Antiseptics and Disinfectants

Disinfectants and Antiseptics

• Disinfectants and antiseptics are antimicrobial agents used to inhibit the growth of or kill microorganisms.
• Disinfectants are used on non-living objects, while antiseptics are used on living tissue.

Effectiveness of Antiseptics and Disinfectants

• Effectiveness can be determined by using a paper disk saturated with an agent on an agar plate inoculated with microorganisms.
• A zone of inhibition (area of no growth) will appear around the disk if the agent is successful.
• Zone sizes are measured in millimeters (mm) and can be affected by incubation time, temperature, and other variables.

Materials

• Broth cultures of 5 microorganisms: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Serratia marcescens, and Bacillus subtilis.
• 5 nutrient agar plates (TSA plates).
• Agents to be tested: mouthwash, betadine, lysol (diluted 1:1), and optional agents (garlic extract, grapefruit-seed extract, zinc, or rubbing alcohol).

Procedure

• Create a "bacterial lawn" by spreading microorganism cultures over the entire agar surface.
• Repeat the process for each of the 5 bacterial cultures.
• Divide each agar plate into four distinct regions using lines on the bottom.

Disinfectants and Antiseptics

• Disinfectants and antiseptics are antimicrobial agents used to inhibit the growth of or kill microorganisms.
• Disinfectants are used on non-living objects, while antiseptics are used on living tissue.

Effectiveness of Antiseptics and Disinfectants

• Effectiveness can be determined by using a paper disk saturated with an agent on an agar plate inoculated with microorganisms.
• A zone of inhibition (area of no growth) will appear around the disk if the agent is successful.
• Zone sizes are measured in millimeters (mm) and can be affected by incubation time, temperature, and other variables.

Materials

• Broth cultures of 5 microorganisms: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Serratia marcescens, and Bacillus subtilis.
• 5 nutrient agar plates (TSA plates).
• Agents to be tested: mouthwash, betadine, lysol (diluted 1:1), and optional agents (garlic extract, grapefruit-seed extract, zinc, or rubbing alcohol).

Procedure

• Create a "bacterial lawn" by spreading microorganism cultures over the entire agar surface.
• Repeat the process for each of the 5 bacterial cultures.
• Divide each agar plate into four distinct regions using lines on the bottom.

Antibiotic Susceptibility Testing

• Ampicillin (AM) testing for Gram-negative microorganisms and Enterococci: 10 μg, with interpretation as sensitive (≤11), intermediate (12-13), and resistant (≥14)
• Ampicillin (AM) testing for Staphylococci and penicillin G-susceptible microorganisms: 10 μg, with interpretation as sensitive (≤20), intermediate (21-28), and resistant (≥29)
• Ampicillin (AM) testing for Haemophilus species: 10 μg, with interpretation as sensitive (≤19), intermediate (20-22), and resistant (≥23)

Bacitracin Susceptibility Testing

• Bacitracin testing: 10 units, with interpretation as sensitive (≤8), intermediate (9-12), and resistant (≥13)

Carbenicillin Susceptibility Testing

• Carbenicillin testing: 50 μg, with interpretation as sensitive (≤17), intermediate (18-23), and resistant (≥24)

Cephalotin Susceptibility Testing

• Cephalotin (CR) testing for susceptibility to cephalotin, cephaloridine, and cephalexin: 30 μg, with interpretation as sensitive (≤14), intermediate (15-17), and resistant (≥18)
• Cephalothin testing for susceptibility to cephaloglycin: 30 μg, with interpretation as sensitive (≤14), intermediate (15-17), and resistant (≥18)

Chloramphenicol Susceptibility Testing

• Chloramphenicol (Chloromycetic) testing: 30 μg, with interpretation as sensitive (≤12), intermediate (13-17), and resistant (≥18)

Clindamycin Susceptibility Testing

• Clindamycin (CC) testing: 2 μg, with interpretation as sensitive (≤14), intermediate (15-16), and resistant (≥17)
• Clindamycin (CC) testing: 2 μg, with interpretation as sensitive (≤16), intermediate (17-20), and resistant (≥21)

Erythromycin Susceptibility Testing

• Erythromycin (GM) testing: 15 μg, with interpretation as sensitive (≤13), intermediate (14-17), and resistant (≥18)

Gentamicin Susceptibility Testing

• Gentamicin (GM) testing: 10 μg, with interpretation as sensitive (≤12), intermediate (13-14), and resistant (≥15)

Kanamycin Susceptibility Testing

• Kanamycin (ME) testing: 30 μg, with interpretation as sensitive (≤13), intermediate (14-17), and resistant (≥18)

Methicillin Susceptibility Testing

• Methicillin (ME) testing: 5 μg, with interpretation as sensitive (≤9), intermediate (10-13), and resistant (≥14)

Neomycin Susceptibility Testing

• Neomycin (NB) testing: 30 μg, with interpretation as sensitive (≤12), intermediate (13-16), and resistant (≥17)

Novobiocin Susceptibility Testing

• Novobiocin (NB) testing: 30 μg, with interpretation as sensitive (≤17), intermediate (18-21), and resistant (≥22)

Oleandomycin Susceptibility Testing

• Oleandomycin (OL) testing: 15 μg, with interpretation as sensitive (≤11), intermediate (12-16), and resistant (≥17)

Penicillin G Susceptibility Testing

• Penicillin G testing for Staphylococci: 10 units, with interpretation as sensitive (≤20), intermediate (21-24), and resistant (≥25)
• Penicillin G testing for other microorganisms: 10 units, with interpretation as sensitive (≤11), intermediate (12-21), and resistant (≥22)

Polymyxin B Susceptibility Testing

• Polymyxin B (PB) testing: 300 units, with interpretation as sensitive (≤8), intermediate (9-11), and resistant (≥12)

Rifampin Susceptibility Testing

• Rifampin testing for N. meningitidis susceptibility: 5 μg, with interpretation as sensitive (≤24), and resistant (≥25)

Streptomycin Susceptibility Testing

• Streptomycin testing: 10 μg, with interpretation as sensitive (≤11), intermediate (12-14), and resistant (≥15)

Sulfonamide Susceptibility Testing

• Sulfonamides testing: 300 μg, with interpretation as sensitive (≤12), intermediate (13-16), and resistant (≥17)

Tetracycline Susceptibility Testing

• Tetracycline (T, TE) testing: 30 μg, with interpretation as sensitive (≤14), intermediate (15-18), and resistant (≥19)

Vancomycin Susceptibility Testing

• Vancomycin (VA) testing: 30 μg, with interpretation as sensitive (≤9), intermediate (10-11), and resistant (≥12)

Operating Room Hand Soap

• The operating room physician recommends periodically changing the hand soap used in the operating room to maintain effectiveness against microbial infections.

Microbial Infections on Cruise Ships

• There have been incidents of travelers getting seriously ill from microbial infections on cruise ships.
• To resolve this issue, specific recommendations would be needed, such as implementing proper hand hygiene practices, ensuring cleanliness of surfaces and equipment, and providing adequate education to employees.

Hexachlorophene

• Hexachlorophene was once an over-the-counter antiseptic but now requires a prescription for concentrations greater than 3%.
• The requirement was likely initiated due to concerns over the potential risks or side effects of high-concentration hexachlorophene solutions.

Kirby-Bauer Method

• The Kirby-Bauer method is used to analyze the effectiveness of antibiotics.
• Objectives of the Kirby-Bauer method include:
  • Defining and providing an example of an antibiotic
  • Explaining the difference between an antibiotic, antiseptic, and disinfectant
  • Performing the procedure for testing antibiotic effectiveness using antibiotic discs on bacterial cultures.

Antibiotic Disc Test

• Antibiotic discs are applied to bacterial culture to test their effectiveness individually or by dispenser.
• The test measures the zones of growth inhibition caused by antibiotics.

Zones of Inhibition

• The zones of inhibition are classified into three categories based on the diameter of the zone:
• R (Resistant): Zone of inhibition with a diameter equal to or less than the inner circle, indicating the antibiotic does not inhibit bacterial growth.
• I (Intermediate): Zone of inhibition with a diameter greater than R, but less than the outer circle, indicating the antibiotic somehow inhibits bacterial growth.
• S (Sensitive): Zone of inhibition with a diameter greater than the outer circle, indicating the antibiotic inhibits bacterial growth very well.

Antibiotics

• Antibiotics are chemicals produced by living organisms to inhibit the growth of or kill other organisms.
• They are sometimes referred to as natural products.

Kirby-Bauer Method

• The Kirby-Bauer method is a technique used to determine the effectiveness of antibiotics.
• Mueller-Hinton agar is used in this procedure because it allows for rapid diffusion of antibiotics through the medium.
• Antibiotic discs are placed on the medium, and the effectiveness of the antibiotic is determined by measuring the zone of inhibition.
• The zone sizes are compared to values on standardized tables to determine whether the microorganism is Resistant, Intermediate, or Sensitive to a particular antibiotic.

Ultraviolet Light and Microbial Growth

• Ultraviolet light is a physical agent used to sterilize surgical instruments, tabletops, and food products, including milk, to decrease microbial growth.

Mechanism of Action

• Ultraviolet light inhibits microbial growth by causing the formation of pyrimidine dimers in DNA.
• Pyrimidine dimers are double bonds that form between adjacent pyrimidines in a strand of DNA.
• Dimers distort DNA structure, thereby inhibiting DNA replication and interfering with subsequent cell division.

Materials Used in the Experiment

• Liquid cultures of Bacillus subtilis, Staphylococcus aureus, Serratia marcescens, and Escherichia coli
• 5 nutrient agar plates (TSA plates)
• Sterile cotton applicator sticks (swabs)
• Index cards
• Ultraviolet (UV) lamp

Experimental Procedure

• Wear protective eyewear (sunglasses) to protect eyes from UV light.
• Label 5 TSA plates with the name of the organism and divide each plate into two halves.
• Label one half "exposed" and the other half "unexposed" and label exposed sides with different exposure times (15, 30, 60, 90, and 120 seconds).
• Dip a sterile swab into the culture tube and spread the culture to create a "bacterial lawn" on each plate.
• Repeat the process for all 4 plates.

UV Light and Microbial Growth Experiment

• The experiment involves exposing plates of Serratia marcescens to different durations of ultraviolet (UV) light: 15 seconds, 30 seconds, 60 seconds, 90 seconds, and 120 seconds.
• The plates are incubated at 37°C for 24-48 hours to observe the effect of UV light on microbial growth.

Procedure

• Dip a sterile swab into the culture tube and spread the culture to create a "bacterial lawn" on each plate.
• Cover the 'unexposed' side of each plate with an index card and place the plate under the UV lamp for the designated amount of time.
• Repeat the process for all five plates.
• Incubate the plates at 37°C for 24-48 hours.

Observations and Results

• Compare the unexposed and exposed sides of the plates to observe the effect of UV light on microbial growth.
• Draw labeled diagrams indicating the number and color of colonies observed on each plate.

Key Concepts

UV Light and Microbial Growth

• UV light inhibits microbial growth by causing the formation of pyrimidine dimers, which prevents DNA replication.
• Exposure to UV light can cause a decrease in the number of Serratia marcescens cells.

Sterilization and UV Light

• UV light is used for sterilization of inanimate objects to kill microbes.
• UV light is applied to objects such as milk for ultra-pasteurization, which provides additional protection against microbial contamination.
• A possible benefit of using both pasteurization and UV light is to ensure more comprehensive killing of microbes.

UV Light and Microbial Growth Experiment

• The experiment involves exposing plates of Serratia marcescens to different durations of ultraviolet (UV) light: 15 seconds, 30 seconds, 60 seconds, 90 seconds, and 120 seconds.
• The plates are incubated at 37°C for 24-48 hours to observe the effect of UV light on microbial growth.

Procedure

• Dip a sterile swab into the culture tube and spread the culture to create a "bacterial lawn" on each plate.
• Cover the 'unexposed' side of each plate with an index card and place the plate under the UV lamp for the designated amount of time.
• Repeat the process for all five plates.
• Incubate the plates at 37°C for 24-48 hours.

Observations and Results

• Compare the unexposed and exposed sides of the plates to observe the effect of UV light on microbial growth.
• Draw labeled diagrams indicating the number and color of colonies observed on each plate.

Key Concepts

UV Light and Microbial Growth

• UV light inhibits microbial growth by causing the formation of pyrimidine dimers, which prevents DNA replication.
• Exposure to UV light can cause a decrease in the number of Serratia marcescens cells.

Sterilization and UV Light

• UV light is used for sterilization of inanimate objects to kill microbes.
• UV light is applied to objects such as milk for ultra-pasteurization, which provides additional protection against microbial contamination.
• A possible benefit of using both pasteurization and UV light is to ensure more comprehensive killing of microbes.

Lysozyme and Nonspecific Resistance

• Lysozyme is an enzyme found in body secretions such as tears, saliva, and mucous, that confers antibacterial activity as part of the immune system's nonspecific resistance.

Antibacterial Activity of Lysozyme

• Lysozyme causes the degradation of bacterial cell walls, specifically affecting peptidoglycan.
• This makes it more effective against Gram-positive bacteria than Gram-negative bacteria.

Effectiveness of Lysozyme Against Different Bacteria

• Lysozyme will be tested against three bacteria: Micrococcus luteus, Staphylococcus aureus, and Escherichia coli.
• The larger the zone of inhibition, the greater the effectiveness of lysozyme in inhibiting bacterial growth.

Exercise Procedure

• Sterile cotton swabs are used to spread bacterial cultures over agar plates.
• Sterile paper disks are saturated with purified lysozyme solution and placed in the center of each plate.
• Plates are incubated at 37°C for 24-48 hours to observe zones of inhibition.

Illness and Its Causes

• Feeling unwell can have various causes, including changes in weather, pollen count, or catching a bug from someone else.
• Signs and symptoms of illness can result from a pathogenic infection or an immune response to a perceived threat.

Immune Response and Fever

• The immune system may release pyrogens, chemicals that increase body temperature, in response to certain pathogens.
• This response creates an unfavorable environment for the pathogen, but it also makes the person feel sick.

Diagnosis and Treatment

• Medical professionals use signs and symptoms to determine the cause of an ailment and prescribe treatment.
• While signs and symptoms can help identify the causative agent of a disease, they are often not unique to a specific disease.
• Direct and indirect diagnostic methods are often necessary to confirm the identity of the infectious agent.

Learn about the differences between antiseptics and disinfectants, their uses, and their effects on microorganisms. Understand the terms microcidal and microstatic in the context of antimicrobial agents.