Candida is the most common cause of disseminated fungal infection in immunocompromised hosts. The failure of therapeutic agents to fully infiltrate biofilm because of the matrix is one reason why biofilms resist to traditional therapies. Our protocol evaluates how phototherapy, using red light, interferes with the growth and arrangement of Candida albicans biofilms.
The methods applied are well established, since the strain used in this study had its extracellular matrix characterized and the light device has been successfully applied to other planktonic microorganism suspensions and biofilms. The greatest advantage of this device is the reduction in treatment time, which makes it more visible for clinical applications. When culturing Candida albicans be aware that antibodies can be acquired.
Therefore, it is important not to use colonies that are more than seven days old, not to start plates at four degrees Celsius, and not to re-streak cells from existing plates. Likewise, a strain should be used prior to 18 hours of overnight growth. When using a spectrophotometer for the first time it is important to first obtain colony farm in units counts, to correlate to optical density values.
Therefore, experiments will start using middle of growth phase in initial cell concentration of 10 to seven cells per milliliters. This cell concentration has been shown to provide the most dependent and stable matter biofilms. To begin, suspend 65 grams of SDA, supplemented with 50 milligrams per liter of Chloramphenicol, in 1, 000 milliliters of distilled water in a beaker.
Place the beaker on a heater to boil, in order to dissolve the medium completely. Then transfer the media to a glass bottle. Place the cap onto the bottle, but do no screw it down completely to ensure the bottle is able to vent.
Place the bottle in an autoclave to sterilize the solution at 15 PSI and 121 degrees Celsius for 30 minutes. Cool the solution to around 45 degrees Celsius. Use a disposable, sterile pipette, to mix well, and pipette 20 milliliters of SDA into sterile Petri plates.
To prepare YNB medium, supplemented with 100 millimolar glucose, mix 6.7 grams of YNB and 18 grams of dextrose to 1, 000 milliliters of ultra pure water in a beaker. Place a stir bar in the beaker and put the beaker on a stir plate to mix. Filter the medium through a 0.22 micrometer vacuum filter system to sterilize.
Prepare RPMI medium by mixing 10.4 grams of RPMI-1640 and 34 grams of MOPS, to 1, 000 milliliters of ultra pure water. Mix in a stirrer without heat and adjust the PH to seven by adding one normal sodium hydroxide. Sterilize the medium using a 0.22 micrometer vacuum filter system.
First, thaw the microorganism Candida albicans SN 425 at ambient temperature. Seed 10 microliters of the stop culture on previously prepared Petri dishes containing SDA, supplemented with Chloramphenicol. Incubate the Petri dishes aerobically at 37 degrees Celsius for 48 hours.
For the pre-inoculum use a sterilized lube to pick 10 colonies of Candida albicans from the Petri dishes and place them into a centrifuge tube containing 10 milliliters of YNB medium, supplemented with glucose. Incubate the tube aerobically at 37 degrees Celsius for 16 hours. Then, prepare the inoculums by adding this starter culture into fresh YNB medium, supplemented with glucose, in a one to 10 proportion for dilution.
Use the spectrophotometer to measure the initial OD at 540 nanometers. Incubate the inoculums aerobically at 37 degrees Celsius for eight hours, and measure the final OD at 540 nanometers. Subtract the final OD from the initial OD to check if the OD of the inoculums are on the mid-log growth phase.
To obtain an inoculum with 10 to the seven cells per milliliter, centrifuge the inoculum for five minutes at 5, 500 times G.Pour the supernatant into a beaker containing sodium hypochlorite and add fresh YNB to concentrate the inoculum to half of the previous volume. Add one milliliter of the inoculum to each well of a 24 well polystyrene plate. Incubate aerobically at 37 degrees Celsius for 90 minutes to allow cell adhesion to the bottom of the wells.
Treat the biofilms for one minute with one milliliter of 0.12%chlorhexidine for the positive control biofilms. For the negative control, treat the biofilms for one minute with one milliliter of sterile, 0.89%sodium chloride. Subsequently, wash the wells two times, one minute each, with one milliliter of sterile 0.89%sodium chloride to remove non-adherent cells.
Then turn on a non-coherent red light and use a power meter to measure the power density of the light. Determine the exposure time based on the energy density requirement of 87.6 joules per square centimeter. Place the plate under the red light.
Keep the distance between the light source and the biofilm to five milliliters to avoid warming the sample. After exposure for one minute, add one milliliter of the sterilized RPMI medium to each well, and incubate the plates at 37 degrees Celsius over night. In the morning, repeat the same washing, light treatment, positive and negative control treatments, and incubation for six hours with RPMI medium.
Again, wash the biofilms twice, expose them to red light, and perform positive and negative control treatments. Aspirate the solution in the wells and add RPMI medium. Repeat the incubation, washing, light treatment, and positive and negative control treatments, until achieving 48 hours of biofilm development.
To start processing, discard the medium and add one milliliter of sterile, 0.89%sodium chloride to the well, and use a pipette tip to scratch the biofilm from the well. Transfer the removed biofilm to a sterile tube. Add another one milliliter of sterile, 0.89%sodium chloride to the well.
Scratch it again and add the suspension to the same tube, for a total volume of 2 milliliters. Vigorously vortex the biofilm suspension for one minute. For CFU analysis, transfer an aliquot of 0.1 milliliters from the biofilm suspension to a micro centrifuge tube containing 900 microliters of 0.89%sodium chloride solution for each dilution.
Dilute the biofilm solution from 10 to the negative one, to 10 to the negative four, in saline solution. Seed 50 microliters of each dilution to SDA plates, and incubate the plates at 37 degrees Celsius for 48 hours. Count the colonies and apply the formula.
For dry weight analysis, first weigh and label micro centrifuge tubes. Add 0.1 milliliters of the biofilm suspension and one milliliter of absolute ethanol into the tubes, and store them at negative 20 degrees Celsius for 18 hours. Then, centrifuge it 10, 000 times G for 10 minutes.
Discard the supernatant and place the tubes in a desiccator to dry the samples for one week. Weigh the micro centrifuge tubes again, and do the subtraction to obtain the biomass weight. In this experiment, after per diem treatments with red light for one minute to Candida albicans biofilms, the CFU was reduced, compared to the negative control treated with sodium chloride.
The outcomes of the biomass of Candida albicans biofilms after daily treatments, show the red light treated group presented similar reduction of biomass to that observed in the positive control group, treated with chlorhexidine. The two groups also showed statistical similarity with each other. While they both showed reduction of the biomass, compared to the negative control treated with sodium chloride.
Inferior amounts of Candida albicans soluble and insoluble EPS, was observed in positive control, compared to negative control. Even though not statistically significant, per diem application of red light numerically diminished the amounts of EPS soluble and EPS insoluble. The negative control and the red light treated group, showed statistical similarity.
Measuring the power density of the light, using a power meter before starting the treatment to determine the exact periods of application. The steps assures that the energy density will always be 87.6 joules per centimeters squared. Use the formula previously cited.
The measurement is made using the same from the light to the bottle of the plates, five millimeters. An association between phototherapy and antifungal drugs can be studied to check if the pretreatment with red light, followed by drug application, improves drug penetration into the biofilm. For the twice daily treatment we adapted our laboratory's protocols, providing an easy, and reproducible biofilm model, that delivers answers regarding viability, dry weight, and extracellular polysaccharide amounts after red light treatment.
The protocol is generic and can be used for testing other therapies, in addition to red light, including drugs, other lights, or the combination of light and drugs. Working with microorganisms requires special equipment, such as safety glasses, button lab coat, and nitro gloves.
