A.Confirmatory Tests
1.Gram Staining
• Bacillus licheniformis Broth Culture
• Glass Slides
• Inoculating Loop
• Bunsen Burner
• Crystal Violet Stain
• Gram's Iodine Solution
• 95% Ethanol (Decolourizer)
• Safranin Stain
• Distilled Water
• Microscope With Oil Immersion Lens (100x)
• Immersion Oil
2.Hot Spore Staining
• Bacillus licheniformis Broth Culture
• Clean Glass Slides
• Inoculating Loop
• Bunsen Burner
• Malachite Green Stain
• Safranin Stain
• Distilled Water
• Slide holder
• Microscope with oil immersion lens (100x)
• Immersion oil
3.Indole Test
• Bacillus licheniformis Broth Culture
• Sterile Filter Paper
• Kovac’s Reagent
• Pipette And Sterile Tips
4.MR-VP Test
• Bacillus licheniformis Culture
• Methyl Red Indicator (For MR Test)
• Barritt’s A Reagent (Alpha-Naphthol) (For VP Test)
• Barritt’s B Reagent (Potassium Hydroxide) (For VP Test)
• Sterile Test Tubes
• Pipettes and Sterile Tips
• Incubator (37°C)
• Test Tube Rack
5.Growth Curve
• Bacillus licheniformis culture
• Spectrophotometer (For Measuring OD600)
• Cuvettes
• Incubator (37°C)
• Sterile Flasks or Culture Tubes
• Pipettes and Sterile Tips
• Sterile Broth Media
• Shaking Incubator
• Conical Flasks
6.Bacterial Transformation
• Bacillus licheniformis culture
• Plasmid DNA
• Calcium chloride (CaCl2) solution
• LB broth and agar plates with appropriate Ampicillin
• Sterile microcentrifuge tubes
• Ice bath
• Water bath (42°C)
• Centrifuge
• Pipettes and tips
7.PCR
• DNA Template
• PCR Primers (Forward and Reverse)
• Taq DNA Polymerase
• dNTPs
• PCR buffer (with MgCl2)
• Sterile Nuclease-Free Water
• Thermal Cycler (for PCR amplification)
• PCR Tubes
• Microcentrifuge
• Pipettes and sterile tips
• Agarose Gel Electrophoresis Setup:
• Agarose
• SYBR Safe (For Staining)
• Gel Tray and Comb
• Power Supply
• UV Transilluminator (for Visualization)
• New England Biolabs DNA Ladder (1kb)
• Loading Dye
B.Growth Curve Analysis
1. Microbial Culture & Storage
• Bacillus licheniformis (stored at 4°C)
• 20% aqueous glycerol solution (for long-term storage at -20°C)
2. Growth Media & Components
Basal Medium:
• Peptone (5 g/L)
• Yeast extract (1 g/L)
• Magnesium sulphate heptahydrate (MgSO₄·7H₂O) (1 g/L)
Fermentation Medium:
• L-glutamic acid (20 g/L)
• Citric acid (12 g/L)
• Glycerol (80 g/L)
• Ammonium chloride (NH₄Cl) (7 g/L)
• Magnesium sulphate heptahydrate (MgSO₄·7H₂O) (0.5 g/L)
• Iron (III) chloride hexahydrate (FeCl₃·6H₂O) (0.04 g/L)
• Dipotassium phosphate (K₂HPO₄) (0.5 g/L)
• Calcium chloride dihydrate (CaCl₂·2H₂O) (0.15 g/L)
3. pH Adjusting Agents:
• 50% NH₄OH
• 3M HCl
4.Glassware & Containers
• 250 mL Erlenmeyer flask (for overnight culture)
• 1 L Flask/fermenter (for batch culture)
• Sterile culture tubes
• 100 mL measuring cylinders
• Pipettes (1 mL, 5 mL, 10 mL)
• Autoclave-safe bottles (for media preparation)
5. Fermentation & Culture Conditions
• Autoclave (121°C, 15 minutes) for sterilizing media
• Water bath (set at 37°C) for thawing frozen cells
• Incubator shaker (37°C, 250 rpm) for overnight culture
6. Microbial Viability Testing
• Nutrient agar plates (for serial dilution & CFU counting)
• Tryptic Soy Agar (TSA) plates
• Sterile dilution tubes (for 10-fold serial dilutions)
• Micropipettes & sterile tips (100 µL, 200 µL, 1000 µL)
• Colony counter (or manual counting)
7. Growth Curve Measurement
• Spectrophotometer (OD600 measurement)
• Cuvettes (for OD readings)
• Sterile 1 mL pipettes (for sample withdrawal)
• Eppendorf tubes (for OD600 measurement samples)
C.Extraction
1.Centrifugation
•Centrifuge
•Centrifuge tubes (suitable for high-speed centrifugation)
2.Precipitation and Purification
•Cold ethanol (absolute ethanol)
•Measuring cylinders (for ethanol measurement)
•Conical flasks or glass beakers (for mixing supernatant with ethanol)
•pH meter (for pH adjustment to 2)
•Hydrochloric acid (HCl) for pH adjustment
3.Dialysis and Lyophilization
•Dialysis tubing (suitable molecular weight cutoff)
•Deionized water
•Beakers (for dialysis setup)
•Magnetic stirrer with a temperature-controlled setup (for dialysis)
•Lyophilizer (freeze-dryer)
•Freeze-drying vials or trays (for lyophilization)
D.Quantification
1.NMR Spectroscopy
•Bruker DRX500 spectrometer (500 MHz)
•Deuterium oxide (D₂O) (as the solvent)
•NMR tubes (suitable for high-resolution spectroscopy)
2.FTIR Analysis
•Mattson FTIR spectrometer
•Durascope accessory
•KBr beam splitter
•Potassium bromide (KBr) pellets or discs
•Sample holder and pressing die (for preparing FTIR samples)
3.Carbon Source and Manganese Sulphate Utilization Studies
•NMR spectrometer (for metabolic analysis)
•Various media formulations for comparative studies
•Reagents for preparing culture media (specific carbon and manganese sources)
4.Gel Permeation Chromatography (GPC)
•Gel permeation chromatography system
•Sodium polyacrylic acid (as calibration standard)
•0.2 M sodium nitrate (NaNO₃) solution
•0.01 M sodium dihydrogen phosphate (NaH₂PO₄) buffer (pH 7)
•Column suitable for aqueous-based polymer analysis
•Pump system with 1 ml/min flow rate control
•Temperature control unit (set at 30°C)
•Polymer Laboratories’ Cirrus 3.0 software (for data analysis)
5.Anion-Exchange Chromatography
•Anion-exchange chromatography system
•Suitable anion-exchange resin
•Buffer solutions (specific composition depending on the study)
•Fraction collector for sample separation