Procedure and Observations for the first set of Avian Sphere Growth Experiments
Notes of Maria Angelella
The following research was carried out as part of UG research at West Chester University, supervised by James Falcone. The ideas to attempt to grow spheres in a gradient gel system were generated in conversations between Eldon Braun, Giovanni Casotti and Jim Falcone. Blaise Frost assisted with the acquisition of and the proper preparation of reagents used.
January 24, 2007
Preparation of 1% Tryptic Soy Broth: Dissolved 0.2509g TSB in 25mL volumetric. The flask was filled to line with DI water. Solution was yellow and not viscous.
January 26, 2007
Preparation of stock solution: The chemicals in Table 1 from MgCl2·6H2O to sodium urate were added to a 200mL volumetric. The volumetric was filled to the line with DI water. This solution was twice the desired concentration. Not all of the constituents dissolved, thus giving the solution a white cloudy hue. Using pH paper, the pH of the solution was measured to be 7.
Table 1
Compound / Source / Amount (g) / mmol/L / g/LMgCl2·6H2O / Fischer Scientific / 0.9759 / 24.00 / 4.88
NaCl / Fischer Scientific / 2.1032 / 180.00 / 10.52
Na2SO4 / Fischer Scientific / 0.5691 / 20.00 / 2.84
Na3Cit / Fischer Scientific / 0.2592 / 4.40 / 1.29
NaOx / Baker & Adamson / 0.0080 / 0.30 / 0.04
KH2PO4 / Fischer Scientific / 0.2728 / 10.00 / 1.36
KOH / JT Baker / 0.0000 / 0.00 / 0.00
NH4Cl / EMD / 0.1062 / 10.00 / 0.53
Urea / Fischer Scientific / 0.3600 / 30.00 / 1.80
Creatinine / Fisher Scientific / 0.4410 / 16.80 / 2.20
1% Tryptic soy broth / Weber Scientific / 4.0068 / 0.00 / 20.00
NaHCO3 / Mallinckrodt / 0.8401 / 50.00 / 4.20
Sodium Urate / Pfaltz & Bauer / 0.0314 / 0.80 / 0.15
Agarose / EM Science / 2.4009
Chicken Serum Albumin / Equitech-Bio Inc. / Variable / Variable / Variable
CaCl2·2H2O / Fischer Scientific / Variable / Variable / Variable
Preparation of supersaturated sodium urate solution: To 134.6068g of DI water, 1.1938g sodium urate was added. Not all of the sodium urate dissolved. Solution was milky white. Put the solution on low-medium heat on the hot plate for about 5 minutes. The solution hardly warmed. Then the solution was placed in the 40ºC hot water bath. Still not all of the sodium urate dissolved.
Preparation of twice concentrated agarose solution: To 149.2722g DI water, 2.4009g agarose were added. The solution was heated in a boiling hot water bath on the hot plate to dissolve the agarose.
Because not all of the stock solution constituents dissolved, the flask was inverted several times before extracting solution in an attempt to transfer the contents uniformly. To four different 50mL volumetric flasks, 25mL of stock solution were added via a transfer pipette. Varying amounts of chicken serum album and CaCl2·2H2O were added, as noted in Table 2.
Table 2
Component / Mass, gTube A
albumin / 1.001
CaCl2∙2H2O / 0.0517
Tube B
albumin / 2.0067
CaCl2∙2H2O / 0.0514
Tube C
albumin / 1.0005
CaCl2∙2H2O / 0.1030
Tube D
albumin / 2.0001
CaCl2∙2H2O / 0.1028
The solutions turned gold upon the addition of the chicken serum albumin. Test tubes B and D were a darker gold than A and C.
The agarose solution was then added, via pouring and plastic pipette, up to the line on the volumetric flasks. To prevent the agarose solution from solidifying it was kept in the 40ºC hot water bath. The agarose was at about 65ºC when added to the flask containing the constituents of test tube A. The agarose gel cooled to about 35ºC after addition to test tube B. The solution started to solidify, so it was heated back up again in the boiling water bath. Enough of the solution didn’t gel to fill the volumetric flasks containing the constituents for test tube C and test tube D. Immediately after adding the agarose solution, the volumetric flasks were inverted several times and then poured into a test tube, filling it about 4/5 of the way. Once filled, the test tubes were left out on the bench top for a few minutes to cool and solidify. Then, they were topped with a half inch layer of the supersaturated sodium urate solution. The sodium urate solution had been sitting in the 40ºC hot water bath. The test tubes were then covered with Parafilm and set in the water bath that was measured to be at 42.5ºC.
OBSERVATIONS
Later that day
Some small bubbles were present in the gel probably due to inverting the volumetric flasks. No dispersion is apparent yet. Water temperature is at 40.5ºC.
February 1, 2007
Test Tube A- No precipitate appears to be present in the gel. White precipitate is present on top of the gel—probably sodium urate.
Test Tube B- Same as A. Also, gel recessed up from the bottom of the test tube about 3cm. Liquid is filling this space on the bottom of the tube.
Test Tube C- Sodium urate and water are present on top of the gel. No precipitate is present in the gel. The gel is recessed up about three times as far as test tube B-liquid is in this space. Also, small circles of white solid are present at the bottom of the test tube.
Test Tube D-Same as C, including white solid.
Water temperature is 40ºC.
February 2, 2007
White precipitate on the bottom of test tubes C and D is twice as abundant today.
February 5, 2007
Test tubes A and B have no change. Test tubes C and D have large, dense, cloudy regions—as if something amorphous is growing. Each has three white spots on the bottom of the test tube. The water bath temperatures is at 37.0ºC
February 7, 2007
Test tubes C and D are almost completely opaque from the amorphous solid growing in the gel. This solid could be bacteria. Test tube A is still transparent, but it appears that some solid is growing in the gel with the same amorphous shape. Test tube B does not appear to have any solids. The water temperature is at 39.5ºC.
February 9, 2007
More white precipitate is growing on the bottom of test tubes C and D. Some white precipitate is on the bottom of B. The water temperature is at 40.2ºC.
February 12, 2007
The water temperature is at 36.5ºC. Test tube A still has not recessed from the bottom. The solid on the bottom of B and D has some yellow in it. Test Tube C is unchanged.
February 15, 2007
The water temperature is at 40.6ºC. Test tube A has white spheres forming at the top of the gel at the interface between the gel and the sodium urate. The white solid in test tubes C and D is likely bacteria. The water has a pungent odor. Test tube D has a small hole in the parafilm and a red solid growing on the top of the sodium urate.
February 16, 2007
Seemingly more spheres have appeared at the interface in test tube A. Some are several millimeters in size. Are the spheres that large or are they agglomerates of smaller spheres? The water temperature is at 41.0ºC.
February 19, 2007
The water temperature is at 40.8ºC. The spheres in test tube A seem to have dispersed further down into the gel. Smaller spheres are about 10 microns. The dispersed spheres seem to still be in the top third of the test tube. See Figure 1.
Figure 1: Photograph of spheres growing at interface of gel and in gel matrix in Test Tube A
The saturated urate solution covering the gel in Test Tube A was poured out and a sample of the solids was prepped (via vacuum drying on a carbon coated stem) for ESEM imaging. One could see what appeared to be clusters of particles. (see Figure 2)
Figure 2: ESEM image of dried solution
Imaging of a cluster shows spheres with sizes ranging from less than 1 micron to over 10 microns. (see Figure 3)
Figure 3: Image of a cluster of particles found in saturated urate solution above gel
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