Hit the right conductivity

The electrical conductivity of your sample vial must be within 1 500–2 200 µS/cm. You control the conductivity of your sample with diluent. Keep the diluent at room temperature!

I dilute 1:1000 or more

When the dilution factor is high enough (1:1000 or more), then the conductivity of your sample stock matters little.

Great, you are good to go already. Use our diluent and keep things at room temperature. See the green boxes just below for details.

Use SBT dilution vial or SBT dilution liquid

Use an SBT dilution vial or SBT dilution liquid. Both have the right electrical conductivity already.

When you dilute your sample (1:1 000 or more), the conductivity of the diluent dominates over that of your sample. This puts the conductivity within the allowed range.

Keep your sample vial at room temperature to avoid surprises

For example, if your sample is refrigerated, wait for it to warm up.

The electrical conductivity of the diluent changes a lot with temperature. A mere 5 °C increase can add about 200 µS/cm.

See Hit the right conductivity for details.

I dilute 1:100 or thereabout

Do you dilute 1:10 or less? See the dedicated section below.

Try with our dilution liquid first (as recommended above). If BactoBox® gives you conductivity errors, then consult this section in detail. You may have to use a custom diluent.

At relatively low dilution factors (1:100 or less), the conductivity of the growth medium comes into play.

Growth media has high electrical conductivity

Most growth media have a relatively high electrical conductivity. Usually around 14 000–20 000 µS/cm at room temperature.

Some growth media have very high electrical conductivity. For example, MRS (used for lactic acid bacteria) has a conductivity of approximately 100 000 µS/cm at room temperature.

Conductivity of diluent

Our diluent consists of two things: Water and PBS. PBS contains salt and this makes it electrically conductive. The more PBS, the higher the electrical conductivity.

Our standard diluent uses 1-in-9 PBS (phosphate-buffered saline). This gives an electrical conductivity of around 1850 µS/cm at 25 ℃.

You reduce the conductivity of the diluent when you reduce the PBS contents. In practice, we often use a dilution factor of 1-in-10 and 1-in-20. Of course, you can use any dilution factor that works for you.

Note that each brand of diluent may have a slightly different conductivity. It might be necessary to adapt the diluent to your room temperature and brands of media

Combined

We pair a high-conductivity growth medium with a low-conductivity diluent.

For most growth media, our regular diluent (1-in-9 PBS) works just fine. However, we need to adjust the diluent recipe slightly (use less PBS) as the conductivity of the growth media increases.

We target a conductivity of approximately 1850 µS/cm. This is right in the middle of the 1 500–2 200 µS/cm allowed range.

Consult the table below to see the diluent that we recommend for each growth medium.

Growth medium	Dilution factor 1:100 for use at 20–23 ℃.
PBS (phosphate-buffered saline)	Use 1-in-9 PBS
TSB (tryptic soy broth)	Use 1-in-9 PBS
BHI (brain heart infusion)	Use 1-in-9 PBS
LB (Luria–Bertani medium)	Use 1-in-10 PBS
MRS (De Man–Rogosa–Sharpe agar)	Use 1-in-20 PBS

Keep your sample vial at room temperature to avoid surprises

For example, if your sample is refrigerated, wait for it to warm up.

The electrical conductivity of the diluent changes a lot with temperature. A mere 5 °C increase can add about 200 µS/cm.

See Hit the right conductivity for details.

I dilute 1:10 or less

Is your sample stock close to the lower limit of detection (30 000 cells/mL) already? Then this section is for you.

You want to use the lowest dilution factor possible to keep the cells/mL concentration above the lower limit of detection. For low dilution factors, the influence of the growth medium increases.

In fact, we want to use a zero-conductivity diluent (no added salts). Preferably, ultrapure water. Here are some examples:

• Milli-Q® Lab Water
• Water for injection (WFI)

We target a conductivity of approximately 1850 µS/cm. This is right in the middle of the 1 500–2 200 µS/cm allowed range.

At low dilutions (1:10 or less), use ultrapure water as diluent.

The table just below shows how many mL of zero-conductivity diluent to add to 1 mL of growth medium. The brackets is the dilution factor (DF) relative to the bacterial concentration of the original sample.

Medium	20.5 °C	22.5 °C	24.5 °C
PBS (phosphate-buffered saline)	7.6 mL (DF 8.6)	7.8 mL (DF 8.8)	8.2 mL (DF 9.2)
TSB (tryptic soy broth)	7.0 mL (DF 8.0)	7.3 mL (DF 8.3)	7.6 mL (DF 8.6)
BHI (brain heart infusion)	7.8 mL (DF 8.8)	8.1 mL (DF 9.1)	8.6 mL (DF 9.6)
LB (Luria–Bertani medium)	9.8 mL (DF 10.8)	10.3 mL (DF 11.3)	10.7 mL (DF 11.7)
MRS (De Man–Rogosa–Sharpe agar)	50.4 mL (DF 51.4)	53.4 mL (DF 54.4)	55.7 mL (DF 56.7)

Keep your sample vial at room temperature to avoid surprises

For example, if your sample is refrigerated, wait for it to warm up.

The electrical conductivity of the diluent changes a lot with temperature. A mere 5 °C increase can add about 200 µS/cm.

See Hit the right conductivity for details.

Example

The ambient room temperature is 22.5 ℃. You have 1 mL of bacteria suspended in TSB. To get an appropriate conductivity, you add 7.3 mL of Milli-Q® water.

Motivation

The conductivity is our contrast agent to distinguish the conductive, salty cytoplasm inside the intact cells from the conductivity in the liquid that surrounds the exterior of the bacteria (the diluent). The electric measurement principle in BactoBox® only works if this conductivity contrast is high enough.