1. Sterilization &Disinfection By Prof. Dr. Zainalabideen A Abdulla, DTM&H., MRCPI, Ph.D., FRCPath. (U.K.)

2. LEARNING OBJECTIVES 1. Differentiate between sterilization, disinfection, and sanitization 2. Differentiate between bactericidal and bacteriostatic agents 3. Explain the process of pasteurization and lyophilization 4. List several methods used to inhibit the growth of microorganisms 5. Identify several factors that can influence the effectiveness of disinfectants

3. Sterilization Destruction or elimination of all microbes including cells, spores, and viruses - Sterile = Devoid of microbial life - Physical or chemical methods in healthcare Dry heat Autoclaving (steam under pressure) Ethylene oxide gas Formaldehyde Radiation (UV, gamma rays)

4. Disinfection The elimination of most or all pathogens (except bacterial spores) from nonliving objects; using: 1. Liquid chemicals (disinfectants) 2. Wet pasteurization Antiseptics: Disinfectants used on living tissues as the skin Sanitization: Reduction of the microbial population to safe levels, e.g. in resturants

5. Microbicidal agents - Suffix: cide or cidal = “killing” Examples: Bactericidal = Kill bacteria Sporicidal = Kill bacterial spores Viricidal = Kill viruses Fungicidal = Kill fungi Pseudomonicidal= Kill Pseudomonas Tuberculocidal = Kill M. tuberculosis

6. Pasteurization - A method to disinfect liquids (e.g. milk) - Is not sterilization - Not all microbes are killed - 63 Celsius 30 minutes 72 Celsius 15 seconds 140 Celsius 4 seconds

7. Microbistatic agents - Drugs or chemicals that inhibit reproduction and metabolism of microorganisms (NOT necessarily kill them) - Freeze drying & rapid freezing: Bacteriostatic - Freeze drying = Lyophilization; preserve m.o.

8. Sepsis: Presence of pathogens in blood or tissues Asepsis: Absence of pathogens - Medical or Surgical aseptic techniques, e.g., hand hygiene, sterile gloves, masks, and gowns Antisepsis: Prevention of infection Antiseptic technique: Use of antiseptics

9. Physical Methods Heat - The most common type of sterilization for inanimate objects able to with stand high temperatures - Effect depends on: 1. Temperature 2. Time - Pathogenic > susceptible than nonpathogenic

10. Thermal Death Point (TDP) Lowest temp. that kill all organism in a standardized pure culture within specified period Thermal Death Time (TDT) The length of time to sterilize a pure culture at a specified temperature

11. Dry heat - 160 – 165 Celsius 2 hours or - 170 – 180 Celsius 1 hour - In oven - Items: Metals, glassware, oils, waxes Incineration: Burning; contaminated disposable materials; must NOT overloaded with protein and moist materials as feces, vomitus or pus Flaming: Bunsen burner flame or electrical heat device for wires, loops or forceps

13. Moist heat 1. Boiling for 30 minutes, e.g. metal (needles), glass. Not effective against spores, viruses and Mycobacterium. 2. Autoclave (Moist heat + Pressure): - 15 psi + 121.5 Celsius for 20 minutes - Kills spores, viruses, and vegetative form - Rubber can be autoclaved - Indicators for completion: Autoclave tape or Solutions of bacterial spores - Keep cans open, bottles covered loosely

17. Cold - Refrigeration: Slows metabolism & growth rate - Slow freezing: Form crystals & rupture cells - Rapid freezing (liquid nitrogen): Keep/Preserve Freeze- Thawing of food: Unsafe (spores vegetative forms

18. Desiccation - Dried clinical specimens and dust may contain viable microorganisms. Radiation - UV: Sterilize vaccines, antisera, toxins May cause skin cancer, eye damage - X-ray, gamma and beta ray: Gamma ray sterilizes food as meat from Salmonella & Campylobacter

20. Ultrasonic Waves - For cleaning delicate equipment - Tank of water + short US waves then sterilize - In dental or medical clinics, e.g. glassware

21. Filtration - Different pore sizes; separate organisms (bacteria, viruses, others) from liquid or gases - Micro-pore filters - High-Efficiency Particulate Air (HEPA) used in operating rooms, patient room to filter air

22. Gaseous Atmosphere - Aerobs/ Microaerophiles: Remove O2 - Anaerobic: Provide O2 Hyperbaric Oxygen Chamber (increased pressure) to kill m.o.

23. Disinfectants (Chemical Agents) - Temporal or permanent - Factors affecting disinfectants: Prior cleaning/proteinaceous materials Organic matter load Bio-burden (microbial) Concentration Time Physical nature Temperature/ pH

25. cont./… Disinfectants - Susceptible: Vegetative state, fungi, protozoa, most viruses - Resistant: Mycobacteria, bacterial endospore, Pseudomonas spp., fungal spores, hepatitis viruses - Degree of resistance (see Table)

27. cont./… Disinfectants - Never use disinfectant if physical sterilization is possible - Do not destroy all bacteria & bacterial spores - Spore/ Mycobacteria/Viruses destruction: Formaldehyde / fumigation Ethylene oxide

28. Disinfectant characteristics Broad Fast-acting Not affected by organic materials Non-Toxic; Evaporation-Concentration Leave residual anti-microbial film Soluble in water Inexpensive Stable Odorless

29. Antiseptics - Chemicals used safely on human tissues such as skin - Reduce numbers of organisms on surfaces - Used at surgical incisions, at pores of folds of the skin - Antiseptic soup + Brushing

30. Factors affecting microbial growth 1. Availability of nutrients: 92 natural elements 2. Moisture: Cells composed of 70-95% water 3. Temperature: Optimum Minimum Maximum cont./…

31. cont./… temperature/ factors - Thermophiles (love heat) e.g. Cyanobacteria, Algae - Mesophiles (moderate temperature) e.g. most bacteria (37 Celsius) - Psychrophiles (love cold) e.g. Psychrotrophs (4 C; bread molds) Psychroduric (warm to very cold)

33. 4. pH - Neutral – slightly alkaline (7.0-7.4); most - Acidophiles (2.0-5.0); human stomach m.o. - Alkaliphiles (> 8.0); Vibrio cholerae

34. 5. Osmotic pressure - Solutes: Dissolved substances - Hypertonic: Higher concentration than in a cell - Osmosis: Movement of solvent (e.g. water) Crenation (shrink): RBC loss water Plasmolysis: Bacterial cell membrane and cytoplasm shrink from cell wall Opposite: Swell (RBC) Plasmoptysis (Bacteria)

37. Halophilic : Salt-loving organisms e.g. Vibrio parahemolyticus Haloduric : Do not prefer salty environment but can tolerate it e.g. Staphylococcus aureus

38. 6. Barometric pressure - 14.7 pounds per square inch (psi)- Normal e.g. most bacteria - Piezophiles: Tolerate very high pressure e.g. Archaea 7. Gaseous atmosphere - Discussed previously

39. Bacterial growth - Increase in number of organisms (not size) - Proliferation/multiplication - Binary fission (two identical daughter cells) - Form colonies (piles) Generation time - Rapid and Slow growers: 20 min. E. coli, Staph., Strept., V. cholera 10 min. Pseudomonas, Clostridium 18-24 hour: Mycobacterium

41. Culture media - Artificial media/ synthetic media - Chemically defined: Known ingredients - Complex medium: Exact contents unknown - Solid + AGAR (polysaccharides from red marine alga) - liquid (broth)

42. Enriched medium Extra rich supply (nutrients) added: Nutrient agar; PLUS: Blood agar: 5% sheep RBC Chocolate agar (lysed RBC- Hb available) - For Fastidious microorganism (complex nutritional requirements) e.g. Neisseria gonorrheae, Haemophilus influenzae

43. Selective medium Inhibitors added to discourage unwanted microbes - MacConkey’s : Selective for gram - Inhibit gram + - Mannitol salt agar: For haloduric

44. Differential medium Differentiate between microorganisms: MacConkey’s agar: Lactose sugar - Lactose fermenter (LF) - Non-Lactose fermenter (NLF) MSA Blood agar: Hemolysis (alpha, beta, gamma) Thioglycollate broth (THIO): O2 gradient

49. Inoculation of culture medium - Streaking - Aseptic technique- Prevent contamination: Professionals Environment Specimens/ cultures/subcultures

51. Incubation - Incubators: CO2 (5% to 10%), Capnophiles Non-CO2 (20% to 21% O2) Anaerobic (Devoid of O2) - Pure culture: One bacterial species - 35 to 37 C

52. Bacterial population count - Contamination of fluid/food - Total number: Spectrophotometry- turbidity - Viable count:. serial dilutions. Aliquots & subcultures No. of colonies X Dilution = Count per ONE ml Q. If 0.1 ml inoculated?- X 10 - UTI (viable count important)

53. Bacterial population growth curve - Logarithm of numbers of bacteria (viable) vs Time - Four phases Lag phase Logarithmic (exponential) phase Stationary phase Death (decline) phase

55. Lag phase - Prepare for cell division - Do not increase in numbers Logarithmic (Log) growth phase - Multiply rapidly; growth rate is the greatest - Usually short

56. Stationary phase - Division slows (nutrients, wastes ) - Numbers of dividing = dying bacteria - Density of culture: Greatest Death (Decline) phase - Death, or spores formed - Change morphology, e.g. filamentous form protoplast/ spheroblast (L-form- no cell wall)

58. Virus cultures - Embryonated chicken eggs, Lab animals, cell culture (monkey, rabbit, human, etc) - Cytopathic effect (CPE); examination Fungal culture - Brain-Heart infusion - Sabouraud Dextrose Agar (SDA) - Anti-microbial agents added, pH 5.6 Protozoa culture: Example Amoeba