1. HOST PATHOGENS INTERACTION…….

2. Origen of microbial flora  The fetus is in a sterile environment during first few days of life  New born……. Many micro organisms

3.  Once established onto or into a particular body site……..  Symbiosis, commensalism, parasitism.

4. Characteristics of indigenous flora Indigenous flora Body sites …same or different flora Resident microbial flora Transient flora

5. Carriers Carrier state Carrier state may be acute or chronic Colonizing….host resistance….microbial flora provide protection

6. Factors that determine the composition of the usual microbial flora Presence of organisms …. Influenced by nutritional and environmental fectors.

7. Composition of the microbial flora at different body sites Human host…colonized by….app. 100 microbs Effectiveness of host…..low incidence of infection Clinical microbiologist must be able to recognize and identify the types of microbs …at diff body sites

8. Usual flora of the skin Skin has numbr of mechanisms...prevent infections Skin…..vide veriety of microbs Reduce but not eliminated by scrubbing and washing

9. Composition of flora….depends… Concentrate….. Moist areas Aerobic diptheroids…moist areas staphylococcus …hair follicles Inhibit pathogenic bacteria P.acnes colonize deep sebaceous glands

10. Usual flora of the mouth Contain larg number of bacteria Many bind… buccal mucosa & tooth surface Bacterial plaques..10 11 streptococci Plaque…..low oxidation potential at tooth surface…growth of anarobes

11. Usual flora of the Respiratory tract Upper….lower tracts Upper….mouth,nasopharynx,oropharynx,larynx Lower….tracea,bronchi,lungs…protected by cilia Usual flora of the mouth….streptocococci Upper….mouth,nasopharynx,oropharynx… microbs show some diff Staphylococcus….30% of normal individuals colonize anterior nares

12. Individuals ….hospitalized…upper r.t…..by gram negative bacteria. Orophyarynx….streptococci A number of species…...s.mutans, s.milleri, s.sanguis, Hospitalizwd patients…..gram negative rods

13. Usual flora of the gastrointestinal tract Comprisis….esophagus,stomach,s.intestine,col on Equiped…defenses & antimicrobial fectors Ingesting organisms Don’t multiply ….esophagus, stomach In ingested food…as transient flora. Most mirobs susceptible...acid ph…destroy…. Exception sper forming becteria,cysts

14. Stomach acidty….reduce Smal intestine….few microbs In the colon….10 8 -10 11 Anarobs…90% large intestine Gram positive cocci, yeasts, pseudomonas Population….antibiotics In some cases …microbs supressed….othr able to proliferate…enterocolititis

15. Role of the microbial flora in the pathogenesis of infectious disease Microbial flora….provide some benefits… symbiotic relationship Some opportunists….when damage..disturbed or change…or immune system Trauma….accidentalor surgical….not part of microbial flora…

16. Host immune response….reduces…due to ….drugs… Lymphoma, leukemia….reduce immune response Microbial flora…initiat an infection….in patients with chronic illnesses

17. Role of the microbial flora in the host defense against infectious disease Beneficial effects Immunological competence Immune system developed or undeveloped Newborn….not developed

18. Micobial flora…block colonization by extragenious pathogens Indigenous flora altered gastroenteritis Pathogenic species C. albcans

19. Microbial flora… Important role in health and diseases Eradication….negative effects Knowledge in…clinical samples

20. Sterile Techniques

21. STERILISATION: any process that effectively kills or eliminates transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) from a surface, equipment, article of food or medication, or biological culture medium. Sterilization can be achieved through application of heat, chemicals, irradiation, high pressure or filtration.fungi bacteriavirusesheatchemicalsirradiation high pressurefiltration

22. STERILISATION Types PHYSICAL METHODS A. Heat : 1. Dry heat 2 Moist heat B. Radiations 1. Ultraviolet radiations 2. Ionizing radiations C. Filtration II. CHEMICAL METHODS

23. Heat sterilization

24. Dry Heat sterilization 1.Dry heat, as the name indicates, utilizes hot air that is either free from water vapour, or has very little of it, and where this moisture plays a minimal or no role in the process of sterilization 1. Hot air ovens : Hot air ovens are electrical devices used in sterilization. The oven uses dry heat to sterilize articles. Generally, they can be operated from 50 to 300 °C. There is a thermostat controlling the temperature. These are digitally controlled to maintain the temperature. Their double walled insulation keeps the heat in and conserves energy, the inner layer being a poor conductor and outer layer being metallic. There is also an air filled space in between to aid insulation. An air circulating fan helps in uniform distribution of the heat. These are fitted with the adjustable wire mesh plated trays or aluminium trays and may have an on/off rocker switch, as well as indicators and controls for temperature and holding time. The capacities of these ovens vary.electricalsterilizationdry heatthermostattemperatureenergyinsulationaluminium

25. Mechanism of killing by dry heat 1.Dry heat kills the organisms by destructive oxidation of essential cell constituents 2.Killing of the most resistant spores by dry heat requires a temperature of about 160 °C for 60 minutes 3.Dry heat is employed for glassware; syringes, metal instruments and paper wrapped goods, which are not spoiled by high temperatures. 4.It is also used for anhydrous fats, oils and powders that are impermeable to moisture.

26. METHODS OF STERILIZATION BY DRY HEAT RED HEAT FLAMING HOT AIR OVEN INFRARED RADIATIONS

27. 1.RED HEAT Inoculating wires, points of forceps and searing spatulas are sterilized by holding them in the flame of Bunsen burner until they are seen to be red-hot.

28. 2. FLAMING This method is used for sterilizing scalpel, mouth of culture tubes, glass slides etc. It involves passing of an article through Bunsen flame without allowing it to become red-hot.

29. 3. HOT AIR OVEN This is the main means of sterilization by dry heat. Exposure at a temperature of 160 °C for 1 hour is generally employed.

30. 4. INFRARED RADIATIONS Source employed is an electrically heated element, the infra red rays are directed on to the object to be sterilized and temperature of 180 °C can be obtained.

31. Steam sterilization A widely-used method for heat sterilization is the autoclave, sometimes called a converter. Autoclaves commonly use steam heated to 121 °C or 134 °C. To achieve sterility, a holding time of at least 15 minutes at 121 °C or 3 minutes at 134 °C is required. Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial spores, which can be quite resistant. It will not necessarily eliminate all prions. For prion elimination, various recommendations state 121–132 °C (270 °F) for 60 minutes or 134 °C (273 °F) for at least 18 minutes. The prion that causes the disease scrapie (strain 263K) is inactivated relatively quickly by such sterilization proceduresautoclavefungisporesprionsscrapie

32. Moist Heat sterilization

33. METHODS OF STERILIZATION BY MOIST HEAT Moist heat can be employed at 1. Temperature below 100 °C 2. Temperature of 100 °C 3. Temperature above 100 °C

34. MOIST HEAT BELOW 100 °C EXAMPLE: Pasteurization of milk In Pasteurization of milk the temperature employed is either 63 °C for 30 minutes or 72 °C for 20 seconds. All nonspore forming pathogens in milk like Salmonellae, M.tuberculosis are killed.

35. MOIST HEAT ABOVE 100°C Sterilization in an autoclave 1.Autoclaving is the most reliable method. It is the method most widely used fo sterilization of culture media and surgical supplies 2.When water is boiled within a closed vessel at an increased pressure, the temperature at which it boils and the steam it forms will rise above 100 °C 3.This principle is used in the autoclave 4.Normally autoclaving is done at 15 lbs. (pounds per sq. inch pressure) and 115 °C for 15 minutes

36. Mechanism of killing by moist heat 1.Moist heat kills the organisms by coagulating and denaturing their enzymes and structural protein. 2.Sterilization by moist heat of the most resistant spores generally requires 121 °C for 15-30 minutes. 3.Moist heat is used for the sterilization of culture media, and all other materials through which steam can penetrate 4.Moist heat is more effective than dry heat 5.Sterilization can be done at lower temperatures in a given time at a shorter duration at the same temperature.

37. FACTORS INFLUENZING STERILIZATION BY HEAT 1.The temperature and time: they are inversely related, shorter time is sufficient at high temperatures.Number of microorganisms and spores: 2.The number of survivors diminished exponentially with the duration of heating 3.Depends on the species, strains and spore forming ability of the microbes. 4.Thermal death point is the lowest temperature to give complete killing in aqueous suspension within 10 minutes 5.Depends on the nature of material: a high content of organic substances generally tends to protect spores and vegetative organisms against heat. 6.Presence of organic or inorganic disinfectants facilitates killing by heat 7.pH also plays an important role in the killing of microorganisms

38. MERITS AND DEMERITS OF HEAT STERILIZATION Advantages of heat sterilization 1. Sterilization is very effective 2. Instruments are standardized to deliver the required effective heat 3. Heat deliver system can be monitored effectively with various controls like pressure gauge, temperature meters etc 4. Established quality control methods available

39. Disadvantages of heat sterilization 1. Steam impermeable materials like fats, oils and powders can not be sterilized by autoclaving. 2. Heat sensitive materials can not be sterilized by heat Examples: 1. Serum can not be sterilized 2. Antibiotics 3. Plastic materials 4. Vaccines 5. Rubbers 3. Presence of organic matters interfere with effective sterilization 4. Dangers of explosion when high pressure is used

40. STERILIZATION BY FILTRATION

41. 1.When fluids are passed through bacteria stopping filters, they are made free from bacteria. 2.It is useful for making preparations of soluble products of bacterial growth such as toxins 3.Liquids that would be damaged by heat such as serum and antibiotic solutions can be sterilized by filtration 4.Efficient filters should be able to retain Serratia marcescens

42. Chemical sterilization

43. sterilization includes both liquid and gas chemicals Chemical Sterilizing With Alcoholal In many experimental procedures, the most effective way to sterilize objects is with ethanol. Either 95% or 70% will work. The latter is actually more effective, but the former is often more convenient.

44. Vapors and Gases formaldehyde, beta-propiolactone and ethylene oxide are not routinely used in laboratory sterilization practices. These sterilants are used in hospitals and commercial facilities where closed systems controlling temperature, humidity, and concentration are required to achieve sterilization using these agents.