Dear Community,
Can you please help me by answering this basic question? I was confused with the answers I found on the internet. Some sources say that xylem stains red and phloem stains blue by TBO, whereas other sources, say completely opposite results.
Hello!!
With safranine, this staining pattern is true!
Although a professor of mine mentioned reverse colors in some scheme of buffers, pH, concentrations...
But, when just TBO Is used, phloem and primary walls stains blue, and xylem or secondary wall don't are stained, they remain translucent! It's very useful.
Best regards!
Hi Tibério Vale
Thank you for your answer. Why the lignified xylem doesn't stain at all in just TBO?
Do you mean that xylem stains red and phloem blue in safranin? Could you write a simple protocol for it?
Thank you!
Best regards,
Yulia
Hi @Yulia Steblyanko!
I am sorry, but talking with my professor, I was a bit wrong...
In reality, both xylem and phloem, secondary and primary walls, react with TBO. But yes, secondary walls become more light coloured.
Indeed, as you said, there is an inversion dependent on buffer, in that case the dark and light colour scheme is reversed
The explanation I would give would be wrong, it has something to do with lignin composition, but I will try to find a certain marvelous book that can help you with this!
Later I show It!
Or even the explanation, if I find this, and if nobody came with one already, haha
The protocol for TBO and safranine, I will confirm, my annotations are not good, and later I paste it here!
Sorry for the confusion!
Best regards,
Tibério
For the case you uses only Toluidine Blue O, you can prepare 0,05% mass/volume of TBO in 100mL of distilled water or phosphate buffer 0,1M. The vantage of the buffer is that you can fine tune the pH and choose one that offer the best colour differentiaton for your samples (people usually uses pHs between 4 and 8).
For the explanation of the staining at chemical level or additional info, the original paper may help:
O'BRIEN, TrP; FEDER, N.; MCCULLY, Mi E. Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma, v. 59, n. 2, p. 368-373, 1964.
For to obtain the red and blue staining, you have to use safranin after TBO, as follows:
*to prepare safranin, dissolve 1g of safranin in 100mL water (I would suggest to make just 10mL for the first try). It is as Luke et al. (1996), citing Kropp (1972)
Prepare 4 little glasses, one with a little of TBO, other with a little of safranin, and another 2 with a little of buffer or distilled water, depending on what you used to make the TBO. Place your sample in the TBO for maybe a minute; after, wash it in the water/buffer glass briefly; place the sample in the safranin glass for just some seconds (!!!) and them wash again in another glass of water/buffer.
Your sample must be already ready for microscopy!
References:
LUQUE, Rebeca; SOUSA, Hildeberto C. de; KRAUS, Jane Elizabeth. Métodos de coloração de Roeser (1972): modificado-e Kropp (1972) visando a substituição do azul de astra por azul de alcião 8GS ou 8GX. Acta Botanica Brasilica, v. 10, n. 2, p. 199-212, 1996.
Kropp, U. 1972. Leitbündel. Mikrokosmos 61: 342-345.
*oh, this book can be really useful, if you can find It: Johansen, D.A. 1940. Botanical microtechnique. New York: McGraw-Hill Book.
*In portuguese, there is
KRAUS, Jane Elizabeth; ARDUIN, Marcos. Manual básico de métodos em morfologia vegetal. 1997 , but I don't know if It is available on Internet or if It can be translated well enough
Best regards, and stay safe,
Tibério
Dear Tibério Vale
Thank you for such a complete answer! I like the protocol with safranin as it gives two differential colors. Regrading TBO in water, the staining indeed is variating and it depends on lignin-content as you mentioned, which means harder to predict in advance the exact color.
All the best,
Yulia
You are welcome, Yulia!!
Hope this help you in your project!
Good research!!
Best regards,
Tibério
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