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Volume 77, 1948-49
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New Methods In Microscopy For The Study Of Small Insects And Arthropods

The main purpose of this paper is to introduce to you some new mounting media for microscopy based on the use of polyvinyl alcohol; but, before dwelling in detail on these I should like to make some general observations on media and methods generally advised in standard texts on microbiology.

The beginner is Invariably instructed to preserve small insects such as Collembola, Thysanura, Thrips, etc. in 70% ethyl alcohol. This, in my experience, is most unsatisfactory, and such small insects should always be preserved in ethyl alcohol of 95% strength; small insects preserved in alcohol of less than 90% strength generally lose their colour and, in about ten years, maceration sets in, more or less spoiling the specimens for study. Arachnida are best preserved in Andre's Fluid, a glycerine-acetic-alcohol mixture, which is far superior to the formalin-acetic-mixtures, such as Pampel's Fluid, generally recommended. Andre's Fluid keeps Arachnid material perfectly preserved and relaxed indefinitely. It is not, however, suitable for most insects as it causes undue swelling of the cuticle, often accompanied by rupture of the intersegmental membranes. I have insects and Arachnids so preserved for ten years and more which are as perfect to-day as on the day when they were captured. Methylated spirit should be avoided as a preservative for small Arthropods, as it causes abnormal shrinkage and the specimens become very brittle.

When making a taxonomic study of small Arthropods under the microscope it is necessary, in order to be able to see the outlines of fine setae or hairs and the structure of delicate sense organs etc., to select a mounting medium of relatively low refractive index. On account of its rather high refractive index, slow rate of setting and the necessity for thorough dehydration Canada Balsam has long been regarded by taxonomists as unsuitable and resort has generally been made to gum arabic-chloral hydrate mixtures, such as the well-known Berlese's Fluid. These media invariably suffer from the disadvantage that sooner or later they show signs of crystallisation which, if allowed to develop, may destroy specimens mounted in them. Furthermore, specimens preserved in alcohol must be brought down to water before mounting. Gilson's Euparal, as prepared by Flatters and Garnet of Manchester, England, and Diaphane, prepared by the Will Corporation, Rochester, U.S.A., are two excellent media into which it is possible to mount direct from alcohol of 95% strength without further dehydration. They have a lower refractive index of 1.483 and set relatively quickly: slides hardened off in a paraffin oven at 56° C. can be used after one hour with Diaphane, and after two hours with Euparal, provided that they are handled with reasonable care. When mounting in these media in order to obtain maximum extension of the specimen it is essential to put the cover glass in place as quickly as possible. The pressure of the cover glass helps to prevent shrinkage of the specimen. Euparal and Diaphane are the most satisfactory media in existence that I know of for the preparation of whole mounts in which it is desired to preserve the colours of the specimens. They do not, however, give mounts in which very fine detail can be readily studied and, to study such things as the sense organs of Collembola, it has always been necessary to clear specimens with caustic potash followed by mounting in a watery medium such as Berlese's. If one proceeds according to the recognised methods advised in most text books much time is spent in preparing a mount by this method. I have found, by experiment, that in the case of small insects such as Collembola or other small Arthropods of not more than 1–2 mm in length, the time of immersion in each down grade of alcohol can be reduced to three minutes; if the material is larger, say up to 4 mm in length, not more than five minutes is necessary. For material preserved in 95% alcohol the first transfer should be into 60% alcohol followed by 30% and then distilled water. Two changes of distilled water are preferable, each of 3–5 minutes, followed by clearing in 10% caustic potash, two further rinsings in distilled water, of 3–5 minutes each, and mounting. No harm appears to be done to even the most delicate specimens by such sudden drops in alcohol concentration and this procedure is in marked contrast to the accepted practice of immersion for periods of an hour or more in each of a gradually descending

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series of alcohol concentrations. In transferring down through alcohols I use small shallow porcelain dishes about 1½ in. in diameter and 1 in. deep. When a specimen is transferred from a higher to a lower percentage alcohol usually it floats in the lower one at first, gradually sinking as the lower strength alcohol replaces the higher one. Potashing such small specimens is most easily done on a cavity slide, which can be warmed over a spirit stove, and the progress of the process periodically examined under a low-power stereo-binocular microscope. It usually takes 3–7 minutes and, after completion, to prevent damage, the specimen should be handled with a fine glass pipette fitted with a rubber teat. By this method, a mount from 95% alcohol is complete in 20–30 minutes, and, if made in Berlese's Fluid it can be examined with an oil immersion lens after 2–3 hours' hardening in a paraffin oven held at 56° C. Considerable shrivelling may occur towards the end of the potash treatment, but after the specimen has been, transferred to water it will commence to expand and all appendages will finally become fully extended.

Since taking up the study of Collembola I have, for several years, been experimenting with mounting methods in an endeavour to perfect a technique capable of giving a preparation which, under the microscope, would render the finest structural details clearly visible without suffering from the bugbear of crystallisation and which, at the same time, would be less time consuming than conventional methods. Although the reduction of time spent in the preparation of a mount to half-an-hour is a great advantage the potash technique is always tedious and fraught with a certain amount of risk to the specimen, particularly when the latter are of the order of 0.1 mm. long. I was rather interested, therefore, in an article which appeared in Science some time ago advocating the use of polyvinyl alcohols as mounting media. Later, I learned from Mr. H. Womersley, of Adelaide, that he had used a medium prepared from medium viscosity polyvinyl alcohol with great success as a mountant following treatment with potash. I was fortunate in securing some samples of low viscosity polyvinyl alcohol from I.C.I. (N.Z.) Ltd., both Type A and Type B and commenced experimenting with various mixtures. These samples were labelled “Elvanol” Type A. 51.A.05 and Type B 70.A.05. It was evident from the outset that polyvinyl alcohol could form a mountant that is the answer to almost all the dreams of the taxonomist in micro-entomology, and I can now offer a range of four formulae giving media of varying refractive indices and possessing other unique properties in varying degrees. All these formulae are really adaptations of Amann's Lactophenol with polyvinyl alcohol as a binder. thus obviating the necessity for sealing the edge of the cover slip. To prepare a set of these four formulae first make up 90 cc. lactophenol by dissolving 45 grams Phenol Detached Crystals B.P. in 45 cc. Lactic Acid B.P. Type Al is made by weighing out into a clean beaker 2.5 grams low viscosity Type A polyvinyl alcohol: dissolve it in 6 cc. distilled water. This makes a stiff white paste to which is added, with vigorous stirring, 30 cc of lactophenol solution. Clear by heating on a water bath. The resulting mountant is a colourless, crystal clear, relatively viscous, oily liquid of refractive index 1.469; it sets sufficiently hard for normal use after eight hours' drying in a paraffin oven held at 56° C. In normal atmosphere at 60–65° F it takes four to six weeks to set. Type A2 is made from the same quantity of the same polyvinyl alcohol dissolved in 10 cc distilled water: add 25 cc lactophenol solution, clear as before; the resulting mountant has a refractive index of 1.458 and sets in about the same time as the previous one. Type A3 with a refractive index of 1.447 is made by dissolving the same amount of polyvinyl alcohol Type A in 15 cc water and adding 25 cc lactophenol solution; but this medium is much slower in setting, requiring several days in the oven to harden it sufficiently for normal use, although about eight hours will harden it sufficiently for observations to be made provided the slide is handled with great care. Type B is made from 2.5 grams low viscosity polyvinyl alcohol dissolved in 10 cc. distilled water; add 15 cc. lactophenol solution. This makes a medium of refractive index 1.438 which sets hard enough for normal use after one hour in the paraffin oven at 56° C. I should mention here that the warming of the mount in. the paraffin oven helps to extend the specimen.

The polyvinyl alcohols are sweet-smelling, creamy-white powders, Type A being whiter than Type B and with a sweeter smell. They are difficult to dissolve in water and this is best accomplished by adding the water drop by drop, stirring the powder into a thick paste. When all the powder is wet the remainder of the water can be added. The resulting “paste” is fluffy and

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white due to occluded air. The media made from Type A. alcohol are progressively slightly less viscous as the amount of water is increased; Type B medium is more viscous than any of Type A. These imountants can be used by the same technique as gum arabie media following potash treatment. I have not yet had them in use sufficiently long to say definitely that they will not crystallise, but according to the chemistry of the ingredients, crystallisation should not be possible. None of my mounts has so far crystallised whereas some variations of Berlese's medium will crystallise in 3–4 weeks.

But the great advantages of these polyvinyl alcohol media lie, not so much in their use following potash treatment, but in their replacing this tediousprocedure through their ability to clear biological material perfectly, and in their tolerance of a wide variety of fluids. Material can be mounted into them direct from ethyl alcohol of any strength, from Andre's Fluid, from Pampel's Fluid, from water, from formalin, from MacGregor's Solution or from life. In this respect I think that they are unique. There is practically no shrinkage whatever after the solutions have ripened, in fact, the tendency is for these media to extend specimens; and hence there is no necessity for undue haste in placing the cover glass, the operator may take his time. I have left a specimen in Type A3 for half-an-hour uncovered without harm. Air bubbles that may be trapped during covering can be driven out by gently warming over a spirit stove, after which the slide should be set aside in a paraffin oven at 56° C. to harden. Type A3 is preferable for extending contorted specimens of Collembola, a process which usually takes 15–20 minutes, after which the cover glass is put in place. All these media completely relax specimens in a few seconds, and with Collembola, much can be done to arrange a specimen in any desired position by “rolling” it with the cover glass; a specimen can be completely turned over by gently moving the cover glass in the desired direction. All these media have a very intenseclearing action on biological material and with Collembola I no longer find it necessary to use potash treatment to clear specimens, not even the tiny colourless species belonging to the genus Megalothorax. The clearing action is stronger with Type A1. formulae than with. Type B, and it is most powerful with Type A1. Delicate specimens may be ruptured by this formula, especially if too much heat is applied over the spirit stove, though this may not necessarily spoil the specimen. This clearing action may sometimes release delicate setae and scales from the cuticle, especially if it is necessary to heat on the stove to drive out air bubbles. To mount delicate specimens where this is likely to occur the following procedure should be adopted:—Place a drop of the medium on the slide and warm it over the stove until all air bubbles are driven off, cool, introduce the specimen, allow it to relax, carefully lower the cover glass and place in the oven to harden. In this way perfect mounts can be made of almost anything. Clearing is exceedingly rapid with Type A formulae, being complete in about one minute, but with Type B it is much slower and continues for about 24–36 hours, though it is never so intense as Type A. Deep pigments are rendered quite transparent and partially bleached; all delicate pigments are destroyed. All these media should be stored away from strong light. There is a slight yellowing in the bulk with aging, but slides are colourless and the specimen is thrown up with a boldness and relief which surpasses anything I have ever seen. I have mounted Collembola of the order of 0.1 mm long in Type A2 of these media and the results are far superior to similar mounts treated with caustic potash and mounted in Berlese's Medium. Several microscopists to whom I have given small samples of Type A3 to try are all most enthusiastic about it and pronounce it better than anything they have previously used. Besides Collembola it has been tried on thrips, mosquito larvae, mites, spiders, opiliones and rotifers with the same outstanding result in each case.

In the Proceedings of the Royal Entomological Society of London recently Lieut. B. Jones gave an account of polyvinyl media he had prepared incorporating picric acid as a fixative and stain. I have prepared a sample according to his formula but I have found that it is not so satisfactory as those I have given. Jones does not state the type of polyvinyl alcohol he used, but his. medium made from low viscosity Type A polyvinyl alcohol lacked the intense clearing power and contained a fine granular precipitate which indicated that the quantities of polyvinyl alcohol, water and lactophenol were not in balance-In the formulae I have given these quantities have been carefully worked out

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for the type of polyvinyl alcohol I was using and tested by experiment; any variation from these amounts may cause this fine granular precipitate to appear. If it should the quantity of lactophenol solution should be increased drop by drop until the precipitate redissolves; the crucial point at which this occurs should be detected by examining samples under the microscope. 2–3 extra cc. of lactophenol over this point will increase both the intensity of the clearing action and the time of setting. If too much is added the medium will not set properly at all. When first prepared these polyvinyl media are, momentarily intolerant of alcohol when contact is first made with specimens transferred from ethyl alcohol, while specimens transferred into them from water will sometimes effervesce considerably for some seconds. No damage, however, results to specimens from either of these effects, which disappear in about 4–6 weeks as the media age and ripen.

The determinations of Refractive Index I have given in this paper were made for me by Mr. J. Finch of the Dominion Laboratory, Wellington, and I should like to express my appreciation of his help in this direction. The determinations were made at a temperature of 20° C.

Bibliography.

Science, 97 (2528). June 11, 1943, p 539–540.

Proc. Ent. Soc. Lond., 21 (10–12), p. 85–86.

[Author's Note: Sine the above paper was read before the Congress I have developed two similar formulae using Medium Viscosity Polyvinyl Alcohol (Elvanol 71—24). These two formulae are prepared by exactly the same procedure as for the light viscosity Alcohol. Type MA] having a stronger clearing action than Type MA1. Formula Type. MA1. Refractive Index 1.425: Polyvinyl Alcohol. Medium Viscosity.

2.5 grams: Distilled Water. 20 cc.: Lactophenol Solution. 25 cc

Formula Type MA2. Refractive Index 1.442: Polyvinyl Alcohol. Medium Viscosity.

25 grams; Distilled Water, 15 cc.; Lactophenol Solution, 30 cc.]