First try to take pure cells from one pure colony

Do your experiments in complete sterile conditions

Do your experiment in triplicate

Agree with the previous answer. Starting from a single colony is the most important aspect. You definitely should do everything in sterile hood. If your strain is resistant to antibiotics, add some for selection and also it could prevent your culture from contamination.

If you run subcultures in shakeflas,k conider the sterile stopper as source of contaminiations. A stopper made from cotton or cellulose must not be wet (e.g. from sterilization - to be coverd with aluminum foil during steam-sterilization in the autoclave)

The stopper must also fit tight into the neck of your shakeflask.

I recommend that you plate out the culture (redardless whether it is aerobic or anaerobic) and then pick up a single colony. Make a very thin suspension (may be having absorbance of 0.2 at 540nm) and observe several fields after suitable staining under a light microscope till you see the same morphological vharacteristics and then subculture it suitably.

The purity of the culture, it is the main feature.

My experience has given me very good results using specific cultures with antibiotics, to obtain pure strains, and applying dilutions retrieve a single cell, reproduce and continue the implementation of an experimental inoculum medium, always observing sterile conditions in containers material, work area and staff