MICROSCOPY AND THE ORGANIZATION OF MICROBIAL CELLS Flashcards
General Features For All Cells (See image on Lecture Slides)
All cells have:
• DNA/RNA
• Plasma Membrane
• Cytoplasm
• Ribosomes
Components of Prokaryotes (see image on slide for detail)
-This includes bacteria and archaea
Cell wall
Cytoplasmic membrane
Nucleoid (not a nucleus)
Cytoplasm
Plasmid
Ribosomes
-Often have a flagella that helps facilitate movement
Components of Eukaryotes (see image for detail)
-Eukaryotes are more complex and have organelles to carry functions and membrane bound nucleus
Cell wall
Cytoplasmic membrane
Mitochondrion
Nuclear membrane
Nucleus
Ribosomes
Endoplasmic reticulum
Cytoplasm
Golgi complex
Microbial Size Comparison
We have to understand that Eukaryotes are the largest cells —> Prokaryotic Cell —> Viruses
-Main thing we have to understand that most microbes are too small and we need microscopes to be able to properly visualize them
Microscopy: Magnification vs Resolution (importance and limiting factors)
• Magnification=enlargement
• Resolution=ability to distinguish two adjacent points
- Resolution tends to be the biggest limiting factor since not being able to distinguish between two cells or components can be very limiting to our understanding of the cell
THE COMPOUND LIGHT MICROSCOPE
One of the kinds of microbes that can be used for microscopy is the compound light microscope
• Magnification: 1000x
• Resolution: 0.2um
This microscope is used to visualize normal sized cells. We can add modifications to use for different specific tasks:
• Bright-Field
• Phase-Contrast
• Differential Interface Contrast (DIC)
• Dark-Field
• Fluorescence
Bright Field and Uses (see image on slides)
When light hits a cell it reflects at a darker shade compared to the background. Bright field is best for:
*Viewing fixed and stained bacterial cells or tissues.
*Observing large microorganisms that naturally scatter light well.
*Differentiating between Gram-positive and Gram-negative bacteria using Gram staining
*Dead fixed cells
Bright field is best for cells that have natural pigment. Also useful for cells which we add the pigment since they scatter light better and appear less transparent in microscope.
Phase Contrast and Uses (see slides for image)
It highlights differences between scattered and non-scattered light. A lot more crisp image is acquired (Almost like “bolding” a cell). It is most useful for:
*Better for live cells (no need to fix and kill cells)
*More crisp to see internal structures of a cell
* Studying cells in their natural, hydrated state, such as bacteria, protozoa, and other microorganisms
Used when staining is not an option
Dark Field and Uses (See image in slides)
Only light that reaches the cell is scattered back. As a result it has a dark background and highlights cells it reflects. Useful for:
*Seeing very small microorganisms
*Seeing the movement structures like flagella
*Dark field can also be used for live cells
Dark-field microscopy is valuable when high contrast is needed without staining or when working with very fine or translucent structures.
Differential Interface Contrast (DIC) and Uses (See lecture slides)
Using DIC we can get a 3-D like image. Prism creates two lights that pass through cell then beams join back in another prism. Useful for:
*Crisp image; not good for structures in the cell
*Good for thicker cells and to see the nucleus
*Useful for live-unstained cells
* Creating a 3D effect, which makes it easier to distinguish the relative depth and structure of the specimen.
DIC is excellent for researchers who need to observe live cells in great detail, especially in areas where phase-contrast might not provide sufficient clarity.
Fluorescence Microscopy and Types (See slides for images)
Shooting light of one color and organism shoots light of another color in response. Some cells fluoresce naturally but we can also add fluorescence in two ways for specificity.
2 Types:
1. Fluorescent Protein (GFP)
2. Immunofluoresce Assay
Protein Florescence (GFP) and Uses:
Great for subcellular localization. Basically add a GFP gene at end of a desired gene sequence and it will fluoresce. Useful for:
*Allows to visualize proteins and sub-cellular structures in real time
*Useful to follow the movement of a protein following a treatment in live cells
* GFP can be used to label organelles (like the nucleus, mitochondria, etc.) to see how different parts of the cell function and interact under different conditions.
GFP has revolutionized cell biology by making it possible to visualize the inner workings of living cells in ways that were previously impossible
Immunofluoresce Assay and Uses (see slides for images)
1 Primary antibody is added and binds to protein of interest (cells have to be dead and fixed)
In IF we use antibodies to target a specific protein of interest.
#2 Excess antibodies are washed off
#3 Secondary antibodies are added and bind to primary antibody (have fluorescent tip that is reflected)
Useful for:
*Visualizing the concentration of a specific protein of interest
* It’s used to identify pathogens or abnormal proteins, as seen in some cancers, autoimmune diseases, or infections
* Researchers use IF to examine if two or more proteins are present in the same location within a cell, which can provide insights into protein interactions and signaling pathways
*Useful to visualize structure of a cell through protein (actin for example in muscle cells)
Electron Microscope Use and Types
Use electrons instead of visible light to image cells and cell structures. Usually used to view structures that are much smaller in structure like viruses since it has higher resolution and magnification.
2 Types:
1. Transmission EM
2. Scanning EM
Transmission Electron Microscope and Use
Needs very thinly sectioned samples stained with special stains (osmic acid, lead salts) for contrast
• Best for internal structures in cells
Scanning Electron Microscope and Use
Gives a 3-D view of cells but only of the external surface of cells
• Better for 3-D images of cells, visualizing surface and external structures
• Specimen coated with heavy metal (gold)
Cell Staining Types
Two Types of Cell Staining:
• Basic Dyes
• Gram Staining
Basic Dyes and Use
Stains entire cell (anything that’s negatively charged).
Useful for:
*To give cell a pigment so that it can be visualized better (bright field)
* This is especially important for cells that are otherwise transparent or lightly pigmented, as they can be difficult to see without staining
Fast and cost effective
Gram Staining and Use
Widely used to visualize and differentiate bacteria - Gram(+) Thick wall vs Gram(-) Thin cell wall
STEPS:
1. Flood the heat-fixed smear with crystal violet for 1 min - Turns all cells purple
2. Add iodine solution for 1 min- Without iodine, the effectiveness of the crystal violet stain would be diminished, making it harder to identify and classify bacterial species accurately
3. Ethanol destain – Destaining G(-) cells but does not wash away in G+ cells because of thick cell wall
4. Counter Stain- Stains G(-) cells pink or red color and now have two cell types with different color stains
Useful for:
- Knowing the Gram classification of a bacterial pathogen can help healthcare providers choose the most effective antibiotics
- Its ability to differentiate bacteria based on their cell wall structure makes it an essential method in both clinical and research laboratories
