---
tags: respirometry sop, macmanes
title: Respirometry SOP

---

# Respirometry SOP


NOTES:
    - FMS must be turned on for at least a half hour before use
    - Still up for debate: Baseline cage should not have bedding or water
        - [ ] ask Barbara to clarify. 
    - Up to 7 experimental cages can be mesured at once because the 8th cage MUST be an empty baseline cage
    - For sable "!" means a changing value, once it stabilizes the "!" goes away
    - Rapid multiplexing of chambers would be below 5 min/cage
    - SS4s should all be at the same flow rate (1800 ml/min as of 1/30/2020)
        * to change turn round metal knob on top of SS4
    -15-30 min to cycle back to the same cage is pretty much the classic way of taking measurements 



THINGS TO RECORD BEFORE EXPERIMENT:
    - mice numbers
    - mice weights
    - cage numbers
    - .csv file used
    - time and date experiment started
    - length of experiment 
    
    
    
TO RUN (AN EXPERIMENT OR ANYTHING):
    - Turn on SS-4’s
    - Turn on MUX
    - Turn on FMS
        * There are three on switches (FLIP THEM ALL UP)
        * Flip the ‘Baseline’ switch in uper left to COMPUTER to start measurements (e.g., the setup file from the computer will control chamber switching)
    - Start manual recording on the video cameras
    - Start logging temperature data with the hobo logger (not necessarily but lets do it anyway)
  
  
  
CHOOSE OR CREATE A NEW CAGE SAMPLING SCHEME:
    -CSV file format:
	Sample#    output code    marker            pause
	0		7		S		0
	300		0		B		0
	600		1		0		0
	900		2		1		0
	.		.		2		0
	.		.		.		0
	.		.		.		0
	3600    	.		.		0
    
    - Explanations:
	sample# = amount of time in seconds to measure each chamber for
	+300s/5min = each chamber measurement thereafter
	3600 = (1*3600)
	3600 = Max Run Length (to be input manually on Digital Control tab)
	1 = number of hours to save one file 
	3600 = # of seconds/hour
    - NOTE: don't make these over three hours becuase files are not written to the disk until the end of the max run length. 
    
    
TO LOAD A SETUP FILE - TO START A NEW RUN
    - Open Expedata
    - File -> Setup Data Acquisition
    - Click "Use the UI" -> Click ok
    - Click “Setup” button in lower lefthand corner
    - Load Setup -> select *.stp file of interest
        Current one: "/Dropbox/Cactus_Mouse_Physiology/respirometry files/setup_files/2020_always_check_sampling_and_digitial_contol.stp"
    - Click the "Sampling" tab, enter the number of minuets it will take for one recording to finish in "Max Run Length" (information comes from the cage_sampleling_scheme csv)
    - Click the "Files Tab" browse for folder you plan to save it, type a root file name, type any notes you want
    - Click the "Digital Control" tab -> the "During Rec" tab -> "Load events" -> "/Dropbox/Cactus_Mouse_Physiology/respirometry files/cage_sample_schemes/8.csv"
        Must correspond with the number of minuets typed into the "Max Run Length"
        Expedata will automaticlly add one second to this. Just click "OK"
    - Click green checkmark to start run (opens new dialoge)
    - Click red arrow to actually start recording. 
    - watch it for 5 minutes to make sure run is actually working. 
        
        
TO CREATE A NEW SETUP FILE:
Set channels before macro use:
    - Open Expedata
    - Open Expedata
    - File -> Setup Data Acquisition
    - Click "Use the UI" -> Click ok
    - Set number of channels to 7
        - O2
        - CO2
        - H2O-WVP
        - BP
        - SS4_FR
        - subFR
        - Respchan
        - Channel 8 (temperature in Degrees C)
    -linear transform all channels (pg 7 manual)
        Example: O2
            * Range = 19 - 21.5
            * A = 19
            * B = 0.5 = (21.5-19)/5 = (max - min) / total # volts
        Repeat for CO2, H2O-WVP, SS4_FR, BP, subFR
        Do not transform the Respchan or Channel 8
    - Go to "Sampling" tab
    - Set 1 second per sample
    - Set the Max Run Length to the total length of your experiment in minutes (e.g., 3600 = (1*3600))
	e.g., If running experiment for 1 hour, this value would be 3600
    - Go to the "Digital Control" tab
Select ‘During Rec’ -> Load Events (select .txt or .csv file created in excel)
	
    
TO TURN EVERYTHING OFF:
    - Flip ‘Baseline’ switch to Baseline to decompress entire system
    - Turn off FMS
    - Turn off MUX
    - Turn off SS-4’s
    - stop manual recording with the video cameras
    - download the hobo logger data to the computer

_______________________________________________________
 
Some Useful Formulas and Calculations:
    - www.respirometry.org for a useful set of water vapor calculators. 
 
    - Saturating Vapor Pressure at Temperature T:
    - Temperature is in degrees Celsius, Vapor Pressure will be in Pascals
	WVP = 611 * 10^ (7.5*T/(237.7 +T))
    - As you can see from the above exponential formula, vapor pressure is a strongly non-linear function of temperature. This is a simple equation: The humidity module in the FMS uses a far more complex proprietary equation.

Relative Humidity from Dew Point Temperature and Actual Temperature:
    - Calculate WVP at dew point using the vapor pressure formula
    - Calculate WVP at actual temperature using the vapor pressure formula
        RH % = (WVPd/WVPa)*100%

Absolute humidity from water vapor pressure:
    - Absolute humidity in kg/m3 = P/(T*Rw)
    - Where P is vapor pressure in Pascals, T is temperature in degrees Kelvin (T +273) and Rw is the gas constant for water vapor . Rw= 461.5 J/(kg*Kelvin).

Conversions of volume units
	- 1 kg/m3 = 1 g/liter = 1 mg/ml
	- 1mg/ml = 1000 ug/ml

Water Loss Rates:
    - Rate of water loss = absolute humidity x flow rate (in same volume units)	
	e.g. 0.001 mg/ml * 100 ml/min = 0.1 mg/min = 6 mg/hour

Mole Fraction of Water Vapor from Water Vapor Pressure:
	- Mole Fraction = Water Vapor Pressure/ Total Pressure where both pressures are in the same units.