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How to Find the pH of Cytoplasm

Potential of hydrogen, or pH, is a measure of the acidity or alkalinity of a liquid. It is important in many fields, including biology, chemistry and environmental science. Measuring pH is simple in most situations. Litmus paper can be inserted into the fluid, and the change in color of the paper identifies the degree to which the fluid is acid or base. In cases where the fluid to be tested is within a cell, other methods have to be devised.

Things You'll Need

2̵7;,7̵7;-bis-(2=carboxyethyl)-5 (and -6)- carboxyfluorescein (BCECF), or other substance that has varying fluorescence under different pH conditions
Fluorescence spectrophotometer with double monochromators, or similar instrument for measuring fluorescence
Centrifuge
Predetermined calibration curves measuring correlation of intracellular BCECF and cytoplasmic pH

Instructions

- 1
  Cool the temperature of the cell sample to 41 to 50 degrees Fahrenheit. This allows for a better absorption rate of the fluorescent material by the cells.
- 2
  Load the cell sample with a fluorescence inducing substance such as BCECF. This is done by adding the BCECF to the cell medium, then stirring the mixture gently for 2.5 hours. The BCECF is absorbed through the permeable membrane of the cells.
- 3
  Separate the cells from the medium and any unabsorbed BCECF by running the sample through a centrifuge. Put the cells into a separate container.
- 4
  Heat the cell sample to 98.6 degrees Fahrenheit for 30 minutes, to approximate the conditions within the human body. If the cells under examination normally live in some other environment than the human body, adjust the sample to the appropriate temperature.
- 5
  Excite the cell sample by stirring. This helps to increase the fluorescent activity.
- 6
  Measure fluorescent intensity of the cell sample using the spectrophotometer. For best results, use excitation light at wavelengths of 500 to 510 nanometers. At these wavelengths, there is a greater spread of fluorescent intensity, so it is easier to differentiate the results. The fluorescent intensity is measured in relative fluorescent units (RFU), a ratio of the brightness of the substance in an excited, fluorescent state to its normal, unexcited state. The higher the RFU, the brighter the substance, in comparison to its normal state.
- 7
  Compare spectrophotometer results to pre-drawn calibration curves. In general, higher fluorescent means higher pH within the sample. With an excitation wavelength of 510 nm, a substance with a pH of 6.4 would show an RFU of about 20, while a substance with a pH of 9.9 would show an RFU of about 55.

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