Technical specification

An image analyzer is indispensable for this technique.
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The following steps must be done:

  1. Prepare slurries of the rock sample
  2. Prepare the bacterial culture
  3. Incubate bacteria with slurries for prolonged periods
  4. Image analysis measurements
  5. Calculation of degradation

Rock sources
As substrates, rocks of diverse character can be used, such as, marble, granite, apatite, basalt, solidified lava, quartz, and limestone. Prepare as follows

If you use stones that were expose to the environment, rock fragments need to be submerged in 1N HCl solution overnight at 2833C to eliminate organic matter.

  1. Rinse several times with de-ionized water.
  2. Dry at 160C for 2 h.
  3. Pulverize rocks in a mill and sieve to particles smaller than 100 m.
Bacteria
Any bacteria capable of weathering rock can be used. It should be a cultivable strain.

Growth conditions

  1. Bacterial strains are initially grown in a general growth medium (nutrient broth, LB, yeast-peptone-glucose, etc. to logarithmic phase, usually with stirring.
  2. Harvest bacteria by centrifugation at 10005000 g for 20 min.
  3. Inoculum is washed three times in sterile, distilled water, and pellets are suspended in saline solution (0.85% NaCl) to a final concentration of 109 CFU ml1.
  4. Flasks containing (in g/l) mannitol, 5; glucose, 10; sucrose, 5, any nitrogen source, 5 (or any other synthetic bacterial growth medium containing carbon and nitrogen sources suitable for your tested strain) and the pulverized rock, 1.5, in 135 ml de-ionized water are each inoculated with 15 ml of a bacterial suspension.
  5. Flasks are incubated for 28 days at 30 C in a rotary shaker at 150 rpm. Samples are taken every week for image analysis.

Image analysis measurements
Weathering of rock particles, expressed as changes in various parameters, is quantified by measuring the number of powdered particles in a sample, particle diameter, and particle surface areas with an image analyzer (we used: Image ProPlus 4.5, Media Cybernetics, Silver Spring, MD) before and after inoculation with bacteria.
  1. The procedure uses 0.5 ml aliquots of a bacteria-rock slurry diluted 1:10 in de-ionized water. This dilution is based on preliminary, empirical determinations of the optimal dilution of suspensions for the image analyzer.
  2. To this concentration, add 0.5 ml 1% agar dissolved in 0.06 M phosphate buffer at pH 7.0 and mix at 45C.
  3. Aliquots of 500 l agar suspension are placed on slides containing a shallow well (23.85 22 1.5 mm; 787 l).
  4. A cover glass is placed on the powdered rock slurry-agar suspension and is slowly pressed down from the sides until the cover glass touches the sides of the slide. This maintains a uniform thickness of agar film.
  5. Direct measurement and counting of particles is performed using the 10X objective lens. We used an Olympus microscope, biological model, tri-ocular BX41 with phase-contrast (PH1).
  6. The image analyzer we used had software Image Pro Plus v. 4.5. Camera model Cool Snap Media Cybernetics connected to a standard computer system (Pentium III, Dell, 750 MHZ), but any other image analyzer will work.
  7. The number of particles and the characteristics in each of five individual fields on each slide is counted.

Calculations of number of particle per sample
The number of particles in a sample was calculated using the following equation:

P = PI x F x D,

where P is particles per ml; PI is the number of particles per image; F is the number of fields per chamber, and D is the dilution factor of the sample.

Here, PI is the data measured by the image analyzer and F is computed from the formula:
F = [volume of counting chamber in mm3 (area x depth)] / [volume of recorded image in mm3 (area x depth)]

An example from our study of calculation:
F = [23:85 x 22 x 1:5] / [1:2943 x 0:9616 x 1:5] = 422 (this study) and D = 20 (this study).
Therefore, P = PI x 8440 (this study).

Sample size and statistical analysis

  1. Samples (500 l) may be used and each should contain up to 50 x 106 particles ml1 of various sizes, but all smaller than 100 m.
  2. Triplicate samples were analytically assayed (three samples from each replicate) and experiments should be repeated 2 or 3 times.
  3. All data is adjusted to normal and tested with ANOVA or Students t test or other statistical analysis of your choice.
  4. Data is ranked from 0.1 to 90 m in diameter for size and 0.9 to 500 m2 for surface area.

Tips

  1. The initial quantity of particles can vary from 0.1100 x 106 ml1 of powdered rock because sieving of pulverized rock controls only the size of the particles, not the amount. Therefore, each sample has its own internal time 0.
  2. Cells of bacteria should be excluded by the software of the image analyzer; the maximum numbers of particles per sample was 100 x106.
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