Apr 28, 2025
Version 2
Magnetic Labeling, Filtration, Immobilization, and Processing of Bacillus cereus Spores for SEM Imaging V.2
- Pedro Fonseca1,2,3,4,
- Wilson Antunes3,5
- 1INESC-MN;
- 2Instituto Superior Técnico, Universidade de Lisboa;
- 3Unidade Militar Laboratorial de Defesa Biológica e Química, Exército Português;
- 4Instituto Nacional de Saúde Doutor Ricardo Jorge, I.P.;
- 5Centro de Investigação, Desenvolvimento e Inovação da Academia Militar (CINAMIL), Instituto Universitário Militar, Lisboa, Portugal
- Advanced Integrated Microsystems Doctoral Program
Protocol Citation: Pedro Fonseca, Wilson Antunes 2025. Magnetic Labeling, Filtration, Immobilization, and Processing of Bacillus cereus Spores for SEM Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb2j71gpk/v2Version created by Pedro Fonseca
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 27, 2025
Last Modified: April 28, 2025
Protocol Integer ID: 184039
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This protocol describes a novel method for labeling Bacillus cereus spores with superparamagnetic nanoparticles, filtering excess particles, immobilizing spores onto membranes, and preparing samples for scanning electron microscopy (SEM) imaging. It combines immunomagnetic labeling, filtration, chemical fixation, dehydration using tertiary butyl alcohol, osmium tetroxide vapor post-fixation, and sputter coating, offering a robust strategy for high-quality SEM imaging of bacterial spores.
Guidelines
- Always maintain cold chain and protect samples from drying out during filtration and labeling steps.
- Perform all fixation and dehydration steps with appropriate ventilation.
- Tertiary butyl alcohol must be completely melted before use.
- Osmium tetroxide handling requires special care: always work inside a fume hood.
- Ensure complete evaporation of tertiary butyl alcohol in the vacuum chamber to avoid sample loss.
Materials
- Bacillus cereus spores (ATCC 14579)
- 100 nm superparamagnetic nanoparticles conjugated to Protein A (nanomag‱-D-spio protein A, Micromod)
- Rabbit polyclonal anti-B. cereus antibody (ab20556, Abcam)
- Phosphate Buffered Saline (PBS) pH 7.4
- PBS with 0.5% Triton-X
- Ultrapure sterile water
- 2.5% Glutaraldehyde + 1% Formaldehyde in 0.1 M Cacodylate Buffer
- Ethanol series (30%, 50%, 70%, 90%, absolute ethanol)
- Tertiary butyl alcohol (≥99.5%)
- Osmium tetroxide solution (for vapor fixation)
- 0.45 µm PES membrane filters (Supor‱ Membrane, PALL Corporation)
- Water filtration system (funnels, manifolds, vacuum pump)
- Orbital shaker
- Glass petri dishes
- SEM metal stubs
- Copper adhesive tape
- Sputter coater (Cressington 108auto or equivalent)
- Scanning Electron Microscope (SEM)
Safety warnings
- Superparamagnetic nanoparticles must be resuspended homogeneously before use.
- Never pipette osmium tetroxide directly; use closed containers.
- Dehydration steps must be performed swiftly to prevent sample collapse.
Use appropriate PPE (lab coat, gloves, eye protection) throughout the protocol.
Before start
- Prepare fresh solutions of PBS 0.5% Triton-X, glutaraldehyde/formaldehyde fixative, and cacodylate buffer.
- Preheat tertiary butyl alcohol to 40°C.
- Turn on sputter coater and SEM equipment to allow warming up.
- Prepare vacuum filtration apparatus and test for leaks.
- Cool down a freezer to -20°C for freezing steps.
- Pre-cut copper adhesive tapes and mount them in metal SEM stubs.
Labeling of Bacillus cereus Spores
Labeling of Bacillus cereus Spores
Grow B. cereus spores following the "High-Yield Sporulation and Purification of Bacillus cereus
and Bacillus thuringiensis Spores" protocol from Antunes & Fonseca (dx.doi.org/10.17504/protocols.io.14egnwxwqg5d/v1).
Harvest spores by centrifugation at 4000 × g, 10 min, wash three times with sterile deionized (DI) water.
Adjust final spore suspension to OD600 = 0.8 (~10⁸ CFU/mL).
Centrifuge spores and resuspend pellet in 100 µL of a 1:50 dilution of anti-B. cereus antibody in PBS 0.5% Triton-X.
Incubate 30 minutes at room temperature (22–25°C) in a rotator at ∼60 RPM.
Centrifuge and resuspend pellet in 100 µL of superparamagnetic nanoparticle suspension (~300 nanoparticles per spore) in PBS 0.5% Triton-X.
Incubate for 30 minutes at room temperature (22–25°C) in a rotator at ∼60 RPM.
Filtration and Immobilization
Filtration and Immobilization
Set up the filtration system with 0.45 µm polyethersulfone (PES) membrane filter.
Note: The filtration setup consisted of 250 mL funnels, 0.45 µm PES filters, suction manifolds, and a vacuum pump - components commonly used in water sample processing for microbiological analysis.
Turn on vacuum pump and add 250 mL PBS to pre-wet the system.
Mix labeled spore suspension with 20 mL PBS 0.5% Triton-X.
Pour the mixture into the funnel containing remaining PBS.
After full filtration, rinse the membrane with an additional 250 mL PBS.
Allow membrane to dry under suction for 2 minutes before turning off the pump.
Fixation and Dehydration
Fixation and Dehydration
Cut a 10 mm × 10 mm section of the PES membrane filter.
Immerse in fixative solution (2.5% glutaraldehyde + 1% formaldehyde in 0.1 M cacodylate buffer) for 30 minutes with moderate orbital agitation.
Wash in sterile DI water.
Dehydrate sequentially in ethanol series (1 minute per step in a petri dish with moderate orbital agitation: 30%, 50%, 70%, 90%, absolute ethanol).
Transfer to melted tertiary butyl alcohol at 40°C in an eppendorf tube for homogenization.
Freezing and Drying
Freezing and Drying
Mount the sample onto SEM stubs with copper adhesive tape.
Freeze the mounted samples at -20°C for 30 minutes.
Quickly place samples in the sputter coater vacuum chamber; evaporate tertiary butyl alcohol under vacuum.
Post-fixation and Sputter Coating
Post-fixation and Sputter Coating
Expose samples to osmium tetroxide vapor for 5 minutes inside a closed Petri dish.
Sputter coat samples with an 8 nm gold layer (10 mA for 15 seconds).
Samples are ready for SEM visualization.