1
Q
Bubonic Plague
A
Caused by Yersinia Pestis (spread by fleas and rodents)
- Infection of the lymph nodes
2
Q
Small Pox
A
Caused by Variola Virus
- cause skin lesions, contagious, airborne
- infect multiple organs
3
Q
Cholera
A
Caused by Vibrio Cholorae
- infection of small intestine, diarrhea, vomiting, dehydration
- contaminated food
4
Q
Robert Hooke
A
First Compound Lens Microscope
- coined the term cell
5
Q
Antonie van Leeuwenhoek
A
Magnifying Lenses (500x magnification)
- first to observe single celled orgs (called animalcules)
6
Q
Germ Theory and its contributors
A
Some diseases are caused by microorganisms
- Florence Nightingale
- Louis Pasteur
- Joseph Lister
They promoted the ideas of sanitation and hygiene
7
Q
Florence Nightingale
A
Founder of professional nursing
- tracked deaths during Crimean war
- found more soldiers died of microbial infection that battle wounds
- improved living conditions for soldiers
8
Q
Louis Pasteur
A
Used swan neck flasks to disprove the spontaneous generation theory
- discovered bacterial fermentation through lactic acid
- developed first artificial vaccine against anthrax
- developed pasteurization techniques for milk
9
Q
Louis Pasteur Swan Neck Flask Experiment
A
- broth boiled to kill bacteria
- no microbes appeared in broth but microbes trapped in curve
- flask tipped to allow broth to connect with microbes
- Microbes multiplied
(env cannot reach broth in the flasks while upward) - if neck was broken off and broth exposed to env then microbes would appear
10
Q
Robert Koch
A
- first to use animal model
- developed pure culture technique
- used technique to prove TB is caused by Mycobacterium Tuberculosis
- discovered agents causing TB, anthrax, cholera
11
Q
Joseph Lister
A
Sterilization
- surgeon
- realized most death was caused by infection
- antiseptic practice during surgery (carbolic acid)
- made surgeons wash hands and wear gloves
12
Q
Koch Postulates
A
1) Microbe is found in diseased and not healthy
2) Microbe can be isolated and grown in a pure culture
3) healthy individual can be inoculated and become diseased
4) microbe is reisolated from new host and matches original microbe
13
Q
Julius Petri
A
Discovered petri dish (a pure culture dish)
14
Q
Angelina and Walther Hess
A
First to develop solid media to culture bacteria
15
Q
Edward Jenner
A
Vaccines
- inoculated children with pus from milkmaids with cowpox
- child did not develop smallpox
16
Q
Discovery of Viruses
A
Dmitri Ivanovsky - found disease causing agent so small it could pass through 0.1microm pore
Martinus Beijeinck - proposed it was a virus
Wendall Stanley - using electron microscopy purified and crystilized agent and identified Tobacco mosaic Virus
17
Q
WWI and Spanish Flu
A
back to back epidemics (spanish flu)
- crowded trenches, moving populations, malnourishment worsened it
- killed 500 mil people worldwide (3-5% world pop)
18
Q
Sir Alexander Fleming
A
Found that the mold Penicillium Notam (secretes penicillin) inhibits the growth of Staphylococcus bacteria
- Mary Hunt found Penicillium Ruben (more efficient) on a canteloupe
Pfizer starts manufacturing penicillin for use in WWII (synthetic antibiotics)
19
Q
Concerns with Infection and Disease
A
1) re-emergence of disease (AMR, evolving disease) (Covid, Ebola, HIV)
2) changing susceptibilities (compromised immune systems, HIV, chemotherapy, immune suppressing drugs, aging)(disruption of microbiome, malnourishment, stress, prolonged antibiotic use)
3) Population Density and Globalization (moving populations)
4) Global warming (viruses proliferate better in warm weather, carriers expand where they live)
20
Q
AMR
A
2 mill cases of drug resistant infections is US/year
- AMR responsible for 2 billion medical care costs
- Escherichia Coli and Staphylcoccus were leading pathogens associated with resistance in 2019
21
Q
Human Health - Microbiome
A
- impact to microbiome largely unknown
- gut linked to brain via nerves
- microbiome linked to metabolic disorders, mental health, cancers, and neurodegenerative diseases
22
Q
Endosymbiotic Theory
A
Starts with lithotrophic prokaryotes
- mitochondria engulfed by prokaryote to allow for respiring proteobacteria, heterotrophic eukaryotes
- chloroplasts engulfed by prokaryote to allow for photosynthesizing cyanobacterium, phototrophic eukaryotes
23
Q
Carl Woese
A
- studied bacteria adapted to live in extreme environments
- 16S ribosomal RNA gene sequencing revealed these were a distinct lineage
- named them archaea
24
Q
Phylogenic Tree
A
- Initially only bacteria and eukarya were known
- archaea branches off the eukarya branch closer to the base
25
Euks vs Proks
Euks
- 80S ribosomal RNA (60S, 40S) - includes 18S subunit
- more densely packed genome
- diploid organism
- linear chromosomes
Proks
- 70S ribosomal RNA (50S) - contain 16S 23S and 5S
- one circular chromosome
- sometimes plasmids
26
Corolus Linnaeus and explain
- decided to name species Genus species (binomial nomenclature)
3 Kingdoms
- animal
- plants
- mineral
Modern: based on DNA sequence similarity
Defining a species:
- Orthologs: high amount of housekeeping genes
- Small subunit rRNA genes: 95% identity = same genus
- determine based off common traits and ecological niche
27
Bacteria (prok)
- nearly every habitat on earth
- mostly beneficial
- most have peptidoglycan cell walls
- photo (cyanobacteria), non photo
- huge diversity
28
Archaea (prok)
- nearly every habitat on earth
- extremophiles
- no known human pathogens
- pseudopeptidoglycan
- chimera btw bacteria and eukaryotes
29
Protists (euk)
Not plants, animals or fungi
Algae:
- uni or multi
- vary widely
- cellulose
- photosynthetic
Protozoa:
- Diverse
- move with cilia or flagella
- some photo, parasitic, or pathogenic
30
Fungi
- uni or multi
- non photo
Yeasts:
- uni
- infections
- food
Molds/ filamentous fungi:
- multi
- hyphae
- decomp and nutrient cycling
- pharmaceuticals (penicillin, cylosporine)
31
Viruses
Acellular
- proteins and genetic material (RNA, DNA)
- inert outside of host
- hijack cells
- can infect all types of cells (even other viruses)
32
Lab Tools
- Petri dish
- test tubes
- bunsen burner
- inoculation loop
- microscopes
- stains and dyes
- growth media (liquid and solid)
33
How much can the eye see?
1m to 100 microm
eye can distinguish between 150 micrometers
34
Light Microscopy (types and anything related)
0.5cm to 100nm
Types:
- brightfield -objects appear as a dark silhouette, limit resolution to 1000x, wet mount allows you to observe cells in natural environment but does not provide much resolution
35
Electron Microscopy
10microm to 0.1nm
- uses beams of e to visualize (in visible spectrum)
- up to 100 000x magnified
Scanning e Microscope:
- detects reflected e and creates an image
Transmission e Microscope
- detects e that move through thin sections of sample to create an image
- e dense regions appear darker
36
Properties of Light
As wavelength increases energy decreases
Light interacts with an object by:
- refracting
- absorbing
- reflecting
- scattering
wavelength (lamda) limits the size of objects we can see
37
Staining Overall (2 types)
Simple Stain: adds dark colour to cells but not env (methylene blue)
Differential Stain: stains one kind of cell but not the another (gram stain)
37
Compound Microscope and Oil emmersion
system of multiple lenses to focus and correct for abberration (10, 40, 100)
Oil emmersion: stops light from bending outside of view of eye piece
38
Microscopy Terms
Resolution:
- distinguish between 2 points
- low res: fuzzy
- affected by wavelength (wavelength must be smaller than or equal to to resolve)
- Numerical Aperture (NA) - higher means increased resolution power NA=nsin(theta), n is the refraction index
Contrast:
- distinguish between structure and environment (gram staining)
Magnification:
- ability of a lens to enlarge the image (x10 for eye piece, xwhatever for the lens)
39
Methylene Blue (procedure)
1) culture on slide (w inoculation loop)
2) spread thin
3) let dry (air)
4) fix slides to cell with methanol (air dry)
5) stain with methylene blue (sit one minute)
6) Wash off stain with water
7) blot off XS water
8)
*kills cells, distinguish between cell and env
40
Gram Staining
Gram + Purple - thicker cell wall with thick peptidoglycan layer
Gram - Pink - thin peptidoglycan layer and outer membrane of lipopolysaccharide
Process:
1) same process for fixing cells to slide
2) add methanol to fix cells to slide (air dry)
3) add crystal violet stain (1 minute)
4) add iodine which binds stain to gram + cells
5) wash off with ethanol (10s) (will wash off stain from gram - bacteria)
6) add safranin counterstain which will stick to gram - bacteria
*kills cells
41
Acid Fast Staining
To differentiate 2 types of Gram + cells
- those that have waxy mycolic acid in cell walls and those that don't
1) apply carbolfuchsin (30 seconds)
2) heat cells to slide with flame
3) decolorize with acid alcohol (15-20s)
4) apply counterstain methylene blue (30s) and rinse XS stain
*acid fast remains red (has waxy mycolic acid
42
Capsule Staining
Certain bacteria and yeast have a capsule
- presence of capsule related to virulence (harmfulness)
- negative staining technique required
- India ink stains surrounding media creating a halo around capsuled bacteria
43
Fluorescence Microscopy and Compounds
- detects "parts" of cells
Compounds: DAPI(stains DNA), FM4-64(stains plasma membrane)
Proteins: GFP, YFP, CFP
- fuse with gene of interest
- the transcribed and translated to the protein fused to GFP
Specificity of compounds and proteins determined by chemical affinity, labelled antibody, DNA hybridization, gene fusion reporter tags
44
Immunofluorescence
Identifies disease causing microbes by observing whether antibodies bind to them
1) antigen is fixed to surface
2) patient serum is added (if antibodies are present they bind to antigen)
3) Second antibody with the fluorescent tag is added
* if antibody is present the second antibody binds to it and shows the fluorescent tag
45
Phase contrast
- looks 3D
- exploits differences in refraction index between cytoplasm and env
- contrasts diff organelles
46
Microbial Growth and Why its important
- exist in communities (less than 1% culturable in pure cultures)
- growth measured in optimal env
Why is it important?
- characterize microbes
- understand impacts (beneficial/ harmful)
- optimize usage of beneficial microbes
47
Unculturable Microbes
99%
- so adapted to environment that we do not know how to grow them in a lab
- depend on factors provided by other species (cohabit niche)
- other live inside other cells (parasites)
- obligately symbiotic (cant survive without partner
48
Essential Nutrients
Must be supplied by environment
Can include Macronutrients (large amounts)
- CHNOPS (carbs, lipids, prots, nucleic acids)
- Mg, Fe, K, Ca (enzyme cofactors)
Micronutrients (small amounts)
- Co, Cu, Mn, Mo, Ni, Zn (components of cofactors for enzymes)
49
Energy Coupling
Catabolic - releases energy and breaks things
Anabolic - uses energy to build things
50
Microbial Growth Cycle
Binary Fission (usually)
- splits into two equal daughter cells
1) DNA Rep
2) cell elongated
3) form divison septum
4) cell seperated
Budding (yeast and others)
- asymmetric
51
Generation Time
In favourable optimal env cells divide at a constant interval
- doubling time
Nt = No x 2^n
n = generations
No - original number
Fastest Gen Time: Clostridium perfringens - 10 minutes
Slowest Gen Time: Mycobacterium leprae (leprosy) - 14 days
factors affecting:
- pH
- Temp
- nutrients
52
Forms of Culture (phases)
Liquid/broth
- in suspension
- pure culture
Applications:
- trying to obtain large number of cells
- looking at secreted products into broth
Solid Media
- gelled/ solidified with agar
- CFU
- develop from same mother cells
Applications:
- separate bacteria
- view separate cultures
- isolate pure culture
53
Types of Culture
Complex/rich media - nutrient rich poorly defined composition
Minimal Defined Media - Only essential nutrients, known conc
Enriched Media - something is added to complex media
(Auxotrophs) - cant make specific nutrients but they need them
Selective Media - only allows growth of one organism
Differential Media - two species grow equally well but exhibit differences due to the media (dye)
MacConkey Media - Selective and Differential
- only gram negative grow (selective)
- only lactose fermenting turn pink/red (only fermentors take up red indicator)
54
Dilution Streaking
Obtain single celled colonies to establish pure culture or estimate number of bacteria
Result:
- seperate single bacterium
- produce visible colony
55
Spread Plating
10x dilution in liquid culture
- small amount (0.1ml) plated on each agar media
- to obtain 30-300 CFU per colony depending on dilution level
- can calculate number of bacteria
Example:
# you count x dilution(to the positive exponent) x 10 = total
56
Counting (why and method)
- quality control
- government regulation
- research
Plate counts (dilution plating)
- long time
Haemacytometry - see diagram in book
- fast
- can calculate volume based off counted cells
- cannot tell if alive or dead
- can stain (if alive dye will not be taken up) (typan blue)
Optical Density
- light will scatter more at higher concentrations
- cells in liquid suspension
- higher concentration higher turbidity
Optical density - OD - light scattered by suspension at a specific wavelength
57
Microbial Growth Cycle
OD as a function of time
In batch liquid culture
- closed system
- no fresh media in
- no waste media out
Lag Phase
- bacteria adapt to env and maturing
- synthesis of RNA and enzymes and other molecules
Log Phase
- cell doubling
- if not limited will continue (remove waste and give more nutrients)
- slope of line is growth rate (depends on conditions)
Stationary Phase
- overall pop plateaus
- growth limiting factors or depletion of nutrients
- could be producing and inhibitory product
Death Phase
- without new nutrients or production of toxic byproducts bacteria will start to die off
58
Chemostat and Continuous Culturing
- continually add nutrients and remove waste
- highly controlled environment
- replicate native environment
- exponential growth
59
Prokaryotic Cells
- 70S ribosomes
- NO mitochondria
- cytoplasmic membrane
- Nucleoid (not membrane bound)
- thick complex cell wall (most)
- Flagellum - motile bacteria
- maybe additional cell structure
60
Pili
- hair like
- exchange genetic material
- adhesion to other bacteria
Sex pili: long, hollow, extensions of cell surface
- can transfer DNA during conjugation
61
Fimbriae
- shorter and thinner than pili
- used for attachment to surfaces (pathogens use pili and fimbriae to adhere)
62
Stalks
- thicker, rigid, extension of cell env and cytoplasm
- secretes adhesion factors to anchor bacterium in
- allow formation of biofilms in water streams
63
Flagella
- in some bacteria and archaea
- long helical appendages from cell membranes
- whip like
- used for motility
- you can stain flagella
64
Bacterial and Archaeal Nucleoid and Chromosomes
- irregular shaped region that contains genetic material
1-2 chsomes
- usually circular chsomes
- haploid (unpaired) - less gen variation and more detrimental mutations
- supercoiled DNA held together with NAPs to form nucleoid (flower)
- if coil is cut is turns into a line
65
Plasmids
extrachromosomal DNA
- small
- circular
- double stranded
- in archaea, bacteria, and some euks
- replicate seperate to the host cells genome
- they are smaller than chsomes
- copy number varies widely per cell
- usually contain additional or advantages genes (not required for everyday survival)
66
Modes of Gene Transfer
Vertical: parent to daughter (binary fission)
Horizontal: transfer between organisms
- almost exclusive to proks
67
Genetic Diversifying
Transformation: cells uptake DNA from env (maybe from a recently lysed bacteria)
Transduction: DNA transfer through bacteriophages
Conjugation: direct DNA transfer through pili
*transposons allow DNA to jump from one region to another
68
Prokaryotic Cytoplasmic Membrane
- diffusion barrier (prevents leakage)
- site of anchor proteins
- selectively facilitates transport in and out
- site of energy production (because no mitochondria)
69
Thermal Classification of Bacteria
Psychrophile - blow 15
Mesophile - 15-45
Thermophile - 45-80
Hyperthermophile - above 80
70
Bacterial Lipid Bilayer Contains
- phospholipids (saturated, oleic acid, cis/trans, cyclopropane FA)
- hopanoids, hopanes (regulate fluidity)
71
Bacteria Phospholipids
- side chains with no branches (tighter)
- glycerol-ester-lipids c=o (hydrolyses)
72
Archaea Phospholipids/bilayer
and why is it stronger?
*a stronger bilayer than bacteria
- isoprene chain with methyl every 4 carbons (cause not bend/ sturdy)
- glycerol-ether-lipids (does not hydrolyse - more stable)
- glycerol group is the mirror image of bacteria
- ends of phospholipids can bind together to create a monolayer (less fluidity)
73
Bacterial Cell Wall and Purpose
Called sacculus
- made of peptidoglycan sugar chains (murein) - to keep cell shape and turgor pressure
- alternating units of N-acetylglucosamine and N-acetylmuramic acid (NAG and NAM)
- NAM is bound to a 4-6aa protein that bind with another on a NAM across to create a cross link
- crosslinks (keep steady) and together
74
Bacterial Crosslink
4 aa and 4 aa
- 2 D-alanines on end of polypeptide second to the end is m-Diaminopemelic acid
- m-D binds with second from end D-ala (peptide bond)
- once crosslink is formed D-ala on end is released
75
Penicillin and Vancomycin to Bacteria
Penicillin - inhibits transpeptidase that cross links the peptides
Vancomycin - binds to D-ala-D-ala and prevents cross bridge formation
76
Gram Positive Bacteria "coating" - firmicutes
*some have tetrapeptides (on NAM) linked with pentapeptides (btw tetrapeptides)
- Glycosyl chains on outside, then s layer, then peptidoglycan layer, then bilayer
- multiple layers of peptidoglycan
- teichoic acids (shorter) and lipoteichoic acids (long and bind to phospholipids) as reinforcement
teichoic acids and lipoteichoic acids are polymers of glycerol
pros:
- very strong
- prevent against osmotic lysis
cons:
- susceptible to things that attack the cell wall (lysozyme) - b/c there is so much of it and peptidoglycan is the outer layer
- more susceptible to antibiotics
77
Gram Negative Bacteria "coating" - proteobacteria
2 membranes separated by periplasm with peptidoglycan inside
Outer Membrane
- lipopolysaccharides (LPS)
- porins (transmembrane for diffusion)
- lipoprotein (anchor peptidoglycan in place)
Thin peptidoglycan layer - 1 or 2 sheets
- attached to outer membrane with lipoprotein
Inner Layer
- diff than prots in outer layer
pros:
- outermembrane manages permeability
- large defense against toxins
- peptidoglycan protected
cons:
- energetically expensive to maintain and build
- usually have larger genomes (more chance of mutations)
78
LPS Complex (permeability barrier)
*very structurally variable
A glycolipid with 3 parts
1) O-antigen - repetitive polysaccaride (composition varies)
- length and kind of sugar can vary
2) Core
3) Lipid A (anchors)
- 6 FA tails in membrane
- disaccharide
LPS - harmless while attached but toxin and activate immune response when released from lysed cell
79
Mycobacteria
Very complex
-Thicker than other proks
- outer layer of hydrophobic phenolic glycolipids
- hydrophobic waxy mycolic acid (2 CH chains of diff lengths) membrane
- arabinan attached waxy mycolic acid layer to peptidoglycan layer
- then phospholipid bilayer
- have porins but very hard to get to
pros:
- waxy exterior
- resistance to dryness, osmotic stress, detergents, antiseptics, antibiotics, phagocytosis by host, immune response by host
cons:
- grows slow
- huge energy expense
80
S layer in prok cell walls
Almost all Bacteria and Archaea have an surface layer
- consists of identical proteins or glycoproteins and encloses wholes surface
- fit like tiles
Positive - on outside of peptidoglycan (before glycosyl chain)
Negative - outside LPS
Function:
- pores
- additional protection
- adherence and biofilm formation
81
Archaea Cell Walls
- no peptidoglycan
- diff types of cell walls or maybe no cell wall
1) Proteinaceous S-Layer
- anchored to cell membrane
- some species only have s-layer
- some also have a protein sheath (fit together like cells)
2) Pseudomurein
- N-acetylalosaminuronic acid (NAT) not NAM
- forms stronger bridges
- still peptide linked
- sometimes methanochondroitin is a cell wall polymer in some archaea
82
Capsules in Bacterial Cell Walls
- in gram positive or negative
- capsule exterior to cell wall
- coat of polysaccharide and glycoproteins
- hard to stain (appear as halos)
- if an s layer then the Capsules is more external
- if on streak plate kinda looks slimy
Functions:
- prevent phagocytosis (immune protection)
- Adherence
- stop dehydration
83
Photoautotroph
Energy from light and inorganic sources (like CO2)
- cyanobacteria
84
Photoheterotroph
Energy from light and organic sources (C from organic compounds)
- can do both (if no sun then energy from sugars)
- most green non sulfur bacteria
*Often organotrophic - using organic comps as a carbon source and an e source
85
Chemoautotroph/ Chemolithotroph
Energy from chemical compounds from inorganic source
- sulfur-oxidizing bacteria
*lithotrophy - mineral oxidation
86
Chemoheterotroph
Energy from chemical organic sources
- most bacteria
- animals
*Often organotrophic - using organic comps as a carbon source and an e source
87
Organotrophy and Autotrophy
- how do they connect
Organotrophy - breaks down glucose from polysaccharides and produces CO2 and H2O which is used by autotrophs
Autotrophy - energy from mineral oxidation or photosynthesis to create glucose which is used in organotrophy
88
Serotyping
Using the length and composition of the O-antigen in LPS to determine which serovors a species is
- same species different strain
- ed E-Coli O157:H7
O is the somatic response severity and H is the flagella immune response
89
Gram + Examples
Bacillus cereus
Streptococcus pyogenes
Staphylococcus aureus
90
Gram - Examples
Escherichia coli
91
Mycobacterium Examples
Mycobacterium tuberculosis
92
Yeast
Saccharomyces cerevisiae
- budding
- fungi
93
Nitrogen Cycle
N2 (in atmosphere)
- Nitrogen fixers (diazotrophs) convert N2 to NH4+ (rhizobium and cyanobacteria
NH4+ (soil)
- Nitrifiers oxidize NH4+ to generate energy (nitrosomonas, nitrobactar)
NO3-
- denitrifiers use oxidized nitrogen forms as alternative e acceptors (paracoccus)
Biology
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