The radiography of ‘soft’ tissues normally requires special X-ray apparatus which generates soft X-rays. Fixation with mercuric chloride, either alone or in mixture, has been found to render such ‘soft’ tissues opaque to those harder X-rays generated by normal clinical X-ray apparatus. Radiographs made by the use of such standard apparatus may be useful in several fields of biological research such as anatomical investigation and illustration, or in the study of the penetration and behaviour of the fixatives themselves.

In work previously recorded (Foxon and Rowson, 1956) the fate of the radioopaque medium, thorotrast, injected into the dorsal lymph space of the frog was followed. The methods used sometimes necessitated the removal, from a freshly killed frog, of the alimentary canal together with the liver and spleen which were then radiographed separately from the rest of the body; after-wards the alimentary canal and associated glands were fixed and sectioned by the usual methods, and the appearances seen in the radiograph were interpreted in the light of evidence provided by a study of the sections. On one occasion after an alimentary canal had been placed overnight in Zenker-formaldehyde solution it was decided that it should be radiographed again. Whereas usually only those parts of the preparation containing thorotrast had shown in the radiograph, on this occasion it was found that the whole alimentary canal had given X-ray shadows of remarkable clarity, the folds of the mucous membrane in the intestine being especially conspicuous. Such a radio-graph is shown in fig. 1, A.

FiG. 1.

(plate). Positive enlargements of radiographs showing: portion of intestine of frog. (Fixed Zenker-formaldehyde. Ilford Process plate. 200 mA. 30 kV 2 25 sec. Focus/film distance 50 cm. Developed I.D. 2 diluted 1 to 2 of water, 6 min at 70o F.) blastoderm with embryo chick. (Fixed Zenker-formaldehyde. Ortholine Cut-Film. 100 mA. 30 kV 5-25 sec. F/f distance 50 cm. Developed as A.) c, a planarian. (Anaesthetized in chloretone. Fixed Zenker-formaldehyde. Ortholine CutFilm. Exposure and development as B.) D, a sheep liver-fluke. (Fixed Zenker-formaldehyde in November 1955; radiographed 21.ii.56. Ilford Process plate, too mA. 3okV 4 5 sec. F/f distance 50 cm. Developed as A.) E, two proglottids of a human tapeworm. (Fixed in formalin, subsequently soaked in Zenker-formaldehyde for 5 days. Ortholine Cut-Film, too mA. 30 kV 6 5 sec. F/f distance 50 cm. Developed as A.) F, the anterior part of an earthworm in side view. (Worm killed in alcohol, then fixed in Zenker-formaldehyde. Ilford Process plate, too mA. 40 kV 3 5 sec. F/f distance 50 cm. Developed as A.) G, a dissection of the anterior segments of an earthworm, dorsal view. (Worm killed in alcohol, then dissected and then soaked in Zenker-formaldehyde for 24 hours. Ilford Process plate, too mA. 35 kV 4-5 sec. F/f distance 50 cm. Developed as A.)

FiG. 1.

(plate). Positive enlargements of radiographs showing: portion of intestine of frog. (Fixed Zenker-formaldehyde. Ilford Process plate. 200 mA. 30 kV 2 25 sec. Focus/film distance 50 cm. Developed I.D. 2 diluted 1 to 2 of water, 6 min at 70o F.) blastoderm with embryo chick. (Fixed Zenker-formaldehyde. Ortholine Cut-Film. 100 mA. 30 kV 5-25 sec. F/f distance 50 cm. Developed as A.) c, a planarian. (Anaesthetized in chloretone. Fixed Zenker-formaldehyde. Ortholine CutFilm. Exposure and development as B.) D, a sheep liver-fluke. (Fixed Zenker-formaldehyde in November 1955; radiographed 21.ii.56. Ilford Process plate, too mA. 3okV 4 5 sec. F/f distance 50 cm. Developed as A.) E, two proglottids of a human tapeworm. (Fixed in formalin, subsequently soaked in Zenker-formaldehyde for 5 days. Ortholine Cut-Film, too mA. 30 kV 6 5 sec. F/f distance 50 cm. Developed as A.) F, the anterior part of an earthworm in side view. (Worm killed in alcohol, then fixed in Zenker-formaldehyde. Ilford Process plate, too mA. 40 kV 3 5 sec. F/f distance 50 cm. Developed as A.) G, a dissection of the anterior segments of an earthworm, dorsal view. (Worm killed in alcohol, then dissected and then soaked in Zenker-formaldehyde for 24 hours. Ilford Process plate, too mA. 35 kV 4-5 sec. F/f distance 50 cm. Developed as A.)

It was realized that this curious result had been obtained because mercuric salts had been absorbed by the tissue of the alimentary canal, and this was confirmed by attempting to radiograph specimens fixed in a non-mercuric fixative such as Bouin’s fluid, which gave no result, and also by using mercuric chloride both with and without acetic acid. These two fixatives gave similar but not superior radiographs to Zenker-formaldehyde.

This property of fixatives containing mercury does not appear to have been recorded before. It would seem to be of importance from two points of view. First, because the affinity between the mercury and different tissues seems to be unequal, it is possible to produce an effect akin to ‘differential staining’ ; and, second, because it would be possible to use this method to investigate the penetration or, alternatively, the removal from tissue of mercury-containing fixatives.

The ‘differential staining’ effect just referred to is well shown in a radiograph of a chick-embryo (fig. 1, B), in which it is very remarkable how the lining of certain of the blood-vessels of the area vasculosa have cast very marked shadows. In Platyhelminthes the alimentary canal seems to show well and sometimes the reproductive organs (fig. 1, C-E). Such radiographs as these may be useful as methods of anatomical investigation and illustration.

Radiographs of ‘soft’ tissues have been found useful by many workers, but, as is well known (see, for example, Barclay, 1951), only very ‘soft’ X-rays (i.e. those generated by X-ray tubes using a comparatively low voltage such as 10 or 12 kV) are of use. As these soft X-rays are harmful to human skin and also lack power of penetration, they are purposely not generated in X-ray apparatus designed for medical work, but it is usually such apparatus that is available to biologists for research purposes. With these X-ray sets it is unusual for a tension of less than 30 kV to be available and the rays produced are much too hard for soft-tissue work. However, the method described here overcomes this difficulty by making the soft tissues much more radioopaque, and thus kilovoltages normal in diagnostic X-ray apparatus can be used. Whether the information conveyed by such radiographs is of value can only be judged in particular instances, but, if it is desired to compare the branching of the alimentary canal in a large number of liver-flukes or planarians, radiographs can be made of several on one plate or film only 24 hours after fixation. By the usual methods, the specimens would have to be carefully stained and mounted and then photographed separately; and to build up a collection of a large number of photographs would take an appreciable time.

The technique now used is as follows : fixation in Zenker-formaldehyde is carried out in the normal way for 24 hours ; if not required for immediate use, the material can be stored in a solution of mercuric chloride 5 g, potassium dichromate 2-5 g, sodium sulphate 1 g, distilled water 100 ml. Successful radiographs of small objects, such as chick-embryos, have been made of material stored in this way for up to three weeks; after this time such specimens become so brittle that manipulation of them becomes impossible. There is also a marked tendency for the radiographs to become spotted, as if the mercury were accumulating in small granular masses. With larger specimens more prolonged storage is possible, and the liver-fluke shown in fig. 1, D had been stored for three months before it was radiographed. Radiographs are usually made, therefore, as soon after 24 hours’ fixation as possible.

When a radiograph is to be made, the specimen is washed in tap-water. In most instances this wash has been rapid with the intention of removing excess fixative from the surface of the specimen. A preliminary experiment has indicated that more prolonged washing gives greater differentiation between various tissues, but further experiments would be needed to demonstrate how useful this process might be.

As sensitive materials, Ilford Process Plates and Kodak Maximum Resolution Plates have been used, and also Ilford Ortholine cut-film. The plate or film is enclosed in a light-tight cardboard box, a sheet of lead to prevent back scatter of radiation being placed under the plate. The plate is laid emulsionside upwards. As recommended by Barclay (1951) for micro-arteriography, a sheet of Styrafoil ‘S’ 1 /rooo in. thick is laid on the surface of the plate and the specimens to be radiographed laid on the Styrafoil by means of a camel-hair brush, care being taken to remove as much of the water from the surface of the specimen as possible. As also recommended by Barclay, if the exposure is to be long (as with Maximum Resolution Plates, where it may have to be built up over half an hour or more, the tube being rested between exposures), a second layer of Styrafoil is laid over the specimens to prevent drying, but this increases the exposure time. Up to this point the procedure has been carried out in the dark room, with the appropriate safe-light. The lid of the box is now placed in position and the whole preparation transferred to the X-ray set with as little disturbance as possible and the exposure made. The radiographic factors used for the illustrations of this paper are given in the legend to the figure.

One modification of this technique which has been tried may be useful. Proglottids from a human tapeworm, which had been fixed for 48 hours in ordinary 5% formalin, were washed in water and then soaked in Zenker-formaldehyde for 5 days, after which radiographs were made. The results are shown in fig. 1, E.

Fig. 1, F, G shows how the anatomy of a larger animal, an earthworm, may be illustrated by this method.

I wish to express my thanks to Mr. M. H. Gregory, technician in this department, for his assistance in the experiments recorded here. He has carried out all the many photographic procedures involved in this research. It is hoped to continue to investigate the potentialities of this method of radiography as circumstances permit.

Barclay
,
A. E.
,
1951
.
Micro-arteriography and other radiological techniques employed in histological research
.
Oxford
(
Blackwell
).
Foxon
,
G. E. H.
, and
Rowson
,
K. E. K.
,
1956
.
Quart. J. micr. Sci
.,
97
,
47
57
.