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Measuring Optical Density

Use this page for measuring the bacterial culture concentration via optical density.

Purpose

When using bacteria in a cell culture or any experiment, it is vital to know how much bacteria and at what concentration you are using. Bacterial concentration in a solution can be measured using a spectrophotometer to measure optical density at the 600nm wavelenth, to quantify this bacterial concentration from a sample.

Safety

• Wear gloves and clean materials
• Spray down all working materials with 70% Ethanol.
• Try not to touch the sides of tubes with pipette tips.
• Do not touch the rims of the flasks or tubes to work with.

Materials

• Rotin Lab’s Nanodrop 2000c spectrophotometer
• Cuvettes
• Open-top tube with bacterial culture
• PBS
• Centrifuge for eppendorf tubes
• Pipette p200 and p1000

Procedure

1. Alliquot the entire open-top (~5mL) tube into an eppendorf tube and with a balance, centrifuge at 4000rpm for 5 minutes.
2. Take out from the centrifuge, aspirate and discard the supernatant gently, replacing with 2.5mL of PBS and resuspend.
3. Centrifuge at 4000rpm for 5 minutes again and discard supernatant again. Add 1mL of PBS and resuspend.
4. Take an empty cuvette and pipette in 1mL of PBS. Place inside the spectrophotometer with the notch facing the arrow.
5. Opening the nanodrop application, select “New workbook” and “Cell Culture”. Run “Blank” at the top left of the application to zero the machine.
6. In a new cuvette, pipette in 900μL of PBS and 100μL of the bacterial solution. Resuspend/mix.
7. Place in the spectrophotometer and click “Measure”.

The desired number is A600, which should ideally line up between 0.3 and 0.8. This is the absorbance at a wavelength of 600nm. From there, the bacterial concentration can be calculated below:

**Insert formula text box and formula for reference.

Quality Control

• Check the clarity of the supernatant and the visibility of the pellet.
• Make sure to complete the methods quickly and accurately.
• Make sure the bacterial culture open-top tube is cloudy.
• Work with the pelleted bacteria gently, as to not disturb.

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