Enumeration Assays: Determining Culture Concentration via Cell Viability

Materials

  • Epitubes 

  • LB broth 

  • Petri dishes 

  • Inoculating loop  

  • Bunsen burner and striker 

  • Inoculating loop 

  • Culture to enumerate (liquid) 

  • Incubator


Enumeration via 10-fold Serial Dilutions

  1. 1. Gather materials and ensure you have sufficient petri dishes for enumeration. Enumeration is done with a liquid culture, so make sure your culture is in that form. 

  1. 2. On each petri dish, label with you name, bacteria contents, date, the dilution factor, and trial number.  

  1. 3. Label each epitube with the dilution factor. For this procedure, we will do 7 dilutions. This will require 7 epitubes, labeled 101 to 107 for 10-fold dilutions. Fill each epitube with 900uL LB broth. Repeat this step if you have multiple trials. 

  1. 4. Add 100uL of the liquid culture you wish to enumerate into the epitube labeled 101. Vortex for 5 seconds. 

  1. 5. From this epitube, remove 100uL and place the fluid into the tube labelled 102. Vortex for 5 seconds. 

  1. 6. Repeat these sequential steps until you have filled the 7th tube with 100uL from the 6th tube. The 7th tube will have 1000uL contained, while each other tube will have 900uL. 

  1. 7. Plate 500uL of each tube onto the respective petri dish. Place the dish in the incubator with the lid facing up for 20 minutes to allow the fluid to be absorbed into the agar. 

  1. 8. After these 20 minutes, flip the plates so the lid is facing down and incubate at 37°C for 24 hours.  

  1. 9. After this period has passed, count the number of cells on readable plates. Readable plates are typically 50-300CFU’s. Make a note of your count on the plate. 

  1. 10. Use this equation to back-calculate the initial liquid culture concentration: 

 

CFU/mL of Culture = (colonies counted) * (dilution factor) / (volume plated)



Serial Dilutions Graphic