The overall goal of this procedure is to form candida albicans biofilms, and examine their susceptibility against antifungal agents. This is accomplished by first forming these fungal biofilms on the wells of 96. Well microtiter plates then antifungals are added to the preformed biofilms and incubated for 24 to 48 hours following this incubation.
A chole metric method that measures metabolic activity of cells within the biofilms is used to measure the activity of these antifungals in order to establish the minimum inhibitory concentration of antifungals against biofilms. Ultimately, results can be obtained that show the ability to form biofilms and antifungal susceptibility testing through a simple and easy 96 well microtiter plate based method coupled to a color metric reading. The main advantage of this technique is that it is easy, relatively inexpensive, highly reproducible, and allows for the formation of multiple equivalent biofilms.
In addition, it is compatible with the 96 well platform technology commonly available in most research and clinical laboratories. The implications of this technique extend towards the therapy of canids. Since very often, these infections are associated with the biofilm formation.
Using proper microbiological handling techniques, retrieve a loop full of cells from a fresh subculture of albicans on a SAU dextrose copperplate Alan and inoculate into a 125 milliliter erlenmeyer flask containing 25 milliliters of yeast pepane dextrose broth placed the culture in an orbital shaker set at 180 RRP M and 30 degrees Celsius, and incubated overnight following the overnight incubation. Pour the yeast cultures into 50 milliliter conical tubes and centrifuge at 3000 RBM for five to 10 minutes. Discard the supernatant vortex.
The pellet vigorously to desegregate the cells. Then add sterile PBS to wash before centrifuging the cultures again. Repeat this washing step one more time than re bend the cells.
In about 20 milliliters of RPMI 1640, medium buffered to pH 7.0. Using a solution of 165 millimolar mops and pre warmed at 37 degrees Celsius from this suspension, prepare a one to 100 dilution using a standard hemo cytometer and a bright field microscope. Count the cells.
Then prepare a final suspension of cells at one times 10 of the six per milliliter in warm buffered RPMI 1640 use. Using a multi-channel pipette at 100 microliters of sea albican suspension into selected wells of a sterile 96 well flat bottom microtiter plate. Leave the wells in column 12 empty as they will serve as negative controls.
After covering and sealing the plate with paraform, incubate it at 37 degrees Celsius for 24 hours to allow the biofilms to form. Following the incubation, gently aspirate and discard the media from each well. Taking care not to disrupt the biofilms formed at the bottom.
To wash at 200 microliters of PBS to each well and drain by carefully inverting and blotting the plate on a stack of paper towels. Repeat this wash step three times. At this point, biofilms are detectable by eye and can be visualized using an inverted microscope using SEM.
We can appreciate that the biofilms consist of an intertwined mixture of filaments and yeast cells. Once biofilm formation has been verified, testing of antifungal agents can commence using buffered RPMI 1640 medium prepare high concentration working solutions of the antifungal agents to be tested. Typical high concentrations are 1024 micrograms per milliliter for fluconazole and 16 micrograms per milliliter for amphotericin B and CASPO fungi beri.
Next, add 200 microliters of antifungal solutions to wells in column one and 100 microliters of buffered RPMI 1640. Medium to wells in columns two through 11. Perform a series of serial doubling dilutions by transferring 100 microliters of the high concentration antifungal solutions from the wells of column one into those of column two.
After gently pipetting up and down to mix pipette 100 microliters from wells of column two into those of column three and so forth until column 10. Do not add any antifungal solution to column 11, as it will serve as a positive control and discard the last 100 microliters from column 10 wells. All wells, except those in column 12.
Should now have a final volume of 100 microliters. Cover the plate, seal it with parfum and incubate it at 37 degrees Celsius for 24 to 48 hours to allow the antifungals to act following the incubation. And after washing the plate three times in PBS as previously described, add 100 microliters of XTT medion solution per well to all wells, including the negative control wells.
In column 12. Cover the plate, wrap it in aluminum foil to protect it from the light and incubate it at 37 degrees Celsius for two hours. To allow color development following incubation, unwrap the plate.
The initially clear XTT solution will have turned orange after being processed by metabolically active cells. Transfer 80 microliters of the colored supernatants to a fresh, flat bottom microtiter plate. Use a colormetric microtiter plate reader set at a wavelength of 490 nanometers to read the plate as shown here In the absence of antifungals, colormetric readings were untreated.
SEA applicant's biofilms in different rows are equivalent. In order to evaluate the effect of the antifungal compounds tested first, subtract an average background value obtained from the negative control wells in column 12 from all the other values. Next, calculate the percent inhibition using the positive controls in row 11.
As the maximal values, the antifungal concentrations at which a 50%or 80%decrease in colormetric readings is detected, are known as the Cecil minimum inhibitory concentrations. These values are typically used as a reference measure of the potency of a compound. Using this simple high throughput method, fungal biofilms can be formed in vitro in 24 hours, and selected compounds can be screened for antifungal activity After its initial development.
This technique paved the way for the researchers in the field of medical mycology to explore the biofilm formation and to examine the activity of the antifungal agents. Again, the cells in biofilms.
