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8/12/2019 (172537095) Microbio Notes
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Scope and History 8/1/2013 7:47:00 PM
Scope and History of Microbiology
The Origin of the Earth
Life forms evolved back 4.6 billion years ago More than 80% of the earths history involved microorganisms Humans only less than a billion years
Anoxygenic phototrophic bacteria no oxygen before, only sulfure
3 bya earth is slowly oxygenated
- 3.8 3.9 BYA = appearance of microbial cells
2 BYA = earth was anoxic & O2 absent, N2 and CO2 were abundant
Phototrophic organisms
Purple sulfure bacteriao Anoxygenic can survive without oxygen
o Relatively simple
o Sulfur as primary growth requirement
Cyanobacteriao They made the work more oxygenic
o Oxygenic
o Circular, simple structures
Early life forms only contain mitochondria.
Cladogram
Shows the relationship of different species Different types and strains and how they have diverged from each
other
Ex.
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LUCA Bacteria Archaea
Eukarya
LUCA Last Unknown Common Ancestor
Life began from mixture of different forms.
Primordial Soup Theory.
Tree of Life
Different domains of life bacteria, archaea, eukaryotes Bacteria and fungi are not the same Fungi is under domain eukaryotes
Domain Archaea are:
Extremophiles - they thrive in extreme environments
High temperature, high salt concentration, etc.Domain eurkarya
Entamoebae Animals Fungi Plants Slime molds
Microbes: the good, the bad, and the yucky
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Common misconception: all microorganisms are bad.
Fact: not all microorganisms are bad.
Microorganisms can be good Helps the progress of society different sectors Helps by preventing diseases
o Food products
But they can also be used as a bioweapon Can also cause disease
Good examples: yakult, cheese, yogurt, cows can eat grass because of
microbes in their stomach
Saccharomyces cerevisige yeast Propionibacterium sp.
Other uses:
Beer fermentation Truffle Most expensive fungi on earth BT corn Bacillus thuringiensis injected into the corn; can
actually that particular plant to resist pathogenesis of a particular
fungi. result: healthy corn; it helps prevent proliferation of fungi
Oil spill some microorganisms can help clean oil; Molds used to heal wounds during the middle ages because
bread molds contain penicillin
Botox paralysis of the muscle caused by a particular organismcalled Clostridium botulinum
Vaccines first made by Edward Jennero He formulated a vaccine against small pox using a cow pox
organism
o Vaccines lessened diseases because of microorganisms
Biofuel made from ethanolBad examples:
Infections Tomb of King Tut full of fungi people died Viral diseases
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Versinia pestis caused a plague in Europe transmitted to ratfleas.
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The Microbes 8/1/2013 7:47:00 PM
July 5, 2013
Bacteria
Algae
FungiViruses
Protozoans
Bacteria
Single-celled No cell nucleus; Lack membrane-bound organisms Photosynthetic derives energy from sunlight Chemoorganotrophic derives energy from organic sources May be motile
E.Coli rod shaped, clusters
Present in small intestines, helps in digestionsEpulopiscium fishelsoni
Some bacteria are actually larger than protozoan. (Bacteria are not always
small) Ex. Epulopiscium fishelsoni is bigger than paramecium.
Thiomargarita namibienesis
Algae
Single/multi-cellular depends on the organism No cell nucleus Numerous membrane-bound organelles Mostly photosynthetic
o Except: Prototheca sp.
Lost its chlorophyll long long time ago. Parasitic. Uses humans to live.
Generally, they are not parasitic (Except Prototheca sp.). Therefore,little or no significance.
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Ex.
Recently, research: algae in biofuels Cyanobacteria Red algae
Fungi
Single/multicellular (mushrooms) Cell nucleus present Intracellular structures Absorbs nutrients from environments Generally not motile Medical significance (secondary metabolites)
o Fungal species have been found to have antibiotic properties
2 types
Macroscopic microscopic
Ex.
Amanita sp. Basicliomycetes Pezizacea Orphiocordyceps sp.
Parts
Haifa like the stem of a plant Conidia hold the spores of the fungi; head Spores reproduction
Protozoans
Under the kingdom Protista Single celled At least 1 cell nucleus Numerous intracellular structures Phagocytic engulf food then eat it
o Eat through phagocytosis
Some are motile Habitat: usually aquatic
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Ex.
o Some are in soil, and parasitic
AmoebaViruses
Acellular Has a head usually surrounded by a protein coat Usually, viruses do not have DNA. They have RNA.
o But there are some exceptions. Some have DNA.
Composed of nucleic acids + few proteins Exhibits living traits once it has infected host
o Dependent of replication
Lytic cycle
Once it infects the host, the virus replicates, then it will burst.Lysogenic
Virus replicates along with the organism Does not kill the organism. Just live alongside that org.
Viroids (NA, no protein coat)
Plant diseasesPrions: (no NA, w/protein)
Mad cow disease
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History of Microbiology 8/1/2013 7:47:00 PM
July 5, 2013
Black Plague
Xenopsylla cheopsiso Enters a flea causes blockageo Flea bites human infects him/her
Unsanitary middle ages
Zacharias Janssen (1590)
Invented the first compound microscope Allowed scientists to look through a lens to discover and explore the
microscopic world
Robert Hooke (1665)
Coined the term cellcellulae (compartments)Anton van Leeuwenhoek (1676-1683)
Looked at pond water animalcules
Spontaneous Generation vs. Germ Theory of Disease
Spontaneous Generation
Life generates itself from non-living matter. Solid, mud, dust, rocks frogs, worms,
1668: Francesco Redi
devised an experiment to prove that Spontaneous Generation isfalse
Maggots spontaneously arose from spoiled meat. Set-ups: spoiled meat inside
o Open jar flies were observed laying eggs on the meat.
Maggots developed.
o Mesh covered jars flies laid eggs and maggots developed on
the net.
o Sealed jar no maggots developed.
Louis Pasteur
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Opposed and dispelled spontaneous generation Pasteurization
o Ex.
o The wine is sterilized. Remove all microorganisms.
o Only one organism. So that that organism will be able to useall the nutrients in that wine.
Rabies vaccineo Using dried spinal cords of rabbits
Silkworm studieso Major step toward proving Germ theory of disease
Pasteur institute1859: Swan Neck Experiment
Broth in straight and swan neck flasks Boiled the broths and exposed into air
o Important to sterilize the broth to kill whatever organism is
there
After a few days,o Straight neck: discolored and cloudy
Airborne fell to the broth unobstructedo Swan neck: clear and same
Airborne microbes were trapped in the curve of theswan neck flasks
Organisms must come from air!Robert Koch
In vitro studies Pure cultures
o Use of potato slices as growth medium
o Defined bacterial colonies
Discovered Myobacterium tuberculosiso Staining
Acid fast bacillio Tuberculin supposedly a vaccine but failed
Isolated Vibrio cholerao Cholera
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GToD and Kochs Postulates
Mere association of bacteria and disease is not proof of cause andeffect
Experimented using Bacillus anthracisPostulates:
1. The suspected pathogen must be present in all cases of the disease and
absent from healthy animals.
2. The suspected pathogen must be grown in pure culture.
3. Cells from a pure culture of the suspected pathogen must cause disease in
a healthy animal.
4. The suspected pathogen must be re-isolated and shown to be the same
as the original.
Kochs Postulate In modern times:
You cannot inoculate the disease to another human. If you use an animal,
you are not sure if the same will happen to a human.
Human diseases not = to animal diseases
July 12, 2013
Ignaz Philipp Semmelweis
The unhappy hero of birthing mothers Hungarian doctor in Vienna 1847: his friend Jakob Kolletschka died of a cut during autopsy
(peurpal fever)
he discovered a connection between a disease and the causativeagent of that disease
o connection between germ and disease
encouraged doctors to wash hands he wanted to set a protocol that one doctor must wash before going
from one patient to another
1865: institutionalizedStreptococcus pyogenes
Causative agent of puerperal fever
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AKA childbed feverJoseph Lister
Founder of antiseptic medicine Prevented post-operative infection
o ward fever
Carbolic acid (phenol) to sterilize and sanitize wounds, surgicalinstruments
Lady Mary Montagu
Wife of the British ambassador She introduced the concept of variolation
o Crude form of vaccination
o Getting a small needle then having a blister fluid from an
existing small pox disease then
o Variolation only if the patient is quarantined
Edward Jenner (1794)
Benjamin Desty He injested cow pox to his children. Then his children did not have
small pox when exposed to persons with this disease
He inoculated the cow pox organism into a childElie Metchnikoff
Father of Natural Immunity White blood cells engulf foreign bodies
Martinus Beijernick
Microbial diversityo Formulation of enrichment culture technique
o Discovered Azotobacter
Virologyo Described the first virus
Tobacco mosaic viruso Refers to specific pathogenic molecules
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Sergei Winogradsky
Diversity of bacteria in soil and water Sulfur and nitrogen oxidizing bacteria
o chemolithotrophs
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Prokaryotic and Eukaryotic Cell 8/1/2013 7:47:00 PM
July 12, 2013
Prokaryotic Eukaryotic
Cytoplasm
Cytoplasmic membraneCell wall (peptidoglycan)
Nucleoid
Cytoplasm
Cytoplasmic membraneCell wall (chitin, cellulose)
Nucleus
Organelles absent ER, mito, chloro, golgi, cytoskeleton
Extracellular structures
(capsule/slime layer, flagella, pili)
Pellicle/test/ shell, cilia
All bacteria are prokaryotes
But not all prokaryotes are bacteria
Prokaryotic Cell Morphology
Shape Size Arrangement
Size
The importance of being small
Faster nutrition uptake Increased evolutionary capacity
Smaller cell = more effective it works
Adaptability and utilization of nutrients in new environments
Shape
Coccus
Rod
Sprillum
Spirochete
Stalk is thinner than hypa
Filamentous
Vibrio
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Overview of Structures
Gram + Gram -
Peptidoglycan thick Thin
Teichoic acid Often present Absent
Lipids Very little present LPS
Lipopolysaccharide
Outer membrane Absent Present
Periplasmic space Absent Present
Cell wall
Functiono
Maintans shapeo Prevents bursting
Peptidoglycan
Peptidoglycan layer is part of the cell wall + has smoother - has rougher domain bacteria differentiation between G+ and G- G+: 90% present, up to 40 layers G-: 10% present Rigid structure Acid-fast bacilli thinner peptidoglycan than G-
o Presence of lipids in cell wall
Impedes passage of nutrients so slow growersGlycan tetrapeptide (familiarize yourself with this structure)
2 sugar derivativeso
N-acetylmuramic acido N-acetylglucosamine
o Linked by beta-1,4-glycosidic bonds
Amino acids (stereoisomers) Lysine or DAP Destroyed by lysozyme
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Teichoic acid
Negative charge in cell surface Passage of ions through cell wall (Ca2+ and Mg2+) Attachment sites for bacteriophages (viruses which will infect
bacteria)
Lipoteichoic acid
Intimately associated with lipids of the membraneOuter Membrane
3 important parts which constitute the LPS layero Phospholipids
o Polysaccharides
o Proteints
LPS is made up of three partso Lipid A
o Core polysaccharide
o O-polysaccharide
O-polysaccharide
Galactose Glucose Rhamnose Mannose
Core polysaccharide connects O-poly to Lipid A
Ketodeoxyoctonate (KDO) Heptoses Glucoses Galactose N-glucosamine
Lipid A constitutes the endotoxin
Glucosamine Phospate
Periplasmic space
Area of cell metabolism Gel-like consistency
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Aqueous Resembles the cytoplasm Contains hydrolytic enzymes, binding proteins or enzymes Contains chemoreceptors The outer membrane prevents diffusion of the proteins present in
the periplasmic space
Gram staining technique
Fixation Crystal violet Iodine treatment (mordant facilitates attachment of the dye to
the cell)
Decolorization (With ethanol)o In G+, the thick cell wall prevents removal of crystal violet.
Ethanol dehydrates the cell wall.
Counterstain with safranin Gram +: purple while Gram -: pink
Cell wall-less Prokaryotes
Mycoplasma (causes pneumonia) Thermoplasma
Cell wall are replaced by sterol
Functions as a cell wall For rigidity
These cells have no definite shape.
They also survive in environment that is osmotically stable.
Archael Cell Wall
Gram variable Pseodomurein
o Lacks peptidoglycan layer
Paracrystalline surface layer (S-layer)Pseudomurein
Beta-1,3-glycosidic bonds
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N-acetylalosaminuronic acid Amino acids present are all L-forms Resistant to lysozyme
Ex of archaea
Methanosarcinao Glucose
o Glucoronic acid
o Galactosamine uronic acid
o Acetate
Halococcuso Sulfate
Cytoplasmic membrane
Also called Fluid mosaic membrane Highly selective barrier Permeability barrier and transport Lipid bilayer Hydrophilic lipids contains glycerol Hydrophobic lipids fatty acids
Functions:
Permeability barrier
Regulate flow of nutrients in and out of the celProtein anchor
Site of transport proteinsEnergy Conservation
Energy is supplied by mitochondria (euk) For prok, there is energy conserved in the cytoplasmic membrane.
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Prokaryotes 8/1/2013 7:47:00 PM
July 19, 2013
Outline:
Internal Cell Structureso Cytoo Ribo
o Nucleoid
o Inclusions
o Endospores
Cytoplasm
Gel like consistency Aqeous solution
o Enzymes
o Proteins
o Carbohydrates
o Lipids
Ribosomes
Protein synthesis There are 2 subunits
o 30S small subunito 50S large subunit
o 30S + 50S = 70S
complete 70S ribosome Svedberd Units (S)
o Sedimentation coefficient
Nucleoid
Eukaryotic have distinct nucleus where DNA is present In prokaryotic, there is only a region called the nucleoid. No
boundaries like the nucleus.
Contains the genetic material of prokaryotes Visible mass of aggregated chromosomes
o DNA, RNA, and protiens
Prokaryotic organisms have circular chromosomes
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o Not true for all organisms
Plasmids
Supplements genetic info jumping-genes they complement the existing genetic material. They add more
genes to the organism.
Inclusions
Granules not bound by any membrane at allo They do not separate from cytoplasm because of its dense
structure very thick
Carbon Storage Polymers
Poly-beta-hydroxybutyric acido Broken down and synthesized if excess C is present
Glycogen
Polyphospate granules
Aka volutin (metachromatic appearance when you stain them they display different colors of intensity)
Accumulates inorganic phospates Stores phosphate to be synthesized into polyphosphates for future
use
Sulfur storage
H2S oxidation:o Chemolitotrophs utilize carbon nutrients
o Autotrophs
Stored in the periplasm (most G-bacteria)Magnetosomes
Contains intracellular particles of iron mineral magnetite (Fe3O4) Magnetotactic alignment of inclusions, not movement per se Surrounded by thin membrane of phospholipids, proteins and
glycoproteins
Morphology is species specific.o Ex. Shape is different for each species.
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Function is unclear but some scientists hypothesize thatmagnetosomes: orientation to low O2 concentrations in aquatic
organisms
Gas vesicles Confers buoyancy to an organism Helps the organism to rise up a water body to get optimum amount
of oxygen/ sunlight depending on what they need
Orientational response to environmental cues Spindle-shaped structure made of proteins Hollow and rigid Found in planktonic blooms and some cyanobacteria
Endospores
Terminal spores Subterminal spores Central spores Highly resistant to heat, radiation, and harsh chemicals
o It is a protective and dormant structure
Produced primarily by Gram + bacteria (double check if + or -)o Bacilus
o Clostridium Remains dormant indefinitely; germinates quickly Parts:
o Exosporium
o Spore coat
o Core wall
o Cortex
o DNA
Several componentso Dipicolinic acid
o Small acid-soluble proteins (SASPS)
Dipicolinic acid + calcium = dehydrates the cell, intercalates
in DNA
SASPS binds to DNA (resists denaturation) & C and energy
source
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Vegetative Cycle and Sporulation stages Endospore activation outgrowth
EXTERNAL STRUCTURES of Prokayrotes
Flagella
Most studied organism for this is E. coli Helical Very thin flagella in prokaryotes Composed of protein subunits flagellin Classified as
o Peritrichous
Inserted around the cell (everywhere)o Polar
Attached to one or both ends of the cello Lophotrichous
Tufts of flagella at one end of the cell Basal body (roptor) model organism Gram -
o L ring embedded in the LPS
o P ring embedded within the peptidoglycan
o MS ring embedded within cytoplasmic membraneo C ring innermost ring; adjacent to cytoplasm
o Rod
Statoro Mot proteins
Generates torque rotate which hgives rice to rotationof rings
o Fli proteins
Acts as motor switch Reverses direction of rotation
Proton motive force acts as a battery Tumble-run movement
o Bundle up tumble to change direction bundle againMicrobial taxes
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Governed by tumble-run movements 2 types
o chemotaxis
movement in response to chemicals run-forward movement (CCW) tumble-stops and jiggles (CW) Towards higher concentrations of attractment
Less tumble, more runs Decreased concentration of repellant increases the runs Temporal factors are detected by the cell
Chemoreceptors membrane proteinso Phototaxis
Scotophobotaxis Observed microscopically Purple bacteria recognizes darkness and turns
back towards the light
True phototaxis Moves up a gradient of light from lowest to higher
concentrations
Axial filaments
Endoflagella Corkscrew rotation Found in spirochetes organisms Treponema pallidum causative agent of syphilis
Pili
Sex pili (conjugation pili)o Pathway for genetic transfer
o Genetic variation (eg. Antibiotic resistance)
o Streptococcus pyogenes
Fimbriaeo Adherence to surfaces increases pathogenicity by enhanced
colonization
Colonization increases ability to infect another cello N. gonorrhoaea, Salmonella sp, Bortodella pertussis
o Pellicle formation in broth cultures
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Air-water surfaceGlycocalyx
Capsuleo Rigid, tight matrixo Excludes small particles
o Outermost layer
o Complex polysaccharide molecules arrange in loose gel
o Increased pathogenicity
o Resist phagocytosis and dessication (removal of water)
o B. anthracis
Slime layero Easily deformed
o Loosely attached to cell wall
o Permeable to particles
o Increased attachment to surfaces
o Resists dessication
o Biofilm: binds cell together
Dental plague
Eukaryotes that are classified as microorganisms Fungi Algae Protozoans
EUKARYOTES
Cell Wall
Rigidity and protection Algae: cellulose and polysaccharides
Cytoplasmic membrane
Sterols (rigidity) Not a site for respiration and energy conservation
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EUKARYOTES Internal Structure
Cytoplasm
Has a cytoskeleton confers structure and additional rigidity Compared to prokaryotes, theres only a small portion of cytoplasm
in eukaryotes bec of presence of other organelles
Cytoskeleton
Network of fibers (microtubules and microfilaments) Confers shape to cell Aids in movement
FUNCTIONS:
Mitosis and cytokinesis Cell communication Transfer of organelles Cell growth and differentation
Nucleus
Nuclear membraneo Regulated entry and exit of molecules
Nucleoplasmo Cellsap (fluid)
Nucleoluso Contains rRNA and proteins responsible for ribosomes
Chromatinso DNA
o Become chromosomes (Aggregated)
o Histone proteins
Mitochondria
20% of the cell used for cellular respiration energy released from organic molecules ATP Cristae: increased surface area Semi-autonomous organelles
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o Origins as a free living organism
Plastids
Contains DNA as well types: chloroplasts, amyloplasts, chromoplast Functions: photosynthesis and storage
Ribosomes
Site of protein synthesis 60% RNA, 40% protein RNA Amino acid proteins
Endoplasmic reticulum
Mnodification of polypeptides Rough ER
o Ribosomes are attached
Smooth ERo Lipid functions
Transitional ERo Associated with Golgi apparatus
Golgi apparatus AKA dicytosomes, Golgi cisternae Packaging and secretion of proteins and lipids Functions:
o Synthesis of non-cellulose component of cell wall
o Storage of proteins, sugars and enzymes
Lysosomes and Peroxisomes
Inclusions: Vacuoles
Up to 90% of the cell occupied by vacuoles (older the plant, thebigger the vacuole)
Contains cell sap (salts, sugars, organic acids water) Recycling of materials within the cell
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Aids in the breakdown and digestions of organelles (mito andplastids)
Storage of fats, startch, and oxygen
EUKARYOTES External Structure
Flagella
Larger and more complex than prokaryotic flagella Whip-like movements Protein subunits: tubulin
Cilia
Shorter and more numerous than flagella In protozoans: coordinated beating for faster movement Paramecium sp.
Pseudopodia
False feet Temporary projections of the cytoplasm Amoeboid movement Slow, inching-along process
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Microscopy and Principles of Staining 8/1/2013 7:47:00 PM
July 26, 2013
Resolution
Microscope not concerned with magnification itself.
What is important is the resolution. Ability of that microscope to separatetwo adjacent organisms.
SIMPLE TYPES of MICROSCOPES
Dark Field Microscopy
Condenser prevents light from passing through Light object on a dark field
Phase Contrast Microscopy
Organisms that cannot be stained Condenser and lenses accentuate small differences Different degrees of brightness
Nomarski Microscopy
Differential interference contrast microscopeo Higher resolution
No halos, 3D-like effect Short depth of field organism must be relatively thin
o Can only penetrate until a particular point
Fluorescence Microscopy
Use of UV light for molecules to release loner wavelength of light Treated with fluorescent dyes
o Fluorochromes (Acridine orange)
Binds to nucleic acids green, orange, yellow Myobacterium tuberculosis Treponema pallidum Naegleria fowleri
ELECTRON MICROSCOPY
Beams of electrons, not light (photons)
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Electromagnets instead of lenses
Vacuum environment
Transmission Electron Microscopy
Better visuals on internal structures Magnification up to 500,000x Thin preparations of specimen (70-90 nm) Shadow casting (heavy metals) Freeze-fracturing
For internal cell structure viewing
Micrographs pictures taken through the microscope
Scanning Electron Microscopy
Surfaces of specimen Magnification up to 100,000x Specimen coated with heavy metals (gold) Electron beam scans back and forth
Atomic Force Microscopy
Tiny stylus is positioned extremely close to the surface of thespecimen
Surface is surveyed Pattern processed by detectors Generated and visualized by a computer Similar to SEM
o Minus the heavy metal coating = live cells
Staining Techniques
Wet Mounts
Visualization of living organisms Addition of carboxymethylcellulose to slow down motility of
organisms
Hanging Drop Disadvantage: gives little or no contrast at all
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Smear and Stains
Basic dyes Acidic dyes + Cells
See lab slides
Simple staining
Differential Staining
Negative
Used for morphological observations Acidic dyes nigrosin, india ink, congo red Background is stained Microorganisms that are sensitive to heating
o E. coli
o P. aeruginosa
o S. pneumonia
o S. epidermidis
Gram
Spore
Lab slides Visualization of spores Endospore walls become permeable to malachite green when
steamed for 5 mins (green structures)
Vegetative cells are then stained with safranin (red-rod shapedcells)
Ziel-Neelsen Staining
Stains acid-fast bacillio Presence of mycolic acids
o Myobacterium sp.
TB and leprosy AFB retains the primary stain, carbolfuschin Does not stain well with Gram stain
Steps:
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Sample (sputum, biopsy, lymph node, etc) Cover with carbolfuschin Hold over the flame until steaming for about 8 mins. Replenish stain
as needed.
Decolorize with acid alcohol (contains HCl) for 15-20 sec. Drain andrinse with water.
Counterstain with methylene blue for 30 sec. Drain and rinse withwater.
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Culture Media Preparation 8/1/2013 7:47:00 PM
Solid and Liquid Media
Solid ex. Agar plate, agar tube Liquid ex. Broth What shows growth of microorganism in liquid media?
o Turbidity tells you that there is a growth of microorganismsbut does not tell you if that is your culture
Pure culture colonies have the same color, shape, etcTypes of Cultured Media
Definedo Contains precise amounts of highly purified organic/ inorganic
components
Complexo Contains dehydrated digests of animal, plant or microbial
products
Casein Yeast Beef Soybeans (in the form of Tryptic soy)
Imprecise nutritional componentsDefined usually clear. Complex is usually colored because of presence of
different components.
Autotrophs can produce their own carbon
Heterotrophs
Complex media
Enriched Mediao Complex base with additional nutrients such as blood and
serum
Chocolate Agaro Usually used for culture of Neisseria and H. influenza
Blood Plate Agaro S. haemolyticus
Selective Media
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Nitrogen Sources
o Contains compounds that inhibit growth of one organism
while supporting the growth of another
MacConkey Agar (MAC)o Bile salts inhibit G+
o Originally red in color (media) Turns yellow if not lactose fermenters
Eosin Methylene Blue Agar (EMB)o Eosin and methylene blue inhibits G+
Mannitol Salt Agar (MSA)o Mannitol and 7.5% salt inhibits NaCl-sensitive G+ and G-
Differential Mediao Contains an indicator
o Determines whether a particular chemical reaction occurred
o Useful for differentiation of organisms (esp. among specieS)
MACo Neutral red dye stains lactose fermenters
EMBo Indicator: Methylene blue differentiates L+ and L-
o Determine whether a sample has E. colli or not
MSAo Indictor: Phenol red - stains mannitol fermenters
o For the culture of G+o Inhibits all G-
Nutrient Requirements
Carbon Sources
Carbon dominant nutrient Needed for synthesis of new cell material Heterotrophic microorganisms
o Glucose, lactose
Autotrophic microorganismso CO2 reduced to glucose
Glycolysis, fermentation, Krebs cycle
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Vitamins
Makes up 13% of the cell-proteins, nucleic acids, cellular material Natural nitrogen: NH3, NO3, N2 Enzyme/ protein synthesis Amino acid production Fastidious organisms usually require the 20 amino acids in medium Some act as nutrient parasites (Disease-causing organisms) Tryptone, peptone
P,S,K,Mg,Ca,Na
P
S
K Mg
Ca Na
o ATP synthesis, phospholipids, and nucleic acids
o PO4 3-
o Protein synthesis, other cell components
o HS, SO4 2-
o Enzymatic activity
o Ribosome, nucleic acid, membrane stability, enzymatic
activity
o Cell wall stability, heat stability of endospores
o Depends on the microorganism: on the nutrient requirement
of that organismTrace Elements
Functions as cofactors in enzymatic reactions Copper, zinc, magnesium, potassium, etc. Cobalt-Vit. B12 synthesis Iron-cellular respiration
o Heme-containing compounds (cytochromes)
o Redox reactions, ATP synthesis
o Fe2+ (anoxic), Fe3+ ( )
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Function as coenzymes Folic acid Vitamin B12 Vitamin K Pathogenic organisms obtain vitamins from humans Complex medium required for in vitro cultivation Streptococcus sp., Lactobacillus sp., Leuconostoc sp.