Final Exam Flashcards
(46 cards)
How did we isolate S. aureus from our noses
- how we can identify it
- swab nares with sterile cotton swab
- Inoculated on blood agar and MSA(Mannitol salt agar)
- On MSA, it turns bright yellow from the fermentation of mannitol (lowered pH)
- On blood agar, S. aureus contains DNase so it produces a hemolysin called alpha toxin that causes a wide, clear zone of beta-hemolysis on blood agar
- pick an isolated beta-hemolytic colony from your blood agar plate, or an isolated yellow colony from MSA plate then inoculated on CHROMagar plate
- It will produces a bright magenta color on CHROMagar in order to confirm that it is S. aureus
Coliform Characteristics
- indicators of fecal contamination in water
- gram-negative
- nonspore forming bacilli(rods) that ferment lactose with the production of acid and gas after incubating at 37’C
- three basic tests to detect coliform bacteria are presumptive, confirmed and completed
Morbidity
- illness due to specific disease
Calculate= (# cases per period)/(pop size @midpoint-given) xK(1,000,000)
Antiserum
- specific proteins in the immune system that identify and neutralize foreign bodies
Positive and negative tests for an agglutination reaction
- Positive:
- if agglutination of the blue latex occurs within 20 seconds in the test circle.
- noticeable clearing of the blue background in the latex will be observed
- indicates S. aureus presence - Negative:
- no agglutination of the blue latex occurs within 20 seconds in the test circle
- no noticeable clearing of the blue background in the test latex
Transformation
- acquisition by a recipient bacterium of small pieces of DNA released from a dead donor bacterium
- acquired pieces of new DNA can give recipient bacterium new characteristics
- cells take up DNA from the environment
Characteristics of pGLO that allow it to transform
- the gfp gene causes jellyfish to fluorescence under UV light
- pGLO plasmid has been genetically modified to carry the gfp gene and ampicillin resistance gene
- Resistance to ampicillin, fluorescence, not a lawn of colonies because not all of the bacteria was able to grow
- the ampicillin resistance gene makes possible the direct selection of transformed cells by glowing in UV light
- pGLO glows under UV light
MBRT
- Methylene blue reduction time
- the time it takes for the methylene blue to become colorless
- The shorter the MBRT, the lower the quality of milk.
Four basic steps to isolate DNA from bacterial cells (DNA extraction)
- extracting DNA from E. coli
1. Centrifuge the cells to concentrate them
2. Add a detergent to break down the cell wall and membrane in order to release the DNA (SDS solution) - sodium acetate solution aids in the removal of DNA associated proteins
3. Add a protease (proteinase K) and heat to denature and digest proteins in the cell (so DNA is not degraded by enzymes)
4. precipitate the DNA from the solution by adding ice-cold alcohol
Purpose of the three replica plates
- mutated bacteria are grown on a nutritionally complete medium
- colonies that grow on the complete medium, but not the minimal medium are auxotrophs
- auxotrophs are identified indirectly because they will NOT grow
- auxotrophs cannot catabolize nutrients or synthesize certain chemicals, which is why they need a complete medium
1. OG on nutrient agar- compare to what grows on nut agar plate
2. Replica on nutrient agar- complete medium to grow on - The bacteria that grows on the replica nutrient plate, but not the streptomycin plate are not strep resistant
3. Replica on Strep agar- minimal medium to grow - shows which bacteria that grow and also grow on the replica nutrient plate are strep resistant
PFU
- each plaque can be designated as a Plaque-forming unit (PFU) and used to quantitate the number of infective phage particles in the culture
- the number of phage particles contained in the original stock phage is determined by:
1. counting the number of plaques formed on the seeded agar plate and multiplying this by the dilution factor
2. for a solid plate count, the number of plaques should range from 30-300
Mortality
- death
Calculate= (# disease related deaths)/(# of infected people) xK(1,000,000)
Fomite
- diseases can be transmitted by contact with contaminated INANIMATE objects called fomites
Presumptive Test Steps
- media and positive test
- specific for the detection of coliform bacteria, gas-producing lactose fermenters
- Media: Lactose fermentation broths with an inverted Durham tube gas vial
1. Add 10mL of water sample to 5 tubes of DSLB (double strength lac broth)
2. Add 1 mL of water sample to 5 tubes of SSLB (single strength lactose broths)
3. Add .1 mL of water sample to 5 tubes of SSLB (single strength lactose broths)
4. incubate at 37’C for 24 hours
A. Positive test: lactose fermenting bacteria that produce 10% or more gas in durham tubes after 24 hrs incubation
B. Doubtful positive test: gas is produced after 48 hours(probably not coliforms)
C. Negative test: absence of gas in all tubes (water is safe)
what does protein araC allow for in the +pGLO plasmid?
- it allows Arabinose to bind, which allows RNA polymerase to bind to the promoter & transcribe genes, which are translated to give ampicillin resistance & green fluorescent protein
- DNA-promoting protein that regulates the arabinose promoter
- Without arabinose, this will not happen
Determine the source of a simulated epidemic
- 3 rounds of shaking hands, whoever had fluorescent light on them was infected
- make a table listing all infected people, then list who they shook hands with
Carrier
- humans who harbor pathogens but do not exhibit any signs of disease
Bacteriophage
- viruses that infect bacterial cells
- they invade a host cell in order to replicate and reproduce
- when a phage particle adsorbs to a susceptible cell, penetrates the cell, replicates and goes on to lyse other host cells, the destroyed cells produce a single plaque in the bacterial lawn
- Certain bacteriophages may only infect certain strains of bacteria.
- The capsid protects and contains the genetic material
- This is attached to a protein sheath that is contractile
- The sheath sits on a base plate to which tail fibers and spikes are attached
why was arabinose added to one of the plates in the pGLO lab?
- bc arabinose is an inducer which allows the fluorescent protein to be expressed
- Production of green fluorescent protein only occurs if RNA polymerase is able to bind to the gene, which only occurs in presence of arabinose
Confirmed Test Steps
- media and positive test
- presence of positive or doubtful pos tests suggests water sample is nonpotable
- have to confirm because results can be due to noncoliform organisms that are not involved in fecal contamination
- if have coliform nonpotable water, if no coliform potable water
- Media: selective and differential mediums (EMB-eosin methylene blue and Endo Agar)
1. Streak each plate for colony isolation (quadrant streak method) from a positive tube
2. Incubate at 37’C for 48 hours
Determine the MPN of a water sample
- Part of the presumptive test
- used to obtain an idea of the number of coliform organisms present, most probable number test
- estimated by determining the number of tubes in each group that show gas following incubation
- use table to find MPN index per 100 mL and 95% confidence limits (lower and upper)
Epidemiology
- the science that deals with when and where diseases occur and how they are transmitted in the human population
Two mutant isolation procedures we used
- what type of mutants are being isolated
- Replica plating: indirect selection
- to isolate streptomycin resistant mutants using the plates from the bacteriophage dilution plates
A. Place the master plate and the uninoculated plate and place reference marks in 12o’clock position and remove the lids
B. Touch the replica plating block to the master plate, without alternating its rotation, touch it to the minimal media(strep agar) and then to a complete medium(nutrient agar)
C. Incubate and calculate the % of colonies that are strep resistant - Gradient Plating
- to isolate streptomycin resistant mutants of E. coli
- double layered agar plate method, observe effects of varying concentration of streptomycin from high to low
- highest conc of strep is found on the overlay
A. In a hot water bath melt two trypticase soy agar(Low strep conc) tubes
B. place a pencil under one end of petri dish and pour tryp into it and allow to solidify
C. add the streptomycin solution to a second tube of tyrpticase soy agar and mix
D. pour molten agar to cover gradient agar layer solidify
E. Add E. coli by spreading over culture of agar surface with a bent rod
F. incubate and count colonies for resistant mutants
Vector
- are insects and other arthropods that carry pathogens
- mechanical transmission: carry on their feet, transfer to food
- biological transmission: bite to infect