Who invented the simple (one lens) microscope?
Leeuwenhoek - discovered microbial world
What are the types of microbes? What type of microbe cannot be seen with a regular microscope?
Algae, prokaryotes, fungi, protozoa, viruses, helminths
Viruses require electron microscopes
Contrast biogenesis and spontaneous generation
Biogenesis - life arises from life
Spontaneous generation - life arises from non-life
What was Needham’s experiment and what did it support?
Boiled gravy and sealed the container. The clear broth turned cloudy - supported spontaneous gen
What was Spallanszi’s experiment and what did it support?
Boiled gravy and sealed container - remained clear as long as seal was not broken
conclusions:
Needham failed to boil long/seal well enough
Microbes from air can contaminate experiments
Suggested spontaneous gen was wrong
What was Redi’s experiment and what did it support?
Observed meat decay - maggots only appeared if flies got to the meat - suggested that spon gen was wrong
What was Pastuer’s experiment for spontaneous generation? What special flask did he use?
Spallanzi’s exp + swan neck flasks
Allowed the broth to have access to air but not the dust - used to defy theory of air being needed for “life force”
If broth touched dust, microbes would form
What were the control and experimental variables in Pastuer’s experiment?
Control variable - allowing gravy to have access to air but not the dust - provided “life force”
Experimental - tilt flask to make gravy touch dust - provided needed contact for microbes to grow
Describe the steps of the scientific method. What is a control group?
Observations - Question/Hypothesis - Experiment - Accept, reject, modify hypothesis - Develop theory
Control group - same condition expert for variable that experiment is trying to test
How did Pasteur discover pasteurization?
Had an experiment to see if adding bacteria or yeast after boiling juice caused fermentation
Bacteria - acid
Yeast - alcohol
To prevent spoiling of the wine, boil juice then add yeast
What did Buchner discover?
Fermentation does not require living cells - enzymes in cell extract can cause fermentation
Discovered metabolism - chemical reactions that occur in organisms
What is germ theory and who discovered it?
Germ theory of disease: germs (pathogens) can cause disease
Discovered by Koch
What are Koch’s postulates and what do they prove?
Prove relationship between microorganism and disease
- agent must be found in all infected cases, absent in non-infected cases
- agent is isolated and grown outside of host
- agent exposed to healthy, susceptible host causes disease
- agent must be found in all experimental hosts
What did Semmelweis suggest?
handwashing to reduce spread of infection in hospitals
- women had 20x higher mortality rate
- believed med students were carrying “cadaver particles” - forced them to wash their hands - mortality rates decreased
What did Listel discover?
developed aseptic techniques in medical procedures.
spraying wounds, surgical incisions and dressings with a phenol solution reduced post-surgical infections
Who discovered the first “antibiotic”?
Salvarsan found an arsenic derivative used to treat syphilis - killed causative agent but not host
Ehrlich’s “Magic Bullet” - possible to kill specific microbes
What did Edward Jenner discover?
Small pox vaccine
took cowpox scab from milkmaid - ground it up - injected into kid - exposed kid to smallpox - did not contract
What did John Snow discover?
cholera epidemic in London was centralized around public water supply on Broad St
What is epidemiology?
study of occurrence, distribution and spread of disease in populations
Who was the founder of modern nursing/techniques?
Florence Nightingale - introduced antiseptic techniques in nursing practices
What are the 4 major processes that characterize living cells?
Growth - increasing in size
Reproduction - increase in number
Responsiveness - ability to react to environment (not all motile)
Metabolism - controlled chemical reactions in living cells (does not have to be constant)
Describe the basic structure of the prokaryotic cells
Nucleoid - aggregate of large, circular chromosome, no membrane bound nucleus
Cell wall surrounding the cell membrane
No membrane bound organelles
Include bacteria and archaea
What is glycocalyx? What is its purpose?
Polysaccharide containing material outside the cell
- Capsule - thicker layer, protects cell from host defenses (phagocytosis), pathogenic - makes harder for phagocytes to engulf
- Slime Layer - loose, thinner structure that prevents dehydration and can be used for adherence to objects
What is the structure and function of flagella?
Filament - outside of cell, rotates, made of flagellin protein subunit
Hook - connects filament to basal body
Basal body - attachment to cytoplasmic membrane and cell wall
Involved in motility and chemotaxis - movement to/from chemical
What are the flagella varieties?
Single - one flagella o the end
Polar tuft - multiple flagella on one end
Paratrichus - Covers the surface of the cell
Endoflagella - on both ends and spiral tightly around the cell, does not protrude. located between cytoplasmic and outer membrane, corkscrews through medium
Discuss the structure and function of pili.
pili - threadlike projections made of protein pilin
Conjucation - involved in bacterial mating - transfers DNA between bacteria cells through conduction pore
Conjugation is what is bringing us to pre antibiotic era
Discuss the structure and function of fimbriae
bristle like pili that allow bacteria to adhere to surfaces
- important in pathogenicity - need to adhere to host
- important in biofilm - bacterial community attached to a surface due to glycocalyx and fimbriae, resist disinfectants and antibiotics
Describe a Gram positive bacterial cell wall structure. Why is it gram positive?
thick layer of peptidoglycan that also contains techioic acids, has negative electrical charge
- peptidoglycan layer attaches to crystal violet dye
- lipotechoic acid - anchors peptidoglycan to cell membrane
Describe a Gram negative bacterial cell wall structure. Why is it gram negative?
thin peptidoglycan layer between inner and outer membrane
outer membrane:
- inner leaflet - made of phospholipids + proteins
- outer leaflet - made of LPS (lipopolysaccharide) endotoxin
- porins - integral proteins that act as protein channels through outer membrane
periplasmic space - between cytoplasmic membrane and outer membrane of Gram (-)
looks pink with Saffron pink
Describe the structure of the peptidoglycan cell wall in prokaryotic cells.
- sugar backbone consists of N-acetylglucosamine (NAG) and N-acetylmuramic (NAM) - glucose derivatives, link by glycosidic linkage
- tetrapeptide of AA on NAM - Ala, Glu, DAP, Ala
- transpeptidase links terminal Ala to another DAP - cross linking to form sheets for cell all rigidity
What is LPS?
lipopolysaccharide - found in Gram NEGATIVE peptidoglycan, endotoxin
O-polysaccharide - highly variable section, can be used to ID
core polysaccharide - similar structure with bacterial genus
Lipid A - immunologically active - can cause fever or septic shock (blood vessel dilation) - released by dead Gram (-) and outer membrane disintegrates
Describe the process of testing an acid-fast bacteria.
cell walls contain waxy lipid called mycolic acid - repels dyes, need to heat to drive stain into acid - turns pink
- Gram stain - turns blue
- mycolic acid prevents cell from drying out
What is the fluid mosaic model?
cytoplasmic membranes are made of phospholipid bilayers that have proteins freely floating laterally
- hydrophilic phosphoric head
- hydrophilic fatty acid tails
- cholesterol- helps cells membranes stay fluid
What is facilitated diffusion?
membrane protein allow molecules to move down their concentration gradient in/out of the cell without the use of energy
- nonspecific channel - range of chemicals that are right size/charge
- permeates - specific channel - binds substrate that causes change in channel shape
What is simple diffusion? Osmosis?
simple diffusion - movement of molecules from high to low concentration (H2O, glycerol, glucose)
osmosis - diffusion of water through specific water channel or through simple diffusion
What is active transport?
use of energy to move molecules against concentration gradient (low to high)
- transport protein acts as ATPase - takes ATP and makes ADP
What are the types of transport proteins for active transport?
uniport - one substance transported at time
symport - 2 substances transported at a time
antiports - simultaneously transport 2 chemicals in opposite directions
What is group translocation?
a type of active transport that alters a molecule are it passes through the membrane to prevent it from escaping the molecule
ex. sugar transport - energy used to add P group to sugar - ready for 1st step of glycolysis
In osmosis, what are the three types of relative medium a cell can be placed in?
isotonic - [same] - no net movement of H2O
hypertonic - high [solute] outside of the cell, causes H2O to move out of the cell, crenation - cannot carry O2
hypotonic - low [solute] outside cell - H2O goes in - can cause cell to balloon up and lyse
What are the structures mentioned for prokaryotic cells?
- nuceloid - contains bacterial chromosome
- ribosome - small protein made of polypeptides and rRNA involved in protein synthesis
- inclusions - reserve deposits that are used to store material (phosphate, energy, lipids)
- fibrae and pili - adhesive and conjugation
What are the structures mentioned for the eukaryotic cells?
- nucleus - contains most of cell’s genetic info
- mitochondria - ATP production
- chloroplast - contains chlorophyll and enzymes required for photosynthesis
- lysosome - comes from Golgi complex, store digestive enzymes, can bind with phagosomes
- flagella - filament-like structures used for mobility made from tubulin
cilia - hair-like structures used for mobility (no prokaryotic cells have CILIA)
What are the transport processes in eukaryotic cells?
endocytosis - uptake of substance into cell
- phagocytosis - uptake of solid substance into the cell
- lysosomes bind to phagosomes - enzymes break down things that have been phacocytized
exocytosis - export of substance away from cell
What is the endosymbiotic theory?
origin of eukaryotic cells - mitochondria and chloroplast are remnants of bacteria
Evidence: shape and size similar to bacteria, DNA looks like bacteria, ribosomes look like bacterial ribosomes
- divide independently from cells, but dependent on cell for proteins
What are the size of micro and nano meters? Which ones is bigger?
Micrometers is bigger
micro: 10x 10^-6
nano: 10x10^-9
What are the three basic principles of microscopy?
- Total magnification - produces enlarged, inverted image. Multiply objective x ocular lens magnification
- Resolution - ability to see two objects as separate. Dependent on wavelength of light + numerical aperture (ability to gather light). Light micro reso = 0.2 um
- Contrast - ability to distinguish specimen from background. Can use different light phases or dye
Describe light/bright field microscopy
background is illuminated.
adv - user friendly, available, inexpensive
disadv - recognize cell but not fine details. lack contrast, need stain
Describe a phase contrast microscope
has special condenser - increases contrast without staining
- uses alignment/disalignment of lightwaves to achieve different contrasts between live organisms + background
- does not require stain, out of sync lightwaves produce 3D effect
Describe dark field microscopy
dark background - bright, live specimen
contains special condenser that blocks light
good for viewing motility, pale, or small colonies
light seen is scattered by the organism
Describe fluorescence microscopy
visualize organisms that are fluoresce against non-fluorescing background
can use dye
light source - UV light - shorter wavelength than visible light
What is immunofluorescence microscopy?
uses antibodies + dye to bind to specific bacteria
- dye binds to antibody, antibody binds to bacteria
- hit with UV light to confirm presence
Describe electron microscopy
uses beam of electrons - wavelength 100,000x smaller than visible light
- light wavelengths too large to see viruses
- much higher resolution (1 nm) and magnification (100,000x)
- focus using magnets
- denser areas= darker
Describe the interference microscope
AKA Normarski scope - type of phase microscope
separates light then recombines it out of shape to produce better contrast
- produces 3D shadow appearance
- done without stain
Why do we use oil immersion?
- improves resolution and magnification
- light travels at different speed in glass and air: light lost due to refraction
- light travels at same speed through class and oil - less light lost, more light goes through objective lens
What is a differential stain? What are some examples
use of 2 types to distinguish cells on the different staining properties of the dyes
Gram, acid-fast, endospore
Describe the process of a smear
used for staining
microbes smeared on glass slide, air dries, and then heat fixed by passing it over the flame 1-2x
- heat fixing desiccates and fixates the microbes to they firmly attach to the side - preserves shape and size of specimen
Describe the process of Gram staining
- prep smear on slide. stain with Crystal violet - primary stain. binds to all cells. rinse with water
- add iodine - mordant that helps stain adhere to cells. rinse with water
- decolorize with alcohol ( > 10 sec). rinse with water
- counterstain with Safranin. rinse with water
Gram positive - purple
Gram negative - pink
Describe the shapes of bacteria
Cocci - sphere Bacillus - AKA rod Cocobacillus - small, in between cocci/rod vibrio - AKA curved rod Spirillum - stiff corkscrew Spirochete - flexible spiral Plemorphic - vary in shape and size
What are some arrangement of cocci
diplococci - two tetrad - 4 sarcinae - cube (8) streptococci - chain staphylococci - bunch
What is an endospore? Describe its relationship to a vegetative cell
dormant form of cell that is resistant to heat, chemicals, and drying in harsh environments due to keratin core
- important for durability and potential pathogenicity
- able to turn back into active vegetative cell when conditions improve - active form of cell that can reproduce and produce toxins
- NO NEW CELLS FORMED IN ENDOSPORE STATE, not considered a reproductive structure
Describe the process of endospore staining
- specimen heat fixed onto slide. paper towel places on top and flooded with malachite green
- heat slide over flame until malachite green steams. do not let malachite green dry out. heat allows malachite green to penetrate the keratin coat
- flush with water.
- counterstain with safranin for 1 minute to stain mother/vegetative cell
endospore - green, vegetative cell - pink
Describe the process of acid-fast staining
- heat fix specimen, place paper towel over and flood with red carbolfuchsin.
- heat slide over steaming water to drive stain past waxy mycolic acid in cell wall
- remove paper towel, cool slide, decolorize with HCl and water - stain retained in acid-fast due to mycolic acid
- counterstain with methylene blue to stain bleached non-acid-fast bacteria
acid fast - red, non - AF - blue
Describe negative staining and what it’s used for
capsule stain
- capsule is negatively charged, repulses acidic stain and forms a halo around cell
- stains background, leaves cell colorless
- can be part of pathogen - inhibits phagocytosis
Contrast the bacteria domain to archaea domain
Both: unicellular, no nucleus
bacteria - straight chain hydro-C cytoplasmic membrane, peptidoglycan
archaea - branched or ring hydro-C cytoplasmic membrane, no peptidoglycan, live in extreme environments, generally not pathogenic to humans
What are extremophiles?
live in extreme environments - archaea
- hyperthermofiles - require 80*C+ to function properly
- halophiles - require extremely saline environments necessary for cell wall integrity
What are the order of taxonomy?
DKPCOFGS - Developed by Linnaeus
What system do we use for nomenclature?
binomial Genus species (G. species)
species - narrowest category of classification
What are some ways of identifying microbes?
- physical characteristics/morphology
- Biochemical tests - microbe ability to ferment carbs, utilize substrates, produce waste
- Nucleic acid analysis - PCR testing - nuclei sequencing and comparison
- % of G + C content: sometimes species with similar content have similar characteristics