Antibiotics and antimicrobial susceptibility lab

In this lab we will test the effect of some antibiotics and some antimicrobial agents, on four
different species of bacteria: E. coli, Bacillus subtilis, Bacillus cereus and Serratia marcescens

I. Antibiotic Testing
Part I: Procedure:
1. You will be working with your lab partner on either the Gram-positive group (Bacillus
subtilis and Bacillus cereus) or the Gram-negative group (E. coli and Serratia
marcescens).
2. Divide the bottom of a petri dish containing the nutrient agar into quadrants by drawing
on the bottom of the dish with a sharpie

3. Label the plate with the name of a specific bacterium species (E. coli, Bacillus subtilis,
Bacillus cereus and Serratia marcescens), one plate for each species.
4. Pipette 100 µl of overnight bacterial broth culture onto the agar surface of the plate
designated for that bacterium
5. Move the plate in a circular motion gently, while it is on the bench, to spread the bacterial
broth
6. Leave it for few min to allow the culture broth to dry.
7. Place one of the four different antibiotic discs (Penicillin, Streptomycin, erythromycin,
and tetracycline) on the agar surface, in the center of each quadrant. use the end of the
sterilized loop to gently press the disc onto the surface of the agar.
8. Incubate the plate at 35֯C for 24 hours.

Part II: Results

1. After incubation, Determine the zone of inhibition around each antibiotic disc by
measuring the diameter of the clearing zone (mm).
2. Collect the data for the antibiotic susceptibility of all four bacterial species to all four
antibiotics.
 Inhibition zone Inhibition zone Inhibition zone Inhibition zone
around Penicillin around around around
(mm) streptomycin (mm) erythromycin (mm) tetracycline (mm)

E. coli 15 No inhibition No inhibition 15

Bacillus subtilis No inhibition No inhibition No inhibition No inhibition

Bacillus cereus 10 No inhibition No inhibition 6

Serratia marcescens No inhibition No inhibition No inhibition 13

Part III: Questions and Discussion

1. Mode of action of the antibiotics: look up the mode of the action (the way antibiotics
work) for each antibiotic in the table in the table and fill in the table below

Antibiotic Mode of action

Penicillin The mode of action for penicillin is by inhibiting bacterial cell wall
synthesis. This is effective on gram positive bacteria.

Streptomycin Streptomycin works by blocking ribosomal subunits from making the

proper proteins, which then results in the death of bacteria. It is a broad-
spectrum antibiotic.
Erythromycin The mode of action for erythromycin is by inhibiting protein synthesis by
binding to the 23S ribosomal RNA molecule in the 50S subunit of
ribosomes. It is a broad-spectrum antibiotic.
Tetracycline Tetracycline inhibits bacterial protein synthesis by binding to the
ribosomes and prevent the association of aminoacyl-tRNA with the
bacterial ribosomes. It is a broad spectrum antibiotics/

2. Based on the mode of action table explain why some antibiotics work for a group and not
for the others and why other antibiotics work on both groups?

The penicillin only works on gram positive bacteria since gram negative bacteria have a
protein layer that protects it’s cell wall from the penicillin attacks.

II. Antimicrobial Testing

Part I: Procedure:
1. You and your lab partner will work with the other group
 2. Divide the bottom of a petri dish containing the nutrient agar into quadrants by drawing
on the bottom of the dish with a sharpie

B. s.
E. coli

3. Label each quadrant with S. m. B. c. the name of bacterium species

(E. coli, Bacillus subtilis, Bacillus cereus and Serratia
marcescens), use abbreviation.
4. You will need three plates of these, named Listerine, Lysol, and the penny.
3. Pipette 50 µl of overnight bacterial broth culture onto the agar surface of the designated
quadrant for each species
4. Streak the bacterial culture out into lawn using the sterile inoculating loop, make sure to
not cross the quadrant designated for each species.
9. In the center of the plate, place the antimicrobial agent designated for each plate, use the
end of the sterilized loop to gently press the penny or the disc onto the surface of the
agar:
• In the first petri dish, place the penny in the middle
• In the second petri dish, place one of the plain discs after soaking in Listerine
• In the third petri dish place one of the plain discs after soaking in Lysol.
5. Incubate the plate at 35 °C for 24 hours.

Part II: Results

1. After incubation, Determine the zone of inhibition (mm), around each antimicrobial agent
by measuring the diameter of the clearing zone, start from the center of the antimicrobial
agent to the end of the clearing zone (radius) then multiply by 2 to get the circle diameter.
2. Collect the data for the antimicrobial susceptibility of all three petri dishes
 E. coli Bacillus subtilis Bacillus cereus Serratia marcescens
radius diameter radius diameter radius diameter radius diameter
Coin “Penny” 10 20 10 20 10 20 10 20

Listerine 4 8 3.5 7 4 8 4 8

Lysol 3.5 7 3.5 7 3.5 7 3.5 7

Part III: Questions and Discussion

For each of the previous antimicrobial agents, look up the ingredients or the material they are
made from, what do you think each one has that give them this antimicrobial character?
*Don’t list the ingredients of Listerine or Lysol

Antimicrobial agent ingredients and explanation

The coin is made from copper and therefore releases copper ions when
Coin “penny” placed in the petri dish with the different species of bacteria. The copper
ions prevent cell respiration, destroy the outer membrane of bacterial cells,
and destroy the viral coat if there is one disrupt the viral coat. Once this
happens, the ions can also destroy the DNA and RNA inside of the
bacteria.
Listerine is said to destroy the cell surface of bacteria, inhibit growth of the
Listerine biofilm, and increase the time for bacteria to grow. The main ingredient
that causes this is menthol. Menthol can kill bacteria due to its
lipophilic characteristics.
Lysol contains peroxide which acts as an antibacterial character.
Lysol