How I make my culture: take a lump from my glycerol stock of my pathogenic strain of Vibrio parahaemolyticus into a 5mL tube of TSB spiked with 2% NaCl. I subculture that after two hours into 95 ml of TSB. Shake 100rpm at 28C. Run culture 15-20 hours before I collect.

- I then measure the OD600 and add more TSB + NaCl so that it reads: 0.5-0.6 nm.

I serially dilute my bacteria with sterile Phosphate Buffered Saline and plate on TCBS agar.

0.6= 2.71 X 10^14

0.52= 1.3 X10^17

That should not be happening! I am finding all single colonies and counting between 30-300 on a plate.

What could be my issue?

thanks in advance!!!