Effect of Fluorophore Labeling

Yeast was suspended in 90 ml of 10 mM phosphate buffer at pH 7.5 and well stirred. Fluorophores were then added. Table 8.1 gives details of the fluorophores. The suspension (now 100 ml in volume) was gently stirred for 30 min to allow the yeast cells to absorb the fluorophores at 20+2°C. The suspension was then washed to remove excess fluorophores and EPS. A washing procedure was used similar to that reported by others [7]. The suspension was placed into two 50 ml centrifuge tubes, and centrifuged at 2500 rpm for 10 min. The supernatant was removed and the yeast pellet was resuspended with buffer. The process was performed three times after which the yeast suspension was further diluted to the required concentration using the same buffer. In all experiments fresh yeast suspensions were prepared on the same day and used within a few hours. 

As the washing procedure removes extracellular material such as EPS, one is only able to use MPM to investigate, with confidence, the behavior of washed cells. 

Fluorophore Color Peak excitation (nm) Peak emission (nm) Item(s) labeled stock cone. (gL ) Volume added Comment 

Calcofluor white (CW) Blue 440 500-520 Cell wall 0.5 10 mL Used in all experiments 

Acridine orange (AO) Alkaline cell green Acid cell red/purple 502 526 Cell acidity 10.0 33 pL Not used in yeast/protein mixtures. Slightly labels the membrane green 

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Figure 8.4 The effect of fluorophore labeling and suspension washing of yeast suspensions. A Unwashed fluorophore labeled suspension concentration 0.50gL , A unwashed nonlabeled suspension concentration O.SOgL, washed fluorophore labeled suspension concentration 0.37gL ,...

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