Overview
This page provides a comprehensive list of materials required for conducting various experiments in a BSL-2 lab, explicitly focusing on Bacillus subtilis and other microbial studies. The materials outlined are essential for multiple microbiological and molecular biology techniques. This list is a practical reference to ensure all necessary equipment, reagents, and consumables are available for successful experimental execution.
Gram Staining
Bacillus subtilis Broth Culture
Glass Slides
Inoculating Loop
Bunsen Burner
Crystal Violet Stain
Gram's Iodine Solution
95% Ethanol (Decolorizer)
Safranin Stain
Distilled Water
Microscope With Oil Immersion Lens (100x)
Immersion Oil
Hot spore staining
Bacillus subtilis 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
Indole Test
Bacillus subtilis Broth Culture
Sterile Filter Paper
Kovac’s Reagent
Pipette And Sterile Tips
MR-VP Test
Bacillus subtilis 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
Growth Curve
Bacillus subtilis culture (LB Broth Media)
Spectrophotometer (For Measuring Od600)
Cuvettes
Incubator (32°C)
Sterile Flasks or Culture Tubes
Pipettes and Sterile Tips
Sterile Broth Media (Luria Bertani)
Shaking Incubator
Conical Flasks
Iturin Extraction
Bacillus subtilis culture (Grown in PDB and LB Media)
6 M HCL
pH Meter
3M KCL (To Calibrate pH Meter)
1M NaOH
Distilled Water
100 % Methanol (97 mg/ml)
Centrifuge And Centrifuge Tubes
0.22µm Filter
Sterile Pipettes and Tips
Incubator
Magnetic Stirrer
Homologous Recombination
Bacillus subtilis Competent Cells
Plasmid DNA (Stock for Campbell)
Electroporation Cuvettes and Electroporator (For Using Electroporation)
Recovery Media
Selection Antibiotic
Agar Plates (With Appropriate Antibiotics for Selection)
Incubator (37°C)
Sterile Pipettes and Tips
Roller Drum
1 mL of 1X MC trp phe
3mM MgSO4
15mL Culture Tube
15mL Conical Tubes
900 µL H2O
100 µL 10X MC
3 µL 1M MgSO4
4 µL trp, and 4 µL phe
Screw Cap Filter
-20°C Freezer
Aluminum Foil
HPLC
HPLC System With Appropriate Detector (Shimadzu)
HPLC-Grade Solvents (Methanol, Water, Acetonitrile)
Iturin A Standard
Iturin A Samples (Extracted from Potato Dextrose Broth And Luria Bertani)
Centrifuge
Pipettes And Sterile Tips
Data Acquisition Software for HPLC (Real-Time Analysis- HPLC-01-system administrator)
Analytical Balance (For Accurate Measurement)
C18 Silica Gel-Packed Column
Distilled Water
Gibson Assembly
Gibson Assembly Master Mix (from New England Biolabs)
PCR-Amplified DNA Fragments (With Overlapping Sequences)
Nuclease-Free Water
Microcentrifuge Tubes
Thermal Cycler (Or Water Bath/Incubator for Incubation At 50°C)
Electroporation Cuvettes and Electroporator (Or Chemical Transformation Setup)
Competent Bacillus subtilis Cells (for Transformation)
SOC Medium (For Recovery)
Antibiotic Selection Plates (for Plasmid Screening)
Pipettes and Sterile Tips
Incubator (32°C for Bacterial Growth)
Oil Emulsion Test (Crude Oil)
Bacillus subtilis Culture
Petrol (As A Substitute for Crude Oil)
Sterile Flasks or Beakers
Petri Dish
Pipettes and Sterile Tips
Distilled Water
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
Zone of Inhibition
Bacillus subtilis Culture
Fungal Test Strains (Candida albicans)
Agar Plates (Pre-Inoculated with The Fungal Cultures-Spread Plate Method)
L Rods
Ruler Or Caliper (For Measuring Inhibition Zones)
Test compounds:
Purified Iturin A
Extracted Iturin from Potato Dextrose Broth
Extracted Iturin from Luria Bertani
Distilled Water (Negative Control)
Methanol (Positive Control)
Amphotericin B (Positive Control)
Sterile Pipettes and Tips
Incubator (32°C for Fungal Cultures)
Minimum Inhibitory Concentration Assay
Candida albicans Culture
96-Well Plate
Eppendorf Tubes and Stand
Amphotericin
100% Methanol
Pure Iturin (<95%)
Sample (Extracted) Iturin
Distilled Water (Negative Drug Control)
Potato Dextrose Broth (Vehicle Control)
MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
DMSO (Di-Methyl Sulfoxide) (To Dissolve MTT)
Spectrophotometer (To Measure Cell Viability)
Sterile Pipettes and Tips
Plasmid Isolation (QIAgen Prep Kit)
QIAprep Spin Miniprep Kit
LyseBlue Reagent (optional)
RNase A (Provided with the kit)
Buffers: P1, P2, N3, PB, PE, and EB
96–100% Ethanol (for Buffer PE Preparation)
Centrifuge (Capable of 13,000 rpm)
1.5 mL Microcentrifuge Tubes
Bacillus subtilis ATCC 13952
GFP Detection
Bacteria From Plates or Glycerol Stocks
Luria-Bertani (LB) Medium
Antibiotics (If required)
1X Phosphate Buffered Saline (PBS) (pH 7.4)
Eppendorf Tubes
96-Well Plate, Clear Bottom
Microplate Reader