Microscopy

Introduction to Microscopy in Microbiology

Microscopy is a foundational tool in microbiology, enabling scientists to observe microorganisms and their cellular structures. The development of the microscope was pivotal in the discovery and study of microbes.Hi Valentina!

It was great to meet you on Thursday and I hope your first few weeks have been going well! 

I wanted to reach out and let you know a little bit about myself before our first formal meeting. I am a junior majoring in Exercise, Sport and Health Science on the Pre-PA track. I am passionate about this route because I want to be an advocate for those who do not seem to have a voice in their own healthcare. I have seen firsthand how easily a patient's concerns can be dismissed, something that should never happen. On campus, I'm involved in the Pre-Med/Pre-PA Club, the Soccer Club, and I work in the Financial Aid Office. This year I have a lot more room in my schedule so I am hoping to add more clubs to my list. Outside of school, I really enjoy traveling and running. I am currently training to run a half marathon in the next few months! Volunteering takes up much of my time, as well. Currently, I volunteer at Gritman Medical Center in the Medical Surgical Unit and at Snake River Community Clinic as a scribe. Finally, I am taking the EMT course this semester through the City of Moscow, with hopes to volunteer with the department in the future. 

As for things to do in Moscow, two of my favorite places to eat are the Alehouse and Moscow Bagel. One World Cafe is also a great place to study. If you need any recommendations, let me know. I have lots! Looking back on freshman year, I would suggest going to office hours and meeting with your professors. You never know when they will give you hints on tests. They are also very good resources for recommendation letters in the future. I would also tell everyone to go to all of the events offered on campus and talk to anyone that is at those events and in your classes. You may find really good friends and study buddies.

As a mentor, I am here to be a resource and support you, whether you need academic support, guidance with pre-health, or just help adjusting to college life. No question is too small! Starting out can be overwhelming but there are many different resources here to support you. You are not alone. I look forward to meeting with you this week to learn more about you and your goals and interests. 

Before our meeting, I would like you to think about any specific goals you have for this semester, what you are wanting from me as a mentor, and how you think I could support you best. 

Finally, I wanted to leave you with a few resources you may find helpful. 

Chas Basic Needs Kits                   208-885-6757                                 TLC, Room 232

Dean of Student's Office                208-885-6757                                TLC, Room 232

West Side Food Pantry                  www. inlandoasis.org                      730 W Pullman Road, Suite 3

Emma SeidelAntoni van Leeuwenhoek (1632–1723) was the first to describe microbes using a simple light microscope.

• Early microscopes used a single lens and could magnify specimens enough to reveal bacteria and the structures of molds.

• Microscopy allows for the visualization of cells, their morphology, and internal structures.

Discovering Cell Structure: Light Microscopy

Light microscopy uses visible light to illuminate specimens, making it possible to study the structure and function of microbial cells.

• Bright-field microscopy is the most common type, where light passes directly through the specimen.

• Resolution is the ability to distinguish two adjacent objects as separate entities. The limit of resolution for a light microscope is about 0.2 μm.

• The numerical aperture (NA) of a lens affects resolution. It is defined as , where n is the refractive index of the medium and θ is the half-angle of the maximum cone of light that can enter the lens.

• Abbe’s equation for minimal resolvable distance: , where is the wavelength of light.

Improving Contrast in Light Microscopy

Contrast is essential for visualizing cells, especially since many microbes are transparent and lack natural pigmentation.

• Staining with dyes (e.g., methylene blue, safranin) increases contrast by coloring cells or cell components.

• Gram stain differentiates bacteria into two groups:

  • Gram-positive: appear purple after staining.

  • Gram-negative: appear red or pink after staining.

• Non-destructive contrast methods include:

  • Phase-contrast microscopy: amplifies differences in refractive index, making cells appear darker against a light background.

  • Dark-field microscopy: illuminates specimens from the side, making them appear bright against a dark background; useful for observing hard-to-stain microbes.

  • Fluorescence microscopy: uses fluorescent dyes or naturally fluorescent organisms; cells emit light of a specific color when illuminated with another color.

Imaging Cells in Three Dimensions

Advanced microscopy techniques allow for three-dimensional imaging of cells and their structures.

• Confocal Scanning Laser Microscopy (CSLM) uses a laser to scan specimens and a computer to compile images from different layers, producing a 3D image.

• Resolution can reach 0.1 μm, and live samples can be imaged.

Probing Cell Structure: Electron Microscopy

Electron microscopy uses beams of electrons instead of light, providing much higher resolution and magnification.

• Transmission Electron Microscopy (TEM):

  • Electrons pass through thin sections of specimens (20–60 nm thick).

  • Specimens are stained with heavy metals to improve contrast.

  • Allows visualization of internal cell structures.

• Scanning Electron Microscopy (SEM):

  • Specimens are coated with a thin film of heavy metal (e.g., gold).

  • An electron beam scans the surface, and secondary electrons are collected to form a 3D image of the surface.

  • Only the surface is visualized, but at high magnification (up to 100,000x).

Microbial Cell Structure and Function

Prokaryotic vs. Eukaryotic Cells

Microbial cells are classified as either prokaryotic (Bacteria and Archaea) or eukaryotic (plants, animals, algae, protozoa, fungi).

Cell Morphology (Shape) of Prokaryotic Cells

Prokaryotic cells exhibit a variety of shapes, which can influence their function and ecological role.

• Cocci: spherical or ovoid

• Bacilli: rod-shaped

• Spirilla: spiral-shaped

• Filamentous bacteria: long, thread-like; can cause problems in wastewater treatment due to poor sedimentation.

• Cell shape is generally a poor predictor of function or identity, but may aid in nutrient uptake (high surface/volume ratio) or motility.

Prokaryotic Cell Size

Prokaryotic cells are generally much smaller than eukaryotic cells, which enhances nutrient uptake and allows for rapid growth.

• Typical size: 0.2 μm to >700 μm (most are 0.5–4.0 μm wide and <15 μm long).

• Small size results in a higher surface area-to-volume ratio, facilitating efficient nutrient exchange.

• Ultra-small cells exist, but there is a lower limit to cell size due to the need to house essential cellular machinery.

The Cell Membrane and Wall

The Cytoplasmic Membrane

The cytoplasmic (plasma) membrane is a critical structure in all cells, acting as a selective barrier and site for various cellular processes.

• Composed of a phospholipid bilayer with embedded proteins.

• Separates the cytoplasm from the external environment.

• Functions as a permeability barrier, protein anchor, and site of energy conservation (proton motive force).

Structure of Bacterial and Archaeal Membranes

• Bacterial membranes contain ester-linked fatty acids in their phospholipids.

• Archaeal membranes have ether linkages and isoprene-based lipids, which can form monolayers or bilayers and are more heat-resistant (important for thermophilic Archaea).

Functions of the Cytoplasmic Membrane

• Permeability barrier: prevents free diffusion of polar and charged molecules; transport proteins facilitate uptake of nutrients and export of waste.

• Protein anchor: holds transport and other proteins in place.

• Energy conservation: site of generation and use of the proton motive force.

Transport Across the Membrane

• Passive transport (diffusion): movement of small molecules (e.g., O2, CO2) down their concentration gradient.

• Facilitated diffusion: uses transport proteins (carriers) for larger or specific molecules (e.g., glucose).

• Active transport: moves molecules against their concentration gradient using energy (often ATP).

Types of active transport:

• Uniporters: transport a single type of molecule.

• Symporters: transport two molecules in the same direction.

• Antiporters: transport two molecules in opposite directions.

Conclusion: Membrane Function

• The cytoplasmic membrane is essential for maintaining cellular integrity, mediating transport, and conserving energy.

• Transport proteins enable cells to accumulate nutrients and expel waste, even against concentration gradients.