ABSTRACT
In vitro tissue culture is shown to be a possible mode of experimentation with the tissues of the Blow Fly larva. Methods are described-whereby the tissues, and the body fluids requisite as culture media may be obtained free from bacteria. The imperfections of the technique are noted and the conclusion reached that a successful technique must depend on the rearing of bacteria-free larvae, for which a method is briefly outlined. It is shown that progress in this part of the work must await further physiological knowledge, particularly in respect to the nature of the body fluids.
INTRODUCTION
Of recent years the methods of in vitro tissue culture have been applied with varying, but often considerable, success to many problems of Cytology, Physiology and Pathology. Vastly the greater part of this work has been carried out with tissues of vertebrate animals and the writer is greatly indebted to the late Professor F. W. Gamble, F.R.S., for the suggestion that the development of a satisfactory technique for the in vitro cultivation of insect tissues could hardly fail to bring to light problems of great interest.
The author’s first intention was simply to develop a technique for the in vitro cultivation of the imaginal discs of the Blow Fly larva, largely with a view to obtaining information on the formation of the mesoderm of the appendages. The wide departure of the work actually achieved from that originally contemplated has been the almost inevitable result of a gradual appreciation of, firstly, the difficulties of the work, and, secondly, the immense possibilities of a successful application of the technique of tissue culture to the problems of insect metamorphosis.
Workers on vertebrate tissues have the initial advantage of access to very detailed information on the composition and functions of the blood plasma, and to some extent even of the embryo extract, which are their customary culture media. Of the composition of the body fluids of insects we are almost entirely ignorant; their function is in many respects a matter more of controversy than of knowledge ; even their microscopical appearance is only tolerably well known in a few instances. Nor is the absence of any detailed information as to the composition of these fluids greatly surprising, since their collection in quantity is in some cases almost impossible, in most extremely tedious, and only in comparatively few instances tolerably easy by means of a technique described herein.
Growth of the imaginal discs of the Blow Fly larva was eventually obtained. The technique and the results were far from perfect but there is little doubt that, even lacking the physiological information alluded to above, successful cultures could have been obtained as far as regards the original aim of observing the formation of the appendicular mesoderm. But even the first cultures, poor as they were, showed a marked difference in behaviour according as the culture medium was larval or pupal body fluid. Rightly or wrongly, it was felt that here, perhaps, lay at least the first step towards the solution of one of the most difficult of biological problems the nature of metamorphosis.
The following, then, was the objective of the work: to find the more important physical and chemical changes in the body fluid of the Blow Fly larva and pupa during metamorphosis and, reproducing these changes separately in artificial culture media, to attempt an analysis by means of single factor variations, of the relation between changes in the composition of the body fluid and the synchronous processes of histolysis and histogenesis. It had not been possible for the writer to investigate more than a few of the more obviously important of the physical and chemical properties of the body fluids in question. Even these have not been investigated as thoroughly as is desirable. In the present stage of the inquiry somewhat superficial information of many of the changes occurring during metamorphosis is considerably more helpful than a detailed and exact knowledge of any one of the changes.
The Blow Fly was chosen for this work as being the most conveniently bred, moderate sized. representative, of that family of insects—the Muscidae—in which the complexity, rapidity, and interest of the metamorphic changes attain their maximum. It has the great disadvantage that only with very special and somewhat difficult technique can its tissues and body fluids be obtained free from bacterial contamination. In point of size, quantity of body fluid obtainable, and ease of obtaining aseptic tissues and fluids, many other insects, particularly among the Lepidoptera, have undoubted advantages over the Blow Fly.
The Blow Fly presents one very great advantage in the rapidity and striking nature of the phenomena of growth during the metamorphic period. The relative constancy of the environment of growing tissues in vertebrates (particularly avian and mammalian) increases the difficulty of discerning the essential factors which promote or inhibit growth and differentiation. Without wishing to stress this aspect of the work (since achievement in this direction is at best extremely problematical, must be subsidiary to the main concept of the work and cannot be aimed at directly) it may reasonably be hoped that progress on the lines indicated in this paper may eventually be of some value not only in the comprehension of the mechanism of insect metamorphosis, but in the wider philosophical consideration of growth in general.
Asepsis is an absolute necessity for successful in ‘vitro tissue culture. In dealing with the vertebrates it is comparatively easy to obtain both sterile tissues and sterile plasma and extracts ; in dealing with such an animal as the Blow Fly very considerable difficulties are encountered at the outset..
Even assuming that the body fluid of the larva is absolutely free from bacteria, and that the surface of the larva can be satisfactorily sterilised—which latter is in fact the case—it is almost impossible to collect the necessary quantity of fluid in a sterile condition. In the first place the time taken over the operation in obtaining the larval fluids would make the chances of asepsis almost negligible, and a single inadvertent puncture of the gut—not easy to avoid consistently with rapid working—would ruin all the fluid extracted. A still more unanswerable objection is that it is impossible to obtain sterile body fluid from the pupae. The pupal body fluid can only be obtained by crushing and centrifuging. In this process the gut is inevitably ruptured and all chance of asepsis in the extract thereby lost. Though the pupal gut is empty of food it is almost inconceivable that it can be bacteria free. (For methods of obtaining larval and pupal body fluids see below, p. 4.)
It seems probable, moreover, that only a very small number of larvae have a body fluid free from bacteria. Twenty-five larvae were surface sterilised in 10 per cent, corrosive sublimate for 15 minutes and after washing in sterile distilled water (12 changes) each larva was tested for surface sterility by washing in a small quantity of sterile salt solution. A drop of this salt solution was then put up as a hanging drop culture. The body fluid of each larva was obtained by pricking with a sterile needle, the utmost care being taken to avoid injury to the gut. The body fluid so obtained was put up as a hanging drop culture. All the surface washing cultures remained sterile ; only one of the body fluid cultures failed to show heavy bacterial contamination after 24 hours’ incubation.
The most obvious solution of the difficulty of obtaining bacteria-free body fluid is the rearing of aseptic larvae and pupae. Only brief reference need be made to this part of the work. Several different methods were tried, the most satisfactory one, and the one eventually adopted, being the use as culture medium of peptone-agar smeared with sterile avian blood. Ordinary agar slopes were used, and one or two larvae reared in each test-tube. The Blow Fly eggs were sterilised in corrosive sublimate (4 parts in 1000, for 5 minutes) before planting on the agar slopes. This technique proved simple and convenient and perfectly aseptic larvae, pupae and imagines were obtained. Bacterial infection in any tube shows up clearly on the agar in nearly every case where it occurs. Nevertheless, the utilisation of such larvae or pupae for tissue culture work was felt to be fundamentally unsatisfactory. Many of the aseptic larvae obtained were obviously abnormal—particularly as regards their fat metabolism ; the method was far from exactly standardised and it was impossible to be sure that apparently normal larvae were not, in fact, abnormal in some respect.
Lacking any physiological standard of normality with which to compare the artificially reared larvae, it was felt strongly that any conclusions based on the use of such larvae for tissue culture could not be other than dubious, and that it was far preferable, if at all possible, to use normally reared larvae for the tissue culture work. All subsequent work on the physiology of normally reared larvae has confirmed this view.
It is, nevertheless, almost certain that eventually the use of such aseptic larvae for tissue culture work would give results of very great value. The first necessity is the elaboration of a standard technique by which “normal” aseptic larvae can always be obtained. It is easy to produce very marked changes in the larval metabolism, and the effect of these changes on metamorphosis and on the behaviour of tissues in culture can hardly fail to be extremely interesting.
METHOD OF OBTAINING BACTERIA-FREE TISSUES
Sterile tissues may be obtained from larvae reared in the ordinary way by the tedious but quite effective method of repeated washing of the fragment of tissue in sterile physiological saline. Twelve changes of saline have been found effective. This method has serious limitations. The tissue most particularly desired for culture experiments was imaginal disc tissue, and for this the washing method of sterilisation was quite effective. The imaginal disc required was cut out as near as possible to the surface end of its peduncle and transferred to the first watch glass of saline. It was left for about two minutes in each change with constant agitation with a needle. Sterile needles were used for each transference to a fresh washing. In practice up to 10 imaginal discs were taken simultaneously through the washing process. After the final washing they were either put up in culture whole, or cut into fragments, depending on the type of growth required (see below) ; well over 90 per cent, of such cultures were free from bacterial infection.
The method was almost equally successful with portions of the supra- and sub-oesophageal ganglia, and of the dorsal vessel. It was markedly less successful with fragments of muscle or fat body, and was almost invariably unsuccessful with the gut and its appendages (the salivary glands, malpighian tubules, and the crop). Fragments of the gut have been washed as many as 20 times and have still shown bacterial infection after incubation in culture for a day or two. It is possible that the gut cells, and perhaps also the fat body cells, ingest living bacteria. Thus, even though all bacteria were removed from the surface, the cutting of the tissue preparatory to implantation would permit the egress of still living bacteria from the damaged cells—even though normally the ingested bacteria are eventually destroyed by the living cell. The cells have not been examined critically to test this view. For the present purpose it is sufficient to know empirically that certain tissues cannot be sterilised by repeated washing, and that these tissues can only be obtained for culture experiments by rearing aseptic larvae.
METHOD OF OBTAINING BACTERIA-FREE BODY FLUID
It is an easy matter to obtain sterile body fluid by filtration through a Berkfeldt candle, but it has proved an extremely difficult matter, and one not successfully overcome, to ensure that the fluid so filtered is unchanged in any way except in being freed from bacterial contamination.
After autoclaving, the Berkfeldt candle must be dried, and it is essential that the drying should be done at a low temperature (30° C.–40° C.) as otherwise it has a very marked effect upon the pH of the filtrate. The nozzle of the candle projects into a small waxed glass tube which is introduced with aseptic precautions into the container after the whole assembled filter has been dried at the temperature mentioned above. Paraffin oil is now poured into the reservoir and sucked through the candle, gradually filling the small glass tube. The candle itself must be kept completely covered with oil and suction is continued until air bubbles, at first numerous, practically cease to emerge from the nozzle of the candle. The fluid to be filtered is then pipetted into the reservoir below the oil which covers the candle. On passing through the candle the fluid is delivered below the surface of the oil in the small glass tube, to the bottom of which it sinks. In this way the body fluid is collected, filtered and stored without once coming into contact with the air. In the absence of this special paraffin oil filtration there is quite enough air in the candle to cause pronounced oxidative changes in the fluid.
The use of the oil has the further and minor advantage, in dealing with small quantities of fluid, that almost the very last traces of the fluid can be forced through the filter. In this respect it is more efficient than the more usual method of covering the candle with a close fitting glass cap. In spite of all these precautions the use of a Berkfeldt candle—the most porous one obtainable—proved very unsatisfactory. A certain sample of pupal body fluid gave a Freezing Point Depression of 1.093° After filtration as described above its Freezing Point Depression was 0.918° C.; a change of 16 per cent. What other changes occur besides this obvious one of change in osmotic pressure is not known.
To anticipate what will be given in detail elsewhere, the osmotic pressure of the body fluid rises during the early stages of pupal life. Assuming for a moment—what is not in fact exactly true—that evagination of the imaginal discs is caused and their growth stimulated by the increase in osmotic pressure, it is obvious that satisfactory results cannot be obtained by using as culture medium body fluids altered to the extent shown above. In the case cited filtration has changed the osmotic pressure of the body fluid to that of a considerably less mature stage.
In the instance given above about 12 c.c. of fluid were filtered. The obvious remedy for the alteration caused by filtration is to discard the first 10 c.c. of fluid passing the filter. In practice this must be ruled out. In dealing with the pupal fluid such a method might be possible, though it would necessitate the rearing of unwieldy numbers of pupae. For the larval fluid such a method is almost out of the question. However essential an exact and trustworthy technique is, methods may become so laborious and time consuming that only research of the utmost importance can possibly justify their use. A technique such as that of “blocking” the Berkfeldt candle with 10 c.c. of fluid in order to obtain a second filtrate unaltered by the filtration would make insect tissue culture a method of research quite impossible for any one unaided worker. The mere mechanical technique of the tissue culture would leave no time for experimentation with varying conditions.
THE PREPARATION AND USE OF COLLODION FILTERS
In the above circumstances attention was turned to the possibility of utilising an entirely different filtration method. Much work has been carried out on collodion membranes of high permeability. A study of the literature shows that the majority of workers in this field have erred on the side of over-elaboration of technique. Eggerth’s method (1921) is a noteworthy exception to this, and the method described below follows in all essential details that described by Eggerth.
A piece of glass tubing about six inches long and of any desired diameter (the greater the diameter the less the strength of the membranes made) is melted down at one end until only a small hole is left. Over this hole a small piece of cigarette paper is placed, painted over with collodion and pressed down firmly. The collodion solution for making the membrane is in a glass tube (3 in. by 1 in.) with a wellfitting bung. The sealed tip of the tube is dipped into the collodion solution three times with drying intervals of about one minute after each dip. After the final dip the collodion tip is allowed to dry for several minutes. This strengthening of the tip of the membrane is essential although it results in impermeability in this region.
It has been found impossible to produce a satisfactory membrane by the use of only one coat of collodion. After many trials with varying numbers of coats, each given various drying times, the following method was adopted as giving the most highly permeable membranes consistent with adequate strength.
The glass tube mould is dipped vertically into the collodion solution and withdrawn steadily and fairly slowly. After withdrawal the mould is held horizontal and rotated rapidly in the fingers for one minute, and then again dipped into the collodion. Three collodion coatings are made in this way, and after the final drying of one minute the mould is held vertically and dipped rapidly into ajar of cold water in which it is held in a vertical position for two minutes. It is then laid horizontally in a dish of cold water, the bottom of which is covered with cotton wool. Here it must be left for at least ten minutes.
The collodion sac is removed by filling the glass tube with water and applying pressure at the open end of the tube while the end of the collodion sac is very gently manipulated with the fingers. By far the safest pressure to use is by blowing with the mouth. The sacs are usually very easily removed. After removal they should be soaked in cold water for at least half an hour.
Considerable difficulty was at first found in attaching the collodion sacs to a suitable holder, but the following simple method has proved extremely satisfactory. A glass tube of slightly greater diameter than that used for the mould in making the sacs is drawn out slightly at one end, so as to provide a narrowed neck about one-third of an inch above the end. The end is smoothed off in the flame. The collodion sac, full of water, is gently worked over the shaped end of the glass tube. This part of the operation is best done under water. The collodion sac is now firmly attached by binding wool round the neck of the tube. With a little practice the membrane is in no way damaged by this apparently drastic treatment, and a perfectly bacteria-tight union is effected between the collodion sac and the glass tube.
The glass tube upon which the collodion sac is tied passes through a rubber bung which fits into a cylindrical glass vessel of suitable size, from which the air can be exhausted by means of a side tube. The tube should be passed through the rubber bung before attempting to attach the collodion membrane.
In testing the sacs a purely arbitrary standard of strength has been used which is perhaps unnecessarily high. Satisfactory filters must be capable of withstanding an internal pressure of 200 mm. Hg for six hours without bursting or showing any leak, the filter during the last four hours being filled with paraffin oil. The necessity for the lengthy testing time arises from the fact that filters have been known to burst after successfully withstanding the requisite pressure for 30 or 40 minutes. As the actual filtration is carried out with an internal pressure of only 150 mm. Hg the test outlined gives an ample margin of safety.
The rubber bung with the attached collodion filter is fitted into the cylindrical glass vessel. The parts must be so arranged that the top of the worsted fixing on the sac is some little way below the exhaust tube of the container. Suction is now gradually applied at the side tube and water drawn through the collodion sac into the container. The sac is replenished by pipetting water into the upper end of the glass tube. When the level of the water in the container is slightly above the top of the worsted binding no more water is put into the collodion sac, which is allowed to empty itself completely into the container. Suction is now increased gradually so that about 15 minutes elapse before the negative pressure in the container is 200 mm. Hg. The filter must now stand at this pressure for two hours. The slightest leak will show as a stream of air bubbles. At the end of two hours liquid paraffin is pipetted down the glass tube until it rather more than fills the collodion bag. The pressure in the container is now7 restored to normal, the container and the outside of the collodion bag carefully dried, and the former filled with paraffin oil. The filter is reassembled, the negative pressure gradually applied until it attains 200 mm. Hg, and the filter left to stand for four hours. The pressure is - then restored to normal and the tested filter autoclaved without emptying the oil from either the container or the collodion sac.
Autoclaving causes some shrinkage in the collodion membrane. Though the strength of this is thereby somewhat increased, its permeability is somewhat reduced. Autoclaving should therefore be at as low a temperature and for as short a time as possible. Fifteen minutes at a steam pressure of 7 lb. per sq. in. has been found uniformly effective in spite of the well-known fact that bacteria suspended in oil have a resistance to heat sterilisation considerably greater than normal. The bacterial contamination is, of course, only very slight, and spore bearing forms are apparently absent or the sterilisation recommended could hardly prove effective.
It is possible, by working in rubber gloves and using the strictest precautions as for surgical asepsis, to make and mount these collodion filters, so that they are strictly bacteria free, and autoclaving is unnecessary. This has twice been carried out with entire success as regards freedom of the filtrate from, bacteria. The method is extremely exacting and laborious and requires strictest attention to minute detail, though the advantage gained is only slight. There is, moreover, always the risk of some slight unnoticed slip in the technique. It is far preferable to be able to autoclave the filter when made.
Several different collodions were used, but by far the most satisfactory found for the purpose was “Necoloidine1.” This was dissolved in alcohol and ether, and lactic acid added to increase the permeability. All four constituents of the solution were tried in widely varying amounts. The final solution which gave the most satisfactory results ‘was as follows.
The permeability of the membranes was tested by using a strong solution of haemoglobin. The filtered solution was compared with the standard by using a colorimeter.
The haemoglobin solution passes through an oiled Berkfeldt filter with no appreciable change, though if passed through a dry filter the change is very marked indeed. With collodion membranes there is an appreciable change. For an unautoclaved membrane made with the above solution in the way already described, the colorimetric reading is 12.9 mm. when the standard solution is set at 10 mm. The value given is the mean for five membranes.
A collodion membrane made in the same way, except that each of the three coats was given a drying time of two minutes instead of one minute, gave readings of 13.5 before autoclaving (mean of four membranes) and 14.4 after autoclaving (mean of five membranes), the standard being set in each case at 10 mm.
The writer is indebted to Miss P. Hunt for carrying out the tests of permeability and tensile strength on the final series of membranes tested.
In most respects the collodion filters must be regarded as unsatisfactory. The greatest degree of permeability obtained is inferior to that given by an oiled Berkfeldt filter, though greater than that given by a dry Berkfeldt filter owing to the smaller degree of adsorption on the collodion membrane. The standard haemoglobin used gave a Freezing Point Depression of 0.851 ° C. After filtration (of 12 c.c.) through an oiled Berkfeldt the value was 0.772 ° C. and after filtration through an autoclaved collodion membrane 0.680 ° C. In view of these results, tests of permeability were not carried out, the body fluids themselves being used as standard solutions.
The method of obtaining sterile body fluids by filtration has thus extremely serious difficulties. The changes noted as occurring on filtration have been only the obvious ones. There may well be changes more difficult of appreciation, yet just as important. It seems that the rearing of bacteria-free larvae will, after all, be necessary before reliable in vitro tissue culture experimentation can be carried out. Knowledge of the physiology of metamorphosis, and of the composition of the body fluids during the period of metamorphosis, is utterly essential for any real progress. Without this the cultivation of insect tissues in vitro degenerates into a mere biological curiosity and will help but little in the understanding of problems of insect metamorphosis. Sufficient knowledge is now available to allow of experimentation along certain lines, and that these lines have not been followed has been due to preoccupation with other aspects of the work felt to be more essential in their ultimate application. With a sound basis of physiological knowledge the technique of tissue culture can hardly fail to be an instrument of the very greatest utility in solving physiological problems which may be almost insoluble by ordinary chemical and physical methods.
IN VITRO CULTURE EXPERIMENTS
The actual culture work will be referred to only very briefly and only as regards the imaginal discs.
Two very distinct types of growth may be obtained by varying the conditions of tissue culture experimentation—”controlled” and “uncontrolled” growth. The latter type of growth is the one most frequently studied and consists in the out-wandering of cells from an implant of tissue into the surrounding medium, where they multiply by mitosis if the medium is favourable. The conditions for good “uncontrolled” growth are a suitable temperature, a chemically suitable medium, and some suitable support for the outwandering cells—commonly the plasma clot. ‘
“Controlled” growth is the growth of an organ or a tissue as an organised unit exhibiting interdependence of its parts. This method of experimentation is a comparatively recent extension of the technique of tissue culture and it is at present impossible to generalise concerning the conditions necessary for its successful achievement. It is to this type of growth that most attention has been paid in the present instance.
“Uncontrolled” growth has not yet been successfully obtained. Fragments of imaginal discs put up in culture medium consisting of larval and pupal body fluid usually showed considerable outwandering of the cells, but mitosis was not obtained. The main objection to the method used was the fluid nature of the culture medium since the body fluid of the Blow Fly larva or pupa does not clot. Agar or gelatin clots proved useless, as supports for the cells, and a chicken plasma clot proved definitely toxic. A sterile clot of fibrin may possibly solve the difficulty of providing support for the cells, but this has not been tried.
More interest was felt in, and more attention given to, the “controlled” growth of entire imaginal discs. The process of metamorphosis in the Blow Fly is extraordinarily complicated. The évagination of the imaginal discs is only one of many striking changes, but it is a change of so definite a nature that its explanation seems to afford some possibility of a starting point in the attack on the problem of the immediate causation of metamorphosis itself. It is from this point of view that the reproduction of this evagination in culture with a view to discovering the causative mechanism of the process has seemed a matter worthy of considerable effort.
For some time the only culture fluid used was the larval body fluid, and no évagination of the imaginal discs was obtained—nor indeed is it now easy to see why any was expected. Cultures were then tried of the leg imaginal discs of mature larvae, using the body fluid of pupae of various ages as culture medium. The discs evaginated in the culture and grew into definite segmented limbs. Growth of the limb was never obtained beyond a stage corresponding to that attained in a fourth or fifth day pupa, and the limbs were not examined histologically.
Imaginal discs from very immature larvae when put up in the body fluid of mature larvae showed slight growth, with no tendency to evaginate. No experiments have been carried out with very immature imaginal discs in the body fluid of pupae, but probably here also there would be no évagination. It may be possible to cultivate an imaginal disc in vitro from a very immature stage right up to évagination and limb formation. Experiments with media of inorganic salts in varying concentrations showed that evagination was not caused simply by the change in osmotic pressure of the body fluid upon pupation. There was some dubious evidence that a change in the salt balance had some relation with évagination. The only point clearly established by the in vitro experiments is that évagination of an imaginal disc is not due to increase in the internal pressure or turgor of the body fluid, since évagination may occur in a fluid medium where no question of directional pressure can arise. Also, the power to evaginate is not an inherent property of the imaginal disc itself, apart from any change in the body fluid surrounding it. Thus, imaginal discs will not evaginate in larval body fluid, though they will remain healthy for several days and capable of évagination if the larval body fluid is replaced by pupal body fluid. Evagination, therefore, depends in part at least on changes in the composition of the body fluids.
One further point of interest may be noted. Even using as culture medium body fluid from mature pupae, from which the imagines were about to emerge, the development of the imaginal discs could not be carried beyond a stage comparable with that attained in a fifth day pupa. It has already been noted that filtration of the body fluids of pupae, the osmotic pressure of whose body fluid is at its maximum (i.e. pupae about seven days old), reduces the osmotic pressure of the filtrate to a value corresponding to that of the body fluid of a younger pupal stage (or equally to that of an older one since the osmotic pressure of the body fluid falls off towards the close of pupal life). The evidence available does not warrant the assumption that change in the osmotic pressure is the dominant factor in conditioning the évagination and growth of the imaginal discs, but it may well be that the partial removal of some substance or substances from the fluids by the filtration inhibits the complete differentiation of the imaginal disc in vitro. If growth and differentiation of the imaginal discs depend directly or indirectly on the concentration of some substance in the body fluid, the partial removal of this substance by filtration would allow growth in vitro to proceed only to that stage where the concentration of the hypothetical substance is the same as its concentration in the filtered fluid derived from pupae of a later stage.
REFERENCE
Manufactured by Necol Industrial Collodions Ltd., Stowmarket, Suffolk. The writer is indebted to the local manager of the firm for advice concerning a collodion suitable for the production of membranes of high grade permeability.