Kristina TurnerMay 10, 2006

Gossypol/Acetic Acid S. aureus SH1000 & COL Susceptibility Testing

Methods

100 mg Gossypol/Acetic acid is solubilized in 1 mL DMSO (dimethyl sulfoxide) in an amber vial and stored frozen to prevent light exposure and keep stable. 5 mg/mL Gossypol/Acetic acid is made by adding 50 L of 100mg/mL stock to 950 uL pH 8 Mueller-Hinton Broth with 10 mM Tris-HCl buffer. Make 1 mg/mL Gossypol/Acetic acid by taking 200 L of 5 mg/mL Gossypol/Acetic acid to 800 L pH 8 Mueller-Hinton Broth and vortex. Make serial dilutions by taking 500 L of 1 mg/mL Gossypol/Acetic acid, adding to 500 L of pH 8 Mueller-Hinton Broth with 10 mM Tris-HCl buffer and vortexing. Repeat this until the lowest concentration tested is reached. Take 500 L of lowest concentration tested and discard to keep at same volume as other test tubes. Add 500 uL of pH 8 Mueller-Hinton Broth with 10 mM Tris-HCl to two test tubes. One of these test tubes will be the growth control with no drug. The other test tube will be the sterility control. Each test tube except sterility control will be inoculated with 50 uL of overnight culture adjusted to O.D. 625nm = 0.1 and vortex. The overnight culture should be an 18 hour culture made by adding 5 colonies of each strain to 5 mL of pH 8 Mueller-Hinton Broth and incubating at 37 Celcius in orbital shaker. Incubate test tubes for 24 hours at 37 degrees Celcius. The lowest concentration with no growth after 24 hours is the minimal inhibitory concentration (MIC).

Divide Mueller-Hinton Agar plate into six sections and take 10 L of each concentration to determine minimal bactericidal concentration (MBC). Allow the drops to dry for an hour in the hood with the lid on. Mueller-Hinton Agar plates poured to a uniform depth of 4 mm in either 150 mm or 100 mm Petri plates should be used with pH 7.2-7.4 Mueller-Hinton Agar. Incubate 24 hours at 37 Celcius and observe results. The concentration with zero to 5 colonies after 24 hours is the minimal bactericidal concentration.

The minimal bactericidal concentration is the serumcidal concentration of the drug and a higher concentration should be given to the patient to help ensure that the bacteria causing the infection are killed. Note that a bacterial sample should be taken from the patient and Gram stained to confirm which bacteria is causing the infection. The identified bacteria should be grown overnight and used for the MIC testing for the drug.

Note that the methods for MIC broth testing are the same as above for 96-well plate testing, with the following changes. Adjust the overnight culture of each strain tested to O.D. 625nm = 0.1. Make 2 x 1mg/mL Gossypol/Acetic acid by taking 800 L of 5 mg/mL Gossypol/Acetic acid and adding to 1.2 mL pH 8 Mueller-Hinton Broth with 10mM pH 8 Tris-HCl and vortexing. Deliver 200 L of the 2 x 1 mg/mL Gossypol/Acetic acid to the first well on the top three rows of wells, skip two wells, and deliver 200 L to the first

well on the bottom three rows of wells. Deliver 100 L of pH 8 Mueller-Hinton Broth with 10mM Tris-HCl to each of the next 7 wells of each of the rows. Add 100 L of pH 8 Mueller-Hinton Broth to the ninth well on each of the rows. Do not add any drug to this well. Skip two wells and add 200 L of pH 8 Mueller-Hinton Broth to the twelfth well on each of the rows. Do not inoculate this well. It will be the sterility control. Make serial dilutions by taking 100 L from the first well and delivering it to the second well. Repeat this for each of the rows and then mix with the pipette tip. Change the tip and repeat this process; changing the tips before making the next concentration. Take 100 L from the eight well on each of the rows. This is the lowest concentration of the drug tested and discarding 100 L of this concentration will allow the wells to have the same volume. The top three rows of wells will be for S. aureus SH1000 and the bottom three rows of wells will be for S. aureus COL. Deliver 100 L of S. aureus SH1000 adjusted culture to the first nine wells on each of the top three rows. Deliver 100 L of S. aureus COL adjusted culture to first nine wells on each of the bottom three rows. Cover the 96-well plate with sterile adhesive covers and incubate for 24 hours without agitation at 37 Celcius. Use a Spectramax 96-well plate reader to take the O.D. readings at 625 nm after shaking for ten minutes to break up any clumps of bacteria. Divide a pH 7.2-7.4 Mueller-Hinton Agar plate into 6 sections. Take 10 L from each concentration and use to make a drop plate. Allow the drops to dry for an hour in the hood with the lid on. Incubate 24 hours at 37 Celcius to confirm the minimal bactericidal concentration (MBC).

Kirby-Bauer disc diffusion testing can also be done to help determine susceptibility to a drug. Use sterile filter discs purchased from Sigma. Concentrations of the drug should be made up as in broth MIC testing. Discs should be completely immersed in the concentrations of drug to be tested and allowed to soak for 30 minutes before being placed with sterile forceps on Mueller-Hinton Agar plates that have been evenly swabbed with cultures of the strains to be tested. Adjust overnight cultures of the bacterial strains being tested to an O.D. 625nm = 0.1. Immerse a sterile cotton swab into adjusted culture until it is thoroughly wet. Remove surplus suspension from swab by rotation against inside of culture tube. Spread entire surface of Mueller-Hinton Agar plate. Even distribution is essential; spread evenly in three directions so that even, confluent growth will result. With plates covered, allow inoculum to dry for 3 to 5 minutes. Flame forceps and use to place each antibiotic surface. Using aseptic techniques, tap each disc gently to allow full contact with agar surface. Immediately incubate inverted plates at 37 Celcius for 18 hours (Medical Microbiology Lab said 35.5 Celcius for 14 hours, so double check). Mueller-Hinton Agar of pH 7.2-7.4 should be poured uniformly to a depth of 4 mm in 100 mm or 140mm Petri plates. This is extremely important for interpretable disc diffusion results, since depth of agar affects the zone of inhibition. Measure zone diameters in millimeters for each concentration and record results. Compare to Kirby-Bauer zone size chart and report organisms as resistant (R), intermediate (I), or sensitive (S) to each concentration and antibiotic used. Sometimes organisms can not be classified as susceptible or resistant and are interpreted as intermediate (I) susceptibility to a drug.