SAFE MICROSCOPIC TECHNIQUES FOR AMATEURS

I - MOUNTING MICROSCOPIC SUBJECTS.

Part 3b - The mixed formulae
PVA-lactic acid and PVA-glycerol mountants

WALTER  DIONI                       Durango (Dgo) Mιxico
All pictures originally captured at 640 x 480 pixels. Many of them are amalgamated with CombineZ. Click on an image to view the master. The title image is part of a Diaptomus antennae mounted in PVA-lactic acid.
Editor's note: Part 3 of this series will be published in four monthly sections. They will be interlinked as they are published.
Part 3a: Introduction; fructoglycerol and modified Brun medium as mountants.
Part 3b below: PVA-lactic acid and PVA-glycerol mountants.
Part 3c: von Apathy's original formula and Lille's medium as mountants.
Part 3d: Gum arabic fructose, glycerinated gum and glycerinated lactic acid mountants.
 

PVA-L.- Polyvinyl alcohol-Lactic acid medium




Lactic acid is not a forbidden substance. It is a clear liquid which has a mild odor and is pleasing to work with. As we saw when I presented lactoglycerol it is considered a powerful clearing agent coming only third to chloral hydrate and phenol. It is also an expensive product of very extensive industrial use, and more or less difficult to get in small quantities. But try old drugstores and suppliers to school laboratories, surely you can get the quantities you need at a reasonable price, as I did.

Probably the first PVA-lactic acid mounting media was the one published by Omar et. al. in 1978. You can prepare your own PVA-lactic mountant, with an RI of approx. 1.39. One professional formula I obtained from several sites on the Web is this:

 PVA………………………….....16.6 g
 Water………………………....100  ml
 Lactic acid…………………..100  ml
 Glycerin……………………….  5  ml




Dissolve PVA in water, add the lactic acid while mixing vigorously. Add the glycerin and leave for 24 hours before use.

My (amateur style) is of this formulation:

O’Glue (See Part 2)…….…….30  ml
Lactic acid……………………...15  ml
glycerin…………………………1.5 ml




It has a very good consistency. PVA-L is a universal mountant. Suitable subjects include small arthropods, parts of the same, microfungi, some algae, some botanical preparations. You can transfer the subject directly from water, alcohol or glycerol to the PVA-lactic media, or if the objects are really dark, you can use a preliminary clearing bath (2 parts lactic acid:1 part glycerin), then transfer your subjects to glycerin. Dilute with water if it is convenient. Another clearing medium could be simply lactic acid at a 1% or 5% strength. Experiment to find the best medium and the time your material needs to be cleared. Some subjects clear in minutes, others require as long as several days. Mount as above.

My “lactic” formula dries very rapidly. In some hours the media will set. As you will see the margins harden enough to clean up as I described for the CPG (commercial PVA glue) medium and seal the preparation with a double layer of nail polish. Some references state that even so sealed the PVA formulas can evaporate solvent, dry out and after some months peel off the glass. I suspect that NPM (nail polish mountant) can have the same behavior.

It is recommended to seal with a really hard sealant. In the USA the Red Glyptal, a varnish to protect electric machinery (also used by palaeontologists to protect fossil bones) is recommended. I have still not found a substitute in Durango, and must use some automotive paints, hoping this helps.
 

PVA-L, mosquito larva head ventral

PVAL, mosquito larva buccal armature

PVA-L, mosquito larva pecten airtube

PVA-L, female Diaptomus antenna

PVA-L, Diaptomus ovisac

PVA-L, Diaptomus ovisac (2)

PVA-L, daphnia

PVA-L,  epithelial cell of leaf underside

PVA-L, fly wing-1

PVA-L, fly wing-2

A few comments about 3 of the above pictures. The diaptomid first antenna is portrayed in my domestic version of the scanning microscope; (Rheinberg plus a vertically displaced central stop somewhat out of center). The copepods were washed from the lactocupric fixative with water and mounted directly to the PVA-L. In spite of this, the eggs don't show the collapse that they suffer with FG and FGL. The ovisac in darkfield was photographed using the same method as for the antenna, but with the central dark stop well centered and adjusted, and I use a Rheinberg filter of another color. (Well... as the black  field doesn't register so black as I'd like it in the original picture, I made a sustitution with the aid of PhotoPaint.)

Commentaries.- Contrary to the CPG, of a lower RI, this lactic formula I prepared has a similar behavior to NPM (nail polish mountant) and gives similar results in the short time. With more time, (one or two weeks) it almost clears the internal tissues and gives a neat view of the chitinized structures. In several weeks the extent of clearing makes it more difficult to discern the thinner structures like spines and setae. You can try to stain the chitinous skeletons by treating them, before mounting, with lactic acid colored with a few drops of methylene blue. (I picked up this trick from the web, but I have not tried it until now.) Or as a last resort use good oblique illumination or phase contrast.

So you must select very well the materials you mount in this kind of mountant. Or try the solution used by Robert Constantine for his Damar mountant. See also my commentaries to the Glycerinated Lactic Gum (to be published in Part 3d).

The literature on PVA mountants has many enthusiastic appraisals and also some totally dismissing opinions. The late G. Ramazzotti, in his monograph about the Tardigrada says that many times (as I have experienced with the O’Glue) the polyvinyl-lactophenol formula he used developed voids under the coverslide, without any known reason. He also states that on the contrary, he had preparations that lasted many years without faults.

I think that if one can obtain a PVA of the recommended density (24 – 32 centipoises), the professional formula is a safe mountant, easy to mix, that merits more additional experimentation. But if you are unable to get it, browse through the art or the office supply houses, as I did. Try the PVA paper glues, they deserve a try.

Some time ago I started to question why the PVA was restricted by professional microscopists to the lactophenol formulas (now additionally restricted to lactoglycerol formulas). So I tried with relative success the CPG adventure (see part 2). Now I propose you indulge in the heresy and design a PVA based media of mild clearing action, a lot less acidic, that could be of a more general application (including those little tardigrada, with his delicate calcified pieces). My own experimental version is below which I've put on the trial to monitor its behavior for the next few months.

PVA-G.- Polyvinyl Alcohol-Glycerol medium
O’Glue…………………10  ml
Borax Water*..……….…4 ml
Glycerol…………………6 ml
*Saturate water with granulated borax (>6 g of borax /100 ml water). Use the supernatant liquor.

Editor's note added Nov 2004. The preparation method of PVA-G is quite critical as apparently the above ingredients can also make synthetic 'slime'. See Howard Webb's Nov. 2004 article .

PVA-G, fly wing-1

PVA-G, fly wing 2

PVA-G, epithelial cell of leaf underside

PVA-G, mosquito larva head

PVA-G, mosquito larva pecten airtube

PVA-G, mosquito larva pecten (1000x)


Comments to the author, Walter Dioni, are welcomed.



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Editor's acknowledgement: Thank you to Ian Walker who prepared the thumbnail images.

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Published in the April 2003 Micscape Magazine.

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