Experimental proof that the fast dividing, hypervirulent bacteria are young bacterial population

In the last blog, I assumed that the fast dividing, hypervirulent bacteria isolated after repeated growth in early exponential phase are young bacterial population. This can be confirmed by doing the same experiment in Saccharomyces cerevisiae.

When yeast cells undergo replication by budding, a bud scar is left behind on mother cell’s surface. Bud scars remain permanently deposited on the surface and get accumulated as mother cells undergo more divisions. Thus, an old mother cell will have more number of bud scars whereas the number will be less in relatively young mother cells. Thus, the bud scars are used as a marker for the number of divisions a cell had undergone or the budding index. The budding index can be calculated by analyzing the cell wall for bud scars using confocal microscopy.

For the experiment, when a normally dividing yeast culture reaches an O.D. of 0.3-0.5, 100 ul of the culture should be withdrawn and added to 3 ml of fresh culture medium and incubated for growth. This process should be repeated 3-4 times. At the end of the fourth cycle, when yeast cells remain in early exponential phase itself, the budding index should be calculated. This can be compared with a yeast culture in one-time exponential phase and also with a culture in stationary phase. If the cells obtained after repeated culturing in early exponential phase are indeed young yeast cells, the number of bud scars will be lower than that in other two.

If they are found to be young yeast cells, the same can be true for bacteria also.

Next- Isolation of slow dividing, small colony forming, hypovirulent, senescent bacteria