Part 3c - The mixed formulae
Gum arabic media

WALTER  DIONI                       Durango (Dgo) México
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 the air tube of a mosquito larva mounted in gum arabic - fructose medium.
Editor's note: Part 3 of this series will be published in three monthly sections. They will be interlinked as they are published.
Part 3a: Introduction; fructoglycerol and modified Brun medium as mountants.
Part 3b: PVA-lactic acid and PVA-glycerol mountants.
Part 3c below: von Apathy's original formula and Lille's medium as mountants. Gum arabic fructose, glycerinated gum and glycerinated lactic acid mountants.


APT.- von Apathy original formula

 Two references on the web claims an RI = 1.52. I think one is an error, the other a copy of the first. All my four old references state a credible 1.42 RI even with the fructose modification discussed later.

gum arabic...................…........50 g
refined sugar...........................50 g
water.......................................40 ml
antiseptic..............................10 ml**

The recipe states that you must use clean drops (stones) of gum arabic. Mix sugar and water and add the gum last. Put in a sealed jar. Completely dissolve it on a stove at 40ºC (from a few hours to days) stirring only occasionally and slowly. Or use a water bath at 60ºC for a more rapid dissolution. Take care not to include air bubbles. Finish adding the antiseptic. The antiseptic is needed because without it the medium develops fungal infections in less than 2 weeks. The amount of water can be raised to 90 ml.

 This is the general method to prepare the gum solutions recorded in the literature. If it is thicker than you want, or if you want to filter the liquid, dilute with enough water. After filtering you must concentrate the medium by evaporating the water on a stove.

 It is a safe and cheap formula. The problems are to get the drops of arabic gum, the difficulty and the time needed to dissolve it…and the frequently reported problem of sugar crystallization. When I was a biology student all the “lipid tissue” slides in our histology lab were stained with Sudan III, and mounted in Apathy’s medium. Many of the slides were more than five years old yet I've never seen a crystallized one. But…

APT, fly wing-1

ATP, fly wing-2

APT, Diaptomus male antenna

APT, Diaptomus 5th male leg

APT, daphnia eggs


APT, epithelial cell of leaf underside

Notes on the quality of the gum based on my trials within the last hour! I insisted with my apothecary on the quality I required of the Arabic gum. He consulted with his suppliers and assured me that the grade he was offering was really Arabic gum, powdered, but pure. I remembered my old days as a young scholar when I bought the powdered Arabic gum and put it in water, only to finish with a sphere of moistened compacted powder enveloped by a shiny bubble of air, and all that work to break it and dissolve (over many hours) my paper adhesive.

I was near to refusing the offer when I remembered my “cocoa trick”. My powdered cocoa, like the Arabic gum with the water, refuse to mix with my cold milk. But I have a trick, you know, I mix the cocoa with the sugar, and later I add the milk. And this works. So I bought some gum. Try this technique:

  1)    In a 30 or 50 ml flask, put 1 part powdered gum, and 1 part sucrose. Seal and agitate thoroughly to obtain a dry homogeneous mixture.
2)    Slowly add portions of the water, mixed with the antiseptic. Any sugar crystals act as micro tunnels that carry the water down the mix. Carefully move the powder with a needle if it stops running.
3)    When all is wetted (it took one minute) leave alone for a time (4-6 hours) the glutinous mass, or put the flask in a bath of water at 60ºC (or a little higher).
4)    Presto! You have your gum arabic solution made in five minutes of not so hard work, and all you need now is to allow some time for all the dirt to precipitate to the bottom (perhaps overnight).
5)    Decant the supernatant liquid and adjust the density to suit your preferences.
von Apathy’s medium isn't compatible with normal histological stains. The pH of the medium is near 4.0 (highly acidic) so stains fade or bleed into the medium. If you only mount unstained materials, or you intend to preserve your slide for only a few weeks, this is not a concern for you. But some modifications claim a better behavior for the formula. Three alternative added chemicals were proposed: potassium acetate (30 to 50 g added to the formula), calcium chloride (10 g) or even sodium chloride, the common table salt, (10 g) It seems that these additions raise the pH to near 7.0

Mount using the technique proposed for Karo. It is not necessary to seal the coverslips. But I do.

** The original formulas for gum arabic or gelatin media always include an antiseptic: thymol crystals (one crystal), phenol (1 g), Mertiolate (10 ml), etc. All are banned as they are either difficult to obtain or are very toxic. Remember that this additive functions just as an antiseptic so you can even eliminate it without affecting the mountant's performance, and take the risk (most improbable) of a bacterial or fungal infection of your slide, as M. Cairn and J.M. Cavanihac had done. Modern alternatives could be Benzalconium chloride, Listerine, sorbitol, or potassium benzoate.

•    As Mertiolate contains mercury bichloride it was banned, and the suppliers changed the formula of the product. For a long time a solution of Benzalconium chloride has been sold under the trade mark name of Mertiolate. It was even colored red as the original product was, which made it unusable as an antiseptic in this formula. Now one can find white reagents under the Mertiolate or Benzalconium Chloride name. When you mix it in the formula you could be worried by the milky cloud it forms. Continue to stir carefully until the mixture becomes clear again.

•    Listerine is a mouth washing solution. Its formula is composed of several disinfectant ingredients that are also used as clearing agents in microscopy. The old formulation had the toxic phenol in its ingredients. It is eliminated in the modern formulation. The published composition is:

menthol..........................42.5 mg
thymol......................…...63.9 mg
methyl salicylate........…..66.0 mg
eucalyptol................…....92.0 mg
ethanol...............…...........22.7 ml
water......................….......77.3 ml

    The Listerine I get has a slight blue green color (turquoise, says my wife), but in the used quantities this is not a problem at all.

LMM.- Lillie medium.- RI = 1.43

I give you one of Lillie’s modifications of von Apathy’s formula. If you use the powdered gum arabic, also use the fructose as crystals. Add fructose and potassium acetate to the powdered gum. Mix well; add the water and the antiseptic.

gum arabic (powder)..…………...50 g
fructose (crystals)……….……….50 g
potassium acetate…………….….50 g
water………………………………..90 ml
antiseptic……………………...…...10 ml

It has a good refractive index and a pH of 6.7.  Most commentaries are as for upper formulas.


As I can’t find drops of gum arabic (and I had not have yet remembered my cocoa trick) I made another trip to the art supply store, and returned with a medium syrupy solution of it as sold for artists to mix their watercolors and for finishing their art works. These formulae are provided as examples for you to develop your own. Some of them do not contain sugar, so you can’t do the old trick. You need the commercial solution.

GAF.- Gum Arabic-Fructose medium (RI probably around 1.42)

Gum arabic solution.........................................30 ml*
(Commercial Karo “Clear” (for babies)) ..........30 ml **

*If the commercial solution contains excess liquid, concentrate it by evaporation.

                                        ** or use Larry Legg’s fructose solution.

I omit the antiseptic because for obvious reasons the commercial solution must have one.
Those concerned about bleeding of histological dyes can include 15 g of potassium acetate (or try the cheaper and ubiquitous sodium chloride…. 1 levelled teaspoonful) to raise the pH.

The fructose substitution for the sugar lowers the danger of crystallization. It is a worthwhile and safe precaution to seal the coverslip to stop evaporation.

Mount directly from water and as if it were pure Karo. File horizontally.

GAF, airtube of mosquito

GAF, mosquito larva head 
nerve ending

GAF, gland in mosquito 
larva body

GAF, ovisac of Diaptomus 
direct mount

GAF, ovisac of Diaptomus 
stepped mount

GAF, Diaptomus male

GAF, ovary of Diaptomus

GAF, epithelial cell of 
leaf underside

GAF, fly wing
Through the skeleton of the head it is possible to discern what is probably the start of the "dendrite" of a nerve cell in contact with a sensitive cell. The other most interesting pictures are the Diaptomus ovisacs (one collapsed after being mounted directly from water, and the other normal, after being worked up through 3 dilutions of glycerin in water) and the cells of the ovary of one of the females, seen through the dorsal exoskeleton.

Commentaries.- This is obviously my version of the Lillie’s medium. The RI, and the appearance of the mounted subjects are good, and it dries fast (in my climate). As it is somewhat liquid (at least in my flask) carefully estimate the drop size to avoid excessive mountant overflow. I challenge you to adapt this formula using powdered arabic gum and fructose crystals.

GG.- Glycerinated  Gum.- (RI about 1.44)

This is a formula in the style of those proposed by Farrant, Lillie, and Dahl.

gum arabic solution..…..………….30 ml
sugar…………………………...…....30 g
glycerin………………………...…....30 ml

If you want, you can add sodium chloride (10 g) or 20 g of potassium acetate.

GG, epithelial cell 
of leaf underside

GG, fly wing-1

GG, fly wing-2

GG, female Diaptomus

GG, Diaptomus ovisac

GG, epizoics on Vorticella
The interesting things to note are the epizoic peritrichs (protozoa in the group of the vorticella) living on the egg sac. They were perfectly fixed by the lactocupric fixative, you can even see the cilia in the open ends of the cells. They were fixed and kept in the fixative for more than a month.

GLG.- Glycerinated lactic gum

lactic acid………………………...25 ml
gum arabic..………………….…..40 ml
glycerin………………….………..20 ml
Karo*………….………………..…10 ml
  *or Larry’s fructose syrup

The above is my recommendation as a substitute for the Hoyer, Berlesse, de Faure and other chloral hydrate based formulae. It is recommended for botanical sections cut with a microtome, including stained ones, or for arthropods or other subjects cleared in lacto-glycerol. Of course this and the PVA-L are the appropriate mediums for chitinized arthropods cleaned of their soft parts with potassium hydroxide or sodium hypochlorite. Small and soft subjects can be overcleared. Try the lactic acid methylene blue staining (one that even I have not tried).

GLG, head of 
mosquito larva 

GLG, pecten in 
airtube of mosquito

GLG, airtube of
 mosquito larva

GLG, epithelial cell 
of leaf underside

GLG, Diaptomus
male antenna 

GLG, fly wing-1

GLG, fly wing-2

In my bottle this formula is highly fluid, and I first suspected it was of little use. But it is really an easy to use media. Drying time depends on the climate I think; it took much more time here in moist weather. If you mount directly some more or less opaque objects, they clear slowly in 8 to 12 hours. Muscles can be made invisible in the medium term. Generally this is the effect that users of this type of formula are searching for. Additionally you can clear your subjects, before mounting, with lactoglycerol, or lactic acid, if they are really dark.

The GLG gives results very similar to the PVA-L. Probably the degree of clearing imparted by each formula could be regulated by changing the quantity of lactic acid included. It dries firm in some hours, but it is a good idea to seal the coverslips.

Mounting in gum arabic media

With an appropriate tool pass the objects from water or an aqueous medium to the slide. With an absorbent paper eliminate all the water you can. Add a very tiny drop of medium, position your subjects as desired with the aid of two needles. Lower over them a carefully estimated drop of the gum media. It is better if it is a little undersized. Apply with care the cover slip. When you apply the weight the gum must reach the borders. You can hurry up the drying process by applying heat with an electric bulb, or with the microwave oven, as explained before.

If you live in a climate with 30% Relative Humidity or less this is all you need. But over 40, 50% RH you need to seal the coverslip.

As this article has run for much longer that I intended, I leave for a future article the discussion of the jelly formulations and of which of the 18 fixatives discussed in this series must be used and when.

Comments to the author, Walter Dioni, are welcomed.


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Published in May 2003 Micscape Magazine.

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