Bacillus sphaericus Rehydration

Aims

We aimed to rehydrate and plate the sphaericus strain bought from BCCM LMG22257. We used nutrient media to rehydrate the cells. The cells are going to be used as a control for high urease activity.

Materials

1. Nutrient Broth
2. Nutrient Plates
3. Pasteur pipette

Procedure

Freeze- dried cultures are supplied in vacuum- sealed ampoules.

1. Make a small file mark on the ampoule enar the middle of the cotton wool plug.
2. Touch the file mark with a red hot glass rod till the glass cracks.
3. Wait a few seconds to allow air ot enter into the amploule.
4. Remove the upper part of the ampoule and the cotton plug.
5. Add approximately 0.5ml of nutrient broth with a sterile Pasteur pipette to the dried material after flaming the open end of the ampoule.
6. Mix the contents gently with the tip of the Pasteur pipette, and transfer the contents to one or more suitable solid and/or liquid media.
7. Allow the cells to rehydrate slowly on solid nutrient media.
8. It is advisable to include a general medium in order to detect possible air- borne contaminants which may have beem introduced during the opening of the ampoule.

N.B. Cultures need at least two times subculturing before they can be optimally used in the experiments. Also note that this procedure has been provided by the BCCM itself for the successful bacterial rehydration and growth.

Conclusion

We have cultured the rehydrated cells onto the nutrient agar plates and have put them at 37°C overnight for their optimal growth.