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81 Cards in this Set
- Front
- Back
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Cultural method
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Grow bacteria using media (solid or liquid)
Streak/isolate to obtain pure bacterial culture Stain Microscope identification |
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Pure culture
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Population of microorganisms composed of a single strain
Obtained through selective laboratory procedures Does not exist in natural environment |
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Non-cultural method
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Using DNA/RNA probes to test for presence/absence of bacteria
Microscope methods detect particular bacteria's absence/presence |
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Immunological method
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Detects antibodies in a patients body fluids
Indirectly detects organism's presence/absence |
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Streaking for isolation
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The most common way of separating bacterial cells
Dilute samples until microorganisms are separated on agar plate After incubation, should see different colonies of different microorganisms Pick one colony off plate and transfer to sterile medium for pure culture |
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Isolation plate
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Used to obtain a pure bacterial culture
The sample is diluted and incubated allowing different colonies of different microorganisms on the plate |
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Selective media
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Designed to favor growth of one type of bacteria over others
Can be chemical defined, pH, and antibiotic |
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Differential media
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Can obtain an isolated colony but it will produce more characteristics to help identify the organism
Ex: pigment produce, morphology |
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Enrichment media
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Use if the number of microorganisms is very low
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Eosin methylene blue
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Selective and differential media
Inhibits the growth of Gram+ bacteria Organisms that can ferment lactose give blue-green shiny colonies |
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What purpose does microscopy have in microbiology
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Initial detection of microbes in clinical specimens
Preliminary or definitive identification of microbes |
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Bright field microscopy
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Standard
Components: Light, stage, condenser objective lens, ocular lens Object is lit by transillumination Two lens system Image goes from objective lens --> ocular In 1000X, distance between any two lines is 1micron Limitations: Image resolution Wavelength of light Angle of light entering lens Can't see viruses Oil immersion reduces reflective indices |
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How can bacterial morphology be examined?
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By observing living unstained organisms in a wet mount
OR By observing killed stained organisms in a heat fixed gram stain |
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Methylene blue stain
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Type of basic type
Dissociates in water into positively charges methylene blue ion and negatively charges chloride ion which is colorless |
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Basic dye
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AKA Positive dye
Cation carries dye which will bind to bacteria Ex: Methylene blue, crystal violet, safranin |
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Acidic dye
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AKA negative stains
Anion carries dye which will be repelled by bacteria Leads to indirect staining Ex: nigrosin, congo red |
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Gram stain procedure
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Fixation --> Crystal violet --> Iodine
Here both Gram+ and Gram- look the same Decolorization with acetone --> Counter stain w/ Safranin Gram+ will look much darker than background Gram- does not have that dark of a background |
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Capsule Stain procedure
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Use skim milk broth culture
Airdry Stain with crystal violet or india ink Wash with copper sulfate Observe with oil immersion microscopy Organism and milk will pick up purple/black dye while capsules remain colorless |
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Endospore Staining
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Stain: Malachite green
Observes green endospores when stained |
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Acid-fast staining
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AKA Ziehl Neelsen Method
Acid-fast cell wall contains glycoplipids like mycolic acid Use stain Carbol fuschin to dye Then decolorize the slide The stain remains - seen as red Used for Mycobacterium and Nocardia |
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Dark field microscopy
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Uses a special condenser so light doesn't directly illuminate the specimen
This allows the specimen to be lit against a back background Good to increase resolution to study thin microbes Bad because you can't study internal structures |
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Phase Contrast Microscopy
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Rarely used
Used to study unstained microbes Obtain 3d image of specimen |
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Electron Microscopy
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Uses beams of electrons and magnetic coils not lenses
Magnification and resolution dramatically improved Used to view topography and morphology of bacterium Types Transmission EM Scanning EM |
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Three domain system
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Proposed by Carl Woese
Classification of microorganisms based on ribosomal RNA (rRNA) structure genes, cell membrane lipid structure, and sensitivity to antibiotics Includes: Eubacteria Eukaryotes Archaea |
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Archaea
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Domain that includes microbes that exist in extreme environments such as high salt content and extreme temepratures
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Eubacterial domain
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Domain that includes cyanobacteria and carbon-eating heterophilic bacteria
This is the microbes we are dealing with |
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Carl Woese
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Proposed the Three Domain System of classifying microbes
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Archaeas
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Don't have peptidoglycan layer
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What are the 4 categories of bacteria?
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Gram+
Gram- Eubacteria lacking cell walls (mycoplasma) Archaeobacteria |
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Gram+ bacteria
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One of the major categories of bacteria
Eubacteria that have cell walls Chemically 50-90% of the cell wall is peptidoglycan Stains purple |
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Gram- bacteria
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One of the major categories of bacteria
Eubacteria that have cell walls Outer membrane + inner thin peptidoglycan layer in periplasmic space (10-20%) |
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Mycoplasma
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One of the major categories of bacteria
Eubacteria lacking cell walls (no peptidoglycan) Therefore staining cannot be used Has plasma membrane and no cell wall One of the smallest microbes to cause disease |
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Archaeobacteria
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One of the major categories of bacteria
Terrestrial and quatic microbes in hypersaline / hydrothermally / geothermally heated environments Very different RNA materia |
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What types of classification are used for bacteria?
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Phenotypic
Analytic Genotypic Classification |
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Phenotypic Classification of bacteria
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Can be based on:
Microscopic morphology Macroscopic morphology Biotyping Serotyping |
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Microscopic Morphology
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Phenotypic classification of bacteria that considers:
shape size arrangement gram staining |
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Macroscopic Morphology
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Phenotypic classification of bacteria that considers:
Hemolytic properties Pigmentation Size and shape of coloines |
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Biotyping
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Phenotypic classification of bacteria that considers:
Fermentation of different sugars (gas production) Enzyme production (Proteases, Lipases, Nucleases) Presence of various aminopeptides Checks for acid/gas and color changes |
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Serotyping
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Phenotypic classification of bacteria based on:
antibodies to detect antigens Subspecies identification Important when microorganism cannot grow or is difficult to grow |
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Why was rRNA used for classification of bacteria?
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Because all ribosomes have the same function of protein synthesis, most of these genes are conserved
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What temperature do most microorganisms grow at?
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Between 27-30degrees C
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What temperature do pathogens grow at?
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37* C - close to body temperature
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What kind of organisms have hemolytic properties
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Alpha - clear zone on agar plate
Beta - small green zone on agar plate Gamma - no hemolysis |
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Analytical Classification
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Using chromatographic methods to identify bacteria
Rarely used Based on: cell wall fatty acid analysis Cell lipid analysis Cell protein analysis Multilocus enzyme electrophoresis |
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Genotypic Classification
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Most commonly used
Extra DNA and look at their properties through PCR Mole percent guanine plus cytosine content determines supercoiling Plasmid analysis Ribotyping |
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What are the shapes most bacteria come in
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Coccus - sphere
Bacillus - Rod Spiral - Helical/corkscrew |
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Coccus
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Spherical or oval shaped bacterium
Various arrangements: Coccus - single cell Diplococcus - two cells Streptococcus - Chain Tetrad - four cells and divides in 2 planes Sarcina - cube - divides in three planes Staphylococcus - cluster of grapes and irregular division |
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Diplococcus
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Pair of cocci
Ex: Streptococcus Pneumoniae (Subtype A,B or C) |
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Streptococcus
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Bacteria that has a chain of coccus arrangement
Ex: Streptococcus pyrogenes |
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Tetrad
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Bacteria that is in a cluster of 2 cocci and can divide in 2 planes
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Sarcina
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Bacteria that are arranged in a cube formation of 8 coci and divide in 3 planes
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Staphylococcus
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Bacteria that are arranged like a cluster of grapes and has irregular division
Ex: Staphylococcus Aureus |
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Bacillus
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Cylindrical shaped bacterium
Always divides in 1 plane Either arranged in two cells (like clusters) or a chain (like sausages) |
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Streptobacillus arrangement?
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Bacilli in chains
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Coccobacillus arrangement
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Bacilli arrangement in clusters similar to coccus
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Spiral bacterium
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Helical bacterium
Types: Spirochetes (skinny squiggly) Vibro (eyebrow) Spirillum (fat squiggly) |
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Spirochetes
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Type of Spiral bacterium
Skinny squiggly Thinnest bacterium Single layer Common cause of lime disease |
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Vibro
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Type of Spiral bacterium
Eyebrow shaped Vibrio cholerae causes diarrhea, dysenteries, and food poisoning |
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Spirillum
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Type of Spiral bacterium
Fat squiggly Thick Spring-like Difficult to grow Easy to differentiate between spirochetes |
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Pleomorphic organisms
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Bacteria that are variable in shape and size because they adapt to the environment
Can be filamentous, square, star-shaped, spindle-shaped, lobed |
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Obligate aerobes
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Needs 100% oxygen to thrive
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Obligate anaerobes
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Cannot survive in oxygen environment
Usually these are oral cavity bacteria |
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Facultative anaerobes
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90% of bacteria fall under this category
Flourish in oxygen-deficient environment but can live and multiply if oxygen is present |
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Bacterial classification of Streptococcus mutans
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Chain of coccus (based on name)
Gram+ 0.5-0.75microns in diameter Polysaccharide capsule No endospore or flagella Not motile Growth enhanced under anaerobic environment Grows in sucrose agar Colonies appear rough, with beads or liquid Produces acid anaerobically GC content 40-41% |
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Bacterial classification of Staphylococcus aureus
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Cluster of coccus (based on name)
Gram+ 0.8-1.0 microns in diameter Cell wall contains peptidoglycan, ribitol teichoic acids, species specific precipitinogen protein Colones are smooth with an entire edge Acid produce aerobic or anaerobically G-C context between 30-39% of total chromosome |
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Ribotyping
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Molecular method based on analysis of restriction fragment length polymorphisms (RFLPs) of ribosomal RNA gene
Use rRNA because it has the same function in most living organisms Used to detect and classify non-cultivatable organisms |
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What are the metabolic requirements of bacteria
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Source of carbon and nitrogen
Energy source Water Various ions |
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Prokaryotic Reproduction
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Reproduce by Binary Fission
Reproduce asexually |
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Generation time
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AKA doubling time
Interval of time between successive binary fissions Varies with different strains of bacteria Shorter doubling means shorter incubation period of disease |
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How can you calculate the number of bacteria in a population at a given time?
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N(t) = N(o) x 2^n
N(o) = starting # of cells n = # of generation cycles (time in minutes divided by replication time) Ex: generation time = 20min Starts with 10 cells (N(o) = 10) Grows for 2 hours (120/20 --> n=6) After 2 hours N(t) = 10 x 2^6 = 640 bacteria |
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What are the phases of bacterial growth
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Lag phase
Log phase Stationary phase Death/decline phase |
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Lag phase
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Bacteria needs some time to adapt to its new environment
May grow in size but not in number No increase in number of bacteria |
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Log phase
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Follows lag phase
Bacteria enter period of rapid growth in number, doubling every interval Log growth |
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Stationary phase
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Bacterial growth towards the end of log phase
Bacteria sense they are running out of nutrients and slows reproduction No increase in number Rate of new bacteria = rate of dying bacteria |
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Death/decline phase
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Stage of bacterial growth when the population starts to die off due to too much metabolic waste production and lack of nutrients
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What phase of growth is Penicillin most effective?
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Penicillin is most effective during the log phase since it only works on growing cells
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At which growth stage do you think bacteria that form spores might begin to initiate sporulation?
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During Stationary phase
Cells sense that they are running out of food so they start to form spores |
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Mitis Salivarius Agar (MS)
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Selective media
Used for cultivation of Streptococcus spp. Inhibits most gram- bacilli and Gram+ bacteria except streptococci |
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Mitis Salivarius Bacitracin Agar (MSB)
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Select medium
Used for cultivation of Streptococcus mutans only Contains Bacitracin antibiotic which Strep mutans is resistant to |
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Aseptic
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Free of pathogenic microorganisms
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Calculate original cell density
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# of Colony forming units /
(Volume plated)(dilution factor) Example: 120 colonies in 10^-5 dilution after plating 0.1ml of diluted sample =(120)/ (0.1ml)(10^-5) Cell density in 0.1ml= 1.2 x10^7 |
