One of the ways to study the morphology of minnow species is through bone and cartilage staining. Bone and cartilage staining is a method of dyeing the bone and cartilage tissue, that allow observation of bone and cartilage without dissecting the specimen. The study of forms and shapes gives information on the biological systems of a species. This is important since a number minnow species is widely being used as model organism in scientific research. By using alcian blue and alizarin red S chemicals, hard tissues of a teleost can be stained. The advantage of staining and clearing is that the skeletal structures and cartilage can be visualised in three-dimension, including minute skeletal elements. According to Connolly…show more content… Instead of using acetic acid, it is replaced with MgCl2 to be mixed with alcian blue. Concentration of MgCl2 is found to be adequate at 20-60 mM. However, it is unsure why a few studies later such as Helland (2009) and Darias et al. (2010) still use acetic acid to stain cartilage in their double staining procedure although it was shown that acid-free double staining produced better staining result compared to the low acid double staining. Nonetheless, this method is the only one that allows PCR genotyping of tissues after staining.
As described by Javidan & Schilling (2004), the full early cranial cartilages pattern of a zebrafish larvae is best labelled after 72 hpf i.e. 3 dpf., though alcian blue first labels the earliest differentiating chondrocytes at 54 hours of development. Meanwhile, alizarin red S first labels the earliest di differentiating bones at 3 to 4 days of development. Walker and Kimmel (2006) stained the the zebrafish larvae at 6.5 days post fertilization…show more content… The translucent flesh allows structures like since the ribs, spines, fin rays and starting point of vertebrae near the skull to be observed with naked eye.
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Figure 9 Indivudual of R.sarawakensis stained using Method 1. Bone stained dark purplish red, and cartilage tissues are more visible at caudal, dorsal, anal and pelvic fin. Skull part stained too dark. Spines were a bit deteriorated. Flesh is not quite translucent at thick part; (A) Full view of specimen. (B) Microscopic view of specimen posterior end. (C) Microscopic view of specimen anterior end.
The following shows the staining result of Method 2 (Helland, 2009) in lateral view of specimens. This method seemed to work better on P.kuchingensis.
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Figure 10 P.kuchingensis stained using Method 2. Bone stained purplish red or pink, cartilage blue very visible especially at fin. Although bones stained with alizarin red, vertebrae cannot be seen clearly since flesh is not quite translucent; (A) Full view of specimen. (B) Microscopic view of specimen posterior end. (C) Microscopic view of specimen anterior end.
