Chemicals for microscopy

Here are summaries and links to discussions for Frequently Asked Questions.

Moderators: Pau, rjlittlefield, ChrisR, Chris S.

Posts: 2634
Joined: Thu Sep 04, 2008 2:03 am
Location: southern New Brunswick, Canada

Chemicals for microscopy

Post by NikonUser »

There have been a few questions on this topic, some of these are useful; from:
http: //
(Update 10/7/2018: the above website no longer exists. See ... plies.html for an archived copy.)

ACETIC ACID, 1%. Mix 1ml glacial acetic acid with 99ml water.

ACID FUCHSIN, ACIDIFIED 0.25%. Mix 0.25g acid fuchsin, 1 ml conc. hydrochloric acid, and 100 ml distilled water.

AMARANTH, 0.1% IN CRICKET SALINE. Add 0.1g amaranth powder to 100 ml cricket saline solution. Amaranth is available from Sigma.

BALAMUTH'S pH 7 BUFFER. For frog rectal contents, especially opalinids. To 1500ml water add 7g sodium chloride (NaCl), 0.4g potassium chloride (KCl), 0.4g magnesium sulfate (MgSO 4•7H 2O), 0.1g potassium dihydrogen phosphate (KH 2PO 4), 0.1g sodium bicarbonate, and 0.2g calcium chloride (Wessenberg, H. 1961. Studies on the life cycle and morphogenesis of Opalina. Univ. California Publ. Zool. 61:315-370.)

BRINE For Artemia cultures. 40 g non-iodized salt per liter of chlorine-free fresh water. Use pond water, spring water, lake water, or aged tap water for this purpose. Tap water should be allowed to sit for at least a week (in a container that provides a large surface area) before use.

CARMINE/SEAWATER SUSPENSION, 0.1%. Mix well 1g powdered carmine powder with 1000ml seawater. Shake well before each use.

CHLOROFORM-SATURATED WATER. An anesthetic for aquatic organisms. Place 5 ml chloroform in 1000 ml water (seawater, pondwater, or tapwater, as appropriate) and shake vigorously. The chloroform will sink below the water after shaking. Use the water, not the chloroform, as needed.

CHLORETONE, 0.2%. Earthworm anesthetic. Dissolve 2g chloretone in 1000 ml pondwater. Chloretone is 1, 1, 1-trichloro-2-methyl-2-propanol, or chlorobutanol, and is available from Ward's Natural Science Establishment.

CHLORETONE, 5%. Anesthetic for freshwater metazoans. Dissolve 50g chloretone in 1000 ml water.

ETHANOL, 5%. An anesthetic for freshwater or marine invertebrates. Add 53ml 95% nondenatured ethyl alcohol to 957ml pondwater or seawater.

IODINE-POTASSIUM IODIDE . Mix 6g potassium iodide, 4g iodine in 100 ml water. Deep blue-black is positive test for starch. Flagella and cilia stain with this solution.

JANUS GREEN-NEUTRAL RED. Mitochondrial stain. 1g neutral red, 0.5g Janus green, 100ml 95% ethanol. Add stain to slide, allow to air dry. Add culture to dry stain on slide.

MAGNESIUM CHLORIDE, ISOTONIC WITH STANDARD SEAWATER. Dissolve 77g MgCL 2•6H 2O in 1000ml distilled water or chlorine-free tapwater.

METHYL CELLULOSE, 10%. To immobilize protozoa. To 10g methyl cellulose add 45 ml boiling water. Let stand 30 min at 10 ° C., add 45 ml cold water and stir until smooth. Bubbles will eventually disappear.
See ... 7&start=15
page 2 for more mixing instructoons

METHYLENE BLUE, 0.5%. Add 0.5 g methylene blue to 100ml water. For nuclei and other organelles.

METHYL GREEN, ACIDIFIED 1%. Excellent nuclear stain but kills cells. 1g methyl green, 1 ml 1% acetic acid, 100ml water. Methyl green without acid is a vital stain and does not kill cells.

NEUTRAL RED, 1%. 1g neutral red in 100ml water.

NIGROSIN, 10%. For flagellate and ciliate pellicle surface pattern. 10g nigrosin, 100g water. Add to preparation on slide and allow to air dry. Observe dry without coverslip.

PHLOROGLUCINOL-HYDROCHLORIC ACID. For lignin, as a test for the presence of wood. Prepare a saturated solution of phloroglucinol (1,3,5 trihydroxybenzene) in 20% hydrochloric acid. Place a drop or two on the slide, mix and add a coverslip. Lignin stains red-violet.

PHOSPHATE BUFFER, 0.01M, pH 6.8. Add 4.58g potassium phosphate and 4.67g sodium dihydrogen phosphate (NaH 2PO 4) to 1000ml water. This makes a 0.5M stock solution. Dilute one part of stock with 4 parts of water to make a 0.01M working solution.

SALINE, EARTHWORM 0.70%. Add 7.0g sodium chloride to 1000ml water.

SALINE, BLUE CRAB. Callinectes Ringer's Solution: 2.444g NaCl, 0.086g KCl, 0.142g CaCl 2, 0.166g MgCl 2•6H 2O per 100 ml water at pH 7.3-7.5 (Pyle & Cronin, 1950).

SALINE, 0.8-0.9% HOMIOTHERM. Add 8-9g, as desired, sodium chloride to 1000ml water.

SALINE, INSECT. Dissolve 9g sodium chloride (NaCl), 0.2g potassium chloride (KCl), 0.27g calcium chloride (CaCl 2•2H 2O) and 4g glucose. Adjust pH to 7.2 with sodium bicarbonate (NaHCO 3) Add enough distilled water to bring final volume to 1000ml.

SALINE, LEECH ISOTONIC (Hedon-Fleig's solution). To 100ml distilled water add 7g sodium chloride (NaCl), 0.3g potassium chloride (KCl), 0.1g calcium chloride CaCl 2), 1.5g sodium bicarbonate (NaHCO 3), and 0.3g magnesium sulfate MgSO 4.

SALINE, 0.5-0.7% POIKILOTHERM. Add 5-7g, as desired, sodium chloride to 1000ml water.

SALINE, WOODRING'S CRICKET. A physiological saline solution for crickets. Dissolve 9.1g sodium chloride (NaCl), 0.52g potassium chloride (KCl), 1.2g calcium chloride (CaCl 2•2H 2O), 0.82g magnesium chloride (MgCL 2•6H 2O) in 1000ml distilled water.

YEAST/CONGO RED SUSPENSION. Paramecium and rotifer food for visualizing feeding. Boil 10g dry yeast with 0.1g Congo red in 30 ml water for 10 min. Cool and store refrigerated until needed. The suspension can be kept frozen indefinitely.

YEAST SUSPENSION Daily Artemia food for maintaining cultures. 1 package baker's yeast suspended in 100 ml fresh water.

[Admin edit RJL, 10/7/2018, to provide URL of archived copy.]
student of entomology
Quote – Holmes on ‘Entomology’
” I suppose you are an entomologist ? “
” Not quite so ambitious as that, sir. I should like to put my eyes on the individual entitled to that name.
No man can be truly called an entomologist,
sir; the subject is too vast for any single human intelligence to grasp.”
Oliver Wendell Holmes, Sr
The Poet at the Breakfast Table.

Nikon camera, lenses and objectives
Olympus microscope and objectives

Posts: 42
Joined: Tue Oct 16, 2012 11:02 am
Location: Bozeman, MT

Post by pittendrigh »

Is CHLORETONE the same as Chorobutanol?

Site Admin
Posts: 21273
Joined: Tue Aug 01, 2006 8:34 am
Location: Richland, Washington State, USA

Post by rjlittlefield »

pittendrigh wrote:Is CHLORETONE the same as Chorobutanol?
"Chorobutanol" looks like a misspelling (missing el).

But NikonUser did write that "Chloretone is 1, 1, 1-trichloro-2-methyl-2-propanol, or chlorobutanol" (emphasis added). provides a more detailed explanation, including a long list of synonyms.


Posts: 1296
Joined: Sat Nov 21, 2009 2:41 pm
Location: Borgholm, Sweden

Post by enricosavazzi »

The link ... plies.html is dead (for me at least).

Site Admin
Posts: 21273
Joined: Tue Aug 01, 2006 8:34 am
Location: Richland, Washington State, USA

Post by rjlittlefield »

enricosavazzi wrote:The link ... plies.html is dead (for me at least).
It has apparently been gone from the web since early 2012.

However, a copy as of Aug 1, 2011, can be obtained from the Internet Archive: ... plies.html

I have edited NikonUser's posting to provide this info for casual readers.


Post Reply Previous topicNext topic