What are microorganisms
"Microscopic forms of life"
I.E. can be seen with a microscope, but not with the naked eye
Why study "Microbes"
they are essential for all life: produce usable nitrogen for plants which can then be used for the rest of the food chain
Microbes aids/functions
digest cellulose in cows and sheep, ecology: recycling, industrial processes, disease aents, models for study of basic biology
Antoni Van Leeuwenhoek
built simple microscope and observed protozoa and bacteria. He called them "animalcules" (sort of like the term molecules)
Compound microscope
was developed around 1820 but it took awhile for it to be refined. It uses more than one lens
How are microbes classified?
fungi: mold & yeast, protozoa, algae, prokaryotes, and viruses (viruses are NOT "organisms", cannot grow on their own)
Key questions of the golden age
1. Is spontaneous generation possible? (life created from nothing)
2. What causes fermentation?
3. What causes disease?
4. How can infection and disease be prevented?
Spontaneous generation
Can life arise from dead organic matter. Theory was first compiled by Aristotle.
Redi's Experiment (1668)
Used flies and dead meat to test the theory of spontaneous generation. Results: larvae appeared if flies in contact with decaying mean. No larvae was produced if the flies were kept out. Therefore, spontaneous generation was not supported.
Needham (1745)/ Spallanzani (1768) also tested for spontaneous generation:
Broth in air-tight flasks
Boiled and incubated
N: growth--used cork to seal (but it was leaky!)
S: No growth if HERMETICALLY sealed. Growth formed if air was allowed.
These experiments appear to refute spontaneous generation
OBJECTION: Air contains "vitalizing power" needed for life
How to deal with the "air" problem in disproving spontaneous generation?
Louis Pasteur's experiment did this
Louis Pasteur's experiment
broth is in swan-necked flasks
Boil-->incubate-->no growth (germs trapped)
Tilted falsk-->incubated-->growth
Pasteur's experiment showed that
air problem was solved, no growth if the flask was kept upright, but there was growth if the flask was tilted. Therefore the concept of spontaneous generation was finally laid to rest.
Pasteur showed that growth was due to
Contamination from the air! In his elegant (simple but beautiful) approach, no "vitalizing power" would be destroyed.
Koch's germ theory of disease
Isolation of "pure culture"
1. Host-several different types of bacteria present in the host
2. Grow the bacteria from host in liquid medium
3. Separate individual types by spreading bacteria on solid media (agar plate). Then incubate
4. Distinct colonies of bacteria on agar plate after incubation.
5. Pick colony of a particular type. Check cell type by microscopy. Grow in broth. Spread on agar. Repeat cycle at least 3 times: pure culture
pure culture
represents a genetically PURE stain because all the bacteria in a colony came from a single bacterial cell.
Koch's lab was separated from others because
first use of agar (better than potato slices)
distinct colony types
allowed isolation of specific microbes: "pure culture"
Pure cultures are essential to determine whether a particular microbe causes a specific disease
Koch's Experiment (CA. 1876)
He used 6 types of bacteria on 6 types of mice. Objective was to show that each type of bacterium caused a SPECIFIC disease. Koch argued that FOUR conditions must be met to prove this
Koch's Postulates
conditions that need to be met to prove that a bacterium caused a specific disease
1. Causative agent found in EVERY case of disease; absent from healthy hosts
2. Putative (thought to be like this or cause) agent can be isolated and can be grown outside the host
3. Disease forms when agent is introduced to healthy host (tentative evidence)
4. Same agent is RE-ISOLATED from diseased experimental host. (complete proof)
Impact of Koch's work
etiology (cause) of many diseases established by his group and later by otehr labs. This list includes: anthrax, malaria, tuberculosis, cholera, tetanus, tobacco mosaic disease, bubonic plague, and yellow fever
Ignaz Semmelweis
studied Puerperal sepsis: post partum disease. He observed that medical students would work from the morgue to the maternity ward without washing their hands. He theorized that "cadaver particles" (today, streptococcus) was what caused these mothers to die. He advised people to wash their hands and came up with a bleach solution. He argued that mortality rates would decrease from 18 to 1%. People called him crazy initially, but he was eventually deemed right.
Joseph Lister
a fan of Pasteur. He treated surgical instruments with boiling water. He also sprayed wounds and dressing with carbolic acid (phenol). This reduced the mortality rate by 70%
Florence Nightingale (during the Crimean War 1854-1856)
introduced hygiene and antiseptic practice. She helped to reduce the transfer of infection. Calculated statistically correlating sanitation vs. low mortality. She also suggested that bathroom facilities be in a different room or area which also helped to prevent infection. When she went back to England, she campaigned to reform hospitals and public health policy. Also, founded the Nightingale school of Nurses (1st of its kind)
Other pioneers in disease prevention
Snow: infection control (cholera in London)
Jenner: small pox vaccine
Ehrlich: chemotherapy (sulfadrugs; organoarsenics; Nobel prize in 1908)
Summary of the Golden age
living things com from other living things
microbes cause fermentation & disease
certain procedures and chemicals can prevent or cure infectious disease
Excellent examples of the scientific method as applied to biological sciences
technological developments of the Golden Age
Agar
Nutrient broth (beef extract, NaCl, peptone, water)
Compound microscope
Staining methods
Sterilization & disinfection
Antiseptics
Modern Age of Microbiology: Basic
1. Microbe centered-bacteriology, mycology, protozoology, virology, parasitology
2. Process centered- Metabolism, genetics, ecology, origin of life (Extra-terrestial microbes?)
Modern Age of Microbiology: Applied
1. Medical-immunology, epidemiology, chemotherapy, gene therapy
2. Environment-pollution, public health, agriculture, transgenic crops (Genetically Modified crops)
3. Industrial-food, pharmaceuticals, recDNA, products AKA genetic engineering technology
recDNA
recombinant DNA technology came from microbiology and affects all areas of biology today. It is defined as DNA sequences that result from the use of lab methods to bring together genetic material from multiple sources.
Eukaryotic cell types
contain a nucleus, have membrane-bound organelles, have complex chromosomes, and are 10-100 um diameter
Prokaryotic cell types
have no nucleus, no membrane-bound organelles, have simple chromosomes, and are 0.2-2.0 um diameter
Prokaryotic structures: Flagella
helical filament made of subunits: single protein species. rigid
Linked via the hook to: basal structure (motor.
Hook has 90 degree bend which allows the helix to point directly away from the cell.
Arrangement of flagella
1. Polar: at one or both ends of the cell
-monotrichous: single flagellum
-lophotrichous: tuft at the end
-amphitrichous: flagella at both ends
2. Peritrichous: all over the cell
Flagella and motility
prokaryotic flagella rotates. There is no whip-like motion but it is rigid. Induces motility: "runs" and "tumbles"
Why do flagella appear on Bacteria?
chemotaxis: attraction to chemicals
phototaxis: attraction to light
sensors in cell membranes
more "runs" when there is attractant
more "tumbles" when it is repellant
Fimbriae aka Pilli
short protein structures which adhesion to tissues via the tips; it has a twitching motility which attach and retract. Other pili racking motility is incremental motility unlike flagella. Gliding motility (100/cell)
Capsule (or slimy layer)
gelatinous sticky substance called glycocalyx (sugar cup) which is a feature of numberous pathogenic bacteria; e.g. Streptococcus. This enables oral bacteria to attach to teeth. This structure allows bacteria to avoid host defense cells
Cell Wall
provides structure and shape to cells, partially protects the cell from osmotic forces, key component in a net-like structure called peptidoglycan
Cell membrane
contains a phospholipid bilayer (40%) and protein (60%)
Functions in:
harnessing energy (metabolism) electro chemical gradient across membrane
solute transport into cells
chemical censors
Prokaryotic cell wall
protects against LYSIS, does not protect against PLASMOLYSIS (used in preservation of foods with salt/sugar), MYCOPLASMA do NOT have a cell wall
Solute Transport: Active
Uphill:against a concentration gradient
requires energy: ATP
specific for particular solutes
uses carrier proteins
Internal structures of cells
cytoplasm: gel-like; 80% water
chromosome: circular DNA (single
ribosomes: protein synthesis
Inclusion: e.g. Storage granules (NOT membrane-bound!)
Microscope types
light microscopy (LM), confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), scanning electron microscopy (SEM)
Light microscopy (LM)
compound: 2+lens
2000 X mag
oil immersion
bright-field
phase contrast
Other-dark field; differential interference
Regular light microscopy
glass to air: light refracted (bent)
Less light enters lens
dim image beyond 1000 X
Oil Immersion LM
glass to oil: light not refracted
more light enters lens
bright image beyond 1000 X
gram stain
based on cell wall structure; cells older than 24 hours appear to gram-ve (pink) when it is actually gram+ve (purple)
Fluorescent Antibody (FAb) Staining
An antigen is a foreign substance (usually a protein) that can trigger the production of specific antibody molecules in an animal. (Ag) Antibodies are made of proteins and react specifically with antigens. UV light: dye flows green. We use this system because it is effective in showing that the antibody is specific to the antigen.
Phase contrast microscopy
Light rays passing through specimen shifted by half wavelength. Rays in phase are brighter; rays out of phase are dimmer. Phase changes due to properties of the medium-which results in contrast. This gives you much better visual detail because of the contrast.
Confocal (laser-scanning) microscopy, CLSM
cells are stained with protein or antibody TAGGED with fluorescent dye. Illumination will be visible with UV laser. Dye glows. Small aperture removes blue:
in focus: bright
out-of-focus: black. Each image is an optical slice down to ~0.5 um. It scans specific planes; builds 3D images
Resolution: 0.4 um
Can observe living organisms in complex communities (biofilms), etc.
Transmission Electron Microscopy (TEM)
rays: electron beams
focused by magnetic lenses
tissue: electron transparent
Metallic stain: electron opague
TEM
stains binds differentially to tissue, electrons are transmitted or stopped. If transmitted, the screen glows. If not, the screen is black. Grayscale image is formed
Scanning EM (SEM)
specimen is coated with metal. focuses electron beam and scans the specimen. The computer reconsctructs images from reflections which has a 3D effect.
Looking at molecules
Atomic Force Microscopy (AFM): 1-10 nm: protein, DNA
Scanning (Quantum) Tunneling Microscopy: 0.5nm-10nm: Amino acids, protein, and DNA
Metabolism overview
organisms harness energy by breaking down molecules. This energy is utilized to build larger molecules and to perform other cellular functions. The chain of reactions used for the above are called "pathways." Enzymes are needed to carry out these reactions under physiological conditions and in a specific manner.
Reactions that require energy are
ENDERGONIC (do not do actions on their own)
\ energy is added in
\
D+E--------------->F
ATP
adenosine triphosphate; formed by a base sugar and three phosphates. It is a high energy molecule. Breaking of phosphate bonds releases energy
A-P~P~P-->A-P~P+P+Energy
Enzymes
allow reactions to occur at physiological temperatures, speed up reactions (catalysts), all are mostly made of protein (sometimes RNA), specific for molecules on which they act, and can be recycled
How enzymes work
1. Substrate-the molecule on which the enzyme acts
2. Active sites-specific for shape of substrate
3. Action: cuts or joins substates
4. Produces end products and enzymes are recycled
In catabolism, glycolysis is where
glucose is broken down
many steps...ATP input (energy investment)
Input: 2 NAD (Electron carrier)
Glycolysis
converting glucose to pyruvic acid. NAD (oxidized form) is used to make NADH (reduced form). It needs NAD back to keep glycolysis going
Fermentation
Glucose
NAD ->NADH | ADP -> ATP
Pyruvic Acid
NADH-> NAD |
Lactic Acid
In this process, pyruvic acid is reduced to lactic acid (or other acids/alcohols)-with the help of NADH which loses H to yield ATP. Thus, NAD is available again and glycolysis can go on...
Fermentation is inefficient
compared to respiration (2 ATP yield per glucose vs. 38 for prokaryotes)
When does fermentation occur?
it doesn't happen everytime there is glycolysis. Facultative anaerobes may undergo respiration or fermentation depending on oxygen availability. Only some organisms are facultative; others are either aerobic/anaerobic.
Types of Fermentation
Depending on type of bacteria, it may yield
Lactic acid: Homolactic
Lactic acid + other acids: heterolactic
Alcohols: alcoholic
End products of metabolism
give "personality" to species. Important for identification of bacterial: Medical diagnosis.
Respiration
breakdown of glucose to CO2 and H2O which gives a maximum release of energy
Glucose
|
Pyruvic Acid
|
Krebs Cycle
|
Electron Transport
Chain
ATP production=substrate level phosphorylation
Krebs cycle
Pyruvic acd converted to Acetyl Co-A. Acetyl Co-A enters the krebs cycle: 4-6 carbon molecules; undergoing oxidation.
Products: NADH, FADH2, ATP, CO2 (by product)
Electron transport chain
electrons and protons from NADH and FADH2 are transported via a series of REDOX reactions. Electrons are lost/gained in redox reactions. Final electron acceptor is O2 (usually
NADH e- negative redox potential
from Krebs -----> | Redox reactions in the
Cycle ----> H | electron transport chain
FADH2 |
Protons<------| Positive redox potential
O2 final electron
acceptor 1/2 O2 + 2H+ 2e -----> H2O
Electron transport
occurs in the cell membrane (bacteria). Electrons move along the membrane via redox reactions
What governs the electron transport process?
redox potential of reactions and position of proteins in the membrane
Results of the electron transport chains
Protons (H) moved across the membrane to the outerside
Hydroxyl ions (OH-) accumulate the inner side of the membrane
Formation of charge gradient across the cell membrane: "battery"
Energy is released when protons move back in a process called chemiosmosis!
Catabolism outline
glucose broken down via: glycolysis/(fermentation), Krebs cycle, electron transport chain
to yield: PMF: ATP (oxidative phosphorylation)
Transport motion
Enzymes and special proteins are essential for key steps
Anabolism
biosynthesis, building up, requires energy (ATP)
ATP--->ADP+ P exergonic
energy
D+E------>F endergonic
What is a PRECURSOR?
in metabolism, these are a substance from which another more mature substance is formed (opposite of breakdown product)
Carbohydrate biosynthesis
glycolysic products are precursors for: starch (plant), glycogen (animal starch), peptidoglycan
Lipid biosynthesis
glycolysis products are precursors for: fat
acetyl co-A is precursor for: fatty acids
Lipids are needed for membranes
Amino acid biosynthesis
Precursors for amino acids:
glycolysis products
Kreb cycle products
Amino acids are needed to synthesize proteins
Nucleotide biosynthesis
precursors of nucleotides:
glycolysic products
Kreb cycle products
Nucleotides are needed to make DNA/RNA
Metabolism summary
Molecules are broken down to release energy: Catabolism
this energy is stored as ATP
ATP is used to build larger molecules from smaller ones: anabolism
Many products of catabolism function as precursors for anabolism
Molecules synthesized include peptidoglycan, proteins, lipids, DNA, RNA
These molecules make up the cell
Metabolism is regulated by
synthesizing/degrading transport proteins, controlling enzyme activity to adjust product concentration, controlling enzyme synthesis, catabolizing the most efficient energy sources first; e.g. glucose is used before lactose
4 nutritional classes of organisms
1. Photoautotrophs
2. photoheterotrophs
3. chemoautotrophs
4. chemoheterotrophs
If mars has life on it, what classes of organisms would you find?
some types of autotrophs-photoautotrophs?
Methanogens
archaea that produce METHANE in wetlands (marsh gas), extremophiles, rumiants, and humans
Some methanogens are hydrotrophic,
that is they use CO2 as the carbon source and H2 as a reducing agent
Why is oxygen harmful to anaerobes?
oxygen releases free radicals which are harmful to organisms. Aerobic organisms have protective enzymes to break down free radicals. Anaerobic organisms DO NOT have these protective enzymes.
Nitrogen fixation
the capability of a few microbes to convert atmospheric nitrogen (N2) to inorganic nitrogen.
Physical requirements for growth
temperature, pH, osmotic pressure, and hydrostatic (water) pressure
Binary fission
chromosome replicates and cells grow in size, a wall or septum is formed, thus 2 cells are formed. Typical division takes 30 minutes. Growth continues until nutrients are depleted OR waste products accumulate to UNFAVORABLE levels
Exponential growth
cells number increases by the same proportion (2X) per unit time. (2,4,8,16,32...)