Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

There are relatively few drugs available to treat mycobacterium tuberculosis, so resistance to any drug is a concern. This video article describes a procedure for testing the antibiotic resistance of M tuberculosis to 12 different microbial drugs simultaneously. Thus, the ability to rapidly detect antimicrobial resistance of M tuberculosis is very important in the effort to control the increase of resistance strains Here to test antibiotic susceptibility.

M tuberculosis is first grown on solid or in broth media, a bacterial suspension. The inoculum is prepared and added to a microtiter plate containing different concentrations of 12 drugs, including first line and second line drugs Following incubation. The extent of growth in each well is determined either manually using a mirror box or using a semi-automated plate reader.

The resulting data indicate the minimum inhibitory concentration for each drug tested and show which antibiotics the tested strain of TB is resistant to. These results aid in choosing the appropriate drug for treatment of tb. Hi, my name is Nancy Ek and I'm director of the Myco Bacteriology Laboratory at the Mayo Clinic in Rochester, Minnesota.

Today we're gonna describe a method for doing antibiotic susceptibility testing for mycobacterium tuberculosis. The main advantage of this technique over existing methods like macro broth dilution is that an MIC is determined for each drug and 12 antibiotics are tested concurrently. Demonstrating the procedure will be Kurt Jude, a quality specialist in my laboratory, Working on a laboratory bench.

Prepare for this procedure by labeling a blood agar plate, three Middlebrooks seven H 10 plates, a saline tween glass bead tube, and a microtiter plate with the patient's name and specimen identification number. Additionally, label the blood agar and seven H 10 plates as purity. Check label one of these seven H 10 plates as one to one colony count and the other as one to 50 colony count.

Then place the tray in the pass through. Next, prepare to work in the level three biosafety BSL three facility by putting on a solid front gown, head cover gloves, shoe covers, and a respirator. Here, a pappa respirator is used.

Then retrieve the tray from the pass through. Next in the BSL three facility, disinfect the surface of the biological safety cabinet. Then place a paper towel soaked with disinfectant inside the hood, approximately six inches from the air vent panel next to the paper towel.

Place inoculating loops, pipetters and tips, an appropriate waste disposal container, a nephi, TER, and a vortex. Once the hood is prepared, place the organism on the paper towel along with a rack with a McFarland 0.5 standard A seven H nine broth with OADC and a saline bead tube. This setup is done from solid, medium.

Prepare inoculum from a solid medium culture from a patient's specimen. Use a sterile loop to scrape the colonies and transfer them into the saline tween glass bead tube. Vortex the suspension for 30 to 60 seconds.

Visually inspect the sample, comparing it to the McFarland standard. The amount transferred should be equal to or greater than the standard. If not, add more of the bacterial sample and vortex.Again.

Once the McFarland standard is matched, allow the inoculum to settle for 15 minutes to allow larger clumps of bacteria to settle. Next, use a nefi TER to measure the density of the suspended bacteria to zero. The nephi ter.

First place a blank saline bead tween tube in the nephi ter. Press the calibrate button to zero the instrument, then place the McFarland 0.5 standard in the Nephi ter and press the calibrate button again. Once the instrument is calibrated, remove the McFarland standard and insert the inoculum.

The inoculum reading should be equal to the McFarland standard reading. If the reading is too low, remove the tube and transfer more bacteria from the plate. Then vortex and read the sample again.

If the reading is too high, add saline tween to the tube vortex. Then read the sample again. Continue adjusting the inoculum as needed until the concentration of the bacteria matches the McFarland standard.

Next, use a 200 microliter pipetter and a long aerosol resistant pipette tip to remove 100 microliters of inoculum from the top third of the tube. To avoid any clumps that are settling to the bottom inoculate, the Middlebrook seven H nine broth slowly vortex the inoculated broth for 20 seconds to plate the inoculum Begin by removing the microtiter plate from the manufacturer's package. If the desiccant is blue or if the foil package is damaged, do not use it.

The plate already contains the 12 antimicrobials to be tested in each well, including the most effective or first line drugs and second line drugs. Carefully pour the seven H nine broth inoculum into a trth taking care not to generate aerosols. Then place the micro titter plate with the label facing out as shown here, and use a 12 channel pipetter to add 100 microliters of the inoculum to each well.

Next, prepare plates for performing colony counts. First, prepare an undiluted plate by transferring a one microliter loop of sample from the positive control well to the seven H 10 plate labeled one to one. Then use the loop to streak the plate as shown here.

Next, to prepare a one to 50 dilution, transfer a one microliter loop from the positive control well and swirl it into the tube with 50 microliters of water streak. One microliter of this dilution on the plate labeled one to 50. Inoculate the purity plates by pipetting 50 microliters of the inoculum from the trth onto appropriately labeled blood agar and seven H 10 plates and streak for isolation.

Then stack another trth on top of the inoculated trth and gently place them into the discard container. Place an adhesive seal on the microtiter plate. Then using the white backing, press firmly on each well to assure adequate ceiling, ensuring that there are no creases.

Disinfect the micro titter plate by wiping it with a paper towel soaked with a tuberculous cidal disinfectant. Then place the microtiter plate in a plastic bag and close it with a heat seal. Finally disinfect all materials, including the nephi, lter vortex, plates and tubes by wiping them with a tuberculous cidal disinfectant.

Here pro spray is used after enough time has passed to ensure disinfection. 15 minutes for pro spray. The plates are taped with scotch tape placed in plastic bags and sealed supplies are removed from the biosafety cabinet and placed in the pass through.

The discard red bag is closed and sealed with a twist tie at this point, removed the gown, booties, and gloves. After proceeding to the ante room and washing hands, remove the respirator and disinfect and store it after washing hands again, put on a fresh gown and leave the BSL three suite. Next, the discard container is autoclaved.

Following this incubate the microtiter plate and the seven H 10 and blood auger plates right side up in the 35 to 37 degrees Celsius incubator with five to 10%CO2 In the BSL two laboratory. This is acceptable because the plate is well sealed and in a plastic bag, mycobacterium tuberculosis generation Time is approximately 12 to 24 hours, so it will be at least a week before growth can be seen as the formation of a cell pellet in the bottom of the control. Well, on day 10, perform a manual mirror read by placing the sealed a ST micro titter blade onto the platform of the mirror box.

Use a desk lamp to aid in visualizing the growth adjust as needed to get the best contrast. Check the growth control wells in this case, H 11 and H 12 by looking at the mirror. If there is growth, the well will appear cloudy or appear to have sediment as shown here.

If there is no growth as shown in this example, the well will appear to be clear. In this case, place the plate back in the incubator and continue incubating for up to 21 days. For each condition, compare the growth to the control.

Well then, for each drug record, the first dilution that has no growth. This is the minimal inhibitory concentration or MIC for the drug to examine results using an automated plate reader such as the vision begin by opening the Myco TB a ST plate software and logging in place the sealed plate with the barcode facing out. As shown here, growth appears as turbidity or as a deposit of cells at the bottom of a well on the computer screen.

First, examine the growth controls. As before. If this is negative for growth, place the plate back in the incubator for up to 21 days.

If this is positive, record the results by touching the first well, that does not show growth on the screen for each drug. In some cases, bacteria, sediment or drugs precipitate to the bottom of the well. This phenomenon known as trailing, is indicated by several wells with the same size pellet and is not actually considered positive growth.

Next, the seven H 10 and blood agar plates are examined to determine bacterial concentrations and assess purity. The blood agar plate should show no growth after 48 hours. Growth on the blood agar plate indicates contaminations with rapidly growing bacteria.

If there is growth, the microtiter test must be repeated. The seven H 10 purity plate should have growth of one organism. Type EM tuberculosis should appear buff, colored, wrinkly, and rough If contaminated, the Microtiter test needs to be repeated from a pure culture.

To assess bacterial concentration, count the colonies on the seven H 10 plate. Then using the table in the accompanying written document, calculate colony counts. Mycobacterium tuberculosis was grown from a patient specimen and antibiotic resistance was assessed as described in this video in the plate shown here, 12 different drugs are present in increasing concentrations with the highest concentrations at the top and the lowest concentrations at the bottom.

The minimal inhibitory concentration, or MIC of the drug was indicated by the lowest concentration of the drug that did not show any growth and is circled in the image shown here using standardized breakpoints established by the Clinical and Laboratory Standards Institute. The data shown here suggests that this strain of TB will be resistant to Isid and Rifampin. In vitro, the high mics seen in several other drugs, including oin, moxifloxacin, streptomycin, and rifabutin suggest the resistance of the EM tuberculosis strain to these agents.

The MIC results should be used by an infectious disease specialist experienced in the treatment of tuberculosis to develop an individualized treatment plan Once mastered this technique takes about 40 minutes to set up one isolate. While attempting this procedure, it's important to remember that this is a respiratory pathogen and once you dot generate aerosols, after watching this video, you should have a good understanding of how to set up mycobacterium tuberculosis, antimicrobial susceptibility testing using the micro broth dilution method. Don't forget that working with mycobacterium tuberculosis can be extremely hazardous, and precautions such as respiratory protection should always be taken where we're required.