessly adjustable needle valve, the paraffin container can be gradually continuously evacuated.

In each case, the one important thing is that the treatment in the paraffin container should be carried out so that the required final vacuum in the container is only reached after a predetermined period of time, so that any hygroscopic dehydration medium remaining in the tissue specimens after the preceding dehydration process does not evaporate abruptly and form bubbles and cause the liquid paraffin to foam. It has been found that the process of evacuating from environmental pressure to the final vacuum should preferably not be carried out more quickly than in 10 minutes, if all risk of damaging the tissue is to be avoided. If larger tissue specimens, e.g. entire human or animal organs are embedded or if a number of tissue specimens are simultaneously embedded in the same container, which will be appropriately large, the time for reaching the final vacuum must be correspondingly increased.

Claims

1. A method of embedding dehydrated tissue specimens in paraffin, which comprises the steps of immersing the specimens in heated liquid paraffin within a container, sealing the interior of the container from the atmosphere and at least partially evacuating the sealed interior of the container, the evacuation being performed so that the maximum evacuation is not achieved until a predetermined period of time has elapsed, said maximum evacuation resulting in a pressure within the container of 0.05 to 0.2 Torr.

2. A method according to claim 1, wherein the evacuation is brought about so that the pressure within the container gradually and continuously decreases during the predetermined period of time.

3. A method according to claim 1, wherein the evacuation is brought about stepwise, the interior of the container first being evacuated to a first pressure comprising a partial vacuum and the resulting partial vacuum being maintained for a period of time, after which one or more further partial evacuation steps are carried out, the resulting partial vacuums of lower pressure being maintained on each occasion, until the maximum evacuation is achieved.

4. A method according to any one of claims 1 to 3, wherein the container is exposed to ultrasonic vibration during evacuation.

5. A method according to any of claims 1 to 4, wherein during the evacuation process, a stream of air is directed on to the surface of the paraffin.

6. A method according to any one of claims 1 to 5, wherein said predetermined period of time is at least 10 minutes.

7. A device for performing a method according to any of claims 1 to 6, wherein said device comprising a hermetically sealable and heatable container for containing paraffin, said container being connected by a pipe to a vacuum pump, there being a control unit for controlling the evacuation power of the pump in dependence on time.

8. A device according to claim 7, in which the vacuum pump is driven by a variable-speed electric motor, wherein the control unit is associated with the electric motor and adapted to vary the speed of the electric motor in dependence on time.

9. A device according to claim 7, wherein at least one electrically controllable aeration valve is disposed in the pipe between the paraffin container and the vacuum pump the control unit being connected to control said aeration valve to effect control of the evacuation power of the pump.

10. A device according to claim 9, wherein two or more electrically controllable aeration valves are disposed in the pipe between the paraffin container and the vacuum pump and the control unit is associated with the aeration valves and constructed so that it opens the valves at the beginning of the evacuation process and then shuts them after successive intervals of time.

11. A device according to any of claims 7 to 10, wherein the control unit comprises an adjustable timer whereby the duration of the process of evacuating from ambient pressure to the maximum evacuation can be optionally varied from 10 minutes to several hours.

12. A method of embedding dehydrated tissue specimens in paraffin substantially as herein described with reference to Figures 1 and 2 of the accompanying drawings.

13. A method of embedding dehydrated tissue specimens in paraffin substantially as herein described with reference to Figures 1 and 3 of the accompanying drawings.

14. An embedded tissue specimen whenever made by the method of any one of claims 1 to 6, 12 and 13.

1 5. A device for performing a method according to any one of claims 1 to 6 substantially as herein described with reference to and as shown in Figures 1 and 2 of the accompanying drawings.

16. A device for performing a method according to any one of claims 1 to 6 substantially as herein described with reference to and as shown in Figures 1 and 3 of the accompanying drawings.

1 7. Any novel feature or combination of features disclosed herein.