Gram Staining
AIM: To determine the Gram reaction and classify Bacillus licheniformis as Gram-positive or Gram-negative based on cell wall properties.
Procedure:
1.Sample Preparation:
Using an inoculating loop, aseptically obtain a small amount of Bacillus licheniformis from the broth culture and spread the sample in a thin, even smear on a clean glass slide. Allow the slide to dry.
2.Heat Fixing:
Once the smear is dry, pass the slide through the flame of a Bunsen burner 2-3 times, smear-side up, to heat fix the cells to the slide. Avoid overheating to prevent distortion of cell structure.
3.Primary Staining:
Flood the heat-fixed smear with crystal violet stain and let it sit for 1 minute. Then rinse the slide gently with distilled water until the excess stain is removed.
4.Mordant Application:
Add Gram's iodine solution to the smear, ensuring it covers the entire surface, and let it sit for 1 minute. Rinse the slide gently with distilled water.
5.Decolorization:
Decolorize by carefully adding 95% ethanol dropwise over the smear for 10-20 seconds, or until the runoff is clear. Immediately rinse the slide with distilled water to stop the decolorization process.
6.Counterstaining:
Flood the smear with safranin stain and let it sit for 30 seconds. Then the slide is to be rinsed gently with distilled water and allowed to air dry completely.
7.Microscopy:
Once dry, add a drop of immersion oil to the smear and observe under the oil immersion lens (100x) of a microscope.
Bacillus licheniformis will appear Gram-positive (purple) and rod-shaped.
Hot Spore Staining
AIM: To visualize and identify the presence of endospores in Bacillus licheniformis using heat and selective stains.
Procedure:
1. Sample Preparation:
Prepare a thin smear of Bacillus licheniformis from an overgrown broth culture on a clean glass slide. Allow the smear to air dry completely.
2. Heat Fixing:
Heat fixes the smear by passing it through the flame of a Bunsen burner 2-3 times, smear-side up.
3.Primary Staining:
Place the slide over a steaming water bath or a heat source. Flood the smear with a malachite green stain and allow it to steam for 5 minutes. Continuously add malachite green to keep the smear moist during steaming.
4.Rinsing:
After 5 minutes, remove the slide from the heat and let it cool for 1-2 minutes. Rinse the slide gently with distilled water to remove excess malachite green.
5.Counterstaining:
Flood the smear with safranin stain and let it sit for 30 seconds. Rinse the slide gently with distilled water and the air dry completely.
6.Microscopy:
Examine the slide under a microscope using the oil immersion lens (100x). Endospores will appear green, while vegetative cells will stain red.
Indole Test
AIM: To differentiate members of the Enterobacteria family.
Procedure:
1.Sample Preparation:
Pipette a drop of Bacillus licheniformis broth culture onto a clean filter paper.
2.Addition of Kovac’s Reagent:
Add 1-2 drops of Kovac’s reagent directly onto the same spot on the filter paper where the broth was applied.
3.Observations:
Observe the color change on the filter paper:
A red or pink color indicates a positive indole test whereas no color change or a yellow color indicates a negative indole test.
Methyl Red (MR) Test
AIM: To determine whether Bacillus licheniformis ferments glucose to produce stable acids.
Procedure:
1.Inoculation:
Inoculate MR-VP broth with a loopful of Bacillus licheniformis from a fresh culture. Incubate the broth at 32°C for 48 hours.
2.Addition of Methyl Red:
After incubation, 1 mL of the culture was transferred into a separate sterile test tube, and then 5 drops of Methyl Red indicator were added to the test tube.
3.Observation:
Observe the color immediately:
The red color indicates a positive MR test, indicating the production of stable acidic end-products from glucose fermentation whereas a yellow color or no color change indicates a negative MR test.
Voges-Proskauer (VP) Test
AIM: To determine whether Bacillus licheniformis ferments glucose to produce neutral end products.
Procedure:
1.Inoculation:
The same broth from the MR test can be used for the VP test:
Transfer 1 mL of the Bacillus licheniformis culture into a clean test tube.
2.Addition of Barritt’s Reagents:
Add 15 drops of Barritt’s A (alpha-naphthol) to the test tube.
Add 5 drops of Barritt’s B (potassium hydroxide) to the same test tube. Gently shake the tube to mix the reagents.
3.Observation:
Allow the tube to stand for 15-30 minutes.
Reddish color at the surface indicates a positive VP test, showing the production of acetoin as a fermentation product whereas no color change or copper color indicates a negative VP test.
Six-hour Growth Curve
AIM: To analyze the growth pattern and calculate the generation time of Bacillus licheniformis under specific conditions.
Procedure:The culture is stored at 4 degrees Celsius. The basal medium contains peptone (5 g/l), yeast extract (1 g/l), and MgSO4·7H2O (1 g/l) at 37 degrees Celsius for 24 hours.
1.Preparation of Overnight Culture:
•The organism should be cultivated with 25 ml of medium in a 250 ml Erlenmeyer flask. After overnight incubation, a sample was drawn and measured for growth.
•The suspension was mixed with 100 ml of 20% aqueous glycerol solution. The suspension was cooled and kept at −20 degrees C until use.
2.Inoculation into Flask:
•The medium was composed of 20 g/l L-glutamic acid, 12 g/l citric acid, 80 g/l glycerol, 7 g/l NH4Cl, 0.5 g/l MgSO4·7H2O, 0.04 g/l FeCl3·6H2O, 0.5 g/l K2HPO4, 0.15 g/l CaCl3·2H2O.
•The pH of this medium was adjusted to 7.5 using NH4OH.
•All media were prepared using de-ionized water and sterilized by autoclaving at 121 degrees C for 15 minutes. The glucose solution was sterilized separately and the vitamin solution was filtered sterilized and added separately to the medium.
3.Fermentation conditions:
•Batch cultures were conducted in a 5 L fermenter with 5% inoculum, prepared by thawing frozen cells at 37°C.
•The fermentation temperature was maintained at 37°C, and the pH was allowed to drop naturally until it reached 6.5, after which it was regulated using 50% NH₄OH or 3M HCl.
•Initially, the stirring speed was 250 rpm, and the airflow rate was 1.0 L/min.
•As γ-PGA production increased, the medium became highly viscous, reducing oxygen transfer.
•To prevent oxygen limitation, the agitation speed was increased to 800 rpm, and the airflow rate was raised to 5 L/min, ensuring oxygen levels remained above 40%.
4.Determination
•Culture monitoring should be performed by aseptically withdrawing 20 ml aliquots at intervals.
•Cell viability was assessed via serial dilution on nutrient agar. 0.5 ml samples were diluted 10-fold up to 10⁻⁷, and 100 µl of each dilution was spread on TSA plates, and incubated at 37°C overnight.
•Colony counts from three plates were averaged for each dilution, and results were expressed as CFU/ml. Each experiment was conducted three times for accuracy.
•The remaining sample was stored at -20°C for further analysis.
5.Measurement of OD600:
•At regular intervals (e.g., every 30 minutes), withdraw 1 mL of the culture from the flask and measure the OD578 to OD620 using a spectrophotometer, preferably, OD600.
•Record the values at each time point.
6.Plotting the Growth Curve:
•Continue taking OD600 readings until the culture reaches the stationary phase (typically 12-18 hours).
•Plot the OD600 values against time to generate the growth curve, showing the lag, log (exponential), stationary, and death phases.
Bacterial Transformation
1.Competent Cell Transformation
•Grow Bacillus licheniformis in LB broth overnight at 37°C under 200 rpm shaking.
•In fresh LB broth, dilute the overnight culture to 1:50, allowing it to reach an OD600 of 0.4–0.6, corresponding to the mid-log phase.
•Transfer the culture to sterile microcentrifuge tubes and put it on ice for 10 minutes.
•Centrifuge at 4°C with 5000 rpm for 10 minutes.
•Discard the supernatant.
•Resuspend the pellet in ice-cold 0.1 M calcium chloride solution and incubate on ice for 30 minutes.
•Pellet the precipitate by centrifugation, then resuspend in a reduced volume of 0.1 M calcium chloride. Keep solution on ice to be used
2.Transformation
•Add 100 to 200 ng of plasmid DNA to 100 µL of competent Bacillus licheniformis cells and mix.
•Allow mixture to remain on ice for 30 minutes.
•Heat shock at 42°C for 90 seconds, return to ice for 5 minutes.
•Add 500 µL of LB broth and incubate at 37°C for one to two hours with shaking to allow expression of antibiotic resistance genes.
•Plate 100 µL of the culture onto LB agar containing the appropriate antibiotic and incubate overnight at 37°C.
3.Verification
•Check for growth of colonies post-incubation, which confirms transformation.
•Validate the presence of the plasmid by carrying out colony PCR, restriction digestion, or sequencing.
Extraction
•The culture broth was centrifuged at 17,000 g for 30 minutes to remove cells.
•The supernatant was mixed with cold ethanol (3:1 ratio) and left overnight for γ-PGA precipitation, followed by further centrifugation at 17,000 g for 15 minutes.
•The crude γ-PGA was purified by dialysis in deionized water and lyophilization.
•To obtain γ-PGA in its free acid form, the supernatant was precipitated at pH 2, dialyzed, and lyophilized.
Quantification
•The polymer isolated from the supernatant was identified using NMR spectroscopy, with H-NMR spectra recorded in D₂O using a Bruker DRX500 spectrometer (500 MHz).
•FTIR analysis was performed using a Mattson FTIR spectrometer with a durascope and KBr beam splitter, over 100 scans in the 400–4000 cm⁻¹ range.
•The study also quantified carbon source utilization, and manganese sulphate utilization using NMR Spectroscopy with the different media used. The medium provided here, in the protocol is the one where Kedia et. al., achieved the highest yield of Gamma-PGA, 50.3 g/L.
•The number-average molecular weight (Mn) was determined using aqueous-based gel permeation chromatography (GPC), calibrated with sodium polyacrylic acid.
•Another study also used Anion-exchange chromatography for the same.
•The GPC analysis was conducted in 0.2 M NaNO₃, 0.01 M NaH₂PO₄ (pH 7), with a 1 ml/min flow rate at 30°C. The data was analysed using Polymer Laboratories’ Cirrus 3.0 software.
Project Timeline for Gamma-PGA Production and Biocement Development
December - March: Identification of Strain and Plasmid
•Research genes of interest and suitable plasmid for Bacillus licheniformis and finalize the strain.
•Finalize selection based on literature review and computational analysis.
•Tentative completion by March end.
•Work in tandem with the initial Dry Lab procedures.
April: Dry Lab and Procurement
•Complete dry lab simulations and modeling (tentative end by April).
•Order strain, plasmid, genes of interest, chemicals, reagents, and lab apparatus (order placement in the last week of April).
•Obtain institutional approvals and necessary permissions.
Mid-April - Mid-September: Wet Lab Experimentation
May - Jun: Standardization of Bacillus licheniformis
•Confirm strain identity using Gram staining, Hot Spore staining, Indole test, and MR-VP test.
•Perform 6-hour, 24-hour, and 48-hour growth curve analysis.
•Optimize growth conditions and media composition.
July: Recombinant Bacteria Analysis and Gamma-PGA Detection
•Perform bacterial transformation with recombinant plasmid.
•Confirm transformation via antibiotic selection, colony PCR, and sequencing.
•Monitor growth curves and optimize conditions for gamma-PGA production.
•Extract and quantify gamma-PGA using NMR/FTIR (HPLC or Ion-Exchange Chromatography as an alternative).
August: Biocement Development
•Mix extracted gamma-PGA with sand following optimized ratios.
•Prepare bricks using standardized methods.
•Conduct strength testing and structural analysis.
•Run 2nd cycle of Gamma-PGA production based on results obtained and enhance optimization.
September: Results and Analysis
•Compile and interpret experimental data.
•Run 2nd cycle of Brick-making and perform strength testing while interpreting data for the first DBTL cycle.
•Compare gamma-PGA production efficiency and bio-cement strength with standard materials.
•Finalize conclusions and prepare for documentation or publication.
References
- Sapkota, A. (2022, July 7). Bacillus subtilis- An Overview and Applications. Microbe Notes.https://microbenotes.com/bacillus-subtilis/
- Tripathi, N., & Sapra, A. (2023, August 14). Gram staining. StatPearls - NCBI Bookshelf.https://www.ncbi.nlm.nih.gov/books/NBK562156/
- Hartline, J. (2023, February 17). Endospore stain. In Microbiology Laboratory Manual. Biology LibreTexts.https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_Laboratory_Manual_( Hartline)/01:_Labs/1.12:_Endospore_Stain
- Aryal, S. (2022, August 10). Endospore staining- Principle, reagents, procedure and result. Microbiology Info.com.https://microbiologyinfo.com/endospore-staining- principle-reagents-procedure-and-result/
- American Society for Microbiology. (2016). Indole test protocol.https://asm.org/getattachment/200d3f34-c75e-4072-a7e6-df912c792f62/indole-test- protocol-3202.pdf
- Rao, P. N. S. (2022). MR-VP tests.https://www.microrao.com/micronotes/pg/mrvp.pdf
- iGEM. (2016). How to Bacillus subtilishttps://static.igem.org/mediawiki/2016/7/74/T-- UBonn_HBRS--How-To-Bacillus-subtilis.pdf
- Kedia, Gopal, et al. "Production of poly-γ-glutamic acid by Bacillus subtilis and Bacillus licheniformis with different growth media." Journal of Nanoscience and Nanotechnology 10.9 (2010): 5926-5934.