Uptake of Neutral Red Dye in Yeast Cells: Lab Report

Abstract

The aim of this laboratory experiment was to investigate the uptake of neutral red dye in Saccharomyces cerevisiae yeast cells using different concentrations of sodium azide as a metabolic inhibitor. The objective was to determine whether higher concentrations of azide more effectively block dye transport into the yeast cells. Contrary to our hypothesis, the results indicated no significant difference in absorbance between various azide solutions and the control group, suggesting that azide had no substantial impact on dye transport through the yeast cell membrane.

Introduction

Cell membranes play a crucial role in the transport of materials into and out of cells. Saccharomyces cerevisiae (Serrano, 1977) possesses a selectively permeable membrane, allowing certain substances to pass through more readily than others (Campbell et al., 2008). Active transport is a process in which cells expend energy, typically in the form of adenosine triphosphate (ATP), to move materials across the membrane (Campbell et al., 2008). ATP production is vital for maintaining cellular pH, which, in turn, affects the uptake of neutral red dye, as a reduced pH impedes its absorption (Repetto, 2008).

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Saccharomyces cerevisiae employs two types of active transporters through its cell membrane: primary and secondary active transporters (Stambuk, 2000). Sodium azide, a metabolic inhibitor, prevents the production of ATP (Rowan University, 2009). In a study involving Escherichia coli, sodium azide inhibited over 90% of ATP production (Noumi, 1987). Given these observations, it was hypothesized that azide, as a metabolic inhibitor, should effectively block dye transport into yeast cells.

Understanding how substances pass through various cell membranes is a common area of study.

Investigating the passage of neutral red dye through a yeast cell membrane offers valuable insights into cellular transport mechanisms. Previous experiments with a 10% azide solution and a control group showed similar absorbance results, except for minor errors at 0% and 2% dye concentrations (Figure 1). These similarities were determined using standard deviation error bars to assess the absorbance of azide and control treatments.

Since azide, a metabolic inhibitor, was expected to block dye transport, this study aimed to investigate whether higher concentrations of azide would more effectively inhibit dye transport. It was predicted that increasing azide concentrations would lead to greater inhibition of dye transport into yeast cells, although there was a possibility that azide would have no significant impact on dye transport.

Materials and Methods

The following materials and methods were employed in this experiment:

• Calculation of azide concentrations (10%, 20%, and 30%)
• Preparation of yeast suspension using yeast growth medium (56mM Glucose, 20mM HEPES, pH 6.8) and Saccharomyces cerevisiae
• Creation of four dye concentrations (0%, 0.5%, 1.25%, 2.5%) from a stock dye solution and yeast growth medium (YGM)
• Preparation of a diluted yeast solution by adding yeast suspension to fresh YGM
• Dispensing of the diluted yeast solution into four 15-mL centrifuge tubes
• Addition of substances to each tube:
  1. Tube 1: Fresh yeast growth medium (control)
  2. Tube 2: 10% sodium azide
  3. Tube 3: 20% sodium azide
  4. Tube 4: 30% sodium azide
• Labeling and preparation of 16 microfuge tubes with four different dye concentrations (0%, 0.5%, 1.25%, 2.5%)
• Transfer of control and azide concentrations into respective microfuge tubes with different dye concentrations
• Incubation of microfuge tubes for 30 minutes to allow transport to occur
• Centrifugation of tubes at 5000 rpm for 2 minutes
• Removal of liquid portion from tubes, avoiding disturbance of pellets
• Resuspension of each tube with fresh YGM (3 times)
• One additional resuspension before transfer of solutions into a microtiter plate
• Measurement of absorbances at 520 nm using a microspectrophotometer

Results

Figure 2 illustrates the relationship between dye concentrations and absorbance in control, 10% azide, 20% azide, and 30% azide solutions. Error bars, calculated using standard deviation, show significant overlap at each point, indicating no substantial difference in absorbance with varying dye concentrations. Notably, the only outlier occurred at 2.5% dye concentration with 20% azide, which warrants further investigation. Overall, the results suggest that azide concentrations did not significantly affect dye transport into yeast cells.

Discussion

The experimental results revealed that higher azide concentrations did not inhibit dye transport as initially hypothesized. This lack of support for the alternate hypothesis suggests that azide had minimal impact on dye transport through the yeast cell membrane. The overlapping error bars across all dye concentrations in the control, 10% azide, 20% azide, and 30% azide solutions imply no significant differences in absorbance between these treatments, further supporting the conclusion that azide did not substantially affect dye uptake.

The presence of a single outlier, observed at 2.5% dye concentration with 20% azide, raises questions about its origin. Possible sources of error include pipetting inaccuracies or the short incubation period (30 minutes) for dye transport. Previous studies have shown that the absorbance of neutral red dye into yeast cells increases with extended incubation time (Repetto, 2008).

It is noteworthy that while azide is known to inhibit various cellular processes, including respiration (Rikhvanov et al., 2001), it did not demonstrate a significant effect in this experiment. This divergence from expectations prompts consideration of potential factors influencing azide's effectiveness. For instance, differences in cellular conditions, such as temperature requirements, may impact the efficacy of sodium azide in yeast cells (Rikhvanov et al., 2001).

Conclusion

Upon completing this laboratory experiment, it became evident that higher concentrations of azide did not effectively block the transport of neutral red dye into Saccharomyces cerevisiae yeast cells. Contrary to the initial hypothesis, azide had no significant impact on dye uptake, as demonstrated by the overlapping error bars across all dye concentrations. The outlier observed at 2.5% dye concentration with 20% azide may have resulted from experimental errors or the relatively short incubation period.

Recommendations

For future investigations into cellular transport mechanisms and the impact of metabolic inhibitors, it is recommended to explore additional variables that could affect the results. Extending the incubation period for dye transport and considering variations in experimental conditions may provide insights into the effectiveness of azide. Furthermore, careful attention to pipetting accuracy and other sources of error is essential to ensure the reliability of experimental outcomes.