Geochemistry & Ecology of Red Mat Systems (GERMS) Undergraduate Summer Research Program
Week Three Microscopy, Enumeration, and Digital Imaging Jana K. Brooks

Mat Layer Processing:

Hillside green mat sample collected 6/28/2004
Hillside green mat sample collected 6/28/2004 Total mat weight: .0361 grams Mat sample was prepared as in week one for microscopy and pigment analysis.
Conical with Filter from Yellowstone Filament samples from filter were retrieved by adding 10ml GTE and agitating using a vortex

Microfuge Tubes for Centrifuging q 8-1 ml aliquots were centrifuged for 10 minutes and 900µl of supernatant (containing no filaments) was discarded q All pellets were combined, resuspended and centrifuged again for 10 minutes q 750µl of supernatant was discarded, and remaining 50µl was used for counting purposes
Cell-Vu® used for grid-based counting q 4µl loaded at a time for viewing q no filaments were seen in our samples

Site: Hillside

50µl suspension of sample and GTE is used for microscopy

Image – Visible Light

10X	40X

Image – F Fluorescence (Filter 2)

10X	40X

Image – R Fluorescence (Filter 3)

10X	40X

Grid-Based Counts: (=no filaments were observed in filter samples)

Replicate 1 (Jen’s)	Replicate 2 (Jana’s)
10X	10X
20X	20X
40X	40X

Chemical Analysis of Acid-Digested Samples for Metal Assessment

Specific Post-Acid Digestion Results: for Hillside

Preparation for Acid-Digested Samples

Rinsing pH meter
Adding NaOH to water sample

Chemical Test	Photo of test	mg/L
Aluminum		0.006
Copper		0.0
Total Iron		0.08
Zinc		0.13

Pigment Analysis

Methanol Extraction:

Methanol Extraction 250µl suspension of sample and GTE is used for Methanol Extraction q extracts the Bchl q gives phylum characteristic
Methanol Extraction Results Peaks at q 442 nm q 616 nm q 665 nm q 769 nm This correlates to: Green Sulfur Bacteria (660-669) And closely to: Purple/Proteobacteria (771) Green Nonsulfur Bacteria (667)

In vivo Bead-Beating:

In vivo Bead-Beating 250µl suspension of sample and GTE is used for Bead-Beating Extraction q extracts Bchl plus membrane protein q gives genus and species characteristic
In vivo Bead-Beating Results Peaks at q 436 nm q 614 nm q 673 nm This correlates the closest with Gram Positive Heliobacteria (670, 788)