Phase I:  Isolation of Bacillus Subtilus

I. Enrichment Media Construction
     A. Concoct a solution composed of the following materials in 2 liters of
          distilled water:
            · 60.0 g soluble starch
            · 40.0 g agar
            · 10.0 g Polypeptone
            · 10.0 g Yeast Extract
     B. Slowly heat solution to a boil.
     C. Autoclave solution for 15 minutes at 15 psi pressure.
     D. Swirl media to suspend starch.
     E. Pour media into petri dishes.
     F. Allow plates to solidify overnight.

II. Isolation and Maintenance of Bacillus subtilus
     A. Obtain two soil samples from the following locations:
           1. Tree by the Microbiology Building
           2. Behind McKeldin Library
     B. Sprinkle Soil Sample one on a plate
     C. Sprinkle Soil Sample two on a plate
     D. Perform a serial dilution of soil samples one and two
           1. For each sample, mix 0.06 g of soil in 1000 mL of saline solution using
               a vortex machine.
           2. Transfer 100 mL of soil solution to a test tube containing 900 mL of
               saline solution resulting in a 10-1 dilution.
           3. Do this eight more times.
           4. Plate 100 ml of the from the 10-1, 10-3, 10-5, 10-7, 10-8, and 10-9 dilution
               tubes.
     E. Incubate all of the tubes for 24 hours at room temperature, 25°C, at a pH
         between 5.5 and 8.5.
     F. The serial dilutions were not successful.
     G. Streak bacteria that have a cream colored wrinkled morphology (Bergey’s
         Manual of Systematic Bacteriology Volume 2 1130).  On the plate with the
         soil sample from the tree by the Microbiology building, a yellow-beige
         colony grew.  On the plate with the soil sample from behind McKeldin
         Library, a coral-like beige colony grew.
     H. Incubate plates at room temperature for 24 hours.
      I. To maintain the bacteria, restreak the plates.
 

III. Identification of Bacillus subtilus
      A. Begin series of tests on both bacterial colonies in order to decipher which
           bacterial colony is truly Bacillus subtilus.  They are the following (the
           expected result is indicated in parentheses next to the test):
              1. Gram Stain (Positive)
              2. Endospore Test (Positive)
              3. Motility Test (Positive)
              4. Catalase Test (Positive)
              5. Thioglycate Test (Negative)
      B. Gram Stain
              1. Soil Sample from the tree by the Microbiology building:
                  Pink and Purple Rods, which indicates that the culture is not pure
              2. Soil Sample from behind McKeldin Library:
                  Gram Positive Purple Rods, as expected

*After consultation with Dr. Smith, we amended the above protocol due to the fact that starch is not the only carbon source in the above media. Thus, we cannot be sure that these bacteria definitely degrade starch.  In fact, after flooding the above bacterial plates with iodine to check that the bacteria do degrade starch, we did not see any changes in the plates.  A clearing around the bacteria would have indicated the presence of a starch-degrader.
 
 

Phase II:  Isolation and Maintenance of Starch-degrading Bacteria

I. Isolation of Starch-degrading Bacteria
     A. Obtain a second soil sample from the tree by the Microbiology building.
     B. Sprinkle soil sample on three of the same plates used previously.
     C. Incubate the plates for 48 hours at room temperature, 25°C.
     D. Properly discard the plate that mold grew on.
     E. Tape a grid onto the back of two new plates. *See Figure 1*
     F. Swab a bacterial colony.
     G. Put it on the same numbered square of the grid on both plates.
     H. Incubate the plates for 48 hours at room temperature.
     I. Despite the growth of mold on the plates, flood one of the plates with iodine.
     J. By comparing this flooded plate to the control plate, identify any clearings
         around the colonies.  This indicates that the bacteria degrade starch.
     K. Streak three different colonies onto new plates.
     L. Incubate the plates for 48 hours at room temperature.
     M. Discard a plate if there is an abundance of mold growth.
     N. Restreak the bacterial colonies.

II. Identification of Starch-degrading Bacteria
     A. Put enough bacterial samples on slides so that a few characterizing tests can be
          performed.
     B. Perform the Oxidase Test
            1. A positive result is indicated by the bacteria turning blue.
            2. A negative result shows no color change
     C. Confirm Bacteria Degrades Starch
            1. Flood Plates with Bacteria
            2. A clearing around the bacteria indicates that the bacteria degrades
                starch.
     D. Perform the Catalase Test
            1. The formation of bubbles indicates a positive result.
            2. No bubbles indicates a negative result
     E. Perform the Simple Stain
     F. Perform the Gram Stain

Figure 1:  This is an example of the grid we used during Phase II of our procedure.  Two of these plates were used, and the same bacterial colonies were swabbed onto each grid, creating two identical plates.  One of these plates was flooded with iodine to determine which colonies contained starch-degrading bacteria.  The other plate was used as a control.  It was left untouched, and then the colonies that degraded starch on the iodine plate were collected  from this control plate and streaked.