Quiz 2 Pages 8-22 Flashcards
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
Salversan
Chemotherapy for syphilis, does not harm the patient
arsenic
Antibiotic penicillin
Clear zones
Scottish
Alexander Fleming
Invented agar
1) solid surface
2) not digested by most Microorganisms
3) withstands high temps
Eilshemius Hesse
Strep
Staph
Pallisade
Chains
Clusters
Based on wall composition
Gram stain
Acid fast stain
Differential staining
What an organism eats and what it releases
Metabolic capabilities/limitations
Carbohydrate utilization
Metabolic byproducts
Ability to ferment
Biochemical tests
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
Using animal responses to organisms for classification
Serology
Anything that illicit an immune response
Antigens
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
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
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
Is the sample susceptible to the virus? Positive Id
Phage
Useful for id of fluorescent bacteria
Add fluorescent due tag to listeria antibody to milk
Flow cytometry
Base composition of cg / at ratio
Genetic Id
Restriction mapping: restriction endonucleases cut at specific sequences, run on gel to find pattern to match to known species
Genetic Id
PCR - polymerase chain reaction
Use primers to amplify a region of DNA to a level that can be run on a gel
Genetic id
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
Make copies of probe DNA
Collect grow muse denature sample
Mix with probe
Detect probe for positive id
DNA probe for known organisms
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
Resolving power = ocular magnification x objective
bright field
Multiple lenses
Light can move through slide, specimen, objective ocular to your eye without any loss to scatter
Oil immersion
Each Time light passes from one medium to another some light is lost
Oil immersion helps
Refraction
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
pH
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
colonies x mls x dilution factor = cells per ml
Extrapolate colony count to the original undiluted sample
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
