mec

Spectrophotometer

4 min read

Procedure

  1. Turn on the spectrophotometer GENESYS20

    • Allow the instrument to warm up for at least 10 minutes if required by the manufacturer.

  2. Set wavelength

    • Adjust the spectrophotometer to 640 nm.

  3. Prepare the blank

    • Fill a clean cuvette with 1 mL (or the required volume) of the same sterile medium used to grow the culture.

    • Wipe the outside of the cuvette with a lint-free tissue to remove fingerprints or droplets.

  4. Blank the instrumen

    • Insert the blank cuvette into the holder, aligning the transparent sides with the light path.

    • Close the lid and press “Blank” or “Zero”.

    • Wait until the display reads 0.000 absorbance (or 100% transmittance).

  5. Measure the sample

    • Mix the microbial culture gently by inversion or pipetting (do not vortex to avoid cell lysis or bubbles).

    • Transfer 1 mL of the culture into a clean cuvette.

    • Wipe the cuvette and place it in the same orientation as the blank.

    • Record the OD₆₀₀ value.

  6. Dilute if necessary

    • If the OD₆₀₀ value exceeds 0.5, dilute the sample (e.g., 1:10 in fresh medium), mix, and remeasure.

    • Multiply the measured OD₆₀₀ by the dilution factor to obtain the true OD₆₀₀.

  7. Clean up

    • Rinse cuvettes thoroughly with distilled water and let them air-dry or store according to lab policy.

    • Turn off the spectrophotometer if not in continuous use.

Notes

  • Always use the same type of cuvette (plastic or glass) for blank and samples.

  • Avoid bubbles and residual droplets—they can alter the reading.

  • Record all values in a lab notebook or data sheet, including dilution factors and time points.

1. The origin of OD₆₀₀

  • OD₆₀₀ (600 nm) became the standard wavelength for measuring bacterial growth, particularly E. coli, because:

    • Early photoelectric colorimeters and spectrophotometers (mid-20th century, e.g., Klett-Summerson colorimeter) used filters centered around 600 nm (“Klett filter #66” was 600 ± 20 nm).

    • At 600 nm, visible light scattering by bacterial cells is high, but absorption by medium components (especially proteins and riboflavin) is minimal.

    • It provided a convenient linear correlation between cell density and light scattering up to OD ≈ 0.8–1.0.

    • Consequently, OD₆₀₀ was widely adopted in bacterial growth protocols, and it became embedded in textbooks and instrument presets.

2. Why OD₆₄₀ (or OD₆₃₀–₆₄₀) is sometimes used

  • Yeast (e.g., Saccharomyces cerevisiae) cells are larger and more refractile than bacteria; they scatter light differently.

  • Early yeast researchers found that measurements at slightly longer wavelengths (620–640 nm):

    • Reduced multiple scattering (less overestimation at high cell densities).

    • Improved linearity between OD and actual cell dry weight or concentration.

    • Minimized interference from colored media or metabolic pigments (like flavins or residual YPD coloration).

  • Some instruments (especially older spectrophotometers in yeast labs) came equipped with a 640 nm or 620 nm filter, not 600 nm, simply because they were originally designed for turbidity or colorimetric assays (e.g., McFarland standards, enzymatic reactions).

  • As a result, OD₆₄₀ became the norm in many yeast laboratories — particularly in Europe (e.g., Scandinavian and German microbiology traditions).

3. Practical differences

  • Both wavelengths measure scattered light, not true absorption.

  • The actual difference in measured OD between 600 and 640 nm is typically small (≈ 5–10%), but not negligible if one is comparing across studies.

  • Empirically, some labs use correction factors: [ \text{OD}{600} ≈ 1.1 × \text{OD}{640} ] (though this varies depending on the spectrophotometer and culture conditions).

4. In the literature

  • E. coli and most bacteria → OD₆₀₀ (historical Klett tradition, standard in molecular biology).

  • S. cerevisiae and other yeasts → OD₆₄₀ or OD₆₃₀ (to improve linearity with biomass and because of larger cell size/scattering behavior).

  • Microalgae and colored cells → even longer wavelengths (e.g., 680 nm) to minimize chlorophyll absorption overlap.

5. Bottom line

The difference is not biochemical, but optical and historical:

OD₆₀₀ comes from bacterial tradition (Klett filter #66). OD₆₄₀ emerged from yeast work to improve scattering linearity and reduce medium interference.

Both are valid, as long as:

  • You consistently use one wavelength within your lab.

  • You report it explicitly (e.g., “Cell density was monitored at OD₆₄₀”) when publishing.

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