Microbiologically Induced Calcite Precipitation by Sporosarcina pasteurii

Abstract

Source: Bhaduri, S., et al., Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii. J. Vis. Exp. (2016)

This video demonstrates the method of enriching Sporosarcina pasteurii with urea and calcium chloride to trigger urease activity, leading to the formation of visible calcite crystals through Microbiologically Induced Calcite Precipitation, or MICP.

Protocol

1. Protocol for Enrichment of Nutrient to Accelerate Precipitation

1. Transfer 9 ml of the prepared culture liquid (cells + medium) into several sterilized centrifuge tubes, each of 10 ml volume.
2. Prepare a 100 ml stock solution of the external enrichment consisting of four components in the following concentrations in fresh medium: Urea: 2 g/L, Ammonium chloride: 1 g/L, Sodium bicarbonate: 212 mg/L, Calcium chloride: 280 mg/L. Carefully measure all the ingredients using an analytical balance and mix all but urea with fresh medium in a beaker and place in the autoclave (121 °C, 15 psi, 15 min).
   1. Following the autoclave, mix the requisite amount of urea (2 mg) with 1 ml of fresh medium and pass it through a syringe fitted with a 0.22 µm syringe filter to complete the enrichment process.
   2. Add 1 ml of this enrichment medium with additives to the sterilized centrifuge tubes containing 9 ml of the prepared culture liquid (cells + medium).
      NOTE: Urea, being degradable at elevated temperatures, cannot be sterilized in an autoclave. Hence, after the other components have been autoclaved (121 °C, 15 psi, 15 min), urea is added last through a 0.22 µm syringe filter.
3. Vortex each tube thoroughly using a mechanical vortexer. Place the enriched liquids (cells + medium + additives) in a non-shaking incubator at 30 °C. Monitor all the units regularly for the initiation of precipitation. Using a light microscope, begin microscopic observations once the onset of precipitation is detected with the naked eye. This is usually between 30 - 36 hours after the start of experiments.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pyrex flasks	Fisher Scientific	S63268	Corning Erlenmeyer
Measuring cylinder	Cole-Parmer	CP08559GC	Cole-Parmer Class A Graduated Cylinder w/Cal Cert,TC;1000 mL,1/Pk
Analytical balance	OHAUS	AX124E	Being used to measure weight of reagents
Autoclave	Brinkmann	58619000
Autoclave tape	VWR	52428864
Aluminum foil	Sigma-Aldrich	Z185140	Being used to seal the flask before placing it in Autoclave
Bacterial stock	Cedarlane	11859	-80°C stock of S. pasteurii, ATCC No. is mentioned against Cat. No.
Mline single-channel mechanical pipettors, variable volume	Biohit	725010	Marketed by VWR under catalog number 14005976
Micropipette tip	Fisher Scientific	212772B	Used for scratching Agar plates
Incubator	Binder	80079098	Microbiology Incubator,BF Series
Shaking incubator	VWR	14004300	VWR Signature Benchtop Shaking Incubators
Parafilm	Sigma-Aldrich	P7793	Being used to seal Agar plates
Urea	Sigma-Aldrich	U1250	Enrichment for nutrient medium
Sodium bicarbonate	Sigma-Aldrich	S8875	Enrichment for nutrient medium
Calcium chloride	Sigma-Aldrich	C1016	Enrichment for nutrient medium