Antimicrobials

1. Antimicrobials IF YOU CAN SEE THIS MESSAGE YOU ARE NOT IN “SLIDE SHOW” MODE. PERFOMING THE LAB IN THIS MODE WILL NOT ALLOW FOR THE ANIMATIONS AND INTERACTIVITY OF THE EXERCISE TO WORK PROPERLY. TO CHANGE TO “SLIDE SHOW” MODE YOU CAN CLICK ON “VIEW” AT THE TOP OF THE PAGE AND SELECT “SLIDE SHOW” FROM THE PULL DOWN MENU. YOU CAN ALSO JUST HIT THE “F5” KEY. Instructor Terry Wiseth

2. Click on the blackboard to view a larger board for discussion. Incubator 370C Agar Plates Swabs Loops Antiseptic Dispenser Microbe Samples Loops Pencil Bunsen burner

3. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C • ANTIMICROBIALSDISINFECTANTS, ANTISEPTICS, AND SANITIZERS • Disinfection is the elimination of microorganisms from inanimate objects or surfaces, whereas decontamination is the treatment of an object or inanimate surface to make it safe to handle. • a) The term disinfectant is used for an agent used to disinfect inanimate objects or surfaces but is generally too toxic to use on human tissues. • b) The term antiseptic refers to an agent that kills or inhibits growth of microbes but is safe to use on human tissue. • c) The term sanitizer describes an agent that reduces, but may not eliminate, microbial numbers to a safe level. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

4. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C • Because disinfectants and antiseptics often work slowly on some viruses, such as the hepatitis viruses, bacteria such as Mycobacterium tuberculosis, and especially bacterial endospores, they are usually unreliable for sterilization (the destruction of all life forms). There are a number of factors which influence the antimicrobial action of disinfectants and antiseptics, including: • 1) The concentration of the chemical agent. • 2) The temperature at which the agent is being used. Generally, the lower the temperature, the longer it takes to disinfect or decontaminate. • 3) The kinds of microorganisms present. Endospore producers such as Bacillus species, Clostridium species, and acid-fast bacteria like Mycobacterium tuberculosis are harder to eliminate. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

5. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 350C 500C 1000C • 4) The number of microorganisms present. The more microorganisms present, the harder it is to disinfect or decontaminate. • 5) The nature of the material bearing the microorganisms. Organic material such as dirt and excreta interferes with some agents. The best results are generally obtained when the initial microbial numbers are low and when the surface to be disinfected is clean and free of possible interfering substances. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Thanks to the work of Louis Pasteur and Joseph Lister, a newly discovered antiseptic is being used by these doctors who are performing an 1871 surgery in Edinburgh, Scotland. A carbolic acid (phenol) aerosol is sprayed with this instrument. It showers an antiseptic mist over the patient to kill pathogens that cause infection, providing a “clean” surgery.

6. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 350C 500C 1000C • There are two common antimicrobial modes of action for disinfectants, antiseptics, and sanitizers: • 1) They may damage the lipids and/or proteins of the semi permeable cytoplasmic membrane of microorganisms resulting in leakage of cellular materials needed to sustain life. • 2) They may denature microbial enzymes and other proteins, usually by disrupting the hydrogen and disulfide bonds that give the protein its three-dimensional functional shape. This blocks metabolism. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 British surgeon Joseph Lister (center), inventor of antiseptic surgery.

7. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 350C 500C 1000C A large number of such chemical agents are in common use. Some of the more common groups are listed below:1) Phenol and phenol derivatives2) Soaps and detergents3) Alcohols4) Acids and alkalies5) Heavy metals6) Chlorine7) Iodine and iodophores8) Aldehydes9) Ethylene oxide gas Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Iodine applied to the skin

8. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C 1) Phenol and phenol derivatives Phenol (5-10%) was the first disinfectant commonly used. However, because of its toxicity and odor, phenol derivatives are now generally used. Examples include Lysol and PhisoHex. Triclosan is a chlorine-containing phenolic antiseptic very common in antimicrobial soaps and other products. Hexylresorcinol is in throat lozenges. A 4% solution of chlorhexidine in isopropyl alcohol and combined with detergent is a common hand washing agent and surgical hand scrub. These agents kill most bacteria, most fungi, and some viruses, but are usually ineffective against endospores. They alter membrane permeability and denature proteins. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

9. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C 2) Soaps and detergentsSoaps are only mildly microbicidal. Their use aids in the mechanical removal of microorganisms by breaking up the oily film on the skin (emulsification) and reducing the surface tension of water so it spreads and penetrates more readily. Some cosmetic soaps contain added antiseptics to increase antimicrobial activity. Laundry powders mechanically remove microorganisms and other materials but are not very microbicidal. Some detergents alter membrane permeability and denature proteins. They are effective against many vegetative bacteria, some fungi, and some viruses. Detergents are inactivated by soaps and organic materials like excreta. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

10. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C 3) Alcohols70% solutions of ethyl or isopropyl alcohol are effective in killing vegetative bacteria, enveloped viruses, and fungi. However, they are usually ineffective against endospores and non-enveloped viruses. Once they evaporate, their cidal activity will cease. Alcohols denature membranes and are often combined with other disinfectants, such as iodine, mercurials, and detergents for increased effectiveness. 4) Acids and alkaliesAcids and alkalies alter membrane permeability and denature proteins and other molecules. Salts of organic acids are commonly used as food preservatives. Desenex is used for dermatophyte infections of the skin. An example of an alkali is lye (sodium hydroxide). Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

11. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C 5) Heavy metalsHeavy metals, such as mercury, silver, and copper, denature proteins. Mercury compounds are only bacteriostatic and are not effective against endospores. Silver nitrate (1%) is sometimes put in the eyes of newborns to prevent gonococcal ophthalmia. Copper sulfate is used to combat fungal diseases of plants and is also a common algicide. Selinium sulfide kills fungi and their spores. 6) ChlorineChlorine gas acts to denature microbial enzymes. Chlorine is used in the chlorination of drinking water, swimming pools, and sewage. Sodium hypochlorite is the active agent in household bleach. Chlorines are used to sanitize glassware, eating utensils, dairy and food processing equipment, hemodialysis systems, and treating water supplies. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

12. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C 7) Iodine and iodophoresIodine also denatures microbial proteins. Iodine tincture and Aqueous iodine solutions are commonly used as a topical antiseptic. Iodophores are less irritating than iodine and do not stain. They are generally effective against vegetative bacteria, Mycobacterium tuberculosis, fungi, some viruses, and some endospores. Examples include Betadine and Isodine. 8) AldehydesAldehydes, such as formaldehyde, denature microbial proteins. Formalin (a formaldehyde gas) is extremely active and kills most forms of microbial life. It is used in embalming, preserving biological specimens, and in preparing vaccines. These kill vegetative bacteria in 10-30 minutes and endospores in about 4 hours. A 10 hour exposure to formalin solution can be used for cold sterilization of materials. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

13. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C 9) Ethylene oxide gasEthylene oxide is one of the very few chemicals that can be relied upon for sterilization (after 4-12 hours exposure). Since it is explosive, it is usually mixed with inert gases such as freon or carbon dioxide. Ethylene oxide is commonly used to sterilize heat-sensitive items such as plastic syringes, petri plates, textiles, sutures, artificial heart valves, heart-lung machines, and mattresses. Ethylene oxide has very high penetrating power and denatures microbial proteins. Vapors are toxic to the skin, eyes, and mucous membranes and are also carcinogenic. Another gas that is used as a sterilant is chlorine dioxide which denatures proteins in vegetative bacteria, bacterial endospores, viruses, and fungi. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

14. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C ANTISEPTIC PROPERTIES OF SPICES Long before the Crusaders introduced the fragrance and taste of the East to the unsophisticated European cuisine, people in India, Asia, and the Far East were trading spices. One of Christopher Columbus's goals in setting sail for India was to bring back pepper, cinnamon, and other flavorful dried herbs, for Europe did not supply the need of its populations for them. Not only do spices enhance the taste of food, the aroma of them arouses the appetite. Spices themselves provide many trace minerals which some populations, especially impoverished or vegetarian ones, depend on to maintain health. History tells us that the European hunt for pepper, ginger, and other "hot" spices grew out of a need to preserve meat, or to disguise the taste of meat which had gotten a little to old to be pleasant by itself. Many herbs and spices MAY have antimicrobial activity:  thyme, clove, cinnamon, garlic, ginger, chamomile, oregano, sage,  echinacea, wasabi, etc.  We will be testing clove oil and various antimicrobial chemicals against different bacteria. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

15. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C • EVALUATION OF DISINFECTANTS, ANTISEPTICS, AND SANITIZERS • A common test for effectivity of antimicrobials is to compare the antimicrobial activity of the agent being tested with that of phenol. The resulting value is called a phenol coefficient and has some value in comparing the strength of disinfectants under standard conditions. • The antiseptics we will be testing are as follows: • 70% isopropyl alcohol3% hydrogen peroxideScope mouthwashListerine mouthwashClove oilPhenol • The bacteria we will be using for the tests are as follows: • Mouth culture • Fecal culture Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

16. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C PHENOL COEFFICIENT The Phenol Coefficient is determined by measuring the effectiveness of Phenol as an antiseptic. For each culture a signature value for the zone of inhibition for Phenol is established by measuring the clear zone surrounding a Phenol test disc. Once this measure is established the Phenol value is defined as 1. Phenol coefficients for other antiseptics in the study are given values based on the zone of inhibition exhibited by Phenol. An example for finding the individual test antiseptic Phenol coefficients is given below.Phenol Zone of Inhibition = 12 mmAntiseptic Zone of Inhibition = 14 mmPhenol Coefficient is calculated as 14 / 12 = 1.17 Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

17. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 500C 350C 1000C Antiseptic Sensitivity In this lab you will be performing two experiments. The first experiment will investigate various antiseptic sensitivity of bacteria cultures from the mouth. The second experiment will investigate various antiseptic sensitivity of fecal bacteria cultures.Click on the Antiseptic Sensitivity Virtual Lab link below.If you have performed the experiment you can click on “END LAB” below. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Antiseptic SensitivityVirtual Lab Print Table 1AntisepticSensitivity END LAB

18. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C ANTIMICROBIALS QUESTIONS 1) Define the following terms: sterilization, disinfection, decontamination, disinfectant, antiseptic, and sanitizer. 2) State why chemical agents are usually unreliable for sterilization. 3) Describe two modes of action of disinfectants, antiseptics, and sanitizers, i.e., how they harm the microorganisms. 4) Name two chemical agents that are reliable for sterilization. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

19. Antiseptic Sensitivity

20. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C • We will be testing two different types of bacterial cultures for sensitivity to six different antiseptics. The bacterial cultures we will be using will be collected from the mouth and fecal cultures. • The types of antiseptics used will be: • AlcoholHydrogen PeroxideScope MouthwashListerine MouthwashClove OilPhenol Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

21. Refrigerator Freezer Incubator Incubator Incubator -100C 00C 350C 500C 1000C In this experiment we will utilize two agar plates which contain the necessary nutrients and moisture necessary for the collected cultures to survive and reproduce. In order to introduce the bacteria to the sterile agar plates we will use sterile cotton tip swabs which can transfer the bacteria from the pure culture tubes to the agar plates. Once the plates are inoculated we will introduce the discs containing the antiseptics to the surface of the agar. Next, we will incubate the plates at 37 OC for 24 hours. An incubator will be used to house these plates so the temperature and moisture will remain stable during the culture time. Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9

22. Refrigerator Freezer Incubator Incubator Incubator 00C -100C 350C 500C 1000C Click on one of the sample locations listed below to collect bacteria and introduce to the broth culture. If you are finished with both experiments you can click on “END LAB” Agar Plates pH = 11 pH = 5 pH = 3 pH = 7 pH = 9 Mouth Culture Fecal Culture Clove oil can be attributed to antimicrobial, antifungal, antiseptic, and antiviral, properties. Clove is an evergreen tree that has numerous medicinal properties. It is often referred as clove bud. Clovewas extensively used in the ancient Indian and Chinese civilizations and it spread to other parts of the world, including Europe, during the seventh and eighth centuries. END LAB

23. Mouth Culture

24. In this experiment we will be swabbing the internal lining of the mouth to collect bacteria, which we will introduce to a broth culture. Click on the Broth Culture Tubes to bring the rack to the table. Click on NEXT when the test tube rack is placed on the table. Incubator 370C Mouth Fecal Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

25. Click on the cotton tip swabs to sample the lining of the interior of the mouth and mix with the broth culture. Click on NEXT when you have inoculated the culture. Incubator 370C Mouth Fecal Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

26. Click on the Agar Plates to bring a plate to the table. When the agar plate has arrived to the table Click on the blue eyedroppers on the shelf to inoculate the plate with the bacteria culture from the mouth. Click on NEXT when the plates have been inoculated. Incubator 370C Mouth Fecal Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

27. Click on the Antiseptic Dispenser to introduce the antiseptic discs onto the surface of the inoculated agar plate. Each of the discs has been labeled indicating the antiseptic present in the disc. Next Click on the agar plate to place it into the incubator. ClickNEXT when finished. Incubator 370C Mouth Fecal Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

28. After 24 hours of incubation we can view the results of growth of bacteria while exposed to the antiseptic discs.We will assume at this point that the plate has been incubated for 24 hours. Click on the incubator to bring the agar plate to the table. ClickNEXT when finished. Incubator 370C Mouth Fecal Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

29. Click on the agar plate on the table to view the plate. Observe the size of the areas around the antiseptic discs in which there is no bacterial growth evident. These areas are called “Zones of Inhibition”. Measure the size of each of these zones by clicking on each of the labeled discs. Record your data for each antiseptic type in Table 1. ClickNEXT when finished. Mouth Culture A P HP C S Incubator 370C L Nose Ear Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Alcohol = AHydrogen Peroxide = HPScope Mouthwash = SListerine Mouthwash= LClove Oil = CPhenol = P How do I measurethe Zone of Inhibition? Table 1

30. At this point you should entered your measurement values in Table 1. Using the Interpretive Zone Chart given to the left, indicate in Table 1 whether the antiseptic tested resistant or susceptible for the culture isolate. Produce a sketch of the agar plate with the indicated clear zones for each of the antiseptics. ClickNEXT when finished. Incubator 370C Nose Ear Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Table 1 Sketch Questions

31. You have now finished this experiment. There are two experiments in this lab involving two separate bacterial cultures. If you have performed both of the experiments you can click on “END LAB” below. If you still need to perform another experiment click on the appropriate link given. Incubator 370C Mouth Fecal Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Mouth Culture Fecal Culture END LAB

32. Fecal Culture

33. In this experiment we will be using bacteria collected from a fecal culture. Click on the Broth Culture Tubes to bring the rack to the table. Click on NEXT when the test tube rack is placed on the table. Incubator 370C Mouth Fecal Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Pencil Bunsen burner

34. Click on the Agar Plates to bring a plate to the table. When the agar plate has arrived to the table Click on the blue eyedroppers on the shelf to inoculate the plate with the fecal bacteria culture. Click on NEXT when the plates have been inoculated. Incubator 370C Mouth Fecal Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

35. Click on the Antiseptic Dispenser to introduce the antiseptic discs onto the surface of the inoculated agar plate. Each of the discs has been labeled indicating the antiseptic present in the disc. Next Click on the agar plate to place it into the incubator. ClickNEXT when finished. Incubator 370C Mouth Fecal Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

36. After 24 hours of incubation we can view the results of growth of bacteria while exposed to the antiseptic discs.We will assume at this point that the plate has been incubated for 24 hours. Click on the incubator to bring the agar plate to the table. ClickNEXT when finished. Incubator 370C Mouth Fecal Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner

37. Click on the agar plate on the table to view the plate. Observe the size of the areas around the antiseptic discs in which there is no bacterial growth evident. These areas are called “Zones of Inhibition”. Measure the size of each of these zones by clicking on each of the labeled discs. Record your data for each antiseptic type in Table 1. ClickNEXT when finished. Fecal Culture A P HP C S Incubator 370C L Nose Ear Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Each mark = 1 mm Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Alcohol = AHydrogen Peroxide = HPScope Mouthwash = SListerine Mouthwash = LClove Oil = CPhenol = P How do I measurethe Zone of Inhibition? Table 1

38. At this point you should entered your measurement values in Table 1. Using the Interpretive Zone Chart given to the left, indicate in Table 1 whether the antiseptic tested resistant or susceptible for the culture isolate. Produce a sketch of the agar plate with the indicated clear zones for each of the antiseptics. ClickNEXT when finished. Incubator 370C Mouth Fecal Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Table 1 Sketch Questions

39. You have now finished this experiment. There are two experiments in this lab involving two separate bacterial cultures. If you have performed both of the experiments you can click on “END LAB” below. If you still need to perform another experiment click on the appropriate link given. Incubator 370C Mouth Fecal Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil Bunsen burner Mouth Culture Fecal Culture END LAB

40. Measurement Instructions

41. At this point you should. ClickNEXT when finished. Incubator 370C Nose Ear Measure this distance Agar Plates Swabs Loops Antiseptic Dispenser Broth Culture Tubes Loops Pencil P Bunsen burner Each mark = 1 mm Measure the diameter of the zone indicated by the clear area of growth around the disc Click here to returnto the experiment

42. Click here to returnto the experiment