Lab 22 - Antibiotic Sensitivity

Question 1

what is an antibiotic?

Answer 1

a drug naturally made by certain fungi or bacteria against competing microbes

Question 2

what is an example of an antibiotic?

Answer 2

penicillin made by Penicillium mold

Question 3

nowadays, what are two things that antibiotics include?

Answer 3

Nowadays, the term includes semi-synthetic and synthetic drugs as well

Question 4

what are sensitive bacteria?

Answer 4

knocked out by antibiotics, aka susceptible

Question 5

what are resistant bacteria?

Answer 5

they survive an attack by antibiotics

Question 6

in a non-resistant cell, what happens when antibiotics enter?

Answer 6

the antibiotics enter the cell and bind to the target

Question 7

in a resistant cell, what happens when antibiotics enter?

Answer 7

1. Increased elimination: the bacterial cell pumps out the antibiotic after it enters the cell.
2. Decreased uptake: The porin proteins prevent antibiotic entry into the cell, so the bacteria don’t allow entry.
3. Alteration in target molecule: The antibiotic cannot bind to the bacteria/target.
4. Antibiotic-inactivating enzymes: the enzymes modify the antibiotic, inactivating it.

Question 8

what are three tests to determine antibiotic sensitivity? AKA what are the 3 antibiotic sensitivity assays?

Answer 8

1. Broth Dilution test.
2. E-test
3. Kirby-Baur Disk Diffusion test

Question 9

what is the broth dilution test?

Answer 9

it exposes bacteria to higher and higher concentrations of antibiotics, and no growth means the antibiotic is effective

Question 10

what is MIC?

Answer 10

Minimum inhibitory concentration - which is the lowest antibiotic concentration that kills or inhibits the growth of a microbe

Question 11

what is the E-test?

Answer 11

tests the MIC as well

Question 12

what is the Kurby-Bauer Disk Diffusion Test?

Answer 12

-When you streak the desired bacterial species all over the plate (3x) to get a lawn.
-Place paper disks with specific antibiotics on the plate.
-Antibiotics diffuse out from the disks creating a concentration gradient - which is higher near the disk

Question 13

what happens in the Kirby-Bauer test as antibiotics diffuse out? where is the MIC?

Answer 13

-As antibiotics diffuse out, they kill or inhibit the growth of bacteria forming clear “zones of inhibition”.
-The concentration at the edge of the zone is the MIC

Question 14

when comparing the diameter of the zones of inhibition to the chart, we can determine what?

Answer 14

we can determine if the bacteria are sensitive to the antibiotic

Question 15

what do the zones of inhibit size in mm mean for the bacteria Cefalothin? (eg. if it is sensitive, intermediate, or resistant) REMEMBER: THESE MEASUREMENTS ARE SPECIFICALLY FOR THE CEFALOTHIN BACTERIA

Answer 15

Cefalothin:

Sensitive: if the zone is bigger or more than 18 mm (this means it can be used in treatment)

Intermediate: if the zone is 15-17 mm (this means it MAY be successful in treatment)

Resistant: if the zone is smaller or less than 14 mm (this means it cannot be used in treatment)

Question 16

can you compare zones of inhibition of two different antibiotics?

Answer 16

No, you cannot because each antibiotic has a different size and chemical properties, so each diffuses at a different rate across the agar

Question 17

do disks of antibiotics contain different concentrations or the same concentration of each antibiotic?

Answer 17

the disks contain different concentrations of each antibiotic

Question 18

what is the purpose of the antibiotic sensitivity lab?

Answer 18

to test different bacteria against several antibiotics and see which ones they are sensitive or resistant to

Question 19

what test did we use for the antibiotic sensitivity lab? what is the medium that we used? what did we streak on the medium?

Answer 19

-Kirby-Bauer Disk Diffusion Test
-Medium: Mueller-Hinton Agar (a special agar)
-We used a sterile swab and streaked the bacteria evenly on the plates, using horizontal, vertical, and diagonal tight streaks.

Question 20

what did we do after streaking the bacteria onto the plate?

Answer 20

we used sterile forceps to place antibiotic disks on the plates. We made sure they are are equidistant from each other, and we only used 5 disks.

Question 21

what did we incubate the plates at?

Answer 21

37 degrees Celsius for 24-48 hours

Question 22

why did the Pseudomonas aeruginosa plate turn green?

Answer 22

due to a diffusible green pigment made by the bacteria

Question 23

where does the zone of inhibition fall in respect to the two circles for sensitive bacteria?

Answer 23

the zone of inhibition falls outside the second circle

Question 24

what does it mean when the disk has no zone?

Answer 24

that means the bacteria is resistant to the antibiotic

Question 25

if bacterial colonies are present within a zone of inhibition, what does that mean?

Answer 25

that means they are resistant mutants in the culture because they grew where others died. (they are numerous, small white dots)

Question 26

what culture was our table given for the lab?

Answer 26

Staphylococcus epidermidis (G+)

Question 27

what are two reasons a bacteria could be resistant from our lab?

Answer 27

1. Innate:
   - Mycoplasma
   - E. coli
2. Acquired:
   -Mutations
   -Gene transger

Question 28

what are narrow-spectrum antibiotics? an example?

Answer 28

Antibiotics that only work for specific bacteria. An example is Penicillin

Question 29

what are broad-spectrum antibiotics? an example?

Answer 29

Can kill more than one type of bacteria. An example is Ampicillin

Question 30

which bacteria was most resistant to antibiotics?

Answer 30

pseudomonas was most resistant to antibiotics

Question 31

explain why a bacterial colony might grow in a zone of inhibition.

Answer 31

these represent mutants because they grew where others died. it developed a mutation to resist antibiotics

Question 32

how did we place the antibiotics onto the plate?

Answer 32

by placing the forceps in alcohol, then putting it over the fire, then picking up the antibiotic disk