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Bacteria Lab Report

1. Introduction

Antibiotics play a large role in killing or hindering the growth of Bacteria. However, these antibiotics select for the resistant strains of bacteria over time, thus leading to reduced antibiotic effectiveness and the need to create new medicines to combat these strains. Rhambia and Gronvall’s (2009) article on antibiotic resistance, states that many disease-causing bacteria have become antibiotic resistant, and continue to divide and multiply with life threatening consequences. This affects the patient or bacteria-infected organism, and often leads to reduced immediate results and longer treatment periods. According to their studies, the methods most commonly leading to antibiotic resistance are mutations creating a conferred resistance to bacteria, genetic transfer of resistance passed between bacterium, and antibiotic-resistant strains deliberately being introduced (Rhambia and Gronvall, 2009). Therefore, over time certain antibiotics are only effective against certain bacteria strains at varying rates.

In this experiment, the effectiveness of three antibiotics will be looked at in killing or inhibiting the growth of the bacteria strain Salmonella typhimurium. The bacteria sample will also be tested by the Gram stain procedure in order to determine if it is Gram-negative or Gram-positive and how antibiotic use varies with respective cell wall differences. The antibiotics being tested are Chloramphenicol, Erythromycin, and Kanamycin. By observing the effectiveness against this bacterial strain, the purpose is to understand what types of antibiotics are still efficient at killing or inhibiting S. typhimurium. If the bacteria sample is Gram-negative, Chloramphenicol and Erythromycin will have the strongest effects. Kanamycin will have the best effect on the sample if it is Gram-positive. The Chloramphenicol will show the strongest effectiveness against S. typhimurium, but Erythromycin and Kanamycin will have no effect against the bacteria.

2. Materials and Methods

2.1 Salmonella Cultures and Antibiotic Disks

An agar plate was prepared a week before performing the actual Bacteriology experiment. Four separate bacterial lawns were arranged by inserting a sterile swab into the S. typhimurium culture and then swabbed into marked off sections within the agar plate for each antibiotic study. Antibiotic dispensers assisted in placing one antibiotic disk in each bacterial lawn. The antibiotics used within this experiment were Chloramphenicol, Erythromycin, and Kanamycin. The fourth bacterial lawn received no disk and was used as a control to compare the other three samples to. The agar plates were incubated at 37°C for 24 hours and then kept at 4°C for 6 days preceding the observation.

7 days after bacteria preparation, the agar plate cultures were removed from incubation and observed to determine S. typhimurium sensitivity and effectiveness against the three antibiotics. The zone of inhibition (Surrounding area of disk where bacteria growth was hindered) were then measured and recorded for each antibiotic.

2.2 Gram Stain Determination

The Gram stain procedure was also performed in order to determine a positive or negative result on S. typhimurium. Three bacteria slides were prepared by smearing the bacteria in a small drop of water with a clean transfer loop and allowed to air dry. The slides were then stained with crystal violet for one minute, gently washed off with water, stained with Gram’s iodine for 1 minute and gently washed off again. 95% alcohol was added drop wise to the slide until the alcohol ran clear into the staining tray. The slides were then counterstained with safranin for 45 seconds and gently washed off with water. After blotting with bibulous paper, the Gram stain results were recorded.

3. Results:

After examining the antibiotic results on the bacteria it was discovered that each antibiotic had varying effectiveness against S. typhimurium (See figure 1)

Figure 1. Agar plate of bacteria and antibiotic results after 7-day incubation. Zones on Inhibition produced by (clockwise from top) Kanamycin, control, Chloramphenicol, and Erythromycin disks.

Chloramphenicol and Kanamycin had the largest zones of inhibition of 21.2 mm and 14 mm respectively. Erythromycin had a zone of inhibition of 11.4 mm, which had little to no effect on the bacteria.  All antibiotics and control group featured very precise zones of inhibition and no fungi were present. Our control group showed no signs of error and provides a strong sample for comparison of the other lawns. Figure 2 displays the average zones of inhibition results of each of the five agar plates.

Figure 2. Average zones of inhibition produced by each antibiotic disk in the bacteria samples. This data is compiled of 5 test results and provides an overview of how effective the antibiotic samples were overall.

After examining and recording the zones of inhibition, the Gram stain procedure was performed on the prepared S. typhimurium slides. The slide remained a blue/purple color until Step 3 of applying the 95% alcohol wash, in which it lost its stain. After the Safranin was applied to the slide for 45 seconds, the smear gained a pink/red appearance, thus confirming S. typhimurium as gram-negative bacteria and a cell shape of Bacilli under a compound microscope at a total magnification of 400x. (Table 3).

Name of Bacteria Results of Gram Stain Cell Shape

Salmonella typhimurium Gram-negative Clumped Bacilli

Salmonella typhimurium

Gram-negative Clumped Bacilli

Salmonella typhimurium

Gram-negative Clumped Bacilli

Table 3. Results of Gram stain procedure and cell shape under microscope. S. typhimurium is gram-negative and had the form of clumped bacilli (rods).

4. Discussion and Conclusion:

After completing this experiment it was found that the Antibiotic Chloramphenicol had the strongest effect at killing or reducing the S. typhimurium growth. Kanamycin also showed strong results, but was not as effective against the strain as Chloramphenicol. Erythromycin performed very poorly against the bacteria strain, and would not be a good antibiotic candidate to fight S. typhimurium. These findings in the lab are also supported by real world studies and research on antibiotic effectiveness against S. typhimurium. Sethi et al. (1976) performed a series of studies on a total of 704 Salmonella strains from around the world. According to their findings of 704 Salmonella strains, 571 (95.4%) were sensitive to Chloramphenicol and 36 of the 46 serotypes tested were completely sensitive to the Antibiotic. Although it appears some Salmonella strains have become resistant to Chloramphenicol, most are still largely affected by certain antibiotic use as shown from our experimental evidence (Figure 2 and Results).

From the lab results we can also see how Chloramphenicol is largely effective against gram-negative bacteria. Due to S. typhimurium being gram-negative, the bacteria has a more complex cell wall, but a thin peptidoglycan cell wall layer that Chloramphenicol can easily bypass. Kanamycin and Erythromycin are less commonly effective against Gram-negative bacteria, leading to poor results in our experiment.

Time and resources put a large limitation in this experiment. Had the samples been available for observation each day, and for more than 7 days it would have allowed a better observation on the performance of each Antibiotic. By testing only three antibiotics it limits the exploration of proper effectiveness on S. typhimurium. If more antibiotics were available, we could ultimately determine which antibiotic properties react best against the selected bacteria. Future experiments could include pooling the entire class or biology lab departmental results and determine an overall trend and how various bacterial samples react accordingly to each antibiotic. This would also give more thoroughly supported evidence on how antibiotic use may vary between gram-positive and gram-negative bacteria.

It’s important to understand that frequent antibiotic use plays a large role in selecting for resistant strains of bacteria. This in turn leads to higher hospital visits, rapid proliferation, and increased medical costs. By studying the antibiotic effectiveness against various bacteria strains and how each behaves, it allows for advancement in preventive medicine and also the production of efficient synthetic and semi-synthetic antibiotics for the future.

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