Hello, I'm Dennis from TG01 and I'm currently in school to complete my Major Project within 10 weeks before I move on to do my SIP, reason being the hospital that I'm attached to didn't have MPs to assign us with. Let me give you a brief overview of my MP.
The aim of my MP is to identify compounds present in tea that possess anti-microbial properties against Staphylococcus aureus. This is achieved by using a whole cell-based assay to test for anti-microbial activity of tea, followed by using High Performance Liquid Chromatography (HPLC) to identify specific compounds.
The cell based-assay in this case is used to test the anti-microbial properties of tea against S. aureus at different concentrations. The assay is made up of a 96-well micotitre plate, consisting of positive control, negative control and as well as different concentrations of tea. Ampicillin is as a standard.
My MP is actually a continuation on the work of a previous student doing her MP last year. She has completed the optimisation of the whole cell-based assay. What I had to do what to carry out some replicate experiments to ensure there was reproducibility of data. After I have ascertained the results I've obtained is reproducible, I can carry on to testing different types of tea for anti-microbial properties. There are several parts to my MP, so I'll be only covering the steps that are involved to determine the IC50 (Inhibitory Concentraion @ 50%) of Ampicillin
Firstly, I had to obtain pure colonies of S. aureus, and this is done by using the streak plate method on Tryptic Soy Agar (TSA). After overnight incubation of 18hours @ 37 degrees celcius, I would then subculture a pure colony of S. aureus 10ml of Tryptic Soy Broth (TSB). Again, it is incubated overnight for 18hours, at 37 degrees celcius and shaking at 150rpm.
Next, I would take 1ml of the culture and subculture it into 49ml of TSB (1:50 dilution). I would then allow the cells to grow to the log phase at the 3rd hour. The log phase has been determined by doing some experiments prior to this, whereby an absorbance curve is plotted using data obtained from the spectrophotometer.
After incubation of 3 hours, I would then use a hemocytometer to obtain the cell density of the culture. The culture was then diluted to obtain a final cell density of 1 X 10^6 cfu/ml. After the final cell density is obtained, the assay can then be set up. The assay consist of the following:
Postive Control: 40ul of cells + 10ul of Ampicillin (10ug/ml)
Negative Control: 40ul of cells + 10ul of autoclaved water
Data points: 40ul of cells + 10ul of Ampicillin (at various concentrations)
The whole microtitre plate incubate overnight at 37 degrees celcius. The results are read the next day using the luminescence method. This is somewhat similar to what we doing in the laboratory using the spectrophotometer. The difference is that the spectrophotometer measures turbity, while in this case, the luminescence is measure. A dye called resazurin is added into the individual wells. Resazurin is an oxidation-reduction indicator which changes from non-fluorescent blue to fluorescent pink by oxidoreductase within living bacteria. Simply put, if there is the presence of S. aureus in a particular well, resazurin would change from blue to pink. If there is absence of S. aureus in a particular well, resazurin remains blue.
The luminescence produced by resazurin is read using a machine which measures the amount of luminescence produced. The data is then plotted into a graph, and the IC50 of Ampicillin can be determined. The IC50 of Ampicillin is the concetration of ampicillin which inhibits 50% of S. aureus, and is useful when used to compare the IC50 of the different types of tea.
Please feel free to ask me questions, I'll do my best to answer them as soon as possible. Thank you.
Regards,
Zhu Zhijie Dennis
0700847G
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10 comments:
Hey Dennis,
Just for curiosity, what are the teas that you are testing for? :)
Rachel Gan :)
Hi Dennis,
just curious about the part where the machine reads the luminescence. You mention that resazurin is converted from blue to pink by oxioreductase within living bacterial right. Does this mean that any living bacterial present will cause the colour change? Is there any chance that the colour change is not caused by S. aureus but something else? Thanks!
Hui Juan
0702012F
Hi! Dennis~
Let's say the IC50 is x, and a tea that you test on has a IC50 more than x. Does that mean the tea has a better antimicrobial effect??
Thanks!!
eriko (0700477C)
Dennis,
You mentioned about counting using the hemocytometer? How do you count microorganisms using the hemocytometer?
Is it like counting cells?
Staphylococcus aureus appears as clusters. So how do you derive the exact cell density? By counting a cluster as 1?
You mentioned that the resazurin is converted from blue to pink by oxioreductase within living bacterial. However isn't it colourimetry instead of luminescence?
Li Yinliang Alex
TG02 0704894E
Group 8
28 July 2009
Dennis,
is it luminescence or fluorescence?
Sorry guys for the really late reply! I'll try my very best to answer any questions you all might have. (:
Rachel: I would be testing four main types of teas, namely black, white, green and oolong tea.
Hui Juan: Yes, there might be chances that another bacteria might have caused the colour change, in the case we say that the assay might be contaminated. To lower the chances of contamination, it is important to adhere to strict aseptic techniques(like we learnt during MCT), such as working in a laminar flow hood when pipetting the tea extracts into the microtitre plate. Also, the assay has to have duplicates, so that the results obtained can be compared to know that the colour change is indeed due to S. aureus, and not by another bacteria due to contamination.
Eriko: Yes, if the IC50 of the tea extract tested is greater than X, it is true that the tea has better antimicrobial effect that ampicillin, which is used as a standard antimicrobial in the assay.
Alex Li: Yes, the hemocytometer is used to determine the cell density of S. aureus just like how we count cells.(It's the same concept as counting cells when we did MCT previously)
It is true that S.aureus appears in clusters, but after the addition of tryphan blue to S.aureus before reading it under a hemocytometer, most of the S.aureus would appear as single cells. However, there would inevitably be clusters. What I would do is that I would try my best to make out how many cells are there in that particular cluster. Of course this would lead to inaccuracies, and to lower the accuracy, I would actually count about an average of 16 squares and get the average of it. In this way, the inaccuracy would be minimized, and the cell density of S. aureus can be better determined.
I'm sorry but it seems that I have a typo error. It should be fluorescence and not luminescence. The fluorescence of resazurin is read by the fluorescence machine at a certain wavelength.
Dr. Alex Lee: Yes sir, thank you for pointing out my error. It should be fluorescence instead of luminescence.
Regards,
Zhu Zhijie Dennis
0700847G
what kind of machine do you use to measure fluorescence? Is it something like the spectrophotometer which measures absorbance? How does it measure the fluorescence?
Sorry about my earlier deleted comment, i couldn't edit what i wanted to ask.
Hello Dennis,
What is the definition of IC50? Using the definition, you may want to re-answer Eriko's question.
:)
Dennis,
Where is your response since August 6? Your posting has been disregarded.
Post again!
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