The aim of this experiment is to assess the efficacy of antibiotics against pseudomonas Aerogen biofilms, under conditions, representative of chronic infections in the cystic fibrosis lung using a defined artificial sputum medium. Bacterial biofilms are grown in 24. Well microtiter plates.
The biofilms are then exposed to antibiotics. Next, antibiotic treated cultures are disrupted using cellulase and incubated with a metabolic dye srin, which is reduced from blue to pink by viable cells. By measuring the absorbance of the wells, the biofilm Cecile cell minimum inhibitory concentration of B-S-M-I-C can be determined to quantify antibiotic susceptibility under near physiological conditions.
The main advantage of this technique over existing methods, such as the determination of minimum inhibitory concentrations based on planktonic culture, is that the bacteria grown in a specialized media containing all the components of sputum, thus promoting biofilm formation. These biofilms are much more resistant to antibiotics. To make the medium first prepare two bottles containing 250 milliliters of sterile water in the first bottle.
Gradually add the stir and then add four grams of fish sperm, DNA to 250 milliliters of sterile water. Stir the solution at room temperature and allow the DNA to dissolve to the second bottle. Gradually add five grams of type two mucin from porcine stomach and stir.
Once the DNA and mucin solutions are ready, dissolve the amino acids in 100 milliliters of sterile water, with the exception of L tyrosine and L cysteine, which should be dissolved in 25 milliliters of water, or 25 milliliters of 0.5 molar potassium hydroxide respectively. Next, makes 5.9 milligrams of DTPA, five grams of sodium chloride and 2.2 grams of potassium chloride in 100 milliliters of water. Combine all the different solutions in a one liter bottle, then add five milliliters of egg yolk emulsion, and top the mixture up to 850 milliliters with water.
Using one molar triss, adjust the pH to 6.9 and bring the final volume up to one liter with water to sterilize the medium, filter it using a vacuum brand ME two diaphragm vacuum pump, and a Millipore stereo top filter unit with a 45 millimeter neck and 0.2 micron pore size. The filtration process can be performed over two days. The A SM medium can be stored at four degrees Celsius for up to one month.
Begin by diluting overnight pseudomona arosa cultures in LB medium to obtain an OD of 0.05. Then further dilute the bacteria at one to 100 in a SM medium plate, 1.8 milliliters per well of diluted bacterial culture in 21 wells of a 24 well tissue culture plate aliquot the same volume of a SM medium in the three remaining wells, which will serve as blank negative controls. Secure the 24 well plate with biofilm, and to allow biofilms to form, place it under micro error fillic conditions, which can be obtained using campi gen gas generation packs in large anaerobic jars.
Incubate the culture on a shaker set at 75 RPM for three days at 37 degrees Celsius after three days, biofilms will appear as a gelatinous mass containing bacterial cells and cannot be disrupted by pipetting. Once the biofilms are formed at 200 microliters per well of antibiotic at serially diluted concentrations. Be sure to set up at least four replicates for each drug concentration to be tested and leave four wells containing biofilms untreated so that they can be used as negative controls.
Then incubate the platers previously for a further 24 hours following the incubation with antibiotics. Use 100 microliters per well of cellulase to diluted to 100 milligrams per milliliter in citrate buffer to disrupt the biofilms, incubate the plates on a shaker set at 150 RPM under aerobic conditions at 37 degrees Celsius for one hour. Following this incubation manual, pipetting may be used to further disaggregate the biofilms if clumps of cells are visible in the medium.
Next, add 100 microliters of rezin per well to quantify the viability of the cells released from the biofilms and incubate the cells for one to two hours. At 37 degrees Celsius, only viable cells are able to metabolize the blue zarin dye to its pink fluorescent form. Following the incubation, transfer the samples into a 96 well plate and measure the fluorescence of the wells using a five 40 nanometer excitation wavelength and a five 90 nanometer emission wavelength.
In order to quantify bacterial viability and drug susceptibility, first, subtract the background fluorescence of the blank control from the other wells. Next, calculate the percentage of inhibition by dividing the mean fluorescence for each concentration by the mean fluorescence of the untreated controls and multiplying by 100. The B-S-M-Y-C is defined as the antibiotic concentration causing a 90%inhibition of metabolic activity as shown in these electron micrographs cells, grown in biofilms differ considerably from bacteria grown in planktonic cultures.
In particular, large amounts of extracellular matrix shown in yellow surround the cells in the biofilm in green, but not in planktonic colonies. In addition, bacteria in biofilms are typically more resistant to antibiotics in this experiment. The minimum inhibitory concentration of tobramycin for pseudomonas aerogen planktonic culture in blue was determined to be approximately four micrograms per milliliter.
In contrast, as shown in red, 100%of bacteria in biofilms survived up to 512 micrograms per milliliter of antibiotic. After watching this video, you should have a good understanding of how to culture bacteria in a defined artificial sputum medium. You should also be able to perform an antibiotic susceptibility test to determine the minimum inhibitory concentration required to inhibit bacterial growth in environment more realistic at cystic fibrosis lung.
