From: PO2::"lhobson@SOL.UVIC.CA" "Louis Hobson" 27-APR-1995 11:58:04.86 To: Multiple recipients of list DIATOM-L CC: Subj: Lugol's solution I re-counted diatoms preserved in acid-iodine (pH=5.5) solution in seawater (pH=6.56 .5 after one year and found between 10 and 60% of frustules remaining. This was surprising to me; however there may be a report regarding this in the literature, which I have missed. I don't want to waste time writing a note for publication if a reference exists. Please, if any of you have information concerning this problem, I would appreciate receiving it. Thank you.u From: PO2::"stoermer@UMICH.EDU" "Gene Stoermer" 28-APR-1995 07:48:27.18 To: Multiple recipients of list DIATOM-L CC: Subj: Re: Lugol's solution I think you would be doing the world a favor by publishing your observations. The phenomenon you mention is well known to people that are very thoughtful phytoplankton work, but we still have people using Lugol's and, worse than that, writing nasty reviews for people that use other methods. In my experience, any counts of diatoms made on Lugol's preserved material that has been held for more than a couple of weeks should be viewed with caution. ***************************************************************** Eugene Stoermer, University of Michigan Internet e-mail address: stoermer@umich.edu Voice Phone: 313-764-5238 Fax: 313-747-2748 ***************************************************************** From: PO2::"root@inbum.sebastopol.ua" "Igor G. Polikarpov" 29-APR-1995 11:53:54.50 To: Multiple recipients of list DIATOM-L CC: Subj: Re: Lugol's solution (and others fixatives) I think that today is good time for analyze of the techniques for preservation for diatoms and other algae (protozoa as well). According to our experience (our laroratory), the Lugos fluid is acceptable for two (as Eugene Stoermer note) or four weeks maximum. It can be more acceptable for flagellates, and not good for coccolithophorids. For our data, the most acceptable fixative (for taxonomic work with diatoms frustules) is glutaraldehyde or formaldehyde (pH 7.0), 2-3 % concentration in sample. Igor Polikarpov -- ****************************************************************************** * Dr. Igor G. Polikarpov Tel: + 380 692 52-59-48 * * Head, Laboratory of Microplankton, Fax: + 380 692 59-28-13 * * Department of Plankton Fax: + 380 692 44-44-77 * * Institute of Biology of the Southern Seas Satellite FAX (INMARSAT) * * 2, Prospekt Nakhimova, 00 871 127 2122 * * Sevastopol 335011, e-mail: * * Crimea, UKRAINE root@inbum.sebastopol.ua * ****************************************************************************** From: PO2::"gfryxell@BONGO.CC.UTEXAS.EDU" "Greta A. Fryxell" 29-APR-1995 13:38:49.15 To: Multiple recipients of list DIATOM-L CC: Subj: Preservation of diatoms We have had problems in preserving diatoms with buffered formalin, as well, especially since we switched from glass storage bottles to plastic, to avoid breakage on cruise and during shipment. The first time we realized what was happening, a beginning student did phytoplankton counts to groups soon after the samples were returned to the lab, intending to get familiar with the groups and counting at least to the dominant species the next semester for his thesis. It was a year before he got back to it, and he couldn't believe what he saw. The centric diatom bloom from the Antarctic (that he himself had counted!) had turned into little bags of chloroplasts, when we were lucky, and little floating chloroplasts all too often. We interpreted this as dissolution of the silica and Jeff Johansen later did careful pH studies, which were not very conclusive. But we always used freshly-prepared buffered preservative according to his directives after that. Diatoms can take a pretty acid medium, but coccolithophorids, etc., cannot. We were trying to get the buffered preservative as near the pH of seawater as possible so we would get everything. I doubt it is really possible to preserve "for everything," and choices need to be made up front. I have tried adding silica to avoid dissolution, but while the theory is fine, precipitation in seawater is hard to avoid. I have fallen back on adding a couple cover slips to every sample (water or net haul), and it seems that the process is slowed down a bit. There is no valid reason that I know why clean cover slips would be more soluble than diatoms--unless it deals with the organic coating most diatoms have (before cleaning). We also use cover slips in samples preserved with glutaraldehyde--particularly nasty stuff. When samples have warmed up (as in coming across the equator), they don't look good at all. So we have used that only when we could keep the samples at room temperature or quite a bit cooler. But the cell contents look intact with glutaraldehyde. When I reach up on the shelf and take down good samples collected thirty years ago and stored in glass bottles, I still think some exchange is going on there! I would welcome a good paper on preservatives...Regards, Greta A. Fryxell Greta A. Fryxell, Department of Botany, University of Texas at Austin Austin, TX 78713-7640. FAX (512)471-3878 From: PO2::"stoermer@UMICH.EDU" "Gene Stoermer" 29-APR-1995 21:12:40.99 To: Multiple recipients of list DIATOM-L CC: Subj: Re: Lugol's solution (and others fixatives) Our experience is similar to Dr. Polikarpov. We generally use glutaraldehyde or glutaraldehyde-paraformaldehyde (Sicko-Goad and Lazinsky) fix. Long term storage is a problem - we continue to use ETOH. ***************************************************************** Eugene Stoermer, University of Michigan Internet e-mail address: stoermer@umich.edu Voice Phone: 313-764-5238 Fax: 313-747-2748 ***************************************************************** From: PO2::"maye0001@GOLD.TC.UMN.EDU" "Paul M Mayer" 1-MAY-1995 08:55:06.22 To: Multiple recipients of list DIATOM-L CC: Subj: ethyl alcolhol Colleagues: With reference to the recent discussions on the internet regarding the appropriateness of fixing/preserving diatom frustules with lugol's solution, is it advisable to put a few drops of 95% ethyl alcohol into vials of cleaned diatom material to prohibit fungal growth? If not alcohol, then should something else be used? Paul Mayer University of Minnesota Conservation Biology St. Paul, MN 55108 From: PO2::"pharg@GSOSUN1.GSO.URI.EDU" "Paul Hargraves" 1-MAY-1995 10:07:23.63 To: Multiple recipients of list DIATOM-L CC: Subj: Re: Lugol's solution Hello Lou ( and others who have preservation problems): Choice of preservatives is always a problem, since many of us want to look at other protists besides diatoms; and seawater imposes its own strictures on the choice. A little known reference is the following: Suzuki, M.T., A.M. Ciotti & C. Odebrecht. 1991. The effect of formaldehyde and iodine as fixatives for phytoplankton and protozooplankton samples from the southern Brazilian coast. Neritica, Curitiba, no.6(1-2):65-71. This paper compares neutralized formaldehyde with acetic iodine, but gives some discussion to glutaraldehyde, OsO4, Bouin's, as well as several pertinent references. We frequently preserve splits of tows with both form. & Lugol's, which is the recommendation of the Suzuki paper. Long-term, voucher samples are another problem. I'm starting a long-term look at the comparative value of several different preservatives in preserving diatom frustule ultrastructure, with & without Si added in different ways,in an artificial community made by mixing a bunch of clones. The results will be available sometime in 2006. Anyone out there doing anything similar? Paul Hargraves From: PO2::"pharg@gsosun1.gso.uri.edu" "Paul Hargraves" 1-MAY-1995 10:22:35.69 To: Multiple recipients of list DIATOM-L CC: Subj: more on preservatives After looking at some older (>20yrs) and more recent(<5yrs) of samples from various places: Glutaraldehyde sucks as a preservative in seawater, particularly if stored at room temp for more than a couple of weeks; you eventually end up with a brown gum. Refrigeration is better, but not for long term. Hexamine-buffered formalin looks O.K. for fairly robust pennates in a sample with lots of microparticulate sandy bits (re Greta F's note on adding coverslip-Si to samples) in a sample from 1972. Paul Hargraves From: PO2::"Hedy_Kling@fwi.dfo.ca" "Hedy Kling" 2-MAY-1995 13:26:25.78 To: Multiple recipients of list DIATOM-L CC: Subj: Re: Lugol's solution HI; The Lugol's formaldhyde works pretty well for freshwater samples. You must kill with lugol's and then add the formaldhyde 30min to a few days later. I find the flagellates are in better shape if you don't add the lugol's and formaldehyde together. If you wait too long to preserve with the formaldehyde the small siliceous scales of chrysophytes have already started to dissolve. It is also useful to make dried mounts and SEM preparations as soon as samples come into the lab. The you have a premanent record of what was in the sample when it we taken. These can be archived and used at a later date if it looks like dissolution is occuring. Even in the formaldehyde lugols preserved samples some dissolution appears after about 5 years in samples from low Si waters. I've looked back at whole water samples preserved in lugols in 1969. Formaldehyde was not added until 1972 after we learned that this was the best way to preserve lugols killed samples. There were no scales of mallomonas left and the Synedra and Asterionella present were showing signs of dissolution. Si content of the water of this lake was very low. Samples taken from an arctic lake in 1978, preserved using Lugols and then formaldehye had very nice perfectly fine Cyclotella bodanica, C. tripartita , and Stephanodiscus alpinus when examined in 1991 but the chrysophyte scales that were there in 1980 were no longer present. I also recently looked at net samples preserved only in formaldehyde. These had been taken by Dr. Kas Patalas for Zooplankton analysis. I dug them out to look at because I needed to check on some diatom taxa. These samples were in very good shape with no signs of dissolution. I could not compare them to the phytoplankton samples as they were lost due to the fact that theyn had been stored in vials with aluminum lined caps. The aluminum liner reacted very badly with the lugol's solution ruining the samples. We have archived phytoplankton samples from many lakes in central and arctic Canada take since 1969. Since 1986 I've been making slides and SEM preparation net and settled whole water samples as soon as they come in as well as archiving the preserved samples. Sincerely Hedy Kling On Mon, 1 May 1995, Paul Hargraves wrote: > Hello Lou ( and others who have preservation problems): > Choice of preservatives is always a problem, since many of us > want to look at other protists besides diatoms; and seawater imposes its own > strictures on the choice. A little known reference is the following: > Suzuki, M.T., A.M. Ciotti & C. Odebrecht. 1991. The effect of formaldehyde > and iodine as fixatives for phytoplankton and protozooplankton samples > from the southern Brazilian coast. Neritica, Curitiba, no.6(1-2):65-71. > This paper compares neutralized formaldehyde with acetic iodine, but gives > some discussion to glutaraldehyde, OsO4, Bouin's, as well as several pertinent > references. > We frequently preserve splits of tows with both form. & Lugol's, > which is the recommendation of the Suzuki paper. > Long-term, voucher samples are another problem. I'm starting a > long-term look at the comparative value of several different preservatives > in preserving diatom frustule ultrastructure, with & without Si added in > different ways,in an artificial community made by mixing a bunch of clones. > The results will be available sometime in 2006. Anyone out there doing > anything similar? > Paul Hargraves > From: PO2::"THERIOT@SAY.ACNATSCI.ORG" 3-MAY-1995 08:38:13.11 To: Multiple recipients of list DIATOM-L CC: Subj: preservation Following Paul Hargraves' recommendation of split samples, I would also suggest that a DRIED, uncleaned (unoxidized) sample be considered. I realize that a number of very flexible and highly 3-dimensional diatoms like Chaetoceros would suffer. But many, (and in freshwater - most) diatoms would come through just fine. Nature has already done the experiment on this one. It is called diatomaceous earth and is reliable for time periods up to and beyond 65 million years, as long as you keep the sample from being buried by 3 miles of dirt and out of contact of hot, molten lava. For those of you who want soft algae preserved, I ask "Why bother?" :) Diatomistically chauvinistically yours, Ed TheRiot From: PO2::"stoermer@UMICH.EDU" "Gene Stoermer" 3-MAY-1995 15:08:35.48 To: Multiple recipients of list DIATOM-L CC: Subj: Re: preservation I sure endorse Ed's suggestion of keeping dried material. It is even better if you freeze dry the stuff. Eliminates a lot of the problems of breakage and distortion that occur with air drying. Freeze substitution might even be better, but it isn't worth the bother for anything we have looked at. ***************************************************************** Eugene Stoermer, University of Michigan Internet e-mail address: stoermer@umich.edu Voice Phone: 313-764-5238 Fax: 313-747-2748 ***************************************************************** From: PO3::"stoermer@umich.edu" "Gene Stoermer" 4-MAY-1995 07:42:28.97 To: "P. Roger Sweets" CC: Subj: Re: Diatom preservation Regarding Roger's message, the breakage problem in clay rich sediments is one fo the reasons we like to freeze dry materials. I'm not the one to be talking about this, but the forces generated by surface tension of water drying around particles in the micrometer to hundreds of micrometer range are pretty impressive. Somebody that knows a little about soil physics might want to comment on this. ***************************************************************** Eugene Stoermer, University of Michigan Internet e-mail address: stoermer@umich.edu Voice Phone: 313-764-5238 Fax: 313-747-2748 ***************************************************************** From: PO2::"FOFW042@UNLVM.UNL.EDU" "susan jensen" 4-MAY-1995 14:02:02.69 To: Multiple recipients of list DIATOM-L CC: Subj: even more on preservation Dear fellow readers, I, too, have found your discussions on Lugol preservation extrememly interesting. I have done work on Charlotte Harbor (Florida) phytoplankton samples preserved in Lugol's (six years'worth of samples)and have found that even the delicate Chaeotceros and Rhizosolenia and Skeletonema have remained in identifyable shape after months of preservation. We do NOT add glacial acetic acid to our Lugols AND we keep the samples refrigerated - in a separate refrigerator because the iodine does seem to get out and discolor the refrigerator lining. Also interesting to note that Charlotte Harbor is estuarine and extremely well buffered. We do appreciate the info and we are currently monitoring our most recent samples for possible enumeration problems. Thanks, susan jensen From: PO2::"root@inbum.sebastopol.ua" "Igor G. Polikarpov" 5-MAY-1995 04:46:50.20 To: Multiple recipients of list DIATOM-L CC: Subj: Re: ethyl alcolhol Concerning ethyl alcohol: We used ethyl alcohol for fixing of benthic diatoms several years. Usually end ethanol concentration in samples was 20 %. We fixed not cleaned diatoms frustules, but living cells from tidal sand flats (from other hand, frustules is abundant component of sediments) and total sand samples. The perfect cells preservation in this solution let me possibility to recommend ethanol for storing diatoms (but not dinoflagellates etc). Igor Polikarpov -- ****************************************************************************** * Dr. Igor G. Polikarpov Tel: + 380 692 52-59-48 * * Head, Laboratory of Microplankton, Fax: + 380 692 59-28-13 * * Department of Plankton Fax: + 380 692 44-44-77 * * Institute of Biology of the Southern Seas Satellite FAX (INMARSAT) * * 2, Prospekt Nakhimova, 00 871 127 2122 * * Sevastopol 335011, e-mail: * * Crimea, UKRAINE root@inbum.sebastopol.ua * ****************************************************************************** From: PO2::"root@inbum.sebastopol.ua" "Igor G. Polikarpov" 5-MAY-1995 07:56:17.15 To: Multiple recipients of list DIATOM-L CC: Subj: Long-term storing experiments Paul Hargraves wrote: [skipped] > Long-term, voucher samples are another problem. I'm starting a >long-term look at the comparative value of several different preservatives >in preserving diatom frustule ultrastructure, with & without Si added in >different ways,in an artificial community made by mixing a bunch of clones. > The results will be available sometime in 2006. Anyone out there doing >anything similar? >Paul Hargraves Yes, my laboratory is developing similar long-term studies of different effects of several preservatives. We don't look at Si adding. The main aim is better understanding of species and groups composition preservation for whole phytoplanktonic communities (mainly diatoms, dinoflagellates, small nude flagellates etc). This work was started in 1995 and first results will be available in 2001. Igor Polikarpov -- ****************************************************************************** * Dr. Igor G. Polikarpov Tel: + 380 692 52-59-48 * * Head, Laboratory of Microplankton, Fax: + 380 692 59-28-13 * * Department of Plankton Fax: + 380 692 44-44-77 * * Institute of Biology of the Southern Seas Satellite FAX (INMARSAT) * * 2, Prospekt Nakhimova, 00 871 127 2122 * * Sevastopol 335011, e-mail: * * Crimea, UKRAINE root@inbum.sebastopol.ua * ****************************************************************************** From: PO2::"maye0001@GOLD.TC.UMN.EDU" "Paul M Mayer" 1-MAY-1995 08:55:06.22 To: Multiple recipients of list DIATOM-L CC: Subj: ethyl alcolhol Colleagues: With reference to the recent discussions on the internet regarding the appropriateness of fixing/preserving diatom frustules with lugol's solution, is it advisable to put a few drops of 95% ethyl alcohol into vials of cleaned diatom material to prohibit fungal growth? If not alcohol, then should something else be used? Paul Mayer University of Minnesota Conservation Biology St. Paul, MN 55108