Purpose:

To learn how to transfer bacterial samples and to test bacterial (Bacillus Coleus) resistance to Erythromycin, streptomycin and penicillin.

Procedure:

• Two test tubes (one containing a bacterial culture, the second containing sterile agar) are held in the left hand. The needle is held in the right hand.
• The plugs from the test tubes are removed with the right hand
• The mouths of the test tubes are flame by passing the through the flame twice
• The transfer loop is heated until it grows orange
• The loop is then cooled by laying it across the surface of the agar in the sterile test tube
• With the loop, a small amount of bacterial culture is removed from the surface of the agar and it is immediately transferred to the surface of the sterile medium
• The loop of each test tube is flamed again. The test tubes are replaced with the cotton plugs that were originally there. The test tubes are placed back onto the rack.
• Test tubes of sterile agar medium are heated in a water bath until the agar melts
• The test tubes are removed from the water bath. They cool until they are safe to the touch, but they do not completely cool so that the agar does not harden again. The transfer is completed rapidly so that the agar will not cool and solidify before it is complete.
• Both the test tube filled with warm agar material and the test tube filled with bacterial culture is held in the left hand. The cotton plugs are removed and the mouth of each test tube is passed through the flame twice. The loop is flames and cooled. A loop full of the bacterial culture is picked up and transferred to the warm agar in the second test tube. The loop is shaken in the liquid a few times and then the transfer loop is removed. The mouths of each test tube are again flames and the cotton plugs are replaced.
• The test tube containing the warm agar is rolled between the palms of the hands so the agar and bacteria will mix. The cotton plug is removed and the mouth of the test tube is flamed. The corner of the sterile petri dish is lifted at an angle of slightly more then 45%. The lifted cover is kept directly over the bottom half of the petri dish in order to avoid contamination as much as possible. The agar is poured into the bottom half of the dish (making sure that the mouth of the test tube does not touch either half of the petri dish). The test tube is then removed and the cover of the petri dish is lowered. The covered petri is then gently moved along the table top in a figure eight pattern so that the gel is distributed evenly. The agar is allowed to cool until it becomes firm.
• The forceps are removed from the 70% alcohol and the tips are moved quickly through the burner flame. The forceps are used to remove penicillin disks from the container. The penicillin disks are placed on one side of the agar medium by rarely raising the top of the petri dish. The forceps are rapidly passed through the flame again and one of the streptomycin disks are picked up. This disk is placed on the other side of the agar surface. The forceps are flamed and replaced into the alcohol. The forceps are rapidly passed through the flame again and one of the erythromycin disks are picked up. This disk is placed on the other side of the agar surface. The forceps are flamed and replaced into the alcohol.
• After a minute, the petri dish is turned upside down and it is allowed to stand at room temperature so that the bacteria will grow. The dish is observed on the next day and on several following days. The color and shape of the bacterial colonies are noted.

Results:

Each antibacterial disk had a ring around it where no bacteria formed (even though some are small). The biggest ring where there were no bacteria was for the erythromycin antibacterial disk (E-15). There were four centimeters of inhibition around the ring the erythromycin disk. There was an area of inhibition 2 ½ centimeters wide around the streptomycin antibacterial disk (S-10). There was an area of inhibition 1 centimeter wide around the Penicillin antibacterial disk. There was also a 2 centimeter zone of inhibition where an erythromycin antibacterial disk was dropped on the agar.

Discussion:

In this experiment 3 antibiotic disks were placed on a growing solution of Bacillus Coleus bacteria and agar. The three antibiotic disks that were placed on the bacteria were Erythromycin, Streptomycin and Penicillin.

All of the rings placed on the bacteria had some kind of ring of inhibition. Erythromycin had the largest ring (4 cm) followed by streptomycin (2 ½ cm) and penicillin (1 cm). Erythromycin was the most effective antibiotic on the bacteria. Erythromycin prevents bacteria from growing by interfering with their protein synthesis. This is what inhibited bacterial growth. The Erythromycin changed the order of the protein synthesis and because of that the right enzymes could not be produced for the bacteria to function properly and grow.

Streptomycin had a smaller ring of inhibition, but the antibiotic was still effective. The way Streptomycin works is that Streptomycin stops bacterial growth by damaging cell membranes and inhibiting protein synthesis. Since the streptomycin affected the cell membrane regular cellular functions (such as transport) could not be produced. Also by effecting the protein chain enzymes that the bacteria needed could not be produced.

Penicillin was the weakest of the three antibiotics. It had a 1 cm ring of inhibition. Penicillin works by effecting the formation of new cell walls during bacterial reproduction. The bacterial cell could not reproduce and therefore died out. Penicillin had such a weak effect on the bacteria because the bacteria have grown to adapt to the penicillin. They know what the penicillin effects and they adapted so that they couldn’t be harmed by the penicillin.

Agar was used in this experiment because it is a great nutrient source for growing bacteria. The bacteria was placed in an incubator (with a temperature of 37 degrees Celsius). There it fed on the nutrients in the agar gel. Agar is such a good nutrient source because it chemically is a polymer made up of subunits of the sugar galactose. This sugar allowed the bacteria to spread and reproduce. The incubator provided the ideal temperature for the growth of the bacteria.

Conclusion: The antibiotic that has the greatest effect on the bacteria is Erythromycin with a four centimeter diameter of inhibition. Next is the Streptomycin with a 2 ½ cm of inhibition. Penicillin had the smallest circle of inhibition with only 1 cm in diameter.

Sources of error: There was an error when the antibiotic disks were being laid onto the agar gel. The Erythromycin disk dropped onto the gel. Where it fell an area of inhibition (with a 2 cm diameter) formed. This was labeled in the results.