Quiz 2 Pages 8-22 Flashcards by Jennifer Walrath

1) Organisms must be present in every case except healthy individuals
2) suspected organism must be isolated and grown In Pure culture
3) isolated pure culture organism must elicit the disease
4) microorganism must be isolated from the newly diseased subject

Provides knowledge of the disease
A starting point for curing infections

Koch’s postulates

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Salversan

Chemotherapy for syphilis, does not harm the patient

arsenic

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Antibiotic penicillin
Clear zones
Scottish

Alexander Fleming

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Invented agar

1) solid surface
2) not digested by most Microorganisms
3) withstands high temps

Eilshemius Hesse

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Strep
Staph
Pallisade

Chains

Clusters

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Based on wall composition
Gram stain
Acid fast stain

Differential staining

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What an organism eats and what it releases

Metabolic capabilities/limitations
Carbohydrate utilization
Metabolic byproducts
Ability to ferment

Biochemical tests

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Studied anthrax
Cultured Microorganisms from blood
Injected them into healthy animals
Infected Animals died 
Isolated and cultured these Microorganisms from dead animals etc...

Robert koch’s experiment

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Using animal responses to organisms for classification

Serology

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Anything that illicit an immune response

Antigens

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Using specific antibodies in a multi well plate
Adding unknown bacterial specimen
If antibody recognizes bacteria positive test
See a color change

Elisa

Enzyme linked immuno absorbant assay

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Run patient protein on a gel
Transfer to filter paper
Wash paper with antibodies to specific bacteria
Antibodies have dye coupled to them
Get color, got disease

Western blot

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Semi-solid matrix of agarose or acrylamide
Run an electrical current
Molecules move based on size and charge
Use DNA fragments of known size to see progress
Visualize by radiation onto X-ray or ultraviolet fluorescence

Gel electrophoresis

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Is the sample susceptible to the virus? Positive Id

Phage

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Useful for id of fluorescent bacteria

Add fluorescent due tag to listeria antibody to milk

Flow cytometry

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Base composition of cg / at ratio

Genetic Id

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Restriction mapping: restriction endonucleases cut at specific sequences, run on gel to find pattern to match to known species

Genetic Id

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PCR - polymerase chain reaction

Use primers to amplify a region of DNA to a level that can be run on a gel

Genetic id

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1) denature DNA with high salt & temp
2) add a single stranded tagged probe
3) run on a gel and look for probe

Nucleic acid hybridization

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Make copies of probe DNA
Collect grow muse denature sample
Mix with probe
Detect probe for positive id

DNA probe for known organisms

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Ask a series of questions by running tests
Each answer reduces the possibilities by 1/2
Identifies organism by it’s characteristics
Useful for pathogens

Dichotomous key

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Resolving power = ocular magnification x objective

bright field

Multiple lenses

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Light can move through slide, specimen, objective ocular to your eye without any loss to scatter

Oil immersion

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Each Time light passes from one medium to another some light is lost
Oil immersion helps

Refraction

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Chromophore is the positive ion Bacterial cells are slightly negative Crystal violet, methylene blue, malachite green & safranin

Basic dyes (stains)

Chromophore is the negative ion negative staining (stains the background) Clear organisms

Acidic dyes (stains)

Basic dye and alcohol, general cell properties

Simple stain

Bacteria will appear different depending on their characteristics

Differential stain

Hans Christian gram 1) heat fix cells to slide 2)add crystal violet 3) wash off excess 4) add iodine as a mordant 5) decolorize with alcohol 6)counter stain with Safranin 7) wash with water 8) blot dry

Gram stain

Purple Retains crystal violet/iodine due to thicker cell walls Sensitive to penicillin and cephalosporins

Gram positive

Pink Colorless after alcohol Counter stained with safranin Thinner cell wall with lipopolysaccArides is disrupted by alcohol so crystal violet and iodine complex wash away Relatively resistant to antibiotics due to the lipopolysaccArides in the cell walls

Gram negative

Binds strongly to bacteria/waxy cell wall I'd for mycobacterium tuberculosis and M. leprae 1) heat fix smear 2) carbolfuchsin dye 3) heat to aid in penetration of the dye 4) wash with water 5) decolorize with alcohol 6) counter stain with methyl blue 7) acid fast organisms retain the dye

Acid fast stain

Examine live specimens | Syphilis diagnosed this way

Darkfield microscopy

Live specimens | Visualize internal structures

Phase contrast

Electron beam passes through a specimens electrons impact fluorescent screen or photographic plate Heavy metals for staining View subcellular structures and viruses Thin slices

Transmission electron microscopy

Electron Beam knocks an electron from the specimens surface These are collected and used to create a 3D inmage on a plate or screen Provide 3D views of intact specimens

Scanning electron microscopy

Temp, pH, osmotic pressure

Physical needs of Microorganisms

-/+ oxygen | C, N, S and Phosphorus

Chemical needs of Microorganisms

Incubators Mesophyles like us Psychrotrophs 0-30 Celsius

Temperature

Most bacteria neutral Yeast & molds 5-6 Heliobacter pylori /80% of ulcers/koch's postulates/don't do research on themselves

Created by the movement of water Membrane Diffusion important too Plasmolysis - plasma membrane pulls away from cell wall, dehydrates bacteria to death, salt or sugar

Osmotic pressure

All Life forms are based on...

Carbon

Can survive with CO2 as their only carbon source

Autotrophs

Require a reduced carbon source ie, glucose methane fatty acids

Heterotrophs

Required for proteins, nucleic Acids and ATP

Nitrogen sulphur phosphorus

Survive with or without oxygen

Facultative anaerobes

Oxygen is toxic

Obligate anerobes

OH- (hydroxyl radical) O2- (superoxide radical) SOD (superoxide dismutase) makes peroxide H2O2 (hydrogen peroxide) catalase makes water and O2 Aerobic and aerotolerant organisms have enzymes that handle these toxic products

Oxygen reduction Produces 3 toxic compounds; all have extra electrons

Isolation and growth of a single organism Aseptic technique Sterile implements and media

How to get a pure culture

Obligate aerobe Obligate anerobe Facultative anerobe

Location of growth in broth indicates oxygen tolerance

Inoculum spread with a sterile loop | Goal is to grow individual colonies

Streak plate

Inserted into an agar slant identifies anaerobic metabolic activity

Stab

Inoculate liquified agar and Pour into a Petri dish Allows for isolated colonies Identifies anaerobic growth ability

Pour plate

Storage

Slant

All components and amounts are known, fastidious organisms, microbiological assays (to determine if a Microorganism is making a vitamin)

Chemically defined media

Exact components and their amounts are unknown i.e. Luria broth, nutrient broth, nutrient agar, chemoheterotrophic organisms

Complex media

Provides favorable conditions for target organism whole discouraging the growth of unwanted Microorganisms Bismuth sulfate agar

Selective media

Allows all organisms to grow but appearances differ according to the species, Eosin methylene blue (e coli is green), Blood agar (streptococcus pyogenes, hemolysis)

Differential media

Using Anaerobic chambers equipt with air locks and filled with inert gas

Culturing anerobes

Utilizing specific growth factors to increase the #'s of desired organism, (Example: enriching for halophiles by growing sample in subsequent increasingly higher salt media)

Enrichment

Time it takes for a population to double Ecoli 22 min 2n Logarithmic under optimal conditions

Generation Time

Lag, log, stationary, death

Bacterial growth curve

Cells adjusting to new media (cell division = cell death)

Lag phase

Exponential growth Shortest generation Time Division exceeds death Kept I. This phase by adding nutrients and removing wastes

Log phase

Cell division = cell death | Nutrient level decreasing and waste level increasing

Stationary phase

Cell decision eclipsed by cell death | pH excessive

Death phase

Extrapolate colony count to the original undiluted sample #colonies x mls x dilution factor = cells per ml

Serial dilution

Filtration, direct microscopic count, most probable #, turbidity

Ways to calculate the population of bacteria

Transmittance of Light shined through a sample onto a receiver Helps determine log phase

Turbidity

Components provide favorable conditions for target organism while discouraging the growth of unwanted Microorganisms

Selective media

Growth of obligate a anarobes

Reducing media