Mouse Gastric pH assessment

Equipment and Solutions:

Microsensor Micrometer (Unisense)

PH-N pH Microelectrode (Unisense) 

            Note: The pH electrodes will typically last 3-6 months with regular use, but can last longer.

REF-NP Reference electrode (Unisense)

50 mL Falcon tubes, or other wide-mouthed containers that can be filled with buffers and fit both Unisense probes simultaneously.

pH Standard Buffers:

            pH 7 (VWR Chemicals BDH Cat # BDH5046-500ML)

            pH 4 (VWR Chemicals BDH Cat # BDH5018-500ML)

            pH 2 (SPEX CertiPrep Cat# PH-BUFF2-500)

70% Ethanol (20-50 mL, for cleaning the probes)

Distilled water (20-50 mL, for cleaning the probes)

Kimwipes (for cleaning the probes)

Dental wax (Electron Microscopy Sciences Cat # 72660)

Surgical tools for necropsy

Procedure:

1. Polarize the microelectrodes: turn on the Unisense Microsensor Micrometer and plug-in the pH and Reference electrodes to the appropriate port. 
2. Submerge the tips of the electrodes in pH 7 standard buffer and allow them to sit for 5-10 minutes or longer to polarize, until the mV reading is stable. 
3. Euthanize the mouse by CO₂ asphyxiation or other approved form of euthanasia. 
4. Using surgical tools, cut open the peritoneal cavity and locate the stomach under the liver. 
5. Excise the stomach by cutting at the esophagus and duodenum (approximately 0.5 cm proximal and distal to the stomach) and place it on a sheet of dental wax or some other hydrophobic surface, to limit drying.  
6. Remove the electrodes from the buffer and clean them by dipping in distilled water followed by 70% ethanol. Gently wipe the electrodes down using a Kimwipe to dry. Do not leave the electrodes dry and exposed for more than 10 minutes at a time. 
7. Insert the reference and pH electrodes in the excised stomach. This can be done by either piercing the gastric tissues with the electrode needles, or by cutting small openings in the forestomach and corpus to insert one electrode in each, which can help limit force on the electrodes and helps extend their working lifespan. 
8. Keeping the electrodes still with the tips inserted into the gastric corpus, allow the mV reading to stabilize and record the value.
9. Between readings, clean the probes with distilled water followed by ethanol as before.
10. To generate a standard curve, take readings of the pH standard buffers by inserting both probes in the solution and waiting for the mV reading to stabilize, rinsing the probes in water and 70% ethanol between each reading. Record the mV reading for each buffer and use these reference values to generate the standard curve. Note: The timing of this pH calibration step is optional and can also be performed only after taking readings from multiple mice. However, we have noticed that some stomach contents (for instance, highly acidic content) can shift the overall pH readings and lead to inaccurate measurements for other animals if using the same standard curve readings taken at the start or end of the process for all animals. We had the best success by taking a set of standard buffer readings after each stomach measurement and generating an individual standard curve for each biological replicate, reflecting the most accurate values for that animal.  
11. Using the best-fit line for the relative standard curve, convert the gastric mV readings to pH values. 

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