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How did we isolate S. aureus from our noses
- how we can identify it

1. swab nares with sterile cotton swab
2. Inoculated on blood agar and MSA(Mannitol salt agar)
3. On MSA, it turns bright yellow from the fermentation of mannitol (lowered pH)
4. 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
5. pick an isolated beta-hemolytic colony from your blood agar plate, or an isolated yellow colony from MSA plate then inoculated on CHROMagar plate
6. 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



- illness due to specific disease
Calculate= (# cases per period)/(pop size @midpoint-given) xK(1,000,000)



- specific proteins in the immune system that identify and neutralize foreign bodies


Positive and negative tests for an agglutination reaction

1. 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
2. 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



- 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



- 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



- 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



- death
Calculate= (# disease related deaths)/(# of infected people) xK(1,000,000)



- 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



- humans who harbor pathogens but do not exhibit any signs of disease



- 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)



- 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

1. 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
2. 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



- are insects and other arthropods that carry pathogens
- mechanical transmission: carry on their feet, transfer to food
- biological transmission: bite to infect



- diseases that are constantly present in the population


Grape Juice Fermentation Procedure

1. 100 ml of grape juice was inoculated with 3mL of yeast culture in an erlenmeyer flask
2. a balloon was attached to the flask and allowed to incubate for a week
3. A positive result of fermentation in the juice was indicated if the balloon filled with air, gas production!
4. The pH was taken before and after and a lower pH indicated fermentation occuring



- the continual source of an infection


Calculate the number of bacteria in a liquid or solid sample based on the number of CFUs on various plates
- what constitutes a “countable” plate

- only count the plates with 25-250 colonies
- if a plate has lower than 25 TFTC (too few to count)
- if a plate has higher than 250 TNTC(too numerous to count)
Calculate the CFUs(colony forming units) per mL
1. count the number of colonies on the plate
2. divide by the dilution factor
ex: if 129 colonies were counted on a 10^-3 dilution do 129/10^-3=1.29x10^5 CFU/mL


Purpose of the gradient plate

- to allow you to isolate a streptomycin resistant mutant from a prototrophic E. coli culture
- the lower, slanted agar medium lacks streptomycin
- the molten agar medium contains the antibiotic strep and will produce a high strep concentration in the surface layer
- the appearance of colonies in a region of high strep conc is indicative of strep resistant mutants


Standard Plate Count (heterotrophic plate count) for
a liquid sample (high quality milk)
- describe dilution scheme

- determines the number of bacteria in the milk sample
- High quality use one 99mL blank, low qual use three 99mL blanks
1. Transfer 1 mL of milk to a 9mL dilution blank (1:10=10^-1)
2. Transfer 1 mL of the 1:10 dilution into a 99mL blank (1:10^3)
3. Label the bottom plates 10^-1, 10^-2, and 10^-3
4. Take .1 mL from the milk and put into 1:10^-1 plate
5. Take 1mL for the 9 mL blank and put into 10^-2 plate
6. Take .1 mL from the 99mL blank and put into 10^-3 plate
7. Incubate and count colonies to determine CFUs per mL in the undiluted sample


The slide agglutination test (serological typing) to identify S. aureus.

- If the test strain of S. aureus is producing coagulase and/or protein A, these antigens will react with the latex reagant(antiserum) and antibodies to cause agglutination of the latex particles
- suspect colony, pos control (S. aureus) and neg control (S. epidermis)
- should see agglutination in S. aureus and none in S. epidermis, compare to suspect colony to det which it is


Heat shock method Steps

- heat shocking, or exposure to heat for a short time can increase the fluidity of the plasma membrane allowing DNA to enter
- used to artificially transform E. coli with plasmid pGLO. which has ampicillin resistance and fluorescence when exposed to UV light
1. two microcentrifuge tubes contain + and - pGLO
2. Transfer of pGLO plasmid into +pGLO
3. incubate in ice for 10 min, then in a water bath for 50 seconds
4. Move the tubes back to ice for 2 min
3. add LB to both tubes, incubate at RT for 10 min
4. spread pGLO+ onto LB/amp and LB/amp/ara plates
5. spread pGLO- onto LB/amp and LB plates
6. incubate for 48 hours
- spread using a spreading rod that was sterilized by dipping it in alcohol, igniting it and letting it drip off



- clumping of bacteria by antibodies
- reaction occurs between particulate antigens and antibodies
-the formation of a visible precipitate (clumping) of the antibody/antigen complex



- a molecule/structure that causes the production of antibodies (once produced, antibody binds to it)


Reductase test

- a test that is used to estimate the microbiological quality of milk
- methylene blue is added to the milk and quality is analyzed by the time determined for decolorization of the dye by the reducing action of the bacteria
1. Good quality milk is decolorized in over 2 hours
2. Low quality milk is decolorized in less than 2 hours
- the faster the methylene blue is decolorized to white the lower the quality of milk



- if the disease spreads to one or more continents
- an outbreak of disease that is prevalent to a whole country or world


calculate the percent of colonies that are strep resistant mutants

= # colonies on strep agar plate/ # colonies on nutrient agar plate x100



- clear areas in an agar medium previously seeded with a diluted phage sample and a host cell culture
- each plaque represents the lysis of a phage infected bacterial cell


EMB vs Endo Agar

1. Levine'sEMB(Eosin methylene Blue)
- selective and differential medium
- Eosin and methylene blue inhibits Gram Pos bacteria
- lactose fermenters(fecal contam): colonies with dark centers
- E. coli: smaller dark colony with green metallic sheen(fecal contaminator)
- Enterobacter aerogenes: larger colony with dark center and light border, "fish eye" appearance
- Non lactose fermenters: pale or colorless
- characteristic for E. coli, major indicator of fecal contam
2. Endo Agar
- selective and differential medium
- Sodium sulfite and basic fuschin inhibit gram pos bacteria
- Lactose fermenters(fecal contam): reddish colonies with possible green metallic sheen
- Non lactose fermenters: clear colorless colonies


Determining the number of bacteriophages in a sample Steps

1. Make a 10^-1, 10^-2, 10^-3 and 10^-4 dilution by adding .5mL of the phage suspension (or subsequent dilut) and transfer it to 4.5 mL TS broth
2. inoculate each with 1mL of dilution plus one loopful of E.coli. Mix quickly and gently.
3. After pouring the inoculated overlay into the plate, swirl the plates before the overlay solidifies.
4. Incubate without flipping over
5. Count the plaques and multiply by the dilution factor
6. refrigerate the plates


Outcomes of a hypothetical transformation

- Determine transformation efficiency by total of transformed cells/amount of DNA used



- when many people in a given area acquire the disease in a short period of time


Standard Plate Count (heterotrophic plate count) for
a solid sample (blended food sample)
- describe dilution scheme

- determines the number of bacteria in the food sample
1. blended food sample is a 1:10/10^-1 dilution
2. put .1 mL of 10^-1 dilution food sample into plate (10^-2)
3. 1mL of 10^-1 dilution to a 99mL blank (10^-3)
4. from the 99mL put 1 mL into a nut agar plate (10^-3)
5. From the 99 mL put .1 mL into a nut agar plate (10^-4)
6. Incubate and count colonies to determine CFUs per mL in the undiluted sample


The effects of lysozyme on bacterial cells
- where lysozyme is naturally found

- its an enzyme that breaks down the beta 1,4-glycosidic peptidoglycan bonds between the NAG and NAM of peptidoglycan in bacterial cell walls
- Cell membrane ruptures through the breaks, resulting in cell lysis and death
- Naturally found in most body fluids/secretions such as saliva and tears.



- the occurrence, rate, or frequency of a disease
Calculate: (# new cases)/(susc pop size) xK(1,000,000)


Describe the general characteristics of Staphylococcus aureus as part of the
normal microflora of human skin.

- forms golden yellow colonies
- a pathogen
- produces coagulase, clots the fibrin in blood
- a test for the presence of coagulase is used to distinguish S. aureus from other S species
- Staphylcoccus is present by observing mannitol fermentation and colony pigmentation
- Gram-positive spherical bacteria that form irregular clusters of cells
- Non-motile, non-spore-forming, and able to grow in high salt concentrations, facultative anaerobes
- Normal microbiota of nasal membranes, hair follicles, skin, and the perineum in healthy individuals
- Developed resistance to many antibiotics, including methicillin (MRSA)