Measuring the concentration of Staphylococcus aureus bacterial suspension using spectrophotometry

There is a technique for roughly measuring the concentration of Staphylococcus aureus bacterial suspension using spectrophotometry. The goal of the experiment is to create a standard curve by simultaneously measuring the absorbance of a bacterial solution and performing a plate colony count. Once the standard curve is established, the absorbance value can be used to indirectly determine the concentration of the bacterial suspension.

The experiment uses a UV Visible Spectrophotometer or Visible Spectrophotometer  with a wavelength of 600nm and a cuvette thickness of 1cm. The blank control and diluent is a 0.9% sterile sodium chloride solution. The bacterial species used is Staphylococcus aureus, cultured in a nutrient broth medium.

The experimental procedure is as follows:

1. Staphylococcus aureus is inoculated into a nutrient broth medium and cultured in a constant temperature incubator at 30-35℃ for 18-24 hours.
2. The cultured bacteria are then diluted with a 0.9% sterile sodium chloride solution in a sterile positive-pressure room to create seven different concentrations of bacterial suspension. This is done by adding 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, and 0.7ml of the nutrient broth culture to 10ml of 0.9% sterile sodium chloride solution.
3. The two methods for determining bacterial concentration are then performed:
   • Plate Colony Counting Method: 0.2ml of each bacterial suspension is spread onto two nutrient agar plates. After incubation at 37℃ for 72 hours, the colonies are counted on plates with 30-300 colonies. The average colony count is used to calculate the bacterial concentration of the original solution, which serves as the standard concentration.
   • Spectrophotometry Method: Five of the original bacterial suspensions are measured for their absorbance at a wavelength of 600nm. A 0.9% sterile sodium chloride solution is used as a blank control, and the absorbance values for each original bacterial suspension are recorded.

A standard curve is then created by plotting the absorbance values against the corresponding standard bacterial concentrations obtained from the plate colony counts. The table below shows the absorbance values recorded for each of the seven original bacterial suspensions: