The overall goal of this experiment is to demonstrate new procedures for making hollow spherical structures of oxide materials using gram negative bacteria. This method can help answer key questions in the bionanomaterial field by showing how microcapsule nanogranial metal oxide material can be formed with the help of bacteria. The main advantage of this technique is that it is environmentally friendly and it has the potential to mass produced.

Weigh out eight grams of LB Lennox Broth powder and place it into a 500 milliliter laboratory bottle with 400 milliliters of water. Next, add a magnetic Teflon stirring bar and stir the contents for 20 minutes. Cap the broth tightly and autoclave the contents at 120 degrees Celsius for 10 minutes.

Allow the solution to cool down to room temperature and then use a pipette to aliquot 12.5 milliliters of the broth into eight 15 milliliter centrifuge tubes. Aliquot the remaining broth into three 100 milliliter laboratory bottles, cap the three bottles tightly, and keep them in the biosafety cabinet. Next, retrieve a cryopreserved stock of Shewanella algae from storage.

Then, in the biosafety cabinet use a stainless steel spatula to remove one milliliter of the frozen material from the storage tube and place it into one of the 12.5 milliliter aliquots containing LB Lennox Broth. Place the sample into a 37 degrees Celsius incubator and incubate the culture for 24 hours. Next, prepare the agar plates.

Dissolve two tablets of LB Lennox Broth with agar into a 100 milliliter bottle containing 100 milliliters of water. Use a stir bar to stir the contents for 20 minutes, and then cap it tightly. Once autoclaved, aliquot 20 milliliters into each of four Petri dishes.

Allow the solution to cool down to room temperature in the hood. And then cover the plates. Label the three bottles prepared with 100 milliliters of LB Lennox Broth with number one, number two, and number three.

Pipette 0.1 milliliters of the resulted bacterial culture into bottle one. Cap the bottle and swing it by hand for one minute to get a homogenous solution. Next, pipette 0.1 milliliters from bottle number one into bottle number two.

Again, cap the bottle and swing it by hand for one minute. Perform the last dilution by pipetting 0.1 milliliters from bottle number two into bottle number three. Cap the bottle and shake it by hand for one minute.

And then pipette 20 microliters of the solution onto to each of the four Petri dishes. Then, place four autoclaved three millimeter diameter glass beads into each of the Petri dishes. Close the lids of the Petri dishes and shake them by hand for one minute.

When finished, turn the Petri dishes upside down and incubate the plates in a 37 degree Celsius incubator for 24 hours. With a stainless steel spatula, pick out the resultant monoclonal bacteria from the four Petri dishes and put them into the remaining seven tubes containing 12.5 milliliters of LB Lennox Broth. Leave the tubes in the 37 degrees Celsius incubator for 24 hours, and then pick out the one with the largest light scattering using the visual colorimetric method.

Place 10 grams of LB Lennox Broth, 10 grams of sodium chloride, and 10 grams of glucose into a 500 milliliter laboratory bottle. Then add water until the volume reaches 450 milliliters. Add a Teflon stir bar and stir for 20 minutes.

Once mixed, qutoclave this solution at 120 degrees Celsius for 10 minutes. Next, measure out 16.5 grams of sodium tungstate dihydrate and place it into a 100 milliliter bottle. Add water until the volume reaches 50 milliliters.

Add a Teflon stir bar and stir the solution for 20 minutes. Sodium tungstate solvent concentration is meant to be high in order to ensure spherical shape of a microcapsule, yet not too high so as to kill the bacteria. Once autoclaved, filter the solution through a fiberglass filter with pores of one micron to get the filtrate.

Pour the filtrate by hand into the 450 milliliter bottle with LB Lennox Broth containing glucose and salt. Then aliquot the resultant solution into 10, 50 milliliter centrifuge tubes. Retrieve the culture containing the monoclonal bacteria and add 50 microliters of it into each of the 10, 50 milliliter centrifuge tubes containing the sodium tungstate media.

Incubate the 10 tubes in a 37 degrees Celsius incubator for 120 hours. Following the incubation, place each of the tubes in an ultra sonicator and sonicate the cells at 20 kilohertz with 150 watts for one hour. Centrifuge the tubes.

And then remove the clear liquid with a pipette. Next, add water. And then sonicate and centrifuge the tubes one more time.

After the second centrification, remove the clear liquid in the tubes with a pipette. Then add 35 milliliters of 99.8%Ethanol and ultrasonicate the samples again. Rinse the minerals by centrifuging them, adding alcohol and sonicating the sample one more time.

Then, remove the clear liquid in the tubes with a pipette and immediately cap the tubes without drying out the sample. This SEM image show the broken hollow shell made to sodium tungstate which was excreted by Shewanella algae using glucose as the carbon source. Granules about 40 to 60 nanometers in diameter hang outside the shell.

The shell itself is made of granules approximately 22 nanometers in diameter. Zooming out, one can see the length to diameter ratio for these hollow shells. These spherical shells have a length to diameter ratio of one to one and were obtained using a high concentration of metal oxianions greater than 100 millimolar in the culture media.

By decreasing the concentration of oxianions in the culture medium to around 20 millimolar, the bacteria produces sodium tungstate shells with length to diameter ratio of three to one. After it's development, this technique help the researcher in the field of bionanotechnology to explore the possibility of making microstructure like microcapsule of a metal oxide material with the of help of a gram-negative bacteria.