Measuring Bacterial Growth by Optical Density

Summary

This video explains methods to measure bacterial growth, focusing on plating and optical density (OD). Plating involves diluting bacteria and counting colonies, but it's time-consuming. Optical density offers a quicker method by measuring how much light passes through a culture, as bacteria scatter the light. Absorbance at 600 nm (OD600) is typically used, with higher OD meaning more cells. The video emphasizes that OD readings are only accurate below a certain threshold and calibration is necessary to correlate OD to cell numbers. OD measurements are widely used for quick bacterial growth estimates.

Takeaways

• 🔬 **Plating Method**: Traditional way to count bacteria involves diluting and spreading them on petri dishes to form colonies.
• 🌐 **Optical Density (OD)**: A faster alternative to plating, OD measures the cloudiness of a bacterial culture caused by bacterial growth.
• 🌟 **Absorbance**: OD is also known as absorbance, which measures the amount of light transmitted through a material.
• 🌡️ **Cuvette Measurement**: OD is measured using a cuvette with a controlled thickness of 1 cm to standardize the light path.
• 📏 **Transmittance**: The ratio of light received to light transmitted through a sample is called transmittance.
• 📉 **Negative Logarithm**: Absorbance is calculated as the negative logarithm of transmittance.
• 🌿 **Light Scattering**: At 600 nm wavelength, light is easily scattered by particles the size of bacteria, which is useful for OD measurements.
• 🔍 **Non-Specific Measurement**: OD measures the presence of particles, not their color or composition.
• 📈 **Calibration**: To relate OD to the number of bacteria, a calibration step involving dilution, plating, and colony counting is necessary.
• 🔄 **Linearity**: OD is linear with cell density, meaning a doubling of OD indicates a doubling of cell count.
• 🌐 **Pre-Existing Data**: For common bacteria like E. coli, the relationship between OD and cell count is often already known, simplifying the estimation process.

Q & A

What is one common method to measure bacterial growth?

One common method is plating, where bacteria are diluted and spread on petri dishes. Each bacterium forms a colony, and by counting the colonies, you can estimate the number of bacteria.

Why is plating considered a time-consuming method?

Plating is time-consuming because it requires multiple dilutions, preparation of plates, and waiting 12 hours or more for the bacteria to grow into colonies.

What alternative method is suggested to measure bacterial growth faster?

A faster alternative method is measuring optical density (OD), which provides an estimate of bacterial growth by measuring the cloudiness (absorbance) of the bacterial culture.

What is optical density (OD) in the context of bacterial growth?

Optical density (OD) refers to the cloudiness of a bacterial culture. As bacteria grow, they scatter light, making the culture more opaque. OD is measured as the absorbance of light passing through the culture.

How is optical density measured?

OD is measured by shining light through a cuvette containing the bacterial culture and comparing the amount of light that passes through to a blank media cuvette. The ratio of light transmitted to light received gives the transmittance, and the negative logarithm of transmittance gives the absorbance.

Why is light at 600 nm commonly used for measuring bacterial growth?

Light at 600 nm (OD600) is commonly used because it is in the green part of the visible spectrum and is easily scattered by bacteria, making it ideal for measuring their growth. Additionally, many bacteria, such as E. coli, don't produce pigments that would interfere with this measurement.

What is the relationship between absorbance and bacterial cell density?

The relationship between absorbance and bacterial cell density is linear. An increase in OD corresponds to an increase in the number of cells, meaning twice the OD equals twice the cell density.

What are the limitations of optical density measurements?

One limitation is that OD measurements are only accurate if some light passes through the sample. If absorbance exceeds a certain threshold, the readings may not accurately reflect the number of bacteria.

How is the relationship between OD and cell count typically calibrated?

The relationship between OD and cell count is calibrated by plating the bacteria, counting the colonies, and comparing the colony count to the OD. This needs to be done only once for specific bacteria, media, and equipment, after which estimates can be made.

What is the typical cell density for E. coli at an OD of 1?

For E. coli, an OD of 1 corresponds to approximately 10^9 cells per milliliter.