For my MP, I have started on doing my 'control' assay on the 96-well microtitre plate. This experiment is to determine the concentration of cells to be use in future experiments.
Yellow section(Row A-D): Positive control
Red section(Row E & F): Negative control(blank)
Blue section(Row G): Negative control(with 100ug/ml of ampicillin)
Green section(Row H): Negative control(with 200ug/ml of ampicillin)
Methods:
1. After taking the OD reading(till the bacteria cells reached the log phase), dilute the cells to get
the concentration of cells needed.
2. Using a multichannel pipette and micropipette,
For Row A-F, add 10ul of autoclave water into each well.
For Row G, add 10 ul of 100ug/ml of ampicillin into each well.
For Row H, add 10ul of 200ug/ml of ampicillin into each well.
For Row A-D and Row G &H, column 1-3, 4-6, 7-9, 10-12, add different concentration of
bacteria cells into the well.
For Row E & F, add fresh TSB as this will be the blank control of the assay
3. After adding all the solution into the wells, incubate the microtitre plate at 37oC overnight.
4. Next day, prepare Resazurin which is a redox active dye.
5, Add 10ul of Resazurin dye into all the wells and incubate it.
6. At 1 hour interval, take the fluorescence reading.
7. After 5 hours, all datas collected are to be sort and plot graphs using those data.
8. From the graph, determine which concentration of cells can give a better sensitivity.
9. In future experiment, the specific concentration of cells will be use.
In the real experiment, the 10ul of autoclave water will be replace by tea extracts.
As for my SIP, other then data entry, I also help to prepare acidulants by measuring 0.6g of citric acid, lime juice, vinegar and tartaric acid, and add 2L of DI water into it, then mix them. Other then helping to prepare media, I also help to check some items in the lab making sure all items are in place. From doing this, I realised how tiring the work of the TSO can be, so make sure the next time you use the lab, put all items back to their original place if not TSO will have a hard time looking for them.
Justina
0605950E
TG01
11 comments:
Hi justina!
ok here's my qn. Why is there so many types of negative control? what's the negative control for?
Thanx!=D
Sharon
Tg01
Hi Justina!
Why are tea extracts used in the experiment? What purpose do they serve?
Thanks ya!
See you soon
Ying Chee
TG01
Hi Justina!
Just wondering..
what's the optimal cell concentration that future experiments requires?
Tan Zhao Rong
Tg01
hello..
wat is a redox active dye?
sutiana
To Sharon:
As this is my control assay, I use different negative control(i.e. blank and different concentration of ampicillin) to see if their reaction is similar. If the results are similar, in future experiments I can just use the blank negative control. Don't waste resources.
To Ying:
Tea extracts use as my project is on tea. Haha.. hmmm.. tea extracts are use instead of the whole tea leaves as the extracts contain most of the chemical substances such as the polyphenols, EGCG etc.
To Zhao:
Well I am still finding out what concentration of cells serve the best reaction. So will answer you when I get the result.
To Sutiana:
Resazurin is a redox active dye. Redox active dye is a oxidation-reduction dye. For Resazurin it is oxidize blue colour at the start, when it is reduce, the colour will turn pink. This dye is use to measure cell proliferation, viability and cytotoxicity.
Hope this help!!
Justina
Hi Justina,
juz a few questions... ...
Juz to clarify, the x-axis and y-axis of the graph are cell concentration and fluorescence readings respectively rite?
U mention "determine which concentration of cells can give a better sensitivity", what u mean by "better sensitivity"? Does sensitivity means sensitive to ampicillin?
Ermm.....from ur post, u mean row A to D only has the bacteria cells (S. aureus rite?) and autoclaved water? So the baterial cells can grow in autoclaved water only? no need nutrients?
Thanks
Lim Xin Ni
Group 9
TG02
Hi Justina
I would also like to see how the graph looks like...if it's not too much to ask for, could you include a simple illustration of how a graph looks like for your next post? It will be great!
Two questions I am rather curious about:
1) Why is there a need to discover the sensitivity by varying the cell concentration first? Can't you just use any amount of cells?
2) Why is there a need to use two negative controls with different ampicillin concentrations for your current experiment?
Hope I did not cause you any inconvenience.
Thank you very much!
Many thanks
Quan Jun
TG02
Group 08
28 August 2008
heys justina,
roughly how long will each control assay take?
U have 3 rows of positive control for different cell concentrations, is to test all the possible range of cell concentration u mentioned during ur first post, all at one shot izit?
Thanks
Jean Leong
Hi xinni:
There will be different graph for different cell concentration. For each graph, the x-axis and y-axis are the time(hour) and the reading respectively.
Better sensitivity meaning which cell concentration can produce better result.
Yup for row A-D only S.aureus and autoclave water. Cells will still be able to grow and nutrients need not be added.
Hi quan jun:
Ok i will include a graph in my next post.
1) So as to see which cell concentration can give a better result. Not all cell concentration can give a good result and another reason is that a known cell conc is needed so that there will be consistency in the future experiments.
2) There are 3 different negative controls. The blank act as the 'control' and 2 different ampicillin conc is use to determine if they will get the same result as the 'blank'
Hi jean:
If you mean the duration of the whole process for control assay, from the beginning till data analysis will take 3-4 days.
Actually there are 4 rows. I divide them into 4 columns so that different cell conc can be added and tested at one shot. (If I get you correctly)
Post a Comment