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The overall goal of this protocol is to induce calcite precipitation in a stagnant culture of Sporosarcina pasteurii. This method can help us answer key biophysical questions about Sporosarcina pasteurii-mediated calcite precipitation, like the pertinent length and time scales. The main advantage of this technique is the fact that it standardizes the complex steps involved in suitably culturing and enriching Sporosarcina pasteurii that ensures reliable calcite precipitation.

To begin, assemble Petri dishes, flasks, Tris base, HCl, agar, Millipore water, and a pH meter. Sterilize all containers by autoclaving at 121 degrees Celsius before use. Prepare one liter of a 0.13 molar aqueous solution of Tris buffer by mixing 15.75 grams of Tris base with one liter of Millipore water.

Add approximately 2.8 milliliters of 50%HCl to adjust the buffer to a pH of nine. Once the culture is ready, it must be suitably enriched with the proper buffer. This step is critical to the success of the experiment, because a failure to provide the proper chemical environment can lead to very long time scales of precipitation or even a complete lack thereof.

Next, divide the one liter of buffer into two parts as follows. In one 400 milliliter aliquot, dissolve eight grams of ammonium sulfate. In a second 400 milliliter aliquot, dissolve 16 grams of yeast extract.

In 100 milliliters of the buffer, mix two grams of ammonium sulfate, and in the last 100 milliliters of buffer, add four grams of yeast extract and four grams of agar. After wrapping the flasks in aluminum foil, autoclave the four solutions. Immediately after autoclaving, set the two 400 milliliter solutions aside.

Combine the two 100 milliliter solutions, mix well, and divide between 10 to 12 Petri dishes. To culture bacterial samples, remove the bacterial stock from the minus 80 degree Celsius freezer and allow it to thaw properly. Then place it along with an agar plate in the biosafety hood.

Select a micropipette of the lowest possible volume and dip a micropipette tip into the Sporosarcina pasteurii stock before using the tip to streak an agar plate. Incubate the plate at 31 degrees Celsius for 48 hours, then visually examine for the presence of single colonies. If none are present, incubate for an additional 24 hours.

Repeat the check and incubations until single colonies are detected. To prepare the final bacterial culture samples, mix together the two 400 milliliter solutions autoclaved earlier in a sterilized beaker, and transfer 125 milliliters into a flask. Perform a visual examination of the agar plate to identify regions with high concentrations of single colonies.

With a micropipette tip, gently nudge and break one of the colonies. Then dip the same tip into the 125 milliliter flask and stir it thoroughly to ensure that a sufficient number of cells are transferred. Place the flask in a shaking incubator at 30 degrees Celsius and 150 rpm for two to three days.

To determine a final cell count, draw seven parallel equidistant bold lines on the bottom of one of the agar plates so that they are visible from the top. Place three small drops of about ten microliters of non-diluted culture into one segment. Add one milliliter of non-diluted culture to nine milliliters of sterilized PBS to obtain a one-to-ten dilution.

Then place three small drops of the dilution onto a second segment of the plate. Transfer the diluted culture to a new flask, and use PBS to dilute an additional ten times to ten to the minus two. Repeat the plating and dilution process until generating a dilution of at least ten to the minus seven to ensure single colonies will be obtained.

Incubate the plate at 31 degrees Celsius for one to two days before counting the colonies in each section of the plate. Transfer nine milliliters of the prepared liquid culture into several sterilized ten milliliter centrifuge tubes. Prepare 100 milliliters of the external enrichment stock solution by using an analytical balance to carefully measure out two grams per liter of urea, one gram per liter of ammonium chloride, 212 milligrams per liter of sodium bicarbonate, and 280 milligrams per liter of calcium chloride.

Into a beaker with fresh medium, combine all the ingredients except the urea, and autoclave. After autoclaving, mix the urea with one milliliter of fresh medium and pass the solution through a syringe fitted with a 0.2 micrometer filter before adding to the enrichment medium. Add one milliliter of the enrichment medium with additives to the sterilized centrifuge tubes containing nine milliliters of the prepared culture.

Vortex each tube and incubate in a non-shaking incubator at 30 degrees Celsius. Monitor all the units regularly for initiation of precipitation. Once precipitation is detected with the naked eye, transfer a small volume of fluid to a high magnification microscopy chambered cover glass system.

Initially view under 4X magnification to observe a wide area of coverage before progressively increasing the magnification. Perform staining and SEM experiments according to the text protocol. When S.pasteurii is cultured following the protocol demonstrated in this video, the bacteria lead to the precipitation of calcium carbonate that settles to the bottom of the tube over time.

This figure shows phase contrast images of a clearly distinguished, rod-like bacterial population within the medium. Here, the well-defined cleavage lines, a hallmark of crystallinity, can be seen. This X-ray photoelectron spectroscopy graph for the same sample contains peaks corresponding to carbonate groups that pinpoint the existence of calcium carbonate.

Shown in this panel is an untreated sponge that easily compresses when subjected to a one kilogram weight. When a sponge was immersed in a calcite precipitant solution with S.pasteurii for seven days and then dried, it hardened and displayed enhanced compressive strength due to pore clogging from calcite precipitation. Once mastered, this technique can be completed in a few hours if all the steps are done properly.

However, although the individual steps take just a few minutes each, there are longer idle periods in between where the sample must be incubated for hours or even days. Following this procedure, several other techniques like additional characterization can be performed to answer other questions about, for example, the statistical distribution of polymorphous forms in calcium carbonate. After watching this video, you should have a proper and clear understanding of how to suitably culture Sporosarcina pasteurii to enrich it properly that ensures calcite precipitation, and also how to observe the process through multi-mode microscopy.

Don't forget that working with high pressure autoclaves can be extremely hazardous and hence, all precautions like complete venting of the chamber and wearing of PPEs must be ensured before performing this procedure.