Hello.
I am studying on the vacuole development of M. oryzae.
I first used neutral red dye. I diluted the dye in phosphate buffer pH 7.4
It stained vacuole well, but It does not work well after several months.
I stored the dye in a refrigerator ( 4℃ ).
Is this too long to store the dye?
Do I need to make fresh dye solution?
Thank you for your attention.
I have looked through a few safety data sheets and Neutral red is supposed to be stable under recommended storage conditions, but at pH higher than recommended (6.8 to below 8.0 - when it is red) it changes colour, and becomes yellow (one of its uses is as a pH indicator) – so check the pH of your solution. If that is not the case – the simplest course of action would be, as you said, preparing a new solution. The solution is light sensitive, and it should be stored at 4 deg C, so using a dark glass bottle is recommended. Obtaining a per-prepared solution (100 ml is not expensive) from a well-established supplier, to check how such solution behaves after long storage, would be an experiment in itself. You need to observe safety measures as it is a suspected mutagen. Good luck with your project.
Title: Neutral Red
CAS Registry Number: 553-24-2; 366-13-2 (free base)
CAS Name: N8,N8,3-Trimethyl-2,8-phenazinediamine monohydrochloride
Additional Names: C.I. Basic Red 5; 3-amino-7-dimethylamino-2-methylphenazine hydrochloride; toluylene red; neutral red chloride; nuclear fast red (basic dye); aminodimethylaminotoluaminozine hydrochloride; C.I. 50040
Trademarks: Kernechtrot; Michrome no. 226
Molecular Formula: C15H17ClN4
Molecular Weight: 288.78
Percent Composition: C 62.39%, H 5.93%, Cl 12.28%, N 19.40%
Literature References: Prepn from N,N-dimethyl-p-nitrosoaniline hydrochloride + toluene-2,4-diamine: Witt, Ber. 12, 931 (1879); DE 15272 (1880), Frdl. 1, 274; Bernthsen, Schweitzer, Ann. 236, 332 (1886); Pokorny, J. Soc. Dyers Colour. 42, 347 (1926). Toxicity data: F. Stolarsky, T. J. Haley, Fed. Proc. 10, 337 (1951). Brief review: H. J. Conn's Biological Stains, R. D. Lillie, Ed. (Williams & Wilkins, Baltimore, 9th ed., 1977) pp 377-380, 596. See also: Colour Index vol. 4 (3rd ed., 1971) p 4446.
Properties: Dark green powder. Absorption max (50% alc): 533 nm. pKa 6.7. Soluble in water or alcohol with red color. Soly in water 4.0%, in abs alcohol 1.8%, in Cellosolve 3.75%, in ethylene glycol 3.0%. Practically insol in xylene. LD50 in mice, rats, rabbits (mg/kg): 142, 112, 97 i.v. (Stolarsky, Haley).
pKa: pKa 6.7
Absorption maximum: Absorption max (50% alc): 533 nm
Toxicity data: LD50 in mice, rats, rabbits (mg/kg): 142, 112, 97 i.v. (Stolarsky, Haley)
NOTE: Not to be confused with Nuclear Fast Red (acid dye) which is C.I. 60760.
Use: As indicator (0.1% in 60% alcohol) for alkalinity of water, urea, nitrite, etc. pH: 6.8 red; 8.0 yellow. Also used for preparing neutral-red paper. As biological stain (study of the Golgi apparatus in cells).
According to the answer of Maria, we (The answerers) need some infos about youir neutral red and your staining. I think you stain the M. Oryzae in vivo and not in VITRO! Please infos of your staining modus and microscopy.
Concerning the vacuoles please read the review :
18. Klionsky, D. J., P. K. Herman, and S. D. Emr. 1990. The fungal vacuole: composition, function, and biogenesis. Microbiol. Rev. 54:266-292. [PMC free article] [PubMed]-
you write " I diluted the dye...". Please give the concentration of it in the buffer. Have you found the pKa value of neutral red in your research? If not , it is 6,8 (siehe above the following infos and the answer of Maria).
Good luck
JRG
Neutral Red solutions have a short shelf life and should ideally be prepared fresh every time is needed and used during no more than one week. The powder, on the other hand, if kept out of direct sun light and on a tightly closed bottle, can last for years.
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