Inhibition of bacterial growth-1

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Inhibition of bacterial growth Objectives  Collect and analyze data to determine the effectiveness of antimicrobial agents on gram-positive and gram- negative bacterial species  Apply scientific reasoning to investigate questions and utilize scientific tools to collect and analyze data (Texas ACGM Student Learning Outcomes)  Use critical thinking and scientific problem solving (Texas ACGM Student Learning Outcomes)  Communicate effectively the results of scientific investigations (Texas ACGM Student Learning Outcomes) Introduction Antimicrobial agents are agents that inhibit the growth of or kill bacteria. There are many different types of antimicrobial agents that are categorized into three main categories: disinfectants, antiseptics and antibiotics. Disinfectants work on inanimate objects to kill bacteria. Since they aren’t meant to be ingested or used directly on humans, they are more toxic than the other types of antimicrobial agents. Antiseptics are antimicrobial agents that are used directly on humans, so they are less toxic than disinfectants. Finally, an antibiotic works directly on bacteria, killing them by interfering with their cellular activities. They capitalize on differences between bacterial and eukaryotic cells, leaving human cells unharmed. Different antibiotics target different cellular structures, affecting bacterial species in different ways. For example, amoxicillin interferes with peptidoglycan production while erythromycin interferes with bacterial ribosomes. This activity will allow you to determine the effect that different antimicrobial agents have on the growth of two different bacterial species: Streptococcus mutans and Escherichia coli . Both species are found as a part of normal human microbiota. S. mutans is a gram-positive bacterium found in the mouth that contributes significantly to tooth decay. E. coli is a rod-shaped, gram-negative organism commonly found in the lower intestine. Though some strains have been known to cause food poisoning, most are harmless, even benefiting their host by producing vitamin K. In order to determine the effectiveness of different types of antimicrobial agents, bacteria are grown in the presence of absorbent discs that are impregnated with antimicrobial compounds. The more effective the compound is, the greater amount of inhibition to bacterial growth. This can be observed as an area of reduced bacterial growth around the disc known as a zone of inhibition (Figure 1). The size of this zone is correlated with antimicrobial activity. Figure 1 Comparison between a zone of inhibition and an untreated lawn culture

Hypothesis Based on what you know about antimicrobial agents, develop a hypothesis that ranks the degree of inhibition that you expect to see from each of the three categories. Procedure Two petri dishes were inoculated with S. mutans and two with E. coli to create an even coating of cells across the surface of the agar on each plate. Seven discs impregnated with different antimicrobial agents and a control were evenly spaced across the surface of the agar on each plate. The dishes were placed in an incubator to allow the bacteria to grow and reproduce. After 24 hours, the plates were removed from the incubator and photographed for data collection. Results Print this page at actual size before taking measurements. DO NOT MEASURE FROM YOUR MONITOR. For each of the two petri dishes containing S. mutans (Figure 2), record the diameter of the zone of inhibition in millimeters on Table 1. The diameter should be measured from edge to edge across the widest point. If a zone of inhibition is not present, record the diameter as 0 mm, disregarding the diameter of the disc. For each treatment, calculate the average diameter of the zone of inhibition across the two replications. Figure 2 Two replicates of Streptococcus mutans treated with six antimicrobial agents plus a control Table 1: S. mutans Category Dish 1 Dish 2 Average 1. Antibacterial soap 11mm 9mm 10mm 2. Bleach 19mm 20mm 19mm 3. Erythromycin 24mm 25mm 24mm 4. Mouthwash 12mm 14mm 13mm 5. Amoxicillin 22mm 19mm 20mm 6. Household cleaner 15mm 15mm 15mm 7. Deionized water 5mm 5mm 5mm

Using what you learned in the introduction, categorize each treatment as an antiseptic, disinfectant, antibiotic or control in Table 1. Calculate the average diameter of the zone of inhibition for each category and create a bar graph in Figure 3. Average diameter by category: contro l 5mm antiseptics 11mm disinfectants 17.25mm antibiotics 22mm Figure 3 Average inhibition by category for S. mutans Repeat the procedure to collect data on the plates containing E. coli (Figure 4). Record the results in table 2.

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Figure 4 Two replicates of Escherichia coli treated with six antimicrobial agents plus a control Table 2: E. coli Category Dish 1 Dish 2 Average 1. Antibacterial soap Antiseptic 10mm 10mm 10mm 2. Bleach Disinfectant 19mm 17mm 18mm 3. Erythromycin Antibiotic 20mm 17mm 18mm 4. Mouthwash Antiseptic 10mm 8mm 9mm 5. Amoxicillin Antibiotic 19mm 16mm 17mm 6. Household cleaner Disinfectant 15mm 14mm 14mm 7. Deionized water Control 4mm 5mm 4mm For the E. coli culture, calculate the average diameter of the zone of inhibition for each category and create a bar graph in Figure 5 below. Average diameter by category: contro l 4mm antiseptics 9.5mm disinfectants 16mm antibiotics 18mm Figure 5 Average inhibition by category for E. coli Summary Questions 1. Identify the dependent and independent variable in this experiment. Remember, the independent variable is the one that is changed or controlled in an experiment. The dependent variable is the one that is tested or measured. It responds to the independent variable.  Dependent Variable is Antibiotic Concentration Independent Variable is Zone of inhibition that we measure. 2. Based on the data you collected, which antimicrobial agent showed the greatest amount of antimicrobial activity? Which showed the least?  Comparing the antibacterial activity we can consider the zone of inhibition value as standard more the zone of inhibition more

3. Different types of antibiotics are known to create zones of inhibition of varying sizes. This is due to several factors including its rate of diffusion, solubility, the type of microbe etc. Because of this, standards have been developed that are used to determine how susceptible a bacterium is to an antibiotic. Using Table 3, classify each species of bacteria as resistant, intermediate or susceptible to each of the antibiotics we tested. Antibiotic Resistant Intermediate Susceptible (< or = mm) (mm) (= or > mm) Amoxicillin <13 14-17 >18 Ampicillin 11 12-13 14 Ciprofloxacin 15 16-20 21 Erythromycin 13 14-22 23 Kanamycin 13 14-17 18 Tetracycline 14 15-18 19 Vancomycin 9 10-11 12 Table 3: Zone diameter interpretive standards for select antibiotics  Streptococcus mutants is sensitive TO ERYTHROMYCIN AND AMOXYCILLIN  Escheritia coli is also sensitive to erythromycin and amoxycillin. 4. Taking into consideration what you learned in the question above, what sort of precautions would you need to take when designing an experiment like the one in this activity? Identify any limitations there are to the conclusions you can draw from data in this type of experiment. 5. Compare any differences in the results between the two bacterial species. What differences in their structure may have contributed to these results? 6. Identify problems or unintended consequences that can result from the prevalence of antimicrobial compounds in our society. (Use outside sources)

 One of the consequences of the prevalence antimicrobial compounds in our society is the longer time period and the harder treatment of these bacteria for example because these bacteria is already an antibiotic resistance. Overusing and not using the antibiotics properly will lead to more complicated and complex problems like having severe infections, sickness and spending more money in medical treatments. We need to address this medical challenge antibiotics properly.

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