Gene Stoermer sent this summary of information about mounting media; individual accounts follow. ************************** In case all of you have not heard enough about mounting media, I supply the following note, based on a list originally prepared by Ben McFarland and updated and revised by Norm Andresen. This is not an endorsement by either Norm or me. Some of these are toxic, and some have different performance, depending on batch. [Gene Stoermer] Mounting Media and sources Adapted and revised by Andresen (and now Sweets) DIATOM MOUNTING MEDIA Media with a high index of refraction are best for mounting diatoms. The higher the index of refraction the greater the contrast of the microscopic image and the better the depth of filed for a given N. A. Diatoms have a refractive index of about 1.43 and would be invisible in a media of this index. With high index media, geater numerical aperture (N. A.) can be used without loss of contrast. Listed below are the best of the resinous media. Many of them, however, are no longer available. It should be noted that many can be made by anyone in the laboratory as their formulae are published. Helle Nielson reminds us that Pleurax can be made using reference 12 below. Hyrax itself has a published formula. Name (References) Index of refraction Availability, Supplier, etc. Hyrax 1,2,8,9,10 1.65 Custom Research and Development P.O. Box 6018 Auburn, California 95604 (916) 885-3341 1990 price $50/o Not Available Walter Ruedrich of Custom R&D was trying to find another manufacturer to take over the prodcution of HYRAX as of 1994... contact him at the company with any questions. Aroclor 5442 5,6,10 1.66 Monsanto Chemical Co. 800 N. Lindberg Boulevard St. Louis, Missouri 63166 Not Available Naphrax 3,4 1.65+ Northern Biological Supply 3 Betts Avenue Martlesham Heath Ipswich IP5 7RH United Kingdom FAX 0044 473612148 Pleurax 7,12 Eileen Cox sends word that Pleurax can be obtained from: Mr Klaus Kemp Blautannen Wickham Way East Brent Somerset UK phone: +44 278 760411 He's also a whizz at mounting individual diatoms as they did in the last century! Eileen's group feels Pleurax is excellent for maintaining colony integrity Carmount 165 1.63-1.64 Cargille Labs, Inc 55 Commerce Road Cedar Grove, New Jersey 07009 Cumar R-9 11 1.63 Neville Chemical Company Neville Island Pittsburgh, Pennsylvania 15225 Meltmount 1.704 (similar to Naphrax) McCrone Accessories and Components 850 Pasquinelli Drive Westmont, Illinois 60559 708-887-7100 *Refractive index given is for the liquid media. When solidified the above media have a slightly higher index of refraction. All of the media use xylene or toluene (toluol) as a solvent except Pleurax. Originally Aroclor 5442 was a solid and had to be heated in order to be used. Pleurax, as originally prepared by Hanna (7), used ethyl or isopropyl alcohol but is now often prepared with iso-amyl alcohol as a solvent. Iso-amyl alcohol must be used in a fume hood. The mounting media can be used in the methods described in references 8, 9, 10 and 11. 1. Hanna, G. D. 1930. Hyrax, a new mounting medium for diatoms. J. Roy. Micros. Soc. 50:424-426. 2. Hanna, G. D., L. A. Penn and P. Ruedrich. 1929. Another synthetic resin useful in microscopy. Science 70:16-17. 3. Flemming, W. D. 1943. Synthetic mounting medium of high refractive index. J. Roy. Micros. Soc. 63:34. 4. Flemming, W. D. 1954. Naphrax: a synthetic mounting medium of high refractive index. New and improved methods of preparation. J. Roy. Micros. Soc. 74:42. 5. McCrone, W. C., R. G. Draftz and J. G. Delly. 1967. The Particle Atlas. Ann Arbor Science Publishers, Inc. Ann Arbor, Michigan. 6. Delly, J. G. 1969. Mounting media for particle identification. The Microscope 17:205-212. 7. Hanna, G. D. 1949. A synthetic resin which has unusual properties. J. Roy. Micros. Soc. 69:25-58. 8. Weber, C. I. 1966. A guide to the common diatoms at water pollution surveillance system stations. F.W.P.C.A. Cincinnati. 101 pp. 9. Weber, C. I. 1966. Methods of collection and analysis of plankton and periphyton samples in the water pollution surveillance system. F.W.P.C.A. Cincinnati. 27 pp. 10. Weber, C. I. ed. 1973. Biological field and laboratory methods for measuring the quality of surface waters and effluents. E.P.A. National Environmental Research Center, Cincinnati. 200 pp. 11. Holmes, R. W., C. J. Wilson and M. C. Amspoker. 1981. Techniques for preparing permanent preparations of cleaned and uncleaned diatoms using Cumar R-9, a Cumarone-Indene resin. Bacillaria 4:21-27. 12. Von Stosch, H.-A. 1974. Pleurax, seine Synthese und seine Verwendung zur Einbettung und Darstellung der Zellwnde von Diatomeen, Preidineen und anderen Algen, sowie f r eine neue Methode zur Elektivffbung von Dinoflagellaten-Panzern. (Pleurax: its synthesis and application to the mounting and clearing for cell walls of diatoms, dinoflagelltes and other alga, and its use in a new method of electively staining dinoflagellate armours.). Arch. Protistenkd. 116:132-141. ************************************* The following people have commented on Alternative mounting media: ALTERNATIVE High Refractive Index mounting media NAPHRAX and DIRAX in export packs with information are available from Northern Biological Supplies Limited 3 Betts Avenue Martlesham Heath IPSWICH United Kingdom IP5 7RH FAX 0044 473612148 NAPHRAX is widely used by European Diatomists. Ref. Mueller, A. 1985 Mikrokosmos vol 2. Some have noted that NAPHRAX may not have the longevity of HYRAX, although reports are inconsistent. Kerstin Williams has sent around a message about another mounting medium called MELTMOUNT: I have for several years now used something called Meltmount. Easy to use, comes in several refractive indeces and is relatively inexpensive. Can't lay my hands on a price list as we speak! Contact McCrone Accessories and Components, 850 Pasquinelli Drive, Westmont, Illinois 60559. Tel: 708-887-7100. Several people have reported information about CUMAR-9 resin Address: Neville Chemical Co., Neville Island, Pittsburg PA 15225. The following info is from Haberyan, Schere, Stoermer, and Stevenson CUMAR_9 is a boat resin, pretty cheap, that comes in pellets, and must ne dissolved in toluene, which they do easily. The slides are optically good. They are dark orange in color (SCHERER), or yellow and burns dark easier than HYRAX when preparing slides (Stevenson). The resin is not terribly pure, there seem to be fine particulates in it (Scherer and Stoermer), but Stevenson did not have these problems. Scherer reports trouble keeping it mixed on the shelf. It seems to become cloudy quickly if exposed to the atmosphere. I had best luck storing the pellets in a dessicator and only mixing small batches. Cloudy slides come clear (temporarily) if heated. Old slides that were clear to start are still clear today. Stevenson has 14 year old slides that are still good today, and are not cracked, but Scherer reports that older slides have fine cracks in the resin. and speculates that these may be ones that were heated too long. The cracks quickly disappear if the slide is reheated. Bottom line: Optically good though a bit tempermental Shelf life: Potentially good if the resin is mixed and handled optimally Good for general microscopy but not recommended for type material. Recommendation: Need to have serious quality control on slide making techniques as people try it. Let's keep each other informed! And John Kingston adds the following note: I am a bit embarrassed by one of the things I implied before, that we should perhaps use hoarded Hyrax for "important" collections. I think it is true in a case such as the one I mentioned with Camburn's types. But the "staying power" of mountants is a big issue for slides from ecological studies, paleoecological studies, etc. One of the major quality assurance problems we have today is huge numbers of uncurated slides, perhaps with inappropriate mounting media. If our work is to be reproducible, if we are to merge data sets and create super databases for many purposes, then the durability of mountants is important for any collection that will contribute to such databases. Melanie Reidinger wrote: has anyone had much success using Norland Optical Adhesive in mounting diatoms? We tried it a few times when I was back at OSU, and down at NKU. From what I remember, the stuff was much cheaper than HYRAX, UV light was used to make permanent slides, color of media wasn't a problem, but we did have trouble with air bubbles during preparation. Recent info from other colleagues is that NOA 60, NOA 63, and NOA 81 have a refractive index of 1.56; NOA 65 of 1.52, NOA 68 of 1.54. Address: Norland Optical Adhesive Norland Products INC. 695 Joyce Kilmer Ave. New Brunswick, N.J. 08902 GEne Stoermer commented: Optical adhesives, as the name suggests, are used to glue glass together. One of the desirable characteristics of such material is that they be the same refractive index as glass, thus invisible when applied to glass. Unfortunately, diatoms are near the same R.I., and also become invisible. Tom WHITMORE wrote to say: Yet another mounting medium. About 12 or 13 years ago, I used Styrax Mounting Medium for a period of time to mount diatoms from Florida lake sediments. I prepared the slides in the same manner that we prepare slides with Hyrax. The slides dried with a distinct orange tinge, but the optical quality of the medium seemed excellent. Looking back at those slides now I find them to be very suitable preparations. The slides appear to be in fine shape, so I suspect the archival potential of Styrax is quite good. What's more, the shelf life of this product seems unbelievable. I have a couple of bottles that may be relicts from Ed Deevey's lab at Yale, and they are obviously still usable. The question arises as to whether the stuff is obtainable any longer. The bottles I have are marked Styrax Mounting Medium, George T. Gurr, Searle Scientific Services, High Wycombe, Buckinghamshire. AN unknown correspondent writes: Great data on mounting media - thanx. One question. I've had good luck using a modified TSM (Karo syrup) with formalin and glutaraldehyde. Dried down the r efractive index seems fine for identifications. Had some slides for quite a wh ile without degrade - Jan Stevenson reported keeping some for a few years at le ast in that mountant. About 15 years ago I used Canada Balsam for permanent slides of Radiolaria sieved from deepsea sediments when I worked in a micropaleo lab. The refractive index was fine for seeing some ofeven the most poorly preserved specimens. It shouldwork for diatoms I would think. We thinned it with xylene and heated the slides to increase its ability to penetrate the tiniest pores. Very few bubbles when the volatiles had escaped and the balsam cooled. I have slides still in good shape. F.A.S. STERRENBERG wrote in October of 1995: Years ago, I experimented with realgar (arsenic trisulphide), which has an RI of >2.0 (!). I sublimated this onto the sample, dried on the cover-slip, a method that was used in the 19th century for Grayson rulings. The rulings were then "scratched" into the realgar. Needless to say that this MUST be done under a hood, and the synthesis of the realgar itself is not to be laughed at. Contrast is awesome, diatoms like Frustulia rhomboides var. saxonica become as easy as pie. Sublimation is very uneven, however, and the preparation looks pretty horrible. More recently, after looking at several 19th-century slides, I have come to the conclusion that the Grail of very high RI is not always really desirable. To check this, I have made some slides with Canada Balsam - about the worst thing you can imagine, but poor lambs like W. Smith had nothing else. I know it sounds ridiculous, but for heavily silicified diatoms like large marine Centrics or the hefty Pinnularias this is actually better than a high RI mountant, where fine detail may be lost in the pitch black shadows! Do we really need a very high RI nowadays? After having worked with museum slides of the 19th century mounted in Canada Balsam, I have become somewhat more tolerant. The type slide of "Gyrosigma spenceri" is a good example: in combination with phase-contrast or DIC the results are not at all bad - see Proc. Acad. Nat. Sc. Phil. Vol 146, Figs 1-6. We've lots of contrast in our optical gadgets now - formerly the mountant was the only option. If you like to tinker, make some preparations of delicate as well as robust diatoms and mount them "dry", in balsam and in a high RI mountant. Then compare the results with phase-contrast or DIC. One plea: NEVER EVER dry the sample on the slide, always on the cover. I have fought with many museum preparations where the sample was dried on the slide and then covered as a dry mount with the slip. These are hopeless, as the spherical correction of a high NA objective then no longer knows its head from its tail... GENE STOERMER then replied I think you are confounding two problems. The "pitch black shadows" are the more result of phase and (to a much lesser estent) DIC than they are of the refractive index of the mountant. All too often, people mistake enhanced contrast for increased resolution and, according to the optical laws as I understand them, you can't have it both ways. You may be able to enhance contrast in low refractive media, but there is no way to recover resolution. The other big advantage of using high RFI media is increased depth of field. I think it would really be great if we could find someone who really understands microscope optics (not the myths thereof) to write an article for one of the journals or home pages. Sadly, this is something not taught in modern universities, at least in this country, and it is a matter of total amazement to me how few people understand what allows decent resolution. If there is anyone out there with a good background in optical physics willing to write a short paper, I am sure several journal editors would be happy to receive it. No salesmen (excuse me, persons) need apply! STERRENBERG's lengthy and interesting response, mostly dealing with optics canbe foundas 'optics.dis' elsewhere on this Web page...