Bright Field: Light source under stage is condensed with the condenser, and goes through objective lens (adjustable) and eyepiece (another lens). Focus can’t be
on center and peripheral at the same time (field curvature). Low contrast (staining required)Phase Contrast: Has better contrast. Change in amplitude results in a
difference in brightness when the light goes through a medium. Annular diaphragm present in the condenser, makes a ring of light on the specimen. When the light
goes through the specimen, scattered light that hits the phase ring increases phase shift and shows that part as dark. Reflected Light/Inverted:Light is bounced
down on a specimen then reflected back up to the eyepiece. Used to see autofluorescent cellular structures,fluorescent dyes, or fluorescent antibodies (helps see
proteins).Uses high intensity UV light,bleaches photos Confocal: Like fluorescence microscopes, but uses laser instead of a Mercury Arc Lamp. Also uses pinhole
so only one focal plane is seen. Laserfocused in a region of the specimen mirrors, pictures of small regions are combined to make a coherent image. Pinhole also
makes it so images of each focal plane can be taken to make a 3d image Electron: Invented because light microscopes refract too much light and makes images
blurred as it gets smaller.200-250 nm is smallest possible by light microscopes. Uses electron lenses. TEM: electrons go through condenser, specimen, objective
lens, then projector to see the image. SEM: electrons focused through condenser and objective lenses, then through scanning coil, then reflected off of the
specimen by heavy metals onto scanners that interpret results Estimate Size: Divide the field number (printed on eyepiece) by the magnification number to get the
field of view diameter. For example, if the field number is 22 and the magnification is 400, the field of view diameter is 0.055 mm. Bacteria: unicellular
microorganisms with cell walls but lack organelles or an organized nucleus Size: 0.2μm in diameter and 2−8μm in length Organelles: not bound by a membrane;
include chromosome and ribosomes Membrane and Cell Wall: plasma membrane; two types of cell wall: gram positive or gram negative - encapsulated by
peptidoglycan Archaea: A single-celled prokaryotic organism with distinct molecular characteristics;structurally similar to gram-positive bacteria, genetic processes
close to eukaryotic cells.Size: 0.7μm up to 4μm Organelles: Ribosomes and DNA Membrane and Cell Wall: plasma membrane; cell wall composed of various
polysaccharides and glycoconjugates Eukaryotic Microbes: Organisms whose cells contain a nucleus and other membrane-bound organelles. Size: usually have
diameters ranging from 10 to 100 μm Organelles: may include mitochondria, chloroplasts, the endoplasmic reticulum, the Golgi apparatus, and lysosomes.
Membrane and Cell Wall: The eukaryotic plasma membrane is a phospholipid bilayer with proteins and cholesterol embedded in it. The cell membrane is an
extremely pliable structure composed primarily of two adjacent sheets of phospholipids. Cholesterol, also present,contributes to the fluidity of the membrane.
Symbiogenesis: Symbiogenesis is the leading evolutionary theory of the origin of eukaryotic cells. Mitochondria, plastids such as chloroplasts, and possibly other
organelles of eukaryotic cells are descended from formerly free-living prokaryotes (more closely related tothe Bacteria than to the Archaea)taken one inside the
other in endosymbiosis. Mitochondria appear to be phylogenetically related to Rickettsiales bacteria, while chloroplasts are thought to be related to cyanobacteria.
Bacterial Cell Shapes: Spherical (Cocci)Rods (Bacilli)Comma (Vibrios)Tightly coiled (Spirochetes)Spiral shaped (Spirilla) Gram Stain procedure: a test that
checks for bacteria at the site of a suspected infection such as the throat, lungs, genitals, or in skin wounds - helps diagnose harmful bacteria 1. Applying a primary
stain (crystal violet).2. Adding a mordant (Gram's iodine).3. Rapid decolorization with ethanol, acetone or a mixture of both.4. Counterstaining with safranin. Grm+
Grm- bacteria: Gram+ stains purple under Gram stain,generally easier to kill, have a thick peptidoglycan bilayered wall; while Gram- stains red. Have a thin layer
of peptidoglycan and a lipopolysaccharide outer layer, often endotoxic; notable examples being salmonella, shigella, and Escherichia. The problem is Acid-fast
bacterium which strain as both,as they have a thin layer of peptidoglycan and a outlier of arabinogalactan Broth: Bacteria are suspended ina liquid nutrient
medium such asLuria Broth in an upright flask.Agar: petri dishes with agar-based growth medium, sometimes with additives, or a dipstick with the same principle
(more cost effective). Long-term storage Stab: similar to agar plates, but are solid agar in a test tube.Bacteria inserted through a needle stabbed in the middle.
Short-term. Culture collections: Collect and catalog lots of cultures. Solid plate culture of thermophilic: higher temps (50-70C). Uses low acyl clarified gellan gum.
Growth curves: a curve that shows various stages of the growth of bacteria. Lag Phase: bacteria prepares for reproduction - active and increasing in size. Log
Phase:( or Exponential phase) This phase is marked by the doubling of the bacterial cells. Stationary Phase: rate of cell growth becomes equal to its rate of
death due to limitation of nutrients. Death Phase: when the rate of death is greater than the rate of formation of new cells. Plate Counts: Estimate population
density of bacteria by counting the colonies of small and dilute portion of the sample. Defined vs. complex media: Defined uses compounds and distilled water to
find the exact growth medium. Complex uses digested products. Differential vs.selective media: Differential uses“indicator media” to show different
microorganisms growing there. Selective kills other microorganisms. Great plate count anomaly: Estimates are that we can cultivate roughly one out of every100
microbes Bacteriostatic vs. bactericidal antibiotics: Bacteriostatic slow bacteria and make it easier to kill. Need to be helped by the immune system.
Bactericidal kill the bacteria directly. Functions of Microbes: Lakes &Oceans: Photosynthetic bacteria(called cyanobacteria) in lakes,ponds, and shallow oceans
supply half of the world’s oxygen; breakdown organic matter into nitrate,phosphate, and other essential nutrients. Soil: soil microbes are bacteria, actinomycetes,
viruses,fungi, nematodes, and protozoa.Production of plant growth regulators and metabolites affecting plant growth and development. Nutrient recycling, break
down crop residues, and stimulate plant growth, inhibiting plant parasites and diseases. Gut Microbiome :supply essential nutrients,synthesize vitamin K, aid in
the digestion of cellulose, and promote angiogenesis and enteric nerve function. Bacteroidetes andFirmicutes are the main bacteria in the metabolism of
undigested food remnants. Bacterial Transcription: Initiation: RNA polymerase recognizes the promoter sequence, facilitating the formation of the transcription
initiation complex. The DNA unwinds, allowing RNApolymerase to start synthesizing RNA. Elongation: RNA polymerase moves along the DNA
template,catalyzing the addition of complementary nucleotides to the growing RNA chain. Termination: Transcription Concludes at a terminator sequence.
Termination mechanisms include Rho-dependent termination, where the Rho factor aids in transcript release, and Rho-independent termination, involving the
formation of a hairpin structure in the RNA. Bacterial Translation: Initiation: The small ribosomal subunit binds to the mRNA at the ribosome binding site. The
initiator tRNA, carrying the amino acid methionine, attaches to the start codon. Elongation: The large ribosomal subunit joins, and the ribosome moves along the
mRNA. During Elongation, tRNA molecules bring amino acids to the ribosome,facilitating peptide bond formation. Termination: Translation concludes at a stop
codon. Release factors promote the detachment of the polypeptide chain from the final tRNA, completing the translation process.Genome Replication: Initiation:
DNA replication begins at the origin of replication, where DNA unwinding creates a replication bubble. DNA polymerase initiates synthesis using RNA primers.
Elongation: DNA polymerase adds nucleotides to the growing DNA strands in the 5' to 3' direction. Termination: Replication concludes when the entire DNA
molecule is duplicated. Termination Mechanisms involve specific termination sequences Bacterial Gene Regulation: Lac Operon: Components: The lac operon
includes the promoter, operator,and three structural genes (Z, Y, A)coding for enzymes involved in lactose metabolism. Regulation: The lac operon is subject to
negative regulation by a repressor protein. In the absence of lactose, the repressor binds to the operator, preventing transcription. Allolactose, an inducer, binds to
the repressor in the presence of lactose, alleviating repression andallowing transcription. Trp Operon: Components: The trp operon consists of the promoter,
operator,and five structural genes (E, D, C,B, A) related to tryptophan biosynthesis. Regulation: The trp operon is negatively regulated by a repressor.In the
absence of tryptophan, the repressor is inactive, allowing transcription. When tryptophan is present, it binds to the repressor,activating it and inhibiting transcription
Fermentation: Microbes are used in the production of various products,such as ethanol, organic acids, andenzymes.Kombucha: Fermented tea produced by
symbiotic communities of bacteria and yeast.Algal Biofuels: Microalgae are harnessed for biofuel production.Bioremediation: Microbes are employed to clean up
environmental pollutants.Phage Therapy: Bacteriophages Are used to combat bacterial infections Endosymbiotic Theory: Some organelles
(mitochondria/chloroplasts) were once prokaryotic microbes Psychrophile (cold): Proteins function optimally in the cold (more α-helix than β-sheet → More
enzyme flexibility). Proteins that bind to ice crystals. More unsaturated fatty acids in the membrane. More G+C in the genome Thermophile (hot): Often has
amino acid substituions that allow enzymes to fold well in hot environments. Saturated fatty acids in membrane (more hydrophobic environment → More stable).
DNA binding proteins make globular structure (resistant to melting) Hyperthermophile (very hot): More ionic IMFs in proteins, no fatty acids in membranes,
instead branching hydrocarbons, use ester linkages to stabilize membrane, lipid monolayer. Uses horizontal gene transfer in the presence of high heat
Acidophile: Need to maintain cytoplasmic pH, so impremeable membrane. Remove excess H+ from cyroplasm via pumps, secondary transport, buffers solution
in cycoplasm (via amino acids) Alkaliphile: Acquire extracellular pH, reduce H+ leakage, ATP synthase does not use ATPase. Produce acid via sugar
