Chapter 2 Flashcards
Microscopy (Light Microscopy)
Uses light to illuminate cells.
Types: bright field, phase contrast, dark field, fluorescence.
Bright field microscope
Specimens are visualized due to differences in contrast between specimen and surroundings.
Two sets of lens form the image
-Ocular and objective lens
-Total magnification= ocular mag x objective mag
Max magnification is 2000x
Resolution
The ability to distinguish between two adjacent objects as separate and distinct.
Determined by wavelength of light and numerical aperture of lens.
Staining
Improving contrast results in a better image.
Staining improves contrast.
-dyes are organic compounds that bind to specific cellular materials
Stains can be very specific and tell us something about bacteria.
Simple Staining
- Prepare the smear.
- Heat fixing and staining
- microscopy
The gram stain
Differential stain.
Used widely in microbiology.
-Bacteria can be divided into two major groups: gram negative and gram positive.
-Gram positive bacteria appear purple, gram negative bacteria appear red after staining.
Steps in Gram staining
- flood heat fixed smear with crystal violet (cells purple)
- Add iodine solution (cells purple)
- decolorize w/ alcohol (gram+ remain purple; gram- are colorless)
- counterstain with safranin (gram+ are purple, gram- are red)
Increased contrast 2: phase contrast
Specimens stand out against background.
- improves contrast of sample without stain
- allows for visualization of live samples
- dark cells on a light background
Increases contrast 3: dark field
Light reaches specimen from side.
Light reaches lens scattered by specimen.
Light specimen against a dark background.
Excellent for observing motility.
Increased contrast 4: fluorescence
Visualize specimens that fluoresce.
-emit light of one color when illuminated with another color of light.
Cells fluoresce naturally or after being stained with DAPI
Used in microbial ecology for enumerating bacteria
Fish 1
Fluorescence In Situ Hybridization
DNA probe attached to fluorescent dye
Identify and count cells
Atomic Force Microscopy (AFM)
Stylus is placed close to specimen.
Measures weak repulsive forces between it and the specimen.
Computer generates image based on data received from stylus.
Confocal Scanning Laser Microscopy (CSLM)
Computerized microscope coupled with laser source to generate a 3D image.
Can focus on a single layer.
Electron Microscopy
Uses electrons instead of photons to image structures.
Two types: transmission electron microscope (TEM) and scanning electron microscope (SEM)
Transmission Electron Microscope (TEM)
Uses electrons.
Uses electromagnets instead of a lens.
Operates in a vacuum.
High magnification and resolution.
Structures can be visualized at the molecular level.
-Proteins and nucleic acids can be seen.
Specimen must be very thin and stained with a heavy metal.
Scanning Electron Microscope (SEM)
Specimen coated with a thin layer of heavy metal.
Electron beams scan the specimen.
Scattered electrons are collected by a detector and an image is produced.
Large specimens can be observed.
Magnification from 15x-100,000x
Basic cell types
Two major classifications
- Prokaryotes: bacteria and archaea spindle shaped)
- Eukaryotes: Everything else (round shaped)
Common to all cells
cytoplasmic membrane
cytoplasm
ribosomes
Cell membrane
Fluid mosaic structure
-phospholipid bilayer
-proteins interspersed in mosaic
Regulates movement of materials into and out of cells
-nutrients enter and wastes exit
Determines boundary of cell
Assists in capturing ATP and capturing proteins
Cytoplasm
Semifluid substance inside of the cell membrane
80% water and 20% carbs, proteins, and lipids
Ribosomes
Site of protein synthesis
Consists of RNA and protein
Often grouped as polyribosomes
Cell wall
Gives structural strength Permeable Outside cell membrane Stronger than cell membrane *Found in plants and most microorganisms *Not found in most animal cells->cytoskeleton
Differences
Eukaryotes
-DNA enclosed in a membrane-bound nucleus
-Cells are generally larger and more complex
-Contain organelles
Prokaryotes
-No membrane enclosed organelles, no nucleus
-Smaller than eukaryotic cells
Viruses
Not considered cells
No metabolic capabilities of their own
Rely on biosynthetic machinery of infected cell
Infect cells of all types
Arrangement of DNA in prokaryotic Cells
Genome: A cells full compliment of genes
Prokaryotic cells generally have a single circular DNA molecule called a CHROMOSOME
-DNA aggregates to form the nucleoid region
-Prokaryotes sometimes have small amounts of extra-chromosomal DNA called plasmids
Plasmids
Genes needed only under some conditions
Genes needed for basic survival located on chromosome.
May be gained or lost by cells.
Eukaryotic DNA
Linear and found in the nucleus
Associated with proteins that fold DNA
Usually more than one chromosome, typically two copies of each chromosome.
During cell division, nucleus divides by mitosis
During sexual reproduction, the genome is halved by meiosis.
Average Genomes
E.coli genome -4.64 milliopn base pairs -4300 genes -1900 different proteins -2.4 million protein molecules Human genome -1000x more DNA than E.coli -7x more genes
Evolution and phylogeny
Evolution: The process of change over time
Phylogeny
-Evolutionary relationships between organisms
-Relationships can be deduced by comparing genetic information
-Phylogenetic tree
-Ribosomal RNA is excellent for determining phylogeny
Why rRNA?
Fundamental to all life Highly conserved -can compare life -similarity indicates degree of relatedness Has more and less conserved regions -Allows for PCR and differences
The domains
Comparative rRNA sequencing has defined three distinct lineages of cells called domains:
-bacteria (prokaryote)
-Archaea (prokaryote)
-Eukarya (eukaryote)
Archaea and bacteria are not closely related
Archaea are more closely related to eukarya than bacteria.
Eukaryotes and multicellularity
Eukaryotic microorganisms are the ancestors of multicellular organisms.
Mitochondria and chloroplasts also contain their own genomes and ribosomes.
-Related to specific lineages of bacteria
Endosymbiosis
Physiological diversity of microorganisms
Phylogenetic diversity in microbial cells is the product of evolution.
Metabolic diversity.
Evolved as we are.
Microbial Growth Capabilities
Temp: -12C-104C
pH: 0-13
Hydrostatic pressure: 0-1400 atm
Microbial Diversity
Morphology (cell size and shape) Metabolism Motility Mechanisms of cell division developmental biology adaptation to environment
All cells need energy (3 sources)
Organic chemicals (chemoorganotrophs) Inorganic chemicals (chemolithotrophs) Light (phototrophs)
Chemoorganotrophs
Obtain energy from oxidation of organic molecules.
Many use oxygen (aerobes).
Some do not use oxygen (anaerobes).
Can break down organic compounds either aerobically or anaerobically.
Most abundant.
Animals are chemoorganotrophs.
Chemolithotrophs
Obtain energy from oxidation of inorganic molecules.
Only in prokaryotes.
Each species of chemolithotroph usually will use only one class of inorganic molecule.
Compounds used by chemolithotrophs are waste products of chemoorganotrophs.
Phototrophs
Use light as an energy source.
Contain pigments that allow light as an energy source.
Oxygenic photosynthesis produce oxygen.
Anoxygenic photosynthesis does not produce oxygen.
Phototrophs do not have to compete for an energy source so they can be found in a wide variety of habitats.
Metabolic options for obtaining energy
Chemicals–chemotrophy–Organic chemicals–chemoorganotrophs AND inorganic chemicals–chemolithotrophs
Light–phototrophy–phototrpphs
Obtaining carbon
All cells require carbon as a major nutrient
Autotrophs
-use CO2 as carbon source
-primary producers
Heterotrophs
-require organic molecules for carbon source
-feed on autotrophs or live off products produced by autotrophs.
Extremophiles
Organisms that inhabit extreme environments.
-hot springs, glaciers, salt water, high pH
Bacterial biodiversity
Many bacteria. Pathogenic prokaryotes. Proteobacteria is the largest phylum. -gram negative -e.coli, salmonella Gram positive bacteria are the second largest phylum. -clostridium, bacillus, lactobacillus Cyanobacteria are relatives of gram positive bacteria. -oxygenate the atmosphere.
Other bacterial groups
Planctomyces group: distinct stalk structure on cells.
Spirochetes: helical shape and include pathogens.
Green non sulfur bacteria: phototrophs that form filaments in hot springs.
Deinococcus: resistant to radioactivity.
Chlamydia: intracellular parasites.
Thermophiles: grow near the BP of water.
-Aquafex and thermotoga.
The domain of archaea
All archaea are chemotrophic.
Many are extremophiles: pH, salt, extreme heat and acid.
Some are chemoorganotrophs and many are chemlithotrophs.
2 phyla of archaea
Euryarchaeota -Methanogens: produce methane -Extreme halophiles: require high salt concentrations. -thermoacidophiles: high temp, low pH Crenarchaeota -hyperthermophiles -line in marine, fresh water and soil
Survey of eukaryotic microbes
Protozoa lack cell walls
-motile
-free living
Slime molds are between protists and fungi
-mold like fruiting body
-resemble protists in their unicellular phase
Lichens
Mutual relationship between two groups of protists
-fungi and algae or fungi and cyanobacteria
