Microbiology WK1

Microbes

Organisms and acellular entities too small to be clearly seen by the unaided eye. Relatively simple in construction (But not function) and lack highly differentiated cells and distinct tissues.

Prokaryotic Cells

Cells that mostly lack a true membrane bound nucleus and organelles. Consist of bacteria and archaea.

Eukaryotic Cells

Cells with a membrane enclosed nucleus, are more complex morphologically, and are usually larger than prokaryotic cells. Include yeast, fungi, and animals among other things.

What are the three phylogenetic domains?

Bacteria, archaea, and eukarya.

Which two phylogenetic domains are the most genetically related?

Archaea and eukarya due to sharing a more recent common ancestor compared to bacteria.

What are the main differences between archaea and bacteria?

Archaea lack peptidoglycan and contain different lipids in their plasma membranes.

What is the prokaryotic cell equivalent to the nucleus and what does it do?

The nucleoid that contains bundled chromosomes and can help give “superpowers” that aid in things like antibiotic resistance.

Why do we prepare and stain specimens?

To increase visibility, to accentuate specific morphological features (cell wall, capsule, flagellum), and to preserve specimens. The microbes we look at are often colorless underneath a microscope.

Fixation

Preserves internal and external structures and fixes them in position. Organisms are usually killed and firmly attached to the microscope slide. The main types of fixation are heat and chemical.

Heat Fixation

Fixation that involves passing a slide over an open flame. Routinely used with bacteria and archaea. Preserves overall morphology but not internal structures.

Chemical Fixation

Fixation that involves bathing slides in alcohol. Routinely used with larger, more delicate organisms. Protects fine cellular substructure and morphology.

What are two common features of microbe dyes?

Chromophore groups (give dye its color) and the ability to bind/stick to cells.

Chromophore groups

Chemical groups with conjugated double bonds that give dye its color.

Dyes

Make internal and external structures of a cell more visible by increasing contrast with background. Can be basic or acidic.

Basic Dyes

Have positively charged groups that bind to negatively charged molecules often found on the surface of bacteria and archaea. Examples include methylene blue, crystal violet, and safranin.

Acidic Dyes

Help to stain background or specific structures. When ionized they have a negative charge and bind to positively charged structures.

Differential Staining

Divide microorganisms into groups based on staining properties. Used to detect presence or absence of structures. Answers a Yes/No question based on specific structures. Examples include gram and acid-fast stains.

Bacterial cell wall functions

Maintains shape, helps protect from osmotic lysis (water rushing in), helps protect from toxic materials, and may contribute to pathogenicity (ability to give disease). Almost all bacteria have a cell wall.

Gram Staining

Most widely used differential staining procedure. Divides bacteria into gram positive and gram negative based on differences in cell wall structure. Gram positive stains purple and indicates a thick peptidoglycan wall and gram negative stains pink or red and indicates a thin peptidoglycan and outer membrane.

Steps of Gram Staining

1. Stain with crystal violet

2. Treat with iodine to form a more complex structure with the crystal violet (traps dye within thicker cell walls)

3. Decolorize with alcohol, any dye that can squeeze through a cell wall will leave

4. Counterstain with safranin to make decolorized cells visible (All cells stained pink but not visible in cells that stayed dyed)

Acid-Fast Staining

A type of differential staining used for identifying members of the genus mycobacterium. Specifically identifies the high lipid content (mycolic acid) in the mycobacterium’s cell walls. Mycobacterium is stained red and non-acid-fast bacteria are stained blue.

How are endospores stained?

Heated, double staining technique. Endospores stained green and vegetative cells stained pink.

How are bacteria with capsules stained?

Through a negative staining technique. Capsules don’t pick up stain. The background is stained and a capsule will have a halo like appearance against the dark background.

How are flagella stained?

Mordant (not iodine) is applied to increase the thickness of the flagella.

Peptidoglycan

Rigid structure that lies just outside the cell membrane. Made from a meshlike polymer of identical subunits forming long strands. Made from two alternating sugars. NAG and NAM along with alternating D and L amino acids.

Peptidoglycan strands form ___ that connect to form ____

Helices, sheets that stack into layers.

Gram positive bacteria have ____ layers of peptidoglycan

200-300

Gram negative bacteria have ____ layers of peptidoglycan

5-10 or less

Gram positive cell wall composition

Mostly peptidoglycan with teichoic acids and some surface proteins.

Teichoic Acids function

Helps maintain the cell envelope by anchoring the cell wall to the plasma membrane. Helps protect from environmental substances. May bind to host cells which can make pathogenic bacteria worse.

Gram positive periplasmic space

Between plasma membrane and cell wall. Much smaller space compared to gram negative. Has relatively few proteins. Mostly enzymes called exoenzymes that aid in the degradation of large nutrients.

Gram negative cell walls

More complex than gram positive. Consist of a thin layer of peptidoglycan (5-10% of cell weight) surrounded by an outer membrane. Contains a larger and more complex periplasmic space (20-40% cell volume). Does not contain teichoic acids.

Gram negative outer membrane is composed of

Lipids, Braun’s lipoproteins, and lipopolysaccharide (LPS)

Gram negative periplasmic space contains

Lots of enzymes and proteins like hydrolytic enzymes, transport proteins, and other proteins.

Braun’s lipoprotiens connect…

Outer membrane to peptidoglycan

Lipopolysaccharides (LPSs) Structure

Molecule found in gram negative cell walls. They consist of a lipid portion (lipid A), a core polysaccharide, and a side chain O antigen. The lipid is embedded in the outer membrane. The core polysaccharide and O side chain extend out from the cell.

Lipopolysaccharides (LPSs) Functions

Contribute to negative charge on the cells surface. Helps stabilize outer membrane structures. May contribute to attachment to surfaces and biofilm formation. Creates a permeability barrier.

The O antigen provides protection from host defenses. Lipid A can act as an endotoxin. Both of these attributes contribute to the pathogenicity of a bacteria.

How is the O antigen dangerous?

When the human body encounters the bacteria initially it creates an antibody. The O antigen can change itself so the human has no acquired immunity. Big part of antibiotic resistance.

How is lipid A dangerous?

When you kill a gram negative bacteria, lipid A is released into the bloodstream as an endotoxin. This can trigger severe inflammatory responses and is a common cause of sepsis.

Gram negative outer membrane permeability

More permeable than the plasma membrane due to the presence of porin proteins and transporter proteins. Porin proteins form channels that small molecules can pass through.