Hello all, its been 5 since SIP/MP started and it was great seeing you all on friday again. This week it is my turn to share my SIP/MP experience to you all.
I am attached to a plant biotechnology lab, and under the TSO, I help in making plant media. I make two kinds of plant media. One has activated charcoal in it and one does not. The steps to making the plant media are similar, therefore I shall explain one, and list the constituents of the other.
Preperation of tissue plant media
1.) Fill up two 3Litre beakers to over 2.5L with MilliQ water.*
2.) Place the beakers on seperate magnetic stirrers.
3.) Add the constituents in the following order after weighing them/determining their volume in each beaker. (MS media formulation (Murashige and Skoog)-6.45g; Myo-30ml; Organic elements-30ml sucrose 60g).*
4.) Top up the water until both beakers are 3L full.
5.) Next the pH is adjusted to a range of 5.2 to 5.21 using the pH meter
6.) Activated charcoal(1.5g) is added next and allowed to be distributed in the media.**
7.) Next 30 vitro vents(containers that will carry the media) are prepared.
8.) 1.6grams of agar powder are then poured into each vitro vent.
9.) Next a marked measuring cylinder specially used to measure for the volume needed for each vitro vent is used and the media is poured to fill up all the 30 vitro vents.
10.) The vitro vents are then covered with a cover and autoclaved.
Constituents for second set of plant media
1.) MS 6.45g
2.) Myo 30ml
3.) Organic Elements
4.) Sucrose 30g
5.) pH 5.2-5.21
30 vitro vents
1.6g agar
*Avoid parallax error by placing the eye onthe same level as the meniscus and read the bottom of the meniscus
**The activated charcoal does not dissolve into the media, therefore it must be thoroughly in the media before it is poured into the vitro vents. And after half the beaker is emptied, place the beaker back on the magnetic stirrer to redistrubute all the activated charcoal that has settled down.
I hope my explanation is clear and easy to understand. If there are any questions, please feel free to ask me. Thanks for taking the time to go through this post.
Johan
0606637G
TG01
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11 comments:
Hey Johan,
Ask u arh, what is the reason for useing activated charcoal for one of the plant media? And how come u need to prepare one with activated charcoal and the other one dun need.?
Thanks.
Zhenling =)
TG02
Hey
So is plant media like those DMEM/RPMI we use to maintain mammalian cells? What is plant media actually for?
And does it need to be prepared under stringent sterile conditions?
One last one, does activated charcoal media provide some sort of extra nutritional effect to the plant cells?
Thanks
Glad
Yo Johan!
I have prepared the tissue plant media before during my DRP. HaHa...! Thanks for refreshing my memories. Btw, I have some questions to ask you:
1)What type(s) of plant can your tissue plant media support?
2)"(MS media formulation (Murashige and Skoog)-6.45g; Myo-30ml; Organic elements-30ml sucrose 60g)."
-What are MS and Myo?
-What are the principles of adding these constituents?
3)Why must the pH be acidic? If the pH is alkaline, how can it affect the growth of plant?
4)Can the constituents of the media be added in any manner? If not, why?
Ok, I think that is all. =) Enjoy your SIP..!
Thankz!
Han Yang
TG01
Hi Johan,
Can you show me a picture of how the invitro vents looks like with the media in it? Will the agar look greyish because of the charcoal?
Why must the plants be grown on the agar? Got any advantages?
Is the agar very soft?
For the preparation of the media is there any reason why u use milliQ water? why not DI water?
Thanks alot
Jean Leong
TG02
hi johan
what reagents do you use to adjust the pH range?
Thanks
Justina
0605950E
TG01
Hi..
after autoclaving, is the media considered ready for the seeds to be planted? so can you water the plants?or you dun have to? i am really curious..how can a plant grow on agar..so the medium acts like the soil issit?are the constituents similar to the soil?
pardon me for the silly questns heh
thanks =)
Nur Farhana
0604834B
Hi Johan
Heya glad u r enjoying ur SIP. I got a couple of questions to ask you
1) Can you explin the purpose of adding activated charcoal to the plant media?
2) Why is the pH of the media acidic? What types of plants specifically u will be growing using this media?
Thanks!! c u tml
From: Benjamin Ma
Class: tg01
0606181F
Hi johan,
upon further reading your post, some doubts started creeping into my mind. I find your post brief but not entirely clear. Hence i have further questions to ask you and hope u wont find it an hassle... It will also help in the understanding of your other peers
1) What do u mean by 'it must be thoroughly in the media'?
2) Why must the beaker be half emptied?
3) Can you explain the materials and methods used in using the pH meter?
4) What are the organic elements in media and their significance?
5)You mean you do not know what plants you will be growing with your plant media?
6)What agar powder u will be using? TSA? LB? plz clarify
7)What aseptic techniques you will be using to ensure no contamination of your plant media?
8)What are the steps to use the autoclave? Andika still haven thought us how to use the autoclave....
9)Why use 30 Vitro vents? and what will your plant media be used for (experiment used by students?)
10) What aspects of mamalian cell technology and microbiologycan you learnt from making the plant media?
Thanks for answering the questions and enhancing our knowledge of your SIP.
From: Benjamin Ma
Class: tg01
0606181F
hello!
i have some similar questions from the rest of them.
what is plant media? what is it used for? how do you grow those plants and how is the plant going to grow in media? i cant imagine it unless its algae.
Thanks.
Yu Mei
TG01
Hi everyone, sorry for the late reply. I needed to look for answers. Here are most of the answer. The are still others that I have to confirm with my TSO.
Q by Zhenling: Activated charcoal is used for these reasons: absorption of inhibitory compounds, absorption of growth regulators from culture media or darkeining of medium.
As activated charcoal is not always needed, therefore 2 different types of media are prepared.
Q by Gladys: Yes, it is the same in the sense that it used to grow and support plants/plant tissue, but it is not a specialised media, just a general media that proveides nutrients for growth. It does not need to be prepared in stringent conditions, but has to be autoclave after completed. AC stimulates cell growth by binding to toxic phenolic compound produced during culture.
Q by Han Yang
Myo is myo-inositol, it is a carbohydrate and us hown to stimulate cell gowth. MS is Murashige and Skoog basal salt formulation, it contains macronutrients, 6 major elements for cell growth.I am not sure why it has to be acidic, but it has to do with the solubility and avaliability of ions in the nutrient media, the ability of agar to gel and subsequent growth of cells.Yes it can be addded in any manner.
Q by Jean: Yes it will look greyish. It contain all the nutrients needed for plant tissue growth in the adequate amount of time, and it is easy to store and carry around. It is as hard as normal agar. It is much more sterile than D.I, water.
Q by Justina: 0.1M NaOH and/or 0.1M HCL
Q by Farhana: Yes, it is ready to be planted. The plants still have to be watered (leaves) to help retain moisture. I am sorry but i cannot show u a picture. Yes it acts like soil and provides a lot of nutrients needed.
Q by Ben
Activated charcoal is used for these reasons: absorption of inhibitory compounds, absorption of growth regulators from culture media or darkeining of medium. I am not sure why it has to be acidic, but it has to do with the solubility and avaliability of ions in the nutrient media, the ability of agar to gel and subsequent growth of cells.
Thanx for reading my post =)
Johan
TG01
More answers =)
Q by ben: throughly means it must be distributed throughly as it does not dissolve. Cos it needs to be poured, then when its halfway empty, the charcoal would have settled. Organic elements are essential amino acids like L-glutamine, L-asparagine and adenine. It is used to stimulate cell growth. Yes, I do not grow the plants. Wearing of gloves and autoclaving. Put the plant media which are in the vitro vents inside the autoclave. Check to see if its 121 degrees celcius and if the time is 15 mins. Make sure the base has water filled to the surface. Close the lid, lock it and press start. 30 vitro vents is enough to contain 2 beakers of 3 litre media.
Q by Yumei
It is media used to support growth of plant/plant tissue.
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