Hi guys,
I'm doing my undergraduate research project on the bactericidal and fungicidal properties of essential oil of Thyme and Clove.
I have run into some issues with the 96-well plate microdilution method for the determination of minimum inhibitory concentration of the oils and I was hoping someone could shed me some light on what I might be doing wrong.
I've attached an excel file with the plate readings.
I prepared the plate by adding 50ug of Mueller Hinton broth and 50ug of S. Aureus with turbidity adjusted to McFarland Standard. (Row B to E, Row A contains only the Thyme Oil)
I started with 100 ug of thyme oil in row A - 1 to 9, and, pipetted 50ug into row B - 1 to 9, and continued this dilution to through row E.
I was expecting the readings to increase as I go from row B to E because the more diluted the oil is in the lower rows e.g row E, the more the bacteria would grow thus giving off higher reading. However, this was not the case, almost all of the readings were higher in row B (where the oil concentration was higher) than in row E and I have no idea as to why this is happening.
I few observations
I didn't dilute the thyme oil, however, the readings for the thyme oil itself, as observed in row A, doesn't seem to be what's trowing off the rest of the readings. The oil has a yellowish color.
The readings for the controls, as you can see in the attached excel file don't seem to be off either, so I really don't understand why the readings for the micro dilution (Row B to E) are so off.
Cheers
Marcus
Hi Marcus,
if I'm right you have no signal for your bacteria in medium (row G)? Was it turbid by looking by eye? If yes, did you measure at the correct wavelength?
To be sure about the cfu/mL you put in, I would propose to also make some dilutions of your input and spread them on MH-medium agarose plates to count if you have really the intended cell concentration.
Here is some really nice paper I used to set up my MIC assay. doi:10.1038/nprot.2007.521
Cheers,
Julia
Hi marcus,
I agreed with Julia that S. aureus was not growth, it's must be turbid.
Since EO do not dissolve in water, you need use some emulsification such as DMSO, Tween 80, Tween 20 ... and then dilute in water or in MHB.
in addition, you can dilute more thyme oil. Maybe these concentrations used was always higher than MIC so the bacteria not growth (as shown in your case).
Here is my
Do you mean µl where you wrote µg?
I don't understand your protocol. It sounds like you may have been diluting the bacteria at the same time you were diluting the thyme oil. That would explain the lack of growth in the later rows.
Also, if columns 1-9 are replicates, there is a lot of variation between them, so there is another problem that must be solved.
I suggest you dissolve the thyme oil in DMSO at and prepare serial dilutions with DMSO as the solvent at 50X the final concentrations in a separate plate. Add 2 µl of these solutions to the assay plate, followed by 100 µl of 1x bacteria suspension. Mix thoroughly and incubate.
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