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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.