Algae SAMPLING
Algal growth can potentially be inhibited by the addition of herbicides such as glyphosate (Round-Up) and diflufenican, which are present in these mesocosms. If algal growth is inhibited, then there will be less biomass and thus less chlorophyll present on the tiles. After using spectrophotometers to measure the absorbance of your samples, you will be able to clearly see which ponds had higher or lower algal growth rates, and possibly a correlation between different treatments!
Materials
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Arm length protective work gloves
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Simple latex gloves
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Bucket of bleach
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Water bucket or tap for rinsing
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Test tubes (1x per mesocosm)
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Toothbrush
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Squirt bottle filled with water
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Funnel
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Vacuum filter pump
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Whatman filter papers
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Centrifuge
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Spectrophotometer
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Ethanol
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Procedures
​Sample collection
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Once equipped with the arm-length work gloves, dip them in bleach and rinse with water.
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Carefully remove the tile from the mesocosm bottom to minimize disturbances to the algae.
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Hold the tile over a test tube fitted with a funnel. Carefully scrape the surface using a toothbrush and rinse into the test tube using the squirt bottle. Make sure to rinse any remaining matter stuck to the funnel into the tube.
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​​This process should be performed in pairs, with the participant equipped with arm-length gloves holding the tile and their partner equipped with simple latex gloves executing the scraping. (see illustration).
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Return the tile to the mesocosm bottom.
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Repeat for all mesocosms. Between samplings, make sure to bleach and rinse the long work gloves, toothbrush and funnel as well as change latex gloves to prevent eDNA contamination (see illustration).
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Freeze samples if you intend do not intend on analysing them on the next day. Otherwise proceed to the steps outlined below.
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Spectrophotometer analysis
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If frozen, defrost the samples. Make sure all tubes are kept in darkness (see illustration).
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Filter each sample with a vacuum filter pump through Whatman filter papers (see illustration).
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Rinse tube and fill with 20m pure ethanol.
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Place the filter paper and sample back into its tube.
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Shake and leave in the fridge for 20 hours.
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Turn on spectrophotometer and leave to warm for 15 minutes.
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Centrifuge at 3000g for 6 minutes (see illustration).
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Adjust zero by pipetting pure ethanol into a cuvette, placing it in the machine’s chamber, closing the lid and pressing zero. Only hold cuvettes by their frosted tips (see illustration).
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Use a pipette to add supernatant from your sample to a clean cuvette, place into the chamber and close the lid.
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Select wavelength: 665.
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Record absorbance.
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Adjust zero with pure ethanol.
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Select wavelength: 649.
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Record absorbance.
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Repeat for all samples.
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Calculating chlorophyll-a and chlorophyll-b
The concentration of chlorophyll a and b can be calculated using the following equations:
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Chl a = 13.70 × A665 - 5.76 × A649
Chl b = -7.60 × A665 + 25.8 × A649
Potential responses
Algal growth rates are known to respond positively to increases in temperature. These increases could be exponential, linear, or eventually plateau after reaching a certain "maximum" temperature. The graph below illustrates these potential response curves.
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ILlustrations
Bleaching between samplings
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It is crucial to bleach and rinse materials that come into contact with either the mesocosm or collected sample to prevent eDNA contamination.
Algal scrape
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While your partner holds the tile, carefully scrape its surface with a toothbrush and rinse the algae into a 50mL Falcon tube using a squirt bottle and funnel.
Keeping samples in the dark
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Light can degrade chlorophyll samples, so it is essential to keep the samples in darkness until they have been read in a spectrophotometer. This image shows the tubes covered in aluminum foil, but any cover (e.g. black garbage backs, dark cloth, etc.) can be used so long as it blocks out any light.
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Sample filtering
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Using a vacuum pump and Whatman filter paper, the excess water can be removed and the algae sample filtered onto the filter paper. This paper will then be soaked in 100% ethanol for 20 hours and then read in a spectrophotometer.
Example: Filter paper soaked in ethanol and centrifuged sample
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The sample must be centrifuged before it can be put in the spectrophotometer. On the left you can see the filter paper soaked in ethanol for 20 hours (extracting the chlorophyll). In the right tube (Eppendorf) you can see there is supernatant at the top of the tube. This will be put in a cuvette and analysed in the spectrophotometer.
Zeroing the spectrophotometer
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Before the spectrophotometer can analyse the chlorophyll samples, you need to "zero" it using a baseline solution. In this case, the cuvette is filled with pure ethanol. It is important to do this each time you measure a new chlorophyll sample.
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You can also see the individual is holding the cuvette by the "frosted" sides, where the light will not go through. If you accidentally hold the cuvette by the transparent side, it needs to be cleaned, as the transparency might be compromised.
