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GrimmHatter 03-18-2011 02:43 AM

Question about simple cytoplasmic staining.
Doing some studying from Carson and Hladik's Histotechnology 3rd edition. So if anyone is familiar with this text, I'd would greatly appreciate some help. I believe 2nd edition is very similar as well.

On page 107, second column, Cytoplasmic Staining....there is FIGURE 6.1 which depicts the structural formula for a simple amino acid and the effect of its isoelectric point (pH 6). I'm confused why the amino acid is listed as "basic (+)" with a lower pH and "acidic (-)" at a higher pH. A simple definition of acid (Lewis or Bronstead-Lowry?) says that an acid is any substance that can donate a H+ ion. This seems to be the case for the amino acid at the lower pH, since the amino and carboxyl groups are both +, meaning it would very readily react with a base, which is a substance that accepts H+. So why the reverse of the classifications in the figure?

The next page (108, final paragraph) even says:

...below the IEP or below pH 6, the net charge on nonnuclear proteins is positive and the attraction is for anionic dyes.
In other words, dyes with a - charge, which, by definition, would readily accept H+, thus defining them as basic dyes. Continuing...

Above the IEP, the net charge is negative and the attraction is for cationic dyes.
Or dyes with a + charge (insert definition of acid here), thus making them acidic dyes.

Substances attracting basic dyes are "basophilic". Substances attracting acidic dyes are "acidophilic".
So, adding up what we've covered so far...basophils would be acids and acidophils would be bases. The book then uses Eosin as an example of a negative dye. Skipping down the paragraph, we have this:

Although eosin is negatively charged [...] if the pH of the dye solution is dropped too low, the -COO group of eosin will combine with hydrogen [I'm assuming from the acid that's been used to lower the pH] and the result is the free acid, uncharged form of eosin.
The fact that too low of a pH causes the negatively charged eosin's -COO group to react with H would tell us that eosin is a basic dye and the point on the tissue with the amino acids present are the acidic constituent that reacts with it (eosin).

So again, in FIGURE 6.1 on 107, why is the amino acid listed as "basic (+)" at the lower pH and "acidic (-)" at the higher pH?

Thanks for any help. Sorry I don't have a picture to post.

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