Narrow Spectrum Antibiotics and Its Effects on Microbes

In 1929, the British Journal of Experimental Pathology received a paper from Alexander Fleming. It was on the antibacterial action of a substance named Penicillin that comes from the cultures of a Penicillium. This paper revolutionized world medicine. The Discovery of penicillin dates back to 1928 when Fleming began a series of experiment on staphylococcal bacteria. Fleming left an uncovered Petri dish near an open window. It got contaminated from outside mould spores. Later he noticed bacteria near the mould colonies were dying. He tested it against many gram-positive pathogens. It was effective in almost all. Before the discovery of penicillin, even minor cuts/wounds turned out to be fatal. Fight with pathogens and harmful viruses has been really long. Scientists and microbiologists have been studying the effects of various substances and different environments on bacteria and pathogens. This shows how important the study of antibiotics is. Here we will conduct a home lab experiment with minimum requirements to study the effects of antibiotics on bacterial culture growth.

Mainly what we will do is that we will take some slices of potato and will culture curd-bacteria (Lactobacilli) on them. Then we will test the effect of antibiotics on these bacteria, making observations and concluding with them.

Step 1: Requirements

• Medium-sized boiled potatoes (used as culture media)
• Scalpel or Knife
• Petri-dish or small glass bowl (drink coasters)
• Food Wrap
• Curd stock solution (prepared with distilled water)
• Tissue paper
• Dropper
• Syringes
• Candles
• Spirit Alcohol/ Sanitizer
• Dustbin
• Distilled water
• Cotton swab (Earbuds)
• Boiled water
• Marker, Transparent Tape, aluminium foil
• Any broad-spectrum antibiotic (like Amoxycillin, Linezolid, azithromycin)
• Gloves and Masks

Biohazard: This experiment involves bacterial and fungal growth, we should take precautions while performing this experiment. Touch nothing with bare hands, if you do wash them immediately. It is understood that microbes can and will make you sick. Immediately dispose of the used syringes and tissues.

Step 2: Protocol (Part 1)

1. Boil some medium-sized potatoes for 20min, and allow them to cool.
2. Clean the Plastic bowl and place a folded piece of wet tissue at the base.
3. Keep the tissue moist with the boiled water. Which prevents the potatoes from rotting and also provides a moist environment to Lactobacillus.
4. Peel the potatoes and cut them into 0.5 cm thick oval slices along their length, maintaining uniform thickness.
5. Place a slice on the wet tissue in one bowl. Do the same for all the other bowls, which is to be used in the upcoming experiment.
6. Prepare a stock solution of curd and soil. Add a certain small quantity of both to boiled water. The weight by volume ratio should be 1g to 100ml. Dilution is important to prevent matt growth.

Step 3: Protocol (Part 2)

1. Five such petri-dish samples are created for the experiment. We take the genus Lactobacillus as our model organism as we can easily find it in curd. We will also see growth from soil samples.

2. Now use the knife that has been sterilized in the flame of a candle beforehand and carefully scoop a well (around 0.5cm in depth) into the middle of the plate. We should carefully make the depth of this well keeping in mind that we don’t puncture the potato from beneath. Also, ensure that no cracks or fissures form on the media.

3. Thereafter streak the surface of one slice of potato with curd. Fill the well of this slice with distilled water(do not overflow). Mark this as our Curd Control (CC for short).
4. Take another slice to do the same except this time add 1ml of 2.5mg/ml solution of antibiotic (Prepare this solution by dissolving one 250mg tablet in 100ml boiled water).
5. Also, make a non-curd control by streaking the potato slice with boiled water.

Step 4: Protocol (Part 3)

1. Cover all the bowls with a plastic lid or kitchen wrap. Be sure not to let the lid or wrap touch the surface of the potatoes.
2. The colonies will take several days to grow. We should store the potatoes at room temperature or above. We can get trouble getting the colonies to grow, we should try to raise the temperature by placing them in the warmest area of the house, such as the bathroom or terrace. Never store the cell culture potatoes in an area where food is prepared. It may be dangerous.
3. Observe the differences between the two types cultured and note them down.
4. We can also try these with the soil stock solution.

Step 5: Results/Conclusion

• After our incubation period is over, we can see a whitewash of bacterial and yeast colonies on the media. Here, we see many small white spots all over our media which are distinctly visible to our naked eye.
• Observe the Curd control
• We see a small zone of inhibition around the well in this 2.5 mg solution sample, giving us the right conclusions regarding antibiotics that can kill the bacteria that comes in their vicinity.
• We see the same zone of inhibition when we used the soil stock solution(3rd last image).

• We can compare these with curd control and non-curd control in which no inhibition region was observed.

• One more thing can be concluded from this experiment, those slices which had antibiotics in their well showed a faster fungal growth compared to the slices which didn't had antibiotics, the second last image shows the slice streaked with soil, it showed less fungal growth. While the last clearly shows a white-colored matt fungal growth. The reason for this may be that fungus and bacteria growing here show a kind of competition for the same nutrient source.

Step 6: References

• Wikipedia contributors, 'Broad-spectrum antibiotic', Wikipedia, The Free Encyclopedia
• Wikipedia contributors, 'narrow-spectrum antibiotic', Wikipedia, The Free Encyclopedia
• Wikipedia contributors, 'Lactobacillus', Wikipedia, The Free Encyclopedia
• Pagan FS. Antibiotics for gram-positive organisms. Br J Hosp Med. 1981;25(1):24-27.
• Kapoor, G., Saigal, S., & Elongavan, A. (2017). Action and resistance mechanisms of antibiotics: A guide for clinicians. Journal of anaesthesiology, clinical pharmacology, 33(3), 300–305. https://doi.org/10.4103/joacp.JOACP_349_15
• https://en.wikipedia.org/wiki/Lactobacillus

• https://www.cdc.gov/drugresistance/threat-report-2...

• Goudard A. & Loreau M. Non-trophic interactions, biodiversity and ecosystem functioning: an interaction web model. Am Nat 171, 91–106 (2008). [PubMed] [Google Scholar]