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WSU / Biology / IM 2200 / What is light microscopy used for?

What is light microscopy used for?

What is light microscopy used for?

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Chapter 2 Book Notes  


What is light microscopy used for?



Microbial Cell Structure and Function

2.1 Light Microscopy  

Light microscopes are used to examine cells at relatively low magnifications.  Electron microscopes are used to examine cells and cells structures at very high  magnification.  

Resolution: the ability to distinguish two adjacent objects as distinct and separate  Resolution is determined by the wavelength of light (shorter is better) and the  numerical aperture (higher is better).  

The total magnification of light microscope is the product of the magnification of the objective and ocular lenses.  

∙ The highest resolution in a light microscope is about 0.2 um. This means that  anything smaller than 0.2 um cannot be seen as distinct and separate.  ∙ The highest resolution in a electron microcope is 0.2 nm.  


What is the purpose of staining?



2.2 Staining 

Many dyes used in microbiology are positively charged and are called basic dyes.  ∙ Ex) Methylene blue, Crystal violet, Safranin We also discuss several other topics like What are the bone stem cells?

These bind strongly to negatively charged cell components

Gram staining: Bacteria can be divided into:

∙ Gram positive- These appear purple-violet after being stained with crystal  violet. These do not get decolorized with ethanol.  

∙ Gram negative- These are counterstained after being decolorized with  ethanol with Safranin. They appear pink.  If you want to learn more check out What happened in nat turner’s rebellion?

DNA Structures 

Eukaryotes

Bacteria/Eukarya

DNA is linear and found in a nucleus  Contains membrane-enclosed organelles Typically 2 copies of each chromosome  Nucleus divides by mitosis (cell division)  Genome is halved by meiosis (sexual  reproduction)

DNA is a single circular chromosome It aggregates in the nucleoid region No membrane bound organells  My have chromosomal plasmids


What is the structure of a dna model?



If you want to learn more check out What is the meaning of the individual level?

2.5 Cell Morphology (Bacteria and Archaea) 

Morphology means cell shape. Major shapes include:

∙ Spherical shaped– Coccus  

∙ Cylinder shaped – Rod

∙ Spiral shaped rods – Spirilla

∙ Others

o Tightly coiled bacteria – Spirochetes

o Have tubes or stalks – Appendaged bacteria

o Filamentous bacteria – forms long chains

Morphology is not used to predict or show physiology, phylogeny or pathogenic  potential.  

Why are some organisms shaped the way they are?  

∙ Optimization for nutrient uptake- small cells have a larger surface to volume  ratio which makes it easier to intake nutrients  

∙ Swimming motility- spiral shaped cells swim easier in viscous environments  ∙ Gliding motility- filamentous bacteria are more suited for gliding  

2.6 Membranes (Bacteria and Eukarya) 

The cytoplasmic membrane surrounds the cytoplasm and separates it from the  environment and has selective permeability.  

∙ Composition If you want to learn more check out What is the scientific study of interactions between organics and their environment?

o Phospholipid bilayer (Fatty acids and glycerol-phosphate components)  o Ester linkages bond hydrophobic side chain to glycerol  o Fatty acids point inwards away from the cytoplasm (hydrophobic)  

∙ The membrane has many proteins. Hydrophobic parts of the protein spans  the membrane while the other sides interact with the environment and the  cytoplasm.  

The outer surface of the membrane interacts with the  

extracellular fluid (environment). The inner surface  

interacts with the cytoplasm.  

Types of membrane proteins:  

o Integral- firmly embedded in the membrane  

o Peripheral- does not go through the membrane, but

is associated with the surface of it  

Archaeal Membranes 

∙ Composition  If you want to learn more check out What is strategic business manager?

o Ether bonds hydrophobic side chains to glycerol  

o Archaeal membranes thus lack fatty acids  

o Hydrophobic side chains are not fatty acids, they are isoprenes 

The membrane can be composed of diglycerol diethers which have 20 carbon side  chains (called phytanyl group). Or diglycerols tetraethers which have 40 carbon side chains. The phytanyl groups that stick out from the glycerol are covalently linked.  This formas a lipid monolayer.  We also discuss several other topics like How do you factor extra credit into a grade?

Monolayers are extremely resistant to heat and are found most in extremist  Archaea.

Some archael lipids contain rings within the hydrocarbon side chain.  ∙ Example) Crenarchaeol is a lipid that has 5 rings.  

2.8 Membrane function (All domains) 

The membrane has 3 major functions:  

1. Permeability – prevents the leakage of solutes into or out of the cell  2. Protein anchor – some of these proteins are enzymes for energy conservation  and others are for transport of solutes into and out of the cell  

3. Energy conservation – it can exist in an energetically charged form,  separating ions for the purpose of energy  

The hydrophobic portion of the membrane is a tight barrier to diffusion of things  located inside of the cytoplasm; especially molecules that are polar and charged.  These molecules must be transported.  

∙ Can H+ diffuse across the membrane? NO! It is charged  

∙ Can water diffuse across the membrane? YES! Water is essential to the cell  and is somewhat polar but is small enough to diffuse. Other examples are O2 and CO2

Because of the selective permeability, transport proteins are essential. Transport  reactions move nutrients from low concentration to high concentration. This takes  energy to do.

∙ Transport reactions are much faster than simple diffusion but they do have a  saturation effect. This means that no matter if there is a lot of substrate  outside of the cell, if all of the proteins are being used up (already  transporting substrate inside of the cell) then the rate cannot go any faster.  

Transport Types 

Active – this type requires energy. It transports solutes against concentration  gradient. (From low concentration to high)  

Passive – this type does not require energy. There is transport along the  concentration gradient. (From high to low)  

∙ Pores (simple channels) in the membranes

∙ Carrier proteins that undergo conformational changes to put substrate across  membrane.  

Transport Types in Prokaryotes  

Simple transport: uses only a membrane spanning transport protein Group translocation: uses a series of proteins in the transport event ABC transport system- uses 3 components:  

1. Substrate binding protein

2. Membrane-integrated transporter

3. ATP hydrolyzing protein

2.10 Cell Walls (Bacteria and Archaea)

Bacteria can be divided into gram-positive and gram-negative. The cell walls of  bacteria have a rigid layer added for strength called peptidoglycan. It is composed  of:

1. N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)  

2. D-alanine and D-glutamic acid  

3. Lysine or DAP  

4. And cross linkages that make it stronger. These differ in gram positive and  gram negative bacteria

Gram-positive cell walls are composed of 90% peptidoglycan. There are usually  several sheets of it stacked upon each other. Their cell walls also contain techoic  acids that are embedded within the wall. Lipotechoic acids are ones that covalently  bound to the membrane lipids.  

Gram negative cell walls only contain a small amount (10%) of peptidoglycan. Most  of their cell wall is composed of the outer membrane or Lipopolysaccharide layer  (LPS). LPS is composed:

1. Lipid A

2. O-specific polysaccharide  

3. Core polysaccharide

The Lipid A is known to be very toxic to animals (endotoxin). Examples of this is  salmonella and certain pathogenic strains of E. coli.  

Gram negative cell walls also have a space between the outer membrane and the  cell membrane called the periplasmic space. In this space is where you would find  proteins that are embedded within the cell membrane.  

Gram staining  

During gram staining, you use crystal violet, iodine, alcohol, and Safranin.  When using the crystal violet, it stains all cells (gram negative and positive) purple.  When you use alcohol, it dehydrates gram positive cells, causing the purple stain to  be trapped inside of the cell. (Making them stay purple.) In gram-negative cells,  alcohol penetrates the lipid-rich outer membrane and extracts the crystal violet,  allowing them to be stained by the Safranin.  

2.12 Archaeal Cell Walls

Archaeal cell walls do NOT contain peptidoglycan. It also does not have an outer  membrane.  

Instead, some of the cell wall consists of a polysaccharide called Pseudomurein. It  is composed of  

1. N-acetylglucosamine

2. N-acetyltalosaminuronic acid  

The most common type of cell wall in Archea is the S-layer. It is also found in some  bacteria. It is composed of interlocking proteins or glycoproteins.

2.13 Cell surface structures 

Many prokaryotes secrete sticky or slimy materials on their cells surface. These are  called either capsules or slime layers.

Traditionally, if the layer is organized into a tight matrix, it is called a capsule. It is  easily defined under an electron microscope. If it is more difficult to see and more  easily deformed, it is called a slime layer.  

Capsules usually stick to the cell wall, some even covalently linked to  peptidoglycan.  

Uses of capsule and slime layers include:  

1. Helps in the attachment of microorganisms to solid surfaces.  2. Acts as virulence factors in certain disease, preventing the cell from  suffereing dehydration

3. Protect against phagocytosis

Other structures on the cell surface include fimbriae and pili. Fimbriae allow for  cells to stick to surfaces. Pili allow for genetic exchange between cells (conjugation)  and helps in the adhesion of pathogen to specific host tissues.  

Type IV pili assist cells in twitching motility.  

Flagella is a structure that assists in swimming. These are helical in shape and have  different arrangements: peritrichous, polar, and lophotricous.  

2.14 Cell Inclusion 

Inclusions function as energy reserves.  

Storing Carbon

One of the most common bodies to includsion in prokaryotes is poly-B hydroxybutyric acid (PHB).  

Glycogen, which is the polymer of glucose, is used as storage for carbon and energy

Storing Polyphosphate

The cell accumulates inorganic phosphate. This is useful for a source of phosphate  for nucleic acid and phospholipid biosynthesis.  

Cells also store sulfur (elemental), carbonate materials such as barium, strontium  and magnesium. Last, they also store magnetosomes, sometimes with particles of  iron oxide.  

2.16 Endospores

Certain bacterias produce endospores. They are highly differentiated cells that are  resistant to heat, harsh chemicals and radiation. They can be thought to be a  dormant stage of bacterial life cycle. They are present only in gram positive  bacteria.  

 They can be found at the end of the cell  

(terminal).  

 Near the end of the cell, (subterminal). Or

near the  

 Center (central.).  

2.17 Flagella 

In polar flagellation, the flagella are attached at one or both ends of a cell. This  allows for the cells to move very rapidly and typically spin around. In peritricous  flagellation, flagella are inserted at many locations around the cell surface.  

Prokaryotes often encounter gradients of physical or chemical agents in nature and  they respond by moving toward or away from the agent.  

Chemotaxis – responding to chemicals

Phototaxis- responding to light  

In the absence of a gradient, cells move in a random fashion that is described as  “runs”. When it stops and changes direction, it is known as tumbling.  

Runs- Flagella rotates CCW

Tumble- Flagella rotates CW, forward motions stops and change directions

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