Cybs' New 'MAX ION' tek

[sabbathin]

Good day. I want to clarify that I am not trying to sound like a smart ass in my first topic. If you were trying to lyse a cell by making it exploding with water in th medium, you would want to use a less concentrated ion medium than inside the cell. That's the principle of reverse osmosis water purification. So, maybe, you could try to use distilled water instead filtered water for the first step, and then add some MgSO4, or even concentrated (boiled for a long time) filtered water, in the rest of the procedure to push the alkaloids into the biphase. Peace .

 

[Infundibulum]

Good day. I want to clarify that I am not trying to sound like a smart ass in my first topic. If you were trying to lyse a cell by making it exploding with water in th medium, you would want to use a less concentrated ion medium than inside the cell. That's the principle of reverse osmosis water purification. So, maybe, you could try to use distilled water instead filtered water for the first step, and then add some MgSO4, or even concentrated (boiled for a long time) filtered water, in the rest of the procedure to push the alkaloids into the biphase. Peace .

Hi sabbathin,

Technically speaking, you are right in that a hypo-osmotic solution would lyse cells as you describe; in practise, this is true for mammalian cells only, as plant, fungal and bacterial cells have cell walls (on top of their cell membranes) that withstand such osmotic pressures. You'd need a plant cell devoid of its cell wall (i.e. a spheroplast) to lyse it by osmosis.

I do not think we know whether that acid cell lysis as described in many teks actually happens, it might though to variable degrees. What most certainly happens is diffusion of dmt from the cells in the aqueous medium by repeated acid boils and straining.

 

[sabbathin]

Thank you for the reply . In caryological karyotype-kind one can use acidic-alkali treatments in conjunction of hipotonic solutions to help lyse cells. Dhamer et al. (2011) used a diluted acidic solution (1N HCl) at 60 ºC per 8 mins to lyse root cells of several Mimosa species for the realization of cytological studies. So, i think the acidic lysis is definitely happening, with the conditions stablished by Cyb (lysis by freeze/thawing + acidic/basic medium) the use of hipotonic medium for the lysis step would be a plus. I think I'll be making an experiment to compare both, the use of distilled water in the first phase and filtered water .

 

[Infundibulum]

Thank you for the reply . In caryological karyotype-kind one can use acidic-alkali treatments in conjunction of hipotonic solutions to help lyse cells. Dhamer et al. (2011) used a diluted acidic solution (1N HCl) at 60 ºC per 8 mins to lyse root cells of several Mimosa species for the realization of cytological studies.

I do not think that this is exactly what they did in the quoted study...in fact you cannot take what this paper says to suggest that dilute acid and warm temperature lyses cell walls This is their methods below:

Somatic chromosome numbers were determined in root
tip cells. Seeds were scarified by a small cut in the testa and
germinated in petri dishes with moist filter paper. When the
roots were around 1 cm in length, they were pretreated
with a saturated solution of paradichlorobenzene for 24 h at 4
C, fixed in 6:3:1 (ethanol:chloroform:acetic acid) for
12–24 h and stored in 70% alcohol below 0
C until required. Slides were prepared by hydrolyzing the roots
with 1 N HCl at 60C for 8–10 min and staining with the
Feulgen technique for 2–3 h (sometimes followed by a 2%
pectinase treatment for 2 min) and squashed in propionic
carmine.

So, i think the acidic lysis is definitely happening, with the conditions stablished by Cyb (lysis by freeze/thawing + acidic/basic medium) the use of hipotonic medium for the lysis step would be a plus. I think I'll be making an experiment to compare both, the use of distilled water in the first phase and filtered water .

It is hard to imagine that no lysis is taking place; even grinding the bark will inadvertently break some cell walls. But all this talk about cell wall lysis is missing the point. we need to ask

1. do you have a method to assess how much cell lysis happens with one method vs another?
2. do you have evidence that efficient cell lysis is what contributes to better yields?

 

[sabbathin]

As for the procedure used in the paper. The Carnoy solution, (EtOH, cloroform, Acetic acid) is for fixation, para-hidroxichlorobenzene is used to stimulate karyokinesis, and the acid treatment is meant for lysing walls and membranes, everything else are tinctions. SWIM been there, donde that for quite a while. The method used to asess the amount of lysed cells is almost the same method than the one in the paper, check through lots and lots of slides under the microscope. But you are right,i got no evidence about number 2 so there's no point on arguing about it . Peace out, moving onto another topic :thumb_up:, thanks again for the reply.