Tropical Parasitology

: 2016  |  Volume : 6  |  Issue : 1  |  Page : 86--88

Phenazopyridine as an innovative stain for permanent staining of trematodes

Mahdi Fakhar, Maryam Ghobaditara 
 Department of Parasitology, School of Medicine, Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran

Correspondence Address:
Mahdi Fakhar
Department of Parasitology, School of Medicine, Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari

How to cite this article:
Fakhar M, Ghobaditara M. Phenazopyridine as an innovative stain for permanent staining of trematodes.Trop Parasitol 2016;6:86-88

How to cite this URL:
Fakhar M, Ghobaditara M. Phenazopyridine as an innovative stain for permanent staining of trematodes. Trop Parasitol [serial online] 2016 [cited 2022 Jan 16 ];6:86-88
Available from:

Full Text


In this research report, an innovative stain for permanent staining of trematodes has been discussed, for the 1 st time. The techniques for staining of helminths are important for parasitologists to confirm identification and also teaching morphology of the helminths throughout student training courses. [1] There are numerous methods for staining of helminths by natural and synthetic dyes. These methods are essential for the permanent preparation of the specimens. [2] Before the era of hematoxylin and synthesized aniline dyes as histological stains, the method of choice to stain tissue sections was one of the natural dyes such as carmine and saffron. Carmine is obtained from the bodies (female) of the insect bug Dactylopius coccus cacti. [3] Today, carmine as a natural dye is used for identification of parasites, especially in taxonomic studies of worms. Although this dye stains the different internal and external structures of a worm, [2] it is expensive and time-consuming. Hence, to seek an inexpensive, rapid, more available stain, phenazopyridine (Php) has been used. In this report, the establishment of novel staining method for permanent staining of trematode has been discussed.

Php (3-phenyldiazenylpyridine-2,6-diamin) is a chemical (C 11 H 11 N 5 ), when excreted into the urine, has a local analgesic effect. It is often used to alleviate the pain, irritation, discomfort, or urgency caused by urinary tract infections, surgery, or injury to the urinary tract. The drug is administered as a tablet, in either 100 mg or 200 mg doses of Php hydrochloride. [4] The tablets have a light red, dark red, or dark violet color. Php is a type of Azo dye. [5] In addition to its generic form, Php is distributed under the following brand names: Azo-Standard ® , Baridium ® , Nefrecil ® , Phenazodine ® . [4] The Php has been used in our laboratory for adult trematode, for a long period. The present method is as an alternative simpler, faster with high resolution and more cost-effective and more available compared to other procedures particularly routine carmine staining. In the present method, internal and external structures of trematodes were stained well-defined. The present staining procedure has the following steps.

Before staining, place the live trematode (while held flat in petri-dish) in tap water at room temperature for 2 h to allow them to relax (relaxation); it causes trematode to emit eggs from their uteri. Then flood them slowly with hot (60°C) A.F.A solution (10 ml commercial formalin, 50 ml 95° ethanol, 5 ml glacial acetic acid, and 45 ml distilled water) and leave them for 2 h (fixation). For staining, grind one 200 mg Php tablet and mix with 5 ml glacial acetic acid and 5 ml distilled water and heat to 50°C for 2 min, cool, and filter. This solution was used as working solution, which can be kept at 4°C. Next, leave the worm in the solution for about 4 min in a small glass dish. Then, wash in tap water and followed by graded alcohol for dehydration, in alcohol 50% for 2 min, 70% (2 min), 85% (2 min), 96% (2 min), and 100% (2 min). Mount in Canada balsam or another mounting medium with cover glasses. While drying at 37°C, after several days, refill edges of the mount with fresh Canada balsam as needed. When dry, remove excess balsam and ring the edges [Figure 1] and [Figure 2]. The Php is available commercially in all pharmacies and drug stores as an inexpensive drug in all countries.{Figure 1}{Figure 2}

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Gillespie SH, Hawkey PM. Medical Parasitology: A Practical Approach. Oxford, UK: IRL Press; 1995.C
2Garcia LS. Diagnostic Medical Parasitology. 5 th ed. Washington: ASM Press; 2007.
3Dapson RW. The history, chemistry and modes of action of carmine and related dyes. Biotech Histochem 2007;82:173-87.
4Wang A, Nizran P, Malone MA, Riley T. Urinary tract infections. Prim Care 2013;40:687-706.
5Thomas BH, Whitehouse LW, Solomonraj G, Paul CJ. Excretion of phenazopyridine and its metabolites in the urine of humans, rats, mice, and guinea pigs. J Pharm Sci 1990;79:321-5.