Question

Transport Across Membranes
Trans-membrane transport can occur in 3 different processes. Simple diffusion through an equilibrium channel facilitated diffusion and active transport. To examine these processes we are going to use yeast cells and some simple model systems.

Staining with Neutral Red
Take a 20ml sample of yeast culture and divide equally into two boiling tubes, Ensure you agitate yeast culture when collecting and dividing to avoid settling.
•   Next add 5ml NaCO3 solution (1% aq) to each tube
•   Heat one boiling tube for 5 minutes at 95℃ in the water bath to kill the yeast.
•   Allow cooling
•   Take samples for methylene blue staining (see below) (use autopipette)
•   Add 1ml dye soltn (Neutral Red 1mg/ml(aq) ) to both boiling tubes (use autopipette)
•   Wait for 15 minutes and observe if there is any change in color

Separation of cells by centrifugation
•   Transfer a 10ml sample from each boiling tube to a 15ml centrifuge tube
•   Centrifuge at 2,500 rpm 5min. (this will be done for you)
•   Compare any difference in color in pellet and supernatant
•   Decant each supernatant carefully into a spectrophotometer cuvette
•   Read the two samples at 520nm in the Visible Spectrophotometer
Staining with Methylene Blue
•   take 100 µl of each yeast sample (agitate beforehand) and place in a microcentrifuge tube
•   add 100 µl of Methylene Blue solution (0.1mg/ml (aq) (use autopipette)
•   incubate for 5 min at RT

Microscope observations
•   prepare slides of all your 4 samples (from before centrifugation!) by placing a drop on a microscope slide and adding a coverslip.
•   observe at high magnification (x400)
•   count the proportion of colored and non-colored cells in a random sample of 100 cells.

Diffusion of Methylene Blue through Agar
•   Add 1 crystal of methylene blue to the surface of an agar plate
•   Record the distance which the color diffuses at 10 min intervals over 1 hour

question:

discuss the differences between living and killed yeast cells .which transport processes do you expect to be present? provide molecular structures of dyes used. Explain the usage of the dyes in this experiment and how this works.

Answer 1

In this case, we need some results in order to fully discuss this. However, we can predict what will happen. In the case of methylene blue, it is a cationic dye that will bind to negatively charged structures of the cell. Generally, it binds to the DNA. It is small enough to enter into the cell through diffusion. However, the cell must be dead because active enzymes within the cell will reduce this dye leading to a colorless observation. So, in the group of dead cells we will observe blue cells while in the case of living cells, we wont observe any color.

On the other hand, we will observe the opposite scenario when we add the neutral red dye. Cells are able to uptake and incorporate the neutral red dye into the lysosomes. IN order to this happen, the cellular machinery must be active. For this reason, we wont observe any coloration in the case of dead cells but we will observe red cells in viable cells.