Use 1:1 000 dilutions in mid-exponential growth stage

As the bacteria multiply, the concentration increases, and a 1:100 dilution is no longer suitable. The maximum concentration in the diluted sample is 5 000 000 total/mL. If this limit is exceeded, an error will display instead of a concentration. In such cases, clean the setup and measure a more diluted sample.

Avoid getting too many particles.

To prevent the "too many particles" error, follow this simple rule of thumb during the exponential growth stage: If the 1:100 diluted sample was above 1 500 000 at the last measurement ~30 minutes ago, it is time to start using a 1;1 000 dilution.

This rule works for all bacteria as long as the sampling frequency is set to 1.5 ^_x generation time.

Do a Clean program after each measurement.

Start a 30-minute countdown to remind you to collect the next sample.

Prepare a 1:1 000 dilution

1. Use a serological pipette to collect a sample from the shake flask.

2. Return the shake flask to the incubator shaker.

3. Start a 30-minute countdown to remind you to collect the next sample.

4. Vortex 30 seconds at max speed to disaggregate bacteria.

5. Make a 1:100 dilution:

   1. Transfer 101 µL of your sample to 10 mL of diluent.

   2. Vortex 10 seconds at max speed.

6. Make a 1:1 000 dilution:

   1. First remove 1 mL diluent from a 10 mL diluent vial for a residual volume of 9 mL.

   2. Then transfer 1 mL of the 1:100 dilution to the vial containing 9 mL of diluent.

   3. Vortex 10 seconds at max speed.

7. Transfer the tubing kit to the 1:1 000 vial.

Measure the diluted samples in Access

1. Click the Measure button in Access.

2. Add metadata:

   1. Dilution: 1 000.

   2. Label: E_coli_growth.

Clean the device

1. Transfer the tubing kit to the disinfection vial.

2. Click Clean.

Summary

The 1:1 000 dilution scheme works well for mid- to late-exponential stage.

Monitor when the diluted sample's concentration surpasses 1,500,000. When this happens, jump to the next section where we will detail how to dilute the sample at a ratio of 1:10 000.

Pro tip: 1-step 1:1 001 dilution.

The described 2-step procedure for a 1:1,000 dilution is reliable with high precision. However, it demands a longer sample preparation time. If you have good pipetting skills, you can save time, sample and diluent by doing a 1-step dilution. Simply transfer 10 µL of sample to 10 mL of diluent to get a 1:1 001 dilution.