The aim of this work is to show how to prepare a highly viable mycobacterium lipid suspension. For into nude mice. We'll retain the bass life from an infected mouse, preferably during early stage of infection.
To guarantee highly viable ary yields, euthanize the animal inside the biological safety cabinet. Hold the rear pulse with hemostatic forceps. Cut them off with seizures and immerse them in 2%Iodine for 20 minutes For decontamination, hold the point position with the foot pad facing up.
Collect the soft tissue Using a scalpel blade, remove the bones of the first and fifth phalanges to facilitate homogenization of the tissue. Place the tissue in a sterile Petri dish. Scrap off epidermis.
Cut the granulomatous tissue in small pieces with scissors and wait for later calculation of yield program of tissue. Transfer the small pieces to a tube containing one ml of Hank's balanced salt solution. Add one additional ml of hank's and homogenize the tissue three times for 15 seconds each time at speed.Four.
Place a cell strainer into a 50 ml sterile conical tube. Pipette the suspension onto the strainer and allow the solution to pass by gravity. To eliminate the debris, add two more MLS of hanks to the suspension and homogenize it again.
Using the same cycle, pass the suspension through this strainer. Rings the strainer by adding hanks. To bring the total volume of the suspension to nine mls, add one ml of 0.5%tripsin solution to the suspension to abut a 0.05%final concentration.
Mix it by gently inverting the tube incubated 37 degrees Celsius for 60 minutes. After incubation, bring the volume of the suspension to four MLS by adding sterile saline centrifuge at 1700 G for 30 minutes at four degrees Celsius, discard the supernat and resuspend the pallet by tapping the tube. Add one ml of sero saline and mix it.
Transfer the suspension to a new tube. Homogenize the vascular suspension using a one ml insulin syringe with a 26 gauge needle while transferring the suspension to a new tube. Perform microbiological control of the suspension by placing two drops of 18 medium, seven H nine brain heart infusion.
11 Stein Genesen incubated 37 degrees Celsius. And then follow routine microbiological control procedures. For code Z staining.
Prepare a one to 10 and one to a hundred. Dilution of the S suspension in saline. Homogenize each dilution before preparing these lights for counting, prepare three slides by drawing three 10 millimeter internal diameter circles on the surface of each slide using a permanent ink marker.
Now surround the circles with the immunohistochemistry pen. Label these lights using a number two pencil prepares lights for the three sary concentrations. Undiluted one to 10 and one to a hundred.
Pipette five microliters of the phenol serum solution and 10 microliters of the sary suspension in each circle, homogenize and spread the suspension evenly throughout the area of the circle. Using the handle of a disposable loop, place these lights on a level table to dry after drying. Fix them by passing these lights three times over the blue flame with the bachelor suspensions facing up.
Perform a code Zuni sustaining. Examine these slides. The three dilutions.
Choose the most appropriate dilution for sary counting. Prepare 200 microliters of killed sary suspension by autoclaving the vascular suspension for 30 minutes at 121 degrees Celsius. Use the L 7 0 0 7 live dead back light bacterial viability kit for fluorescent staining of microbacterial lipid.
Prepare a want to 10 dilution of solution A and one to 20 dilution of solution B.Right before use. To each 200 microliters of undiluted vascular suspension original and killed, add 3.6 microliters of the diluted solution A and six microliters of the diluted solution B.After 15 minutes of incubation in the dark, stfu the suspension at 10, 600 G for five minutes at four degrees Celsius. Remove the super natin with the pipette reus.
Suspend the pellet in 50 microliters of 10%Glycerol, homogenize it by pipetting up and down. Place eight microliters of each suspension onto a glass slide and cover with a small cover slip. Protecting it from light.
Observe suspensions under fluorescent microscope. Prepare suspension with the concentration of 10 to seven BA lip per ml, qu one ml of freezing medium in each cryo vial. Then add 150 microliters of the S suspension to the one ml of the freezing medium and homogenize it by inverting the tube.
Place the cryo vial inside the freezing container. Maintain the container at minus 80 degrees Celsius for at least 24 hours. Transfer the tubes to a storage box and maintain them at minus 80 degrees Celsius for thawing.
Thaw the frozen suspension in a water bath set at 37 degrees Celsius. Transfer the suspension to a conico tube containing 20 miles of sterile saline cent. Refused to concentrate.
The baline, followed the Z sustaining protocol for counting and the fluorescent staining protocol for variability determination. Prepare suspension contain 10 to eight BA per meal, homogenize the suspension before inoculating each animal. Pull a hundred microliters into an insulin syringe and inoculate 30 microliters into the rear foot pads of each animal in the viability determination.
The green fluorescence is observed in all BAI and red fluorescence only in that bai. When that BAI are stainless control, the same intensity of green and red BAI light is observed. Inoculation of M Lepar frozen for different periods yield 10 to 1000 times increased in the number of BAI recovered after seven months.
As a final remark, we would like to add that since IL leid does not grow in vitro, this protocol allows for a fast and easy way of maintaining viable yl Leid available for further uses in the laboratory.
