Title: Detection of β-Lactamase Production by Staphylococcus aureus or E. coli
Aim: To detect the production of β-lactamase enzyme by Staphylococcus aureus or E. coli using the iodometric method.
Introduction:
β-lactamases are enzymes produced by bacteria that hydrolyze β-lactam antibiotics, rendering them ineffective. These enzymes play a significant role in antibiotic resistance, making their detection crucial in clinical microbiology and biotechnology. Staphylococcus aureus and E. coli are common β-lactamase-producing bacteria that contribute to multidrug resistance.
Theory:
β-lactamase enzymes break the β-lactam ring of antibiotics such as penicillins and cephalosporins, inactivating their antibacterial properties. Their presence can be detected using the iodometric method, where the degradation of penicillin leads to a color change due to iodine-starch interaction.
Principle:
β-lactamase hydrolyzes β-lactam antibiotics, releasing acid byproducts. In the iodometric method, the degradation of penicillin prevents iodine from binding with starch, leading to decolorization.
Requirements:
- Staphylococcus aureus or E. coli cultures
- Nutrient agar plates
- Penicillin solution
- Iodine-starch solution
- Sterile cotton swabs
- Inoculation loop
- Incubator (35–37°C)
- Sterile water
- Petri plates
Procedure:
- Prepare and autoclave nutrient agar medium at 121°C for 15 minutes.
- Pour the medium into sterile Petri plates and allow it to solidify.
- Using a sterile inoculation loop, streak the test bacteria onto the nutrient agar plate.
- Alternatively, use a sterile cotton swab to evenly spread liquid culture over the plate surface and Label the plates properly.
- Incubate the plates at 35–37°C for 18–24 hours.
- a. Iodometric Method: Add a few drops of penicillin solution to the bacterial growth.
- After 5 minutes, add iodine-starch solution and observe color changes.
- A disappearance of the blue-black color indicates β-lactamase activity.
Observation:
- In the iodometric method, a decolorized zone indicates β-lactamase production.
- No color change suggests the absence of β-lactamase production.
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| Fig: Decolorized zone indicates β-lactamase production |
Result:
The presence of decolorization (iodometric method) confirms β-lactamase production in S. aureus or E. coli.
Conclusion:
The β-lactamase detection test is a crucial method for identifying antibiotic-resistant bacteria. The presence of β-lactamase in S. aureus or E. coli suggests resistance to β-lactam antibiotics, highlighting the importance of alternative treatment strategies in clinical microbiology and biotechnology.
