Aspects of Mannheimia haemolytica cryopreservation

Abstract

Mannheimia haemolytica causes severe pneumonia and septicemia in ruminant animals. The study aims to create an effective protective media for the long‐term storage of microbial cultures of M. haemolytica in a veterinary laboratory. The protective nutrient media were based on Hottinger's nutrient broth. Glycerol and ethylene glycol were used as cryoprotectants. Two protective media were obtained. Medium #1 consisted of nutrient broth (90%) and glycerin (10%), and medium #2 had nutrient broth (90%) and ethylene glycol (10%) in it. A homogeneous suspension 1.0 McFarland units turbidity was prepared from a pure culture of microorganisms (3.0 x 108 CFU/ml). The suspension was added to ampoules with an appropriate protective medium. The inoculated media were placed in a cryogenic storage dewar in liquid nitrogen at ‐196 ° C. The viability of M. haemolytica was determined by inoculation on blood agar and counting the number of living bacteria. The initial inoculum concentration of M. haemolytica was 3.0x107 CFU/ml. After 18‐20 hours of cultivation, it becomes 7.0x109 CFU/ml. The survival and safety of bacteria depend on the cryoprotectant. The results of the experiment showed that 10% glycerol provides 98.6% survival of M. haemolytica. The number of viable cells after cryopreservation was 6.9x109 CFU/ml. Ethylene glycol showed a 95.7% survival rate. The number of viable cells after cryopreservation was 6.7x109 CFU/ml. Without cryoprotectant, M. haemolytica showed a 40.0% survival rate with only 2.8x109 CFU/ml viable cells after defrosting. M. haemolytica causes turbidity and sediment formation at the bottom of the tube on Hottinger broth. After growing on semi‐liquid media (semi‐liquid agar with the addition of glucose or serum), bacteria show bipolar staining in smears. On Hottinger broth, with the addition of 10% defibrinated ram blood M. haemolytica formed small dewy colonies with an even edge without a hemolysis zone. The final species identification was performed by the proteomic method using MALDI‐TOF‐SM. All used media keep microorganisms' cultures in a stable, viable state. Thus, they can be successfully used for the storage of bacteria at low‐temperature conditions.