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What are some important lab safety protocols?
- lab doors must be closed
- tables must be disinfected
- closed shoes, no skirts/shorts worn
- pull back long hair
- do not remove cultures
- be careful with your surroundings
- report spills and accidents
- anything contaminated goes in bio-hazard
- contaminated cultures go in autoclaving bin (remove tapes)
- used microscope slides in special jar
What do you do if something in the lab spills?
- 1. Don't move. inform your neighbors and the instructor of the spill
- 2. cover the spill area and all contaminated objects with paper towels
- 3. saturate the towels with wavicide and let sit for 20 minutes
- 4. Put on barrier gloves and clean up the spill with more dry paper towels. Discard al waste in a bio-hazardous waste container
- 5. Wash hands
Label the parts of the microscope
How do you do a gram stain?
- 1. Prepare thin smears of bacteria as directed by your instructor
- 2. Stain with crystal violet for 1 minute
- 3. Gently wash the excess crystal violet off the slide with water
- 4. Stain with iodine for 1 minute
- 5. Gently wash the excess iodine off the slide with water
- 6. Decolorize by gently washing the slide with ethanol for 10-15 seconds
- 7. Immediately wash the ethanol off the slide with water
- 8. Counterstain with safranin for 45 seconds
- 9. Gently wash the excess safranin off the slide with water
- 10. Blot the slide dry (top and bottom) with bibulous paper. Make sure top and bottom of slide are absolutely dry before placing slide on microscope stage. Observe under oil-immersion
How can you tell if a stain is gram negative or gram positive? Give one genus example of each
- If the stain is a dark purple, it is positive.
- If the stain is pink, it is negative.
- Example positive:Bacillus
- Example negative: Salmonella
What causes the gram stain to stay purple or be pink?
- The MO is stained by it's cell wall
- -the much thicker, crosslinked wall of the gram positive bacteria hold onto the crystal violet once they have iodine as a mordent
- -the thinner peptidoglycan wall of the gram negative is decolorized and therefore turns pink
How can you tell from an Endospore stain if endospores have been produced? What genus produces endospores?
The malachite green in the stain sticks to the spores if the stain is done correctly.
- Clostridium is a spore-former
How can you tell from an acid fast stain if the bacterium is indeed acid fast? What is one genus of acid fast bacterium?
The bacteria that is "Acid fast" will be pink as the thick waxy layer of mycolic acid is not decolorized.
- Mycobacterium are acid-fast
How would you use the aseptic technique to transfer a broth to a sterile medium?
- 1. Flame your loop or needle
- 2. Uncap your test tube and hold cap in opposite hand
- 3. pass end of test tube through flame
- 4. Inoculate loop with brother
- 5. Pass end of test tube through flame and cap it
- 6. If transferring to another test tube, flame the edge. If transferring to a plate, clam-shell the plate
- 7. Inoculate medium
- 8. If test tube, flame edge of tube again and cap. If plate, close plate.
- 9. Flame loop or needle.
How do you do a streak plate? How can you tell if you have a pure culture?
- You should have single colonies at the end
- -you can test this with a stain
- -or with a new culture and check for purity under scope
Is MSA agar differential and/or selective? How does it work?
- Manitol Salt Agar-both selective and differential
- -the level of salinity makes it selective for Staphylococcus
- -it differentiates between S. aureus and other Staph by the color change
contains: carbohydrate manitol, sodium chloride, and pH indicator phenol red
Is EMB agar differential and/or selective? How does it work?
Eosin Methylene Blue
-both selective and differential
The dyes inhibit Gram + growth, making it selective for Gram - bacteria
sugars and dyes help differentiate gram - bacteria
- coliforms like E. Coli will be shiny green
- -by breaking down the sugars produces acid which make show dark marks
others will be the same color as agar
Is HE (Hektoen enteric) agar differential and/or selective? How does it work?
Bile salts inhibit most gram+ bacterica to make it selective
lactose fermenting bacteria (coliforms) will have pigmented colonies (ORANGE)
- non-lactose fermenting bacteria will be the same color as the agar
- -salmonella will have black rings or dots in the middle
- ecoli on left, salmonella on right
What specifically grows well on MSA? Why? How can you tell?
- only staphylococcus will grow on MSA because of the high salt content
- -S. auerus will turn the agar yellow
What is the indicator for S. aureus on MSA? Why does it do this? What about other Staphylococcus species?
S. aureus will turn MSA yellow. This happens when the MO ferments the manitol. This produces acid and lowers the pH. The pH indicator phenol red then turns yellow.
What grows well on EMB? Why?
Only gram - bacteria, due to the dyes
What is the significance of the color of the colonies on EMB?
Colonies with a green sheen are coliforms. These coliforms are from fecal contamination and are often indicators of much worse things in your sample
What grows well on HE? Why?
Only gram - bacteria will grow due to the bile salts
What do the pigment of colonies mean on HE?What if they have dark central spots?
If the colonies are the same color as the agar, it is pathogenic, such as shigella
If the agar has turned orange, it is a coliform
If there is dark rings or spots on the colonies, it is salmonella due to the Hydrogen Sulfide being produced
What is the difference between alpa-hemolytic, beta-hemolytic, and non-hemolytic colonies on blood agar?
Beta hemolysis is evidenced by clearing in the agar. This happens as the red blood cells are lysed by the MO
How does an anaerobic jar work?
- The jar contains a packet that releases CO2
- -once the MO are placed inside, the satchel is opened and the lid is clamped shut
- -it creates an anaerobic environment for MO to grow
- -indicator strips inside the jar turn blue when O2 is present, to let you know the conditions inside are indeed anaerobic
How would you perform a catalase test? How would you interpret the results?
- 1. Aseptically place MO on a clean slide
- 2. Put a drop of peroxide on the MO on the slide
- IF BUBBLES ARE PRODUCED IT IS CATALSE POSITIVE
- -no bubbles means catalase negative
How would you perform an OF glucose fermentation test? How would you interpret the results?
- OF glucose for fermentation:
- 1. Aseptically stab inoculate the OF glucose tube with bacterium
- 2. place mineral oil on top of medium at least 1cm deep
- -prevents O2 from entering medium forcing MO to use fermentation if possible
If glucose is fermented, it will go from blue/green to yellow as acids are produced
How would you perform an OF glucose oxidation test? How would you interpret the results?
- 1. asceptically stab inoculate the of glucose tube with bacterium
- 2. do not add mineral oil. This allows the 02 into the medium for the MO to use
bacteria that can use glucose by oxidation will produce acids that will lower the pH and turn the medium yellow
How would you perform a SIM test? How would you interpret the results?
- 1. Aseptically stab inoculate the SIM deep
- 2. incubate for at least 48 hours
- If the result is positive the MO will produce hydrogen sulfide which will turn the medium black
How would you perform a Simmons Citrate test? How would you interpret the results?
1. Aseptically inoculate the simmons citrate slant with very little bacteria in a fishtail pattern
If the MO is able to utilize the citrate as its only carbon source, it will grow. The acid produced will lower the pH of the medium and cause it to turn from green to blue
How would you perform a Nitrate test? How would you interpret the results?
- 1. Aseptically incoluate the nitrate broth with the MO. Incubate for 48 hours
- 2. After incubation, add the entire contents of nitrate agent A vial and the entire contents of nitrate vial B and then gently swirl nitrate broth to mix. Do this in a fume hood!
- 3. If no color develops, add a small quantity of zinc powder to the culture
- Turns red after A and B are added-the MO has reduced the nitrate to nitrite and this is a positive result for nitrate reduction
- Turns red after zinc addition- there is nitrate still in the tube and it is negative for nitrate reduction
- No color after zinc addition-the nitrate is now gone from the broth and therefore it is positive for nitrate reduction
How would you perform a motility deep? How would you interpret the results?
1. Aseptically stab inoculate the motility deep and incubate
this medium contains a dye that turns red in the presence of bacteria. if the original line of inoculation is the only thing red, it is negative. If there are radiating red lines from the center, it is positive for motility
How does a phenol red broth work? What changes the color?
phenol red broth is a pH indicator to indicate the reduction of carbohydrates. If acids are produced, the broth will turn yellow as the pH lowers. If the pH is raised as bases are produced, it will turn a bright pink.
What is a transformation plate and how does it work? What was the purpose of the Ampicillin?
In a transformation plate we are transforming E. coli with 2 plasmids by shocking it with temperatures
The first plasmid is ampicillin resistance. We use a plate with ampicillin added to make it selective for those that have transformed the resistance plasmid
The second plasmid is a flourenscent marker. This shows us which MO have been transformed.
What is the importance of satellite colonies on a transformation plate?
- On the transformation plate, white satellite colonies can grow near the new ampicillin resistant MO
- -the MO destroy the ampicillin allowing other non-transformed MO to grow
How would you interpret a water filtration test? What colonies are looking for to determine if the water is unsafe?
- In a water filtration test we are looking for coliforms
- -they show up with a green metallic sheen
- -other MO are the same color as the agar
- -we use Endo agar
If you find coliforms in your water, there is a good chance there is something worse in there too!
How would you analyze a Kirby-Bauer test? How do you know what is resistant, intermediate, and susceptible?
A kirby-bauer test is analyzed using the zones of inhibition around the antibiotic where the MO does not grow
Measure the zones of inhibition and then check the chart to see if it is resistance, intermediate, or suscpetible
What is the Method of Action for Penicillin?
- -Inhibits cell wall synthesis
- -interferes with the peptidoglycan cross-linking chains by inhibited the catalytic enzymes
- -effective on gram+ bacteria
What is the Method of Action for Ampicillin?
- -effective on both gram + and gram - bacteria
- -interferes with cell wall synthesis
- -inhibits the enzyme transpeptidase which interferes with peptidoglycan synthesis
What is the Method of Action for Polymyxin B?
- -disrupts the cell membrane
- -changes the permeability of the membrane
- -causes leakage of the cell
- -effective on Gram - bacteria
What is the Method of Action for Choramphenicol?
- -effective on both gram + and gram - bacteria
- -inhibits protein synthesis
- -irreversibly binds to the receptor site on the 50S ribosomal unit and therefore interferes with the forming of polypeptide chains
What is the Method of Action for Streptomycin?
- -effective against gram - bacteria
- -binds to the 30S ribosome to prevent protein synthesis
- -the mRNA cannot read the new shape of the 30S ribosome and therefore no new proteins are synthesized
What is the Method of Action for Sulfisoxazole?
- -effective against both gram + and gram - bacteria
- -interferes with the coenzyme transport to prevent it from successfully producing folic acid
- -without the necessary folic acid to replicate DNA, the cell dies
What is the Method of Action for Tetracycline?
- -effective against both gram + and gram - bacteria
- -inhibitor to the codon-anticodon interaction to inhibit protein synthesis in the ribosome 30S
What is the Method of Action for Trimethoprim?
- -inhibits the cells from synthesizing folic acid
- -the cells lacks what it needs for necessary nucleotides and DNA replication