Dear Sir. Concerning your issue about the preparation of 10% buffered formalin or Trump’s fixative for polyclads fixation. For histology the standard preferred fixative is 10% neutral buffered formalin (NBF). To make a histological fixative for this we need a 10% solution** of this stock formalin i.e. 1 part of the stock formalin with 9 parts water, preferably distilled. This makes an unbuffered formalin solution, which will have a pH of 3-4. If used unbuffered the acidity can react with haemoglobin in the tissues to produce dark brown acid formaldehyde haematin precipitates, which complicate histological interpretation. To adjust the 10% formalin solution to a neutral pH you would need to mix in quantities of a buffer, typically sodium phosphate. A recommended recipe is as follows: 100ml Formalin (37-40% stock solution) 900ml Water 4g/L NaH2PO4 (monobasic) 6.5g/L Na2HPO4 (dibasic/anhydrous) 10% formalin can also be referred to as formal or formol. A common example is formal saline which is another method of fixative preparation using sodium chloride and sodium phosphate to buffer as follows: 100ml Formalin (37-40% stock solution), 900ml Water , 9g NaCl , 12g Na2HPO4 (dibasic/anhydrous). I think the following below links may help you in your analysis:

https://www.surrey.ac.uk/sites/default/files/Formalin-Fixative.pdf

 http://stainsfile.info/StainsFile/prepare/fix/fixatives/formalin-buffered.htm

http://www.ihcworld.com/_protocols/histology/fixatives.htm

Thanks

the following link may help you

http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/FORMALIN.PDF

Where only a standard stock solution of formalin* is available it is typically 37-40% formaldehyde (a gas) in aqueous solution and unbuffered. To make a histological fixative from this we need a 10% solution** of this stock formalin i.e. 1 part of the stock formalin with 9 parts water, preferably distilled. This makes an unbuffered formalin solution, which will have a pH of 3-4. If used unbuffered the acidity can react with haemoglobin in the tissues to produce dark brown acid formaldehyde haematin precipitates, which complicate histological interpretation. To adjust the 10% formalin solution to a neutral pH you would need to mix in quantities of a buffer, typically sodium phosphate. A recommended recipe is as follows: 100ml Formalin (37-40% stock solution) 900ml Water 4g/L NaH2PO4 (monobasic) 6.5g/L Na2HPO4 (dibasic/anhydrous) 10% formalin can also be referred to as formal or formol. A common example is formal saline which is another method of fixative preparation using sodium chloride and sodium phosphate to buffer as follows: 100ml Formalin (37-40% stock solution) 900ml Water 9g NaCl 12g Na2HPO4 (dibasic/anhydrous) * Formalin is formaldehyde gas dissolved in water and reaches saturation at 37-40% formaldehyde. This can therefore regarded as 100% formalin ** 10% formalin actually represents10% of the 37-40% stock solution. The actual amount of dissolved formaldehyde in the 10% formalin is therefore only 3.7-4.0%.

V1S1 = V2S2

formaldehyde mixed 1:10 in phosphate buffer (sodium hydrogen phosphate).

I've used seawater as a buffer with formaldehyde.

Formaldehyde mixed 1:10 in phosphate buffer.

Recipe:

https://www.surrey.ac.uk/sites/default/files/Formalin-Fixative.pdf

[LONG post]

Just for the records and in case of further readings and necessity for Information:

Just came across this thread - unfortunately it may / is too late for timely clarification of one part of the question by Alejandro Catalá Jimenez :

Since in the original question was asked also for "Trump's fixative" (its name has nothing in common with the former US-President with same surname....) also termed: Trump's '4CF-1G'-fixative (which is known to be a +/- stable fixative for Light Microscopy (Histology) and Electron Microscopical studies also for field studies out in the wild: human tissue, small and higher animals, plants, [aquatic] invertebrates, etc.).

(Quote) "McDowell’s andTrump’s 4F:1G is recommended as a primary fixative, both for light and electronmicroscopic samples. This fixative was originally developed for kidney perfusions, so it is a good choice for organ and whole-body perfusions (eg, for peripheral nerves, reproductive tissues, CNS, kidneys, and lungs).(end quote, cf: https://journals.sagepub.com/doi/pdf/10.1080/01926230290166823).

That particular fixative especially in histological as well as routine-TEM-applications has been recommended since it is known for its relative stability and effectivenness. Usually it consists of ["4CF-1G"] 4 parts 'CF' =commercial(ly available) formaldehyde and 1 part 'GA' (commercially aavailable glutaraldehyde), buffered either with appropriate (0.2, 0.1M) sodium-cacodylate buffer or even sodium-phosphate buffer. The ready to use solution also available from some suppliers (in liters, gallons) but can be produced easily by oneself too (using GLP-SOP's / recipes). For the interested reader some website URL's or links to related articles:

• Original publication: McDOWELL-TRUMP 1976: Comparative Study McDowell EM, Trump BF. Histologic fixatives suitable for diagnostic light and electron microscopy.

PMID: 60092. Arch Pathol Lab Med. 1976 Aug;100(8):405-14.

Histologic fixatives suitable for diagnostic light and electron microscopy

E M McDowell, B F Trump

· PMID: 60092 cf.: Article Histologic Fixatives Suitable for Diagnostic Light and Elect...

[ NB: delete '_x_' as inserted in between 'https:' and '// pubmed...' prior to use in your browser: https:_x_//pubmed.ncbi.nlm.nih.gov/60092/ ]

Abstract

The merits of formaldehyde, formaldehyde-glutaraldehyde combinations, and glutaraldehyde in phosphate buffers have been compared as fixatives that will give easy and satisfactory preservation of tissues for routine automated histologic processing and yet keep them suitable for electron microscopical studies after prolonged storage at room temperature. We recommend a combination of 4% commercial formaldehyde and 1% glutaraldehyde in a buffer of 176 mOsm/liter. Tissue sections should not exceed 3 mm in width, and tissues to be examined by electron microscopy should be taken from the outside of the tissue sections. All special stains performed at light microscopical level gave satisfactory results. The fixative should be stored at 4 C and is stable for at least three months. The superior cross-linking features of glutaraldehyde are retained, while the concentration of glutaraldehyde is low enough not to substantially obscure the PAS reaction.

• Recipe [© Cold Spring Harbor Protocols] Modified Trump’s universal fixative , Cf: http://cshprotocols.cshlp.org/content/2010/8/pdb.rec12288.full

(Quote):

• „Trump’s Fixative is an excellent fixative for tissues to be examined by electron microscopy. Cytoplasmic organelles are well fixed and distinct. Other subcellular components are visualized easily and with precision. Available in Gallon, Liter and Prefilled Jars.“ (end quote)

Cf: https://www.cancerdiagnostics.com/Trumps-Fixative

• Trump's Fixative, Electron Microscopy Science Supplier: Electron Microscopy Sciences ‚A combination of Sodium Cacodylate, Formalin, and Glutaraldehyde. Ready to use.‘ Cf: https://us.vwr.com/store/product/17336794/trump-s-fixative-electron-microscopy-science

Also see:

Copyright: Copyright © Microscopy Society of America 2000

Carson, F. (2000). Formaldehyde as a Fixative for Light and Electron Microscopy. Microscopy Today, 8(5), 30-31. doi:10.1017/S1551929500065238

PDF from Publisher: @ https://www.cambridge.org/core/journals/microscopy-today/article/formaldehyde-as-a-fixative-for-light-and-electron-microscopy/C2F354EA2BA94B28DF7199FD9C17C53D

Abstract

Since Blum discovered its hardening properties in 1893, formaldehyde has become the most widely used fixative in the world for specimens to be examined by light microscopy. However, since most commercial preparations of formaldehyde contain methanol, a protein precipitant, formaldehyde has been considered an unsatisfactory fixative for tissues to be examined by electron microscopy. In 1973, Carson et al. , described a parallel study comparing the electron microscopic results of fixation with paraformaidehyde vs. formaldehyde. They found that there was no difference in the preservation of ultrastructural morphology provided that the buffer systems were identical. In 1976, McDowell and Trump described a fixative combining commercial formaldehyde and glutaraldehyde (4CF-1G).Both of these fixatives are dual purpose fixatives and preclude the selection of tissue for electron microscopy prior to fixation. They can both be prepared in large quantities and used for routine surgical specimens. The fixative containing formaldehyde alone does not need to be refrigerated and is stable for months; whereas, the formaldehyde-glutaraldehyde mixture should be refrigerated.

(NB: can be found also (without full text at: Article Formaldehyde as a Fixative for Light and Electron Microscopy

Most outstanding SOP can be considered to be

https://journals.sagepub.com/doi/pdf/10.1080/01926230290166823

• Armamentarium Suggested Standard Operating Procedures (SOPs) for the Preparation of Electron Microscopy Samples for Toxicology/Pathology Studies in a GLP Environment

MICHAEL J. DYKSTRA, 1 PETER C. MANN, 2 MICHAEL R. ELWELL, 3 AND SHELLEY V. CHING4

1 Microbiology, Pathology and Parasitology Department, College of Veterinary Medicine, Raleigh, North Carolina 27606, USA

2 Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina 27709, USA

3 Pfizer Global Research and Development, Groton, Connecticut 06340, USA, and

4 SVC Associates, Inc., Apex, North Carolina 27502, USA

in: TOXICOLOGIC PATHOLOGY, vol 30, no 6, pp 735–743, 2002 Copyright (C) 2002 by the Society of Toxicologic Pathology DOI: 10.1080/0192623029016682 3

ABSTRACT We provide a set of Standard Operating Procedures (SOPs) for preparing samples for electron microscopic evaluation that allow storage of samples in the primary fixative for at least 17 years without noticeable degradation, do not compromise the ability to prepare the same samples for standard light microscopic evaluation, and provide tips for orientation of samples that may be necessary for evaluation. Guidelines for proper sample size, buffer composition, and fluid concentrations during processing are given. The impact of these procedures on specimen quality, ability to produce truly comparable samples for drug development studies, and ways to minimize time spent by technicians preparing these samples during necropsies is evaluated. Although many laboratories routinely employ most of these techniques, this compilation will facilitate the simultaneous light and electron microscopic preparation by the pathologist of comparable specimens that can be stored long-term at 4 C in McDowell’s and Trump’s 4F:1G fixative (4F:1G).

Keywords. Sample preparation; TEM; SEM; SOPs.

Cf: https://journals.sagepub.com/doi/pdf/10.1080/01926230290166823

Also:

Cf: https://www.researchgate.net/post/Which-is-the-storage-temperature-of-glutaraldehyde-grade-II-25-in-H2O:

the Question by Barbara Loi, IMC International Marine Centre, 24th Jan, 2019, Use of GA and FA for fixation of 'for fish larvae' and my answer on "4CF-1G" , Wolfgang H. Muss, dated: 24th Jan, 2019, with links (URL's) to articles with 'marine applications' and enclosed pdf's:

• 3.06 MB McDOWELL&TRUMP_1976_ fix_Why_4CF-1G Fixative works for me,Histo&!EM_by DYKSTRA_MJ_in_Micr.Today18_02(March,pp50-53)(works collection WHMuss10-04-23_for RG-19-01-24pdf).pdf
• 29.89 KB McDowell-Trump 4CF-1G(1976)_Karnovsky Fix_orig&half strength)_16%-FA fr.PFA_&osmolarities(Lab f.Adv.E&L-Optical Meths,ex web12-04-11_ex workscoll.WH.MUSS_for RG19-01-24pdf).pdf