Question

LAB EXERCISE #6: Kirby-Bauer Disk Diffusion Test for Antibiotie Sensitivity Determination The Kirby Bauer Test is an agar diffusion test that is used to determine the effectiveness of antibiotics killing various species of bacteria. Filter paper disks saturated with the antibiotic of interest are placed a Mueller-Hinton agar plate on which bacteria that has been isolated from a clinical sample has been sarcad. The antibiotic then diffuses from the disk and if the bacteria is sensitive to the antibiotic, a clear zone will appear where the bacteria fail to grow. This clear zone is known as the zone of inhibition PROCEDURE 1. Label the bottom of a petri dish with the name of one of the bacteria at your bench (Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, or Staphylococcus epidermidis). 2. Label the bottom of the remaining petri dishes with the name of one of the other bacteria. 3. Divide the bottom of each plate into 4 regions. Each quadrant will receive a disk containing one of the antibiotics to be tested (amoxicillin/clavulanic acid (AmC), erythromycin (E), ciprofloxacin (CIP), and polymyxin B (PB)]. The disks are already labeled with the abbreviation for each antibiotic. 4. Gently mix one of the culture tubes to resuspend the bacteria. Use a sterile pipet tip to transfer 100 ml of the bacteria to the center of the appropriate petri dish. 5. Spread the cells across the entire plate with sterile beads (keep the plate on the lab bench and slide it around for best coverage), discard the beads into a labeled container, and cover the petri dish. 6. Repeat steps 4 and 5 with the other four bacterial cultures and their designated plates. 7. Sterilize forceps using an alcohol swab. Pick a sterile antibiotic disk from its dispenser and place it in one quadrant of a plate. Tap down lightly on the disk. Repeat this procedure for all remaining plates and antibiotics. 8. Keeping the plates upright so the antibiotic disks remain in place, incubate the plates at 37°C for 48 hours.

can you please help me with Qs 1-6

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Answer 1

1. Inorder to study the antibiotic sensitivity assay one has to get the pure culture of that bacteria to study the effect of antibiotic on that bacteria. So clinical sample of that bacteria is streaked on the plate and a single colony was isolated and pure culture was developed prior to the study of antibiotia assay.

2. Staphyloccus aureus is more sensitive to the antibiotics because the diameter of the zone of inhibition is high (45mm). As it is a gram positive bacteria effected by cell wall synthesis inhibiting antibiotics.

3. Pseudomonas aeruginosa is more resistant to antibiotics. The zone of inhibition is 7mm (amoxicillin), 7mm (erythromysin) is very less and is resistant to antibiotics,may be due to the cell wall compositon as it is a gram negative bacteria, not allowing antibiotics to get in the cell wall.

4. Gram positive bacteria are more sensitive to antibiotics ( Staphyloccus aureus, Staphylococcus epidermidis) than to gram negative bacteria (E.coli, Klebsiella, Pseudomonas) . As gram positive bacteria possess peptidoglycon in their cell wall, prone to antibiotics ,so the zone of inhibition is high. Comparitively gram negative bacteria are resistant to antibiotics with respect to their zone of inhibition. Because gram negative bacteria are impermeable to antibiotics incomparision with gram positive bacteria.