The overall goal of this procedure is to demonstrate a method of growing m tuberculosis biofilms on an air media interface, and determining the frequency of drug tolerant basi in the biofilms. This is accomplished by first preparing the appropriate media and inoculums. The second step is to grow m tuberculosis biofilms in 12 world plates.
Then a test antibiotic is added at a desire concentration, and the frequency of drug tolerant persisters. In M tuberculosis, biofilms can be determined. Ultimately, the technique can be exploited to help understand the basis of biofilm development and to decipher the mechanisms of drug tolerance In M tuberculosis, The main advantage of growing in vitro biofilm culture of microm tuberculosis in detergent free media is that the spontaneous growth of the bassline complex multicellular architecture reveals unique properties of the organism not seen in platonic culture.
These include surface attachment, intercellular interactions, synthesis of accessor matrix, and physiological adaptation within the heterogeneous microenvironment of this structure. Importantly, these information will be highly relevant in understanding the basis of M tuberculosis, persistence in certain specialized niches such as cavities, where an exuberant growth of basi in accessory environment are likely to occur in multicellular structures. The implications of this technique extend towards shorter therapy of tuberculosis because identifying and targeting factors associated with biofilms could reduce the frequency of drug tolerant subpopulations in biofilms.
In this protocol, a standard composition of soans media is used to culture M tuberculosis. However, when growing biofilms of other mycobacterial species use their appropriate specialized media, prepare the primary inoculums of M tuberculosis. In 125 milliliter plastic bottles containing 15 milliliters of liquid media.
Between 80 growth. The cultures under standard conditions until their OD 600 is between 0.7 and 1.0, which takes about a week At this density, the culture can be used directly as an inoculum at a one to 100 dilution. With the inoculum prepared, add 600 microliters to 60 milliliters of sawtons media.
After gently mixing the inoculum into the media, dispense 4.5 milliliters of the mixture into each well of a 12. Well plate, cover the plate and wrap it several times with param. Then incubate the plate in a humidified incubator at 37 degrees Celsius.
After five weeks of undisturbed growth, proceed with testing for the drug tolerant persisters. After five weeks of maturation, the media volume will have reduced to about three milliliters per well into four wells. Inject an antibiotic of interest just beneath the biofilm on the media.
Four other wells should be injected with the solvent alone and the last four wells should be left alone. This provides statistical strength and the proper controls swell the plate gently so that the antibiotic is thoroughly diffused in the media. Now cover the plate.
Put fresh layers of paraform around the plate and incubate the plate for between one to seven days according to the experiment. At the end of the incubation, add tween 80 to each. Well swell the whole plate gently to disperse the detergent uniformly.
Then incubate the plate at room temperature for 15 minutes. After 15 minutes, use a pipette to mix the content of each well until it is uniform. Then transfer the culture to a 15 milliliter conical tube.
Centrifuge the conical tube, and resus. Suspend the pellet in five milliliters of fresh wash buffer, spin down and resus. Suspend the cells in wash buffer three more times.
Then keep the cells in wash buffer overnight on a rocker in a cold room. Although low temperature was originally developed for EMS magmatic to minimize its growth during dispersion and use for M tuberculosis as well. The slow growing species can most likely be rocked at room temperature without any impact on the results.
Rocking at room temperature could be necessary if working in a BSL three facility The next day, prepare a syringe to homogenize and transfer. Each suspension fit a micro pipette tip to a sterile syringe. Cut the broad end of the tip to size, connect it to the syringe, and wrap the connection with para film.
Pass the chilled cell suspension through the tip fitted syringe and deposit the content in a clean 15 milliliter conical tube. Repeat this step five to six times continually replacing the tube until the suspension looks very homogenous. Now, prepare serial dilutions of each suspension and plate each dilution on an agar plate.
After incubating the plates for three weeks, determine the frequency of persisters in the biofilm population by comparing plate colony numbers on the antibiotic treated plates and the control plates. When cultured in a bottle. During the first week, M tuberculosis grew at the base of the bottle.
By the end of the second week, patchy growth of bacteria on the air media interface was seen, which became even more visible at the end of the third week. Attachment of the bacteria to the wall of the container was also observed, and growth of the culture occurred primarily on the air media interface. The liquid beneath the surface growth was clear.
By the end of the fifth week, the structure had fully matured. In a 12 well format after five weeks of incubation, a robust biofilm at the air media interface was seen in each well l on plates that were not wrapped completely. Differential biofilm growth was observed as well as significant media evaporation.
Bacterial growth on these plates was stalled. The number of viable bacilli in biofilms was quite reproducible and varied with different antibiotics. For example, in rifampicin, a bacter cydal antibiotic, a rapid decline in the viability during the first three to four days was followed by a persistent phase in which a small percentage of the population remain completely recalcitrant to antibiotics, irrespective of the concentration of the antibiotic or the time of exposure.
The same procedure can be used in screening of mutants in order to identify genes that are involved in attachment of mycobacterium tuberculosis to the biofilm surface growth of bacteria on the interface and maturation of Pecos. Don't forget that working with M tuberculosis can be extremely hazardous and precautions such as the use of biosafety level three containment should always be taken while performing this procedure.
