SAFE MICROSCOPIC TECHNIQUES FOR AMATEURS I - MOUNTING MICROSCOPIC SUBJECTS. 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.

 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.

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.

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

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.

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.
 

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 larva	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 antenna
GAF, ovary of Diaptomus	GAF, epithelial cell of  leaf underside	GAF, fly wing

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.
 
 

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

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 antenna	GG, Diaptomus ovisac	GG, epizoics on Vorticella

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 larva	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.
 
 

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.