Dear Vinay Mohan Kandpal many factors might cause this change. First, have you checked the quality of culture medium components? After cooling, has culture medium gelled? Are you sure that 121 CG is the temperature used for autoclaving? Is it your first time preparing this formulation? If not, what were previous results? What about the pH?

Darkening of the medium is related to the burning of sucrose (caramelization), but I have never seen such a dark MS medium. Check the parameters of the autoclave, the concentration of each components and, as Ricardo Julian Licea-Moreno mentioned, quality of chemicals.

(NAA is auxin, BAP is cytokinin)

Browning the medium is related to too much temperature which cause burning the sucrose. But in your case it's too much brown. Maybe one of your chemicals does not have good quality

Vinay Mohan Kandpal

I don't think that is due to contamination. We have autoclaved many contaminated medium. They don't look like that. It is from charted medium component(s).

Did you check the pH?

Anyway, had this brown color only after autoclaving contaminated medium with plant pieces on them. Especially small twigs...

Some important parameters for preparation of medium for plant cell and tissue culture are: water should be double distilled to remove impurities, inorganic and organic compounds required must be AnalaR Grade; gelling agents be of high quality (example Didcot agar), vitamins and auxins added after autoclaving and cooling medium to 25C under asceptic conditions. These solutions are adjusted to the desired pH and then filter sterilized and then added to autoclaved-cooled medium under asceptic conditions. Flaw in any of these steps may result in mess you are facing.

Difco Bacter Agar

Before Autoclave, try a sample with normal boiling till your agar dissolves and let it cool down, and check the change in color of your media. If it shows change in color then you have to again recheck your composition, pH, and distill water used. If there is no change in color, then there may be a problem with your autoclave temperature settings.

There may be two main reasons, first is your quality of MS ingradients. Like many times in lab, most of using HiMedia ready made MS media and that may be expired. So first check quality of MS media and better prepare macroelements and micro elements stock solutions and they prepare working media from stocks. And check for sugar also. Second check for your autoclave settings. Most of time we using automatic pressure releasing autoclave, but due to disfunctional automatic timer, media autoclave for longer period. And that the reason of caramelisation of sucrose. Best luck

It is not sense that your media become so dark. This browness wil become for othes substances that your put into the media or you have been taken more time dor the sterilisation. Other reser sould be concerning the reciempts not so clean

or sucrose become so brown, maybe pH wouldo be wrong.

Treat to make another media very carefully as well time, temperature and pression.

Good luck

Well, according to eleven answers sending to you or experts, those recommendations are very good. Maybe you do not have enough experience in media preparation.

By the way, it is somebody in your laboratory that can help you? Otherwise, it is necessary to find basic bibliography and read very carefully what shoul be your mistake.

Good luck

Ricardo Julian Licea-Moreno Sir, the quality of the chemicals was satisfactory. No, the media didn't gel even after 2-3 days and was in an aqueous state with some charred precipitate at the bottom of the bottles. Sir, the autoclaving temperature was 1210 C and 15 atm for 15 min but the autoclave was preheated to some 550 C before use. the pH of the media before the addition of agar was 5.9

This was my first time preparing this media, after taking suggestions from here I again prepared the media and it turned out to be perfect. So I couldn't rectify my mistake about wrong stock preparation or incorrect composition or poor quality may be due to high temperature in autoclaving the browning may have occurred?

The new media prepared after autoclaving has been attached below. Thank you for your valuable suggestions!!

Monika Kamińska Ma'am, I have checked for the parameters mentioned above and I was able to get the desired results, thanks to your valuable advice. I have a little doubt about the use of the word Caramelization of sucrose. Can we call it charred media here? Is there any technical aspect related to it?

Regards,

Vinay

S. Wenkart Ma'am the media autoclaved was the M.S media and not a contaminated media to be disposed of. So there were no plant parts in there!

M. K. Razdan Sir, Thank you for the detailed precautions. After preparing the media again with proper precautions I was able to get the desired results.

Regards,

Vinay

Oinam Santosh Sir, Vinod Kumar Jangid Sir Thanks for pointing out these common mistakes while preparing media. I again prepared the media keeping them in mind and found the correct results. Thanks for the valuable advice!!

Regards,

Vinay

Perea Dallos Margarita Ma'am your idea about the long duration of sterilization of media could be right as I prepared the new media again keeping the mentioned points in mind and this time I was able to get the desired results, thanks to your motivating answers.

The pH of the media was 5.9 then and yes I'm new to wet lab techniques. I was able to learn so many things in this way.

Thank you, Ma'am.

Regards,

Vinay

Vinay,

I am very happy because, you have the solution with your media preparation.

Any mistake in your laboratory is gives worries to your research.

Finally your MS media looks very good. Congratulations

Well, your media after sterilization become brown, you have any mistake during your procces, maybe time of autocalving, because your sugar conditions are caramelized or maybe, you added another compounds to the media like pluoroglucinol.

I recommended to you to prepare again the media slowly and check step by step to be sure what is your mistake.

Good luck,

Vinay Mohan Kandpal, Dear, the only issue of browning of the medium is due to over heating. Just autoclave your medium in another good autoclave, you will understand the issue.

Concerning your browning media after autoclavation, the time and temperature of it because of sacarose. But maybe you added any other elements that could be turned your browning media like pluorogucinol or another component.

Check again and you will find the mistake.

Good luck,

Revisar los parametros del proceso de esterilizacion, temperatura, presión y tiempo, alguna otra opción, son los constituyentes del medio de cultivo que estén oxidados o caducados

Check again your media preparation, maybe you added another compound or you autoclave doen¨t work. Check again pH, time, temperature and pressure of your equipment.

Welcome,

you may have added another substance to the media that changed the color of the medium, or that the temperature of the autoclave was too high, which led to the caramelization of sucrose, or that Concentration of sucrose was high. It is preferable to re-prepare the media and check the ingredients.

Good luck

It’s the indication of breakdown of sucrose but this is too dark, check your autoclave parameters and chemicals also

There is diffent ways to understand the reason of your browing media:

- Should be sucrose, temperature of autoclave (very hingh), also if you add any other chemical in your media as Ploroglucinol, maltose.

Check and you will see.

Check all your chemical you added to the media, slowly you can find which salt can give your mistake.

I think the addition of sucrose and the high temperature resulted in the turning of the MS media to brownish colour. It happened because of oxidation of sucrose. Vinay Mohan Kandpal

Reference:-

Caramelization reaction is oxidation of carbohydrates or sugar resulting in the development of the brown colour and caramellic flavour when heated at high temperature. The temperature of reaction depends on the type of sugar present in the food