Geochemistry & Ecology of Red Mat Systems (GERMS) Undergraduate Summer Research Program
Week One – Practice Representative Water Analysis Jana K. Brooks

In preparation for our field research in Yellowstone, the first week was spent becoming familiar with all procedures that would be required in later weeks. We used the Luckimute watershed to do the site documentation and water analysis, and previously collected mat samples from sites in Yellowstone were used for pigment and microscopy studies.

Site Documentation

Luckimute Watershed Helmick State Park Methods: The following readings were taken at collection site: pH paper (dip into liter container and remove immediately to read): pH 5.5 Digital thermometer (measured at the site of water collection): 19.9 °C

Field Chemical Test Methods and Results (AccuVac)

Colormeter DR 1880 Methods: 4L of water were collected into 2L bottles. For each test, the colorimeter was zeroed using a filled 10ml sample cell. For each test, the appropriate program was selected, and corresponding filled AccuVac inserted and read. *Always wipe glass containers prior to inserting into Colormeter
Results:	(mg/L)
Total Chlorine	0.0
Chromium	0.01
Molybdenum	0.2
Nitrate	0.9
Nitrite	0.003
Sulfate	0.0
Sulfide	0.0

Water Filtering

Water Filtering Apparatus Methods: The apparatus was assembled as seen here. Methods were aseptic. Once assembled, 1L of mixed sample water was added to the glass chimney and then the vacuum was turned on. After filtration was completed, the filter paper was transferred to a sterile 50 ml conical and labeled.
Nate Labeling Conical Methods: For each liter filtered, the following information should be recorded into the side of the conical: q Date q Site Name q 1^st liter, 2^nd liter, etc.

Representative Pigment Methods and Results:

Methods: Prior to being able use a mat sample for microscopy or pigment analysis, specific preparations must be made. For week one, I was using sample Jcl-02 Bott 053g of sample Using a disposable + mortar and pestle, the 550 µl GTE sample is ground until . evenly suspended.
50µl for Microscopy q 20µl view with microscope	250µl for Methanol Extraction q extracts the Bchl q gives phylum characteristic	250µl for “In vivo” Extraction q extracts Bchl plus membrane protein q gives genus and species characteristic

Results for Methanol Extraction Spectrophotometric Analysis Peaks at q 665 nm q 764 nm This correlates to: Green Sulfur Bacteria (660-669) And closely to: Purple/Proteobacteria (771) Gram positive Helicobacteria (756)
Results for “In vivo” Extraction Spectrophotometric Analysis Peaks at q 743 nm q 889 nm This correlates to: Purple/Proteobacteria (830-890) And closely to: Green Sulfur Bacteria (745-755)

Representative Microscopic Analysis Methods and Results:

Methods: Basic and Fluorescence microscopy was used to analyze the mat suspension.

Image – Visible Light

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Image – F Fluorescence

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Image – R Fluorescence

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Discussion

From a sample containing unknown bacteria, we were able to deduce the species, genus and phylum with the aid of absorption spectrum data. To further document the sample (however in this case a different water source was used), a chemical analysis was performed on the water as well as basic and fluorescent microscopy on mat samples to capture a visualization of the mat community. Applied to our studies in Yellowstone, these procedures will leave us with a very specific description of the mat itself, as well as the surrounding environment.