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Safety Warning |  |
Always be careful when using chemicals. Many of the ones we use in fungal microscopy are rather "nasty" being Corrosive, Toxic, Caustic, Irritant or a combination of these. Keep them away from children and pets. Wash your hands after using chemicals or if you get any on your skin. The Stains really do stain!!
It’s a good idea to keep all the bottles on a tray to limit the effects of any spillage. Don’t leave the tops off the bottles especially if they’re unattended. Having said that they are not going to leap out of the bottle and attack you! Just use common sense and good practice. Don’t leave used slides lying about. A useful thing to do is have an old margarine tub with some water and detergent in it. When you have finished with your slides push the cover slips off with a probe into the tub and drop the slides in. This dilutes the "nasties" and makes it easy to clean the slides afterwards.
What follows is a guide to microscopical use but it is not intended to be definitive. There are all sorts of reagents that have been used over the years.
Water
Very useful, easy to get and free! Use if you wish to know the natural colour of your subject. The addition of a tiny quantity of detergent or, even better, photographic wetting agent helps to "wet" the subject. Some spores can be a nuisance in this respect by floating on the water and then escaping sideways when you try and put a cover slip on top.
I find adding about 10% of glycerol (glycerine) aids in preventing the slide drying up prematurely.
Congo Red
This stain comes as a powder but is normally used as a 1% solution in water or strong ammonia.
The ammoniacal form seems best to me, as the plain water version is rather unsatisfactory. I find this a really useful stain and tend to look at everything with this first. If I had to pick only one stain to use this would be it. Very good for seeing clamp connections. Sometimes it is more effective to wash out the excess stain from around the subject to improve the contrast. This is easily done by putting a drop of 10% ammonia on one edge of the cover slip and touching the opposite edge with a scrap of rolled up paper towel or tissue.
The liquid is drawn towards the paper and the ammonia is pulled under the cover slip taking the excess dye with it. Repeat as necessary until the desired result is achieved.
(See also Ammonia)
Melzer's soln.
A well-known mycological reagent. This consists of chloral hydrate, iodine, potassium iodide and water. Its effectiveness is based on the fact that iodine reacts with starchy substances to produce an intense dark blue-black colour. Often used macroscopically to determine if spores are "amyloid", i.e. turning a blue-black colour.
The three reactions to material mounted in Melzer's soln. are: -
- Amyloid or positive, as above.
- Dextrinoid, a brownish to red-brown reaction.
- Inamyloid or negative, a yellow to hyaline reaction.
Melzer's soln. is very good at showing the ornamentation of Russula & Lactarius spores. It is also useful for ascomycetes as it "blues" the tips of some asci ( J+ in the B&K book).
Be aware that Melzer's soln. does not like being mixed with alkaline substances. This means that if you are working with dried material and re-hydrating with any of the normal reagents, which are all alkaline, you could have problems. I have successfully rinsed off rehydrated material with water.
Cotton Blue
Also called aniline blue. Used dissolved in lactic acid, glycerol and phenol "lactophenol cotton blue". This is another generally useful stain. I find this works well to show the structure of ascomycetes. I notice that B&K use this to make permanent mounts.
The literature talks of spores etc., which take up this stain to a greater extent than other structures, they are said to be "cyanophilous". I did find Pholiota flammans recently and the cystidia took up this stain strongly, just like Phillips said they did!
Cresyl Blue
Used as an aqueous soln. approx. 1%. This, as its name suggests, is a strong blue stain. However, certain hyphae and spores turn a reddish violet colour with this stain instead of blue. For example the stipe hyphae of Mycena. This is a "metachromic" reaction.
Sulphovanillin
This is a solution of vanillin in strong sulphuric acid. The main use is in staining the cap cystidia of certain Russula species where it produces colours varying from black to purple-grey. The soln. is sometimes made up in advance when it turns black but still works. The formula is 5mg. of vanillin crystals dissolved in 6ml. of 80% sulphuric acid. Some people make it up on the slide by adding a tiny amount of vanillin crystals to a drop of 80% sulphuric acid and stirring with an acid resistant tool (e.g. glass or stainless steel). A pale straw coloured soln. is what’s required. It is important to dissolve all the crystals otherwise they can crack the cover slip when you press it down.
I tend to make up a small amount (5 or 6 drops) in a watch glass and use that. Use great care when handling sulphuric acid as it is extremely corrosive! I ruined a favourite T-shirt at a recent Russula workshop by burning several holes in it! Never attempt to make up dilute acid from concentrated unless you are sure of what you are doing. The dilution process can cause the acid to boil and spit!!
Dispose of unused sulphovanillin promptly, including any slides with it on. You will find that the stained material lasts only a short while anyway, maybe only 10 mins, as the strong acid quickly destroys it.
Carbol fuchsin
A strong stain in solution with phenol. Mostly used in determining "fuchsinophile hyphae" in some Russulas. Needs to be used in conjunction with 10% hydrochloric acid. See Rayner’s keys to the Russulas, page 36, for the method.
Clemencon’s soln.
This is used to rehydrate dried material and make it easier to cut sections. There are other reagents you can use but this is very popular with mycologists. The formula is 80ml of 96% ethanol (or industrial methylated spirit), 20 ml of concentrated ammonia and 1gm of glycerol. The dried material is soaked in this until it is sufficiently softened. It is then removed and allowed to dry for a while when it should be "waxy" and able to be sectioned. A final soak in 10% ammonia may help to expand the section.
Acetocarmine
Used to stain the contents of the basidia in certain agarics. Such basidia are then spoken of as being "siderophilous". The method is described in the B&K book Vol.3 p14. It is rather complex and not for anyone unused to handling chemicals.
Ammonia
Apart from its inclusion in other reagents it has some specific uses, causing colour changes to various fungi, see the books. Sometimes just the vapour is needed. Be aware it is very pungent, like the old fashioned "smelling salts"so keep the bottle away from your face when you open it. It helps to keep the bottle in a cool place and to replace the top promptly as the ammonia eventually escapes into the air.
A 10% soln. is sometimes used to rehydrate dried material or to "expand" it after rehydration with Clemencon’s soln.
Potassium Hydroxide & Sodium Hydroxide
These are both strong alkalis and very caustic. Usually used as a 3-5% soln. in water. Sometimes used to rehydrate dried material and also to soften hard material, like some bracket fungi. Here again there are some specific colour reactions with certain fungi.
Guaiac
A saturated soln. of gum guaiacum (a resin from a tropical tree) in 70% ethanol. Mainly of use macroscopically as it gives a colour reaction with some fungi.
Ferrous sulphate
The famous "iron salts" which many mycologists carry as a large crystal for use in the field. A 10% aqueous soln., which is acidified with sulphuric acid, seems to work better than the crystal even if it’s not quite so handy. Essential for Russulas! No microscopical use but I thought you might like to know!
In Conclusion
I hope you find the above useful and that it will encourage you to try different reagents. Unfortunately chemicals are difficult for the amateur to obtain nowadays. The days are gone when you could go to the local chemist and get two ounces of cyanide just by signing the poison book. However some things are available. Local chemists frequently stock household ammonia (which is 10%) and glycerol (glycerine). Sodium hydroxide is often sold as drain cleaner.
Brunel Microscopes can supply Congo red powder and ready made up Lactophenol cotton blue.
I got a 7lb bag of ferrous sulphate from a garden centre, it cost 90p!
As the NWFG is associated with the BMS we are sometimes able to get chemicals from them to distribute to members but supplies are limited. However it’s a perk of membership!
Finally, be careful with chemicals and you will find them very helpful in your mycological work.
Useful reading
The "Swiss Books" Fungi of Switzerland by Breitenbach & Kranzlin vols. 1-3.
Keys to the British Species of Russula by R.W.Rayner.
How to Identify Mushrooms to Genus III: Microscopic Features Mad River Press Inc.85.
Moser’s Keys to the Agarics (contains many formulae and methods).
