WALTER  DIONI                       Durango (Dgo) Mexico

Brachionus sp. in a cluster of micro algae. It was fixed with Lactocupric and mounted in Glycerin 24 months ago. Rheinberg illumination (black center, clear green periphery). The illumination emphasizes the mastax trophi and the architecture of the lorica.
Click on the image to see it full size.

Pictures were taken at 640 X 480 with a Motic DC-3 camera, integrated in my National Optical Microscope, and reduced or cropped for their insertion in the article. Some pictures can be clicked on to see a bigger version. The width of the picture with each objective (on the pictures not cropped of course) is as follows: 4x = 3400, 10x = 1333, 40x = 340 and 100x = 133 microns. Equipment and the filters used were described in a previous article.

I wish to specially recognise the author of the small but splendid Neat Image Demo software, and to Jean Marie Cavanihac who brought it to my attention.


Due to the real concern about the medical risks associated with the chemicals used in microscopy laboratories, I presented and discussed in a series of Micscape articles (see below) 20 safe mounting mediums. These 20 formulas provide mounting mediums for all the materials which may be of interest to the amateur. I present here a critical synopsis of the most important results, more than 20 months after first using the majority of them, and my selection of the most useful ones.

As I said before, they can replace for the amateur, whether a novice or advanced, the high priced Canada Balsam.

Leaving apart the Damar Gum, all the mediums I review do not need any hydrocarbon solvents and therefore have two main advantages: 1) they are safe, 2) they are cheap.

As I stated in my Conclusions for the series: Leave the Canada Balsam for the professional taxonomists who seek legitimately the “HYP”, (hundred years of permanence) for the preparations of their type species, and have many moments of recreation by making your collection of preparations look really professional.

List of the experimental mounting mediums, and the article
in which the formula can be consulted

The presentation, the formulas and images that show the aspect of freshly mounted subjects can be seen in the following links:

 Part 1. - Liquid mediums

www.the microscopy-uk.org.uk/mag/artdec02/wdmount2.html

Part 2. - Simple drying mediums:

 www.the microscopy-uk.org.uk/mag/artjan03/wdmount3.html

Part 3. - formulas: Fructoglycerol and modified Brun's medium:

www.the microscopy-uk.org.uk/mag/artmar03/wdpart3a.html

Part 3b: PVA-lactic acid and PVA-glycerol mountants:

www.the microscopy-uk.org.uk/mag/artapr03/wdpart3b.html

Part 3c: The gum arabic mediums

 www.the microscopy-uk.org.uk/mag/artmay03/wdpart3c.html

Parts 4 and 5. - The glycerin jellies and conclusions:

www.the microscopy-uk.org.uk/mag/artaug03/wdpart4.html

With some inevitable repetition, my actual opinion on the use of those formulas is presented here..

Aw, water disinfectant: aqueous solution with an additional fixative.

Indicated when you want to examine and preserve for future examination under the microscope your collections of micro invertebrates and micro algae, mixed (or not) with very fine detritus. Fix them using 20% of Gala 20 or Lactocupric added to your sample in the field. For a short term examination you don’t need to wash the samples.

Try to obtain a very thin preparation. Normally it is used for a provisional mounting planned to last for weeks or some months at the maximum.

But because it allows pictures to be taken easily, and because the elasticity of the coverslip lets you move and turn the individuals observed by applying light pressure with a dissection needle, it is one of the most useful techniques that the amateur must master.

Aw mounts are a very useful for the rapid screening of a new sample.

As you see, it is only a refinement, but a very useful one, of the wet-mount method. By its nature, even well sealed, the duration of these preparations is only two or three months. Because of this there are no examples here.

Many advantages of this technique are shared by Glycerol, Fructose or PVA-G (see below) employing in this case a stepwise technique to avoid the excess of osmotic pressure that these products can exert on the micro invertebrates, all very fragile, and obtaining with a supplementary effort, a permanent preparation.


1. - One Monostyla closterocerca (by the shape of the foot) caught in the detritus, one can see the mastax. 2.- a spore (probably from Alternaria a pathogenic fungi) 3.- two euglenas, a coarse grain of paramylon, in one of them, and the well preserved chloroplasts in the two cells, can be seen. 4. - The anterior end of the Brachionus of the first picture, with a objective X40, you can see clearly the cilia of the crown. 5. - An arcella (may be A. vulgaris). Optical section showing both nuclei opposed at the extremes of a diameter. The test was already transparent when I saw the preparation for the first time. All from the same glycerin preparation. (The picture of Brachionus was not cropped). All in brightfield.

PG, pure glycerine

Glycerin is a difficult, but very useful permanent mounting medium. It is also the standard to mount nematodes.

It exerts a great osmotic pressure on living biological materials or even on fixed but delicate ones, having as a result the distortion and collapse of the individuals. To avoid this it must be introduced very gradually.

The methods to do this were discussed in the original article. With materials correctly fixed GP guarantees an almost realistic aspect of the subjects.

1. - A bdelloid rotifer, contracted as they always do, but I have studied it alive and it is Philodina roseola. 2.- even if chloroplasts are not seen I think this is a Chaetophora. 3. - two very small unknown peritrichs. 4. a chlorophyta, one can see the nuclei in the cells. 5.- a cyanobacteria, probably Scytonema
 (1 and 2 with the obj. X40, others X100). Brightfield

The pictures show micro-algae and some invertebrates mounted in glycerin. The sealing of the coverslip must be very carefully done. There is always the risk of infiltration. My preparation after 24 months has a bubble over almost ¼ surface of the slip. Clearly 1) my sealing was not perfect and 2) the materials were not in pure glycerin. Water has evaporated. But the remainder of the preparation provided splendid examples of the combined effect of lactocupric and glycerin. See later my discussion of PVA-G.

Two diatoms at the end of a gelatinous stalk, x 40.
A cyanobacteria, probably Gloeocapsa. The cell groups are encapsulated in jelly.
A unicellular cyanobacteria, with a jelly envelope.

MMF, Mounting mediums with Fructose

Pure, the fructose can be found as a powder in the sweeteners section of supermarkets. (If you bought a bottle (500 g -1 kg), make donations to your colleagues, it will be sufficient for tens of them and will last a long time.)

This can be prepared according to the formula of Larry Legg (see his article in Micscape).

Onion skin, colored with Gentian Violet. x 40,

Both pictures from materials mounted in Karo 60%.
Dorsal (longitudinal) and legs (oblique) mucscles seen through the chitinous carapace of a copepod x 40. Rheinberg, blue center, orange annulus.

Fructose is suitable for histological sections (probably for fungi also) and for many of the invertebrates like small insects, entomostracans, or acarina. Another source, more convenient and cheap, is the concentrated corn syrup (with 76% fructose) which is sold in the Americas for breakfast under the trade name of Karo. There must be other suitable trade marked products also, but you must read the labels to be sure that the syrup does not contain saccharose (sucrose, common sugar) which is a potential source of crystallizations (see the von Apathy critique). Karo does not crystallize, and it is recommended as a standard method by many researchers for algae (marine or fresh water). It is also good for higher plants histological sections as well.



The articulation of a fly wing. Brightfield
Obj. X 10. Karo 60%.

""Gills" in the tail of a mosquito larva . Darkfield, disc of 12 mm. Obj. X 10. Karo 60%.

My preparations are thin, dry, the coverslips do not move, do not have any crystallization, and even the Gentian Violet stain is preserved.

Fructose receives subjects directly from water, or glycerin; if you have materials preserved in alcohol, transfer them gradually to water. Like the glycerin, the fructose is capable of collapsing very sensitive materials. A microtechnique employing three or four stages can be necessary.

It receives and maintains many dyes, and preserves very well chlorophyll in plant samples which were fixed with GALA or Lactocupric.

It is one of the easiest and safest mediums. I think that Fructose and PVA-G should be your standards.

CPG-pva, commercial pure glue, based on PVA

For handicrafts there are sold some transparent and syrupy glues made of PVA (Polyvinyl Alcohol, not to be confused with Polyvinyl Acetate - also PVA - the latter is the basis of students' glues) it can be bought in handicraft stores, and in my experience it is not difficult to find. The product is transparent and has the consistency of honey and can be diluted with 30% of water. Some selected materials (you must test) can be mounted in this adhesive of 70% commercial PVA glue, directly from water or from a 1: 3 dilution of glycerol in water. The slides must be sealed because if they are not, the pure adhesive tends to dry and peels from the glass in a few months. I do not like the refractive index of the pure adhesive, the glycerin improves it a lot (see PVA-G).


Cladocera. Darkfield . obj. X10

Perigastric gland. Mosquito larva. COL-D1. PVA-G. Obj. X10.

Caudal end of the same larva. COL-D1. obj. X10 - PVA-G.

You do not know the exact molecular weight of the prime materials, or the exact concentration of the adhesives. But you are amateurs, and if you buy a tube of 50 ml of adhesive for one dollar, as I do, you would have PVA for your life (buy two if you anticipate a long time life.)

I encourage the search for this commercial adhesive because the PVA sold for microscopy is very expensive and difficult to obtain and dissolve. Glues are really very cheap and ready to use.

A cyclopoid copepod, Dark field x10.
The postabdomen of a cladocera
brightfield. x40

These 3 pictures from PVA-G mounts.
A small chlorophyte Two optical sections stacked.

PVA-G. - Polyvinyl Alcohol with glycerol

It receives materials practically from all the fixatives if you take care to wash them very well. For particularly fine materials use a two or three step sedimentation technique. It is clear, transparent, has a very good index of refraction, good clearing ability, good behavior when drying, and lasts a long time, and it's not necessary to seal it.

It receives and maintains the color of the most usual dyes that the amateur can employ, and also the chlorophyll of plants. And you can mount in it practically all subjects, including pollen (with or without additional dyes). I am a fanatic of PVA-G and recommend that this becomes your normal mounting medium.

One acarina - the “little red spider "of Hibiscus
  Darkfield disc of 12 mm. obj X10. PVA-G
A detail of the head of the same. Brightfield. Obj. X40. PVA-G.
This preparation is only 6 months old. It is included here to show the versatility of the PVA-G medium.

For those difficult materials such as samples mixed with detritus, which we discussed in the article on PG, a step by step method can be used with PVA-G with very good results. This gives you a preparation with the benefits (or better) of a glycerol mount, but solid, dry, and likely to be filed easily and examined even with the oil immersion without problems. I use a product labeled "ITOYA" Paper Glue. (They have an Internet site.)

019-PVA-G 020-PVA-G

Epidermis of Aptenia cordiflora - 24 months after. Obj. X100- Brightfield. PVA-G

Pollen of Petunia grandiflora. Fast Green FCF, Brightfield. obj. X100 - PVA-G
This is an 18 month old slide.

Note: Some correspondents say that the PVA glues are difficult to find in Europe. After the article in Micscape was published I visited the site of Marcel Lecomte, a Belgian mycologist who sells chemical products for microscopists. If you do not find the cheap adhesives of PVA (or if you want as a reference a well standardized professional material, at a reasonable price) I think that the formula can be prepared thus with his m-23a PVA solution:

Polyvinyl alcohol (m 23a)...........30 ml

Water saturated with borax........12 ml

Glycerin...........................................18 ml

This includes a polyvinyl product which has 1% of phenol as an included preservative; it is an advantage.

Four images of clusters of micro algae fixed with Lactocupric and mounted in PVA-G, more than 20 months ago. They are included to compare them with the quality of the Glycerin and the PVA-G mounts.
The sample was mounted using the PVA-G step by step technique as if it were glycerin. All the pictures in brightfield and with the X100 immersion objective.

PVA-L. - Polyvinyl alcohol with lactic acid.

This, and the now not recommended GLG, were proposed as a replacement for the formulation of Hoyer. Hoyer's is a powerful clearing agent / mounting medium with arabic gum, chloral hydrate and phenol, both forbidden. The lactic acid clears fast and is very penetrating, and the formula was promising, but its refractive index of 1.43, is disappointing and much lower than that of glycerin (1.47). The PVA-L is a good clearing medium which acts quickly, but if PVA-G is allowed time, it must be more powerful, for the same materials. Of course, the preparations which I have, with PVA-L, are dry, firm, and clear. Certain materials like aphids cleared up excessively, for example. But some of the slides had an additional problem which I do not know what to ascribe it to. They had small crystals strewn under the coverslips.

025-PVA-LThere is no chemical reason which I can discover that justifies this fact and I believe simply that one needs to be very neat when one washes the coverslips, the slides, and also sourcing the fixatives or other reagents added to the subject.

For the moment I must recommend the use of PVA-G, and to continue some experiments with PVA-L. Here two successful examples of mountings with PVA-L.

The left-hand picture is from a small "thrip" (an insect of the order Thysanoptera) captured on a Gerbera jamesoni flower. It was fixed with alcohol 70%, washed in water and mounted with  PVA-L, 8 months ago. The preparation is perfectly dry, firm and clear, without crystals. Notice the particular wings of this insect. Obj. X10. Illumination through the COL-D3. Six pictures were stitched for this image. The background was blurred with Photo Paint.

NPM.- Nail Polish Mountant

Nail polish is well established as a sealant for coverslips. But it can also be employed as a synthetic resinous mounting medium. It is the most expensive of the mediums discussed here. But certainly it is the easiest to find.

The common solvent for all nail varnishes is acetone, butyl acetate or many other commercial solvents.

Generally, to employ it as a mounting medium, it is wise to dilute it a little with its solvent to make sure that it runs well under the coverslip.

Very delicate materials to be mounted must be completely dehydrated by some alcohol stages (30, 50, 75, 96, and 96 a second time, could be a suitable series). One can mount in the NPM from the 96% alcohol. In more than 20 months the preparations which I prepared show that they do not retract, that very thin preparations are possible and that, more importantly, they preserve the dyes applied to the mounted subjects.

026-PVA-L 027-PVA-L 028-PVA-L
Wings of the thrip over black background. Click it to see the original
Obj. x10 - PVA-L

One aphid found on basil.

The abdomen of the same aphid.

Actinophrys sol - a heliozoan, fixed with AFA, colored with Fast Green FCF, the actinopodes are retracted. Obj. X40. NPM. End of wing of a mosquito. Obj. X10. NPM.

I have mounted leaf epidermis, hairs, scales of butterfly, and parts of insects, algae, protozoa and rotifers. The worst subjects were the cylindrical branches of filamentous algae, which were flattened by dehydration with alcohol.

Upper surface of the wing of a mosquito. Obj. X40 - NPM
Leg joint of a mosquito. Obj. X40 - NPM

Most difficult are the thick preparations, because one must continuously replace varnish that retracts from the border, until the material consolidates. One needs to put some wedges (paper, pieces of broken coverslip or of aluminum foil, hairs, and so on) around the subject to avoid crushing it.

Permanence of the mounts depends perhaps on the quality of the varnish. A microscopist who used it tells me that, contrary to my results, his preparations peeled from the glass in a few months. Perhaps the slides and the coverslips must be carefully degreased? I have used a transparent varnish with the Revlon trademark. I consider now a good general precaution is to seal the coverslip with acrylic cement.

Gum Damar (Dammar)

Being a natural resin of vegetable origin, Damar shares with balsam the property of long life. It is almost a HYP. Read the description of its preparation and techniques for use in the previous article.

Gum Damar- fly wing, obj. X 10 Gum Damar - surface of the wing of a fly. Obj X 40

It is naturally a very good choice, but for the adult amateur. There is a protozoologist who recommends it as the best medium to mount gregarines (a form of parasitic protozoa).

 Kaiser glycerin jelly

 It is the best formulation of the group of mediums of glycerin jellies, even if you double the quantity of water in my formula.

Brood pouch of a Daphnia (the same one included in the former article) - x10 - Kaiser Glycerine Jelly.
One embryo from the left-hand picture. x40

II raised my objections to this much advocated medium in the previous article. Nothing has changed.

The Fisher’s borax jelly is the most difficult formula, although it is much recommended (for pollen especially). Even with double borax and sufficient disinfectant, this formula becomes easily soiled with fungi and bacteria. I do not use it.

You must recall that to make a very good inclusion in the jellies it is better to soak your subjects previously in glycerin, although not always needed.

 (If you have the equipment, or you cannot obtain PVA, experiment with the jellies, but most certainly you will have to make your apparatus. If you are enthusiastic of the jellies, try to build the heater of Jean Legrand or the heating plate of Jean Marie Cavanihac, both are able to allow a complete handling of your subjects in the melted form before applying the coverslip. Don't hurry and be aware of burns.)

Some people say that the jellies are the mediums of choice to mount pollen. But try using PVA-G (with or without additional dyes). I believe that the glycerin jelly is not better, even if this needs a change of mentality in the professionals.

von Apathy Syrup

 It is a popular arabic gum syrup with saccharose (common sugar).

See my comments and preparation techniques in the corresponding article.

Apathy was always blamed for the crystallization of saccharose. In the very thin preparations of tissues colored with Sudan, for the demonstration of the lipids, which I employed as a student in my university many years ago, I never saw the crystals. I think now that possibly they were using the Lillie modification (here adapted as GAF).

24 months after preparation, the antenna of a copepod. Von Apathy syrup. Obj. X40. Brightfield.

Crystals of saccharose on the margin of the coverslip
von Apathy syrup, obj x 10- Polarized light.

But after 24 months all the preparations which I prepared with this syrup show crystallization which are formed along the coverslip margins and grow towards the center, even if it is sealed with nail varnish. Because crystallization is much more likely in the unsealed preparations, I think that a very good cement, applied carefully, can possibly avoid the problem.

The formula does not dry fast, but it dries. The subjects included are well preserved. The only problem is the crystals. Try to seal the margins.

GAF - Arabic Gum with fructose.

I've noted that GAF, which incorporates fructose (I use Karo, see the formulas), does not suffer the crystallization problem. GAF is my formula to replace the Lillie's medium, derived from Apathy’s by incorporating fructose in place of saccharose. The results are comparable with PVA-G, even for the sensitive protozoa. But the drying of the edges is much slower and in wet climates could require the assistance of a furnace. But it does dry.

039-GAF (038)
040-GAF (039)
Articulation of a fly wing, GAF, obj X10. Cells seen through the chitin of the back of a copepod, GAF, obj X40.

There remains only two serious problems which I've encountered with GAF. One is that it needs a disinfectant to preserve it from fungal infections (I've employed successfully 10% of Listerine), and two, that in parts of my preparations of cladocera with embryos, or fatty copepods, there is oil loss to the environment ruining the aspect of the preparation. I think that this can be prevented by passing materials charged with lipids through an alcoholic bath, and again to water before mounting them.

"Gills" of a mosquito, illumination with a Rheinberg stop with red and blue quadrants, black center. Obj. X10 - GAF.
Oblique illumination of the antenna of a copepod, obj. X40.

But, before you engage in GAF preparation and its use, you must remember that the results are very similar and no better than mounting in fructose or in PVA-G. If you find Karo or fructose in your supermarket you do not need GAF. It is the same one if you obtain PVA.

Norland 61.

I am not a diatomist. But it is clear to me that this synopsis must include this synthetic resin which has a refractive index of 1.58, making it useful for the mounting of diatoms. It is sold to repair cracked front lamp glasses of cars. Probably the cyanoacrylate resins which must be UV treated (the intense indirect light of the sun is a good source of UV) could have more or less the same index. I never have used it but it is frequently recommended by serious amateurs.

Other formulas

I have additionally prepared and discussed in earlier Micscape articles:

AG, alcoholic glycerol
FG, medium of glycerol and fructose
FGL, glycerol, lactic acid and fructose

GLG, arabic gum, lactic acid and glycerin
GG, Glycerol-Arabic Gum

FJ, Fructose Jelly
Borax Fisher's Jelly

They all have a good performance and are useful for short term work. But they do not harden at all, they need to be very carefully sealed, and by no means are they better than those discussed above. I’ve discarded their use.



Thus, you have a useful and required medium for temporary mounts: Aw. Four mediums of choice for the average amateur: FMM (home made solution of fructose, or preferably commercial fructose syrup of the Karo type), PVA-G, NPM, and the Kaiser’s Jelly. And a medium for adults: Gum Damar. If you can not find fructose or PVA, use the very good but a little more difficult GAF. Two good alternative mediums: PVA-L, for a similar result to the PVA-G; PG, difficult to seal, for certain subjects it is similar to PVA-G, (for the moment PG must be considered the standard for the nematodes, in the future we must compare it with the PVA-G or the PVA-L). We also have a special medium for diatomists: Norland 61. So the present review reduces the list of the most useful amateurs' safe mounting mediums from twenty to nine (postponing my opinion on PVA-L pending additional experiments).



Larry Legg's  articles in Micscape

Marcel Lecomte site  http://users.skynet.be/Champignons_passion/main.htm

Jean Legrand   CONSTRUCTION D'UNE HOTTE DE SECHAGE http://www.microscopies.com/DOSSIERS/Magazine/Articles/JLegrand-Hotte/Etuve.htm

Jean Marie Cavanihac A Slide Heater For Glycerin Jelly Mounting  and http://prodelos.no-ip.com/beyond/contents4.php

Comments to the author, Walter Dioni , are welcomed.


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