Begin by growing mycobacterium paratuberculosis K-10 strain in T25 flasks containing enriched medium for two weeks at 37 degrees Celsius. Assess the colony growth regularly through visual inspection. Grow 200 milliliters of the enrichment culture in a 500 milliliter erlenmeyer flask in a shaking incubator for one week.
Place the bacterial suspensions at 70 degrees Celsius for five to 10 minutes to inactivate them and centrifuge them at 3000G for 10 minutes. Wash the pellets in 20 milliliters of PBS twice and centrifuge the suspension. Transfer the pellet to a sterile container and freeze it with liquid nitrogen.
Transfer the frozen pellet to a lyophilizer chamber immediately. After lyophilization transfer it to a bio cleaning bench. Using a stainless steel spatula dislodge the dried MAP lumps adhering to the inner walls of the container and pulverize them to a fine powder.
Weigh the pulverized cells and manually place them in an aseptic 10 milliliter vial.
