Bio3124 Lecture #6

1. Bio3124Lecture #6 Control of Microorganisms

2. Definitions • sterilization destruction /removal of all viable organisms • disinfection killing, inhibition, removal of pathogens disinfectants usually chemical used on inanimate objects • sanitization reduction of microbial population to levels deemed safe • antisepsis prevention of infection of living tissue by microorganisms • antiseptics chemical agents, kill or inhibit growth of microorganisms when applied to tissues • Chemotherapy: internal use chemicals to kill or inhibit microbes within host tissues

3. Definitions… • Antimicrobials: • -cidal agents: agent kills, commonly called germicides • kills pathogens and many nonpathogens but not necessarily endospores • include bactericides, fungicides, algicides, and virucides • -static agents: agent inhibits growth • include bacteriostatic and fungistatic

4. Microbial Death microorganisms are not killed instantly death curves are exponential Plot: log of survivors vs antimicrobial exposure time The slope: average death rate Bacterial Death Curve Survivors( log of CFU/ml) Survivorsx109 (CFU/ml) Time

5. Effectiveness of Antimicrobial Agent Activity Depends on: • population size • population composition vegetative vs dormant • concentration • duration of exposure longer exposure  more organisms killed • temperature higher temperatures usually increase killing • local environment e.g., pH, viscosity and concentration of organic matter organisms in biofilms are physiologically altered and less susceptible to many antimicrobial agents

6. Methods in controlling microorganisms Two major methods are used, • Physical methods • Heat • Moist heat sterilization (autoclaves) • Pasteurization • Dry heat sterilization (ovens, incinerators) • Low temperature (refrigeration, freezing) • Filtration (for heat labile liquids) • Irradiation (UV and ionizing radiation) • Chemical methods • Disinfectants and antiseptics (phenolics,alcohols, aldehydes, gases… etc) • Chemotherapeutic agents (internal use)

7. Moist Heat Sterilization • above 100oC , requires saturated steam under pressure (autoclave) • effective against all types of microorganisms and spores • degrades nucleic acids, denatures proteins, and disrupts membranes

8. The Autoclave or Steam Sterilizer • Autoclave • 121°C, 15 psi (2 atm) for 20 minutes • Kills all bacteria • Kills endospores • Clostridium botulinum • Botulism • Bacillus anthracis • Anthrax

9. Pasteurization • Louis Pasteur and Claude Bernard (1862) • does not sterilize • logarithmic reduction of germs rather than killing them all • Most often ~5 log reduction; milk, beer, apple cider, fruit juice and other beverages • Procedures • High temperature short time: holding milk at 72 C for 15-30 seconds • Ultra high temperature: exposure to ~130 C for a fraction of second • Double pasteurization: 68C for 30 minutes followed by cooling and again heating at 68C for additional 30 minutes (spores germinate, killed upon entry to vegetative stage)

10. Dry Heat Sterilization • less effective • Clostridium botulinium spores killed in 2-3 hours • Ovens: higher temperatures & longer exposure time • (160-170oC for 2 to 3 hours) • oxidizes cell constituents and denatures proteins • Bench-top incinerators • inoculating loops • Institutional incineration

11. Measuring Heat-Killing Efficiency • To develop standards for killing efficiency: specially important for industrial settings to develop SOPs • decimal reduction time (D or D value) time required to kill 90% of microorganisms or spores in a sample at a specific temperature • One log reduction

12. 106 Δt DT= log N1-logN2 105 100oC 1 log 104 D100 103 Time Kinetics of thermal reduction D is the time required for one log reduction (90% kill) Can be calculated using: Δt: total exposure time N1: initial population N2: population size after treatment # Bacteria T= applied Temperature

13. Δt 15 15 DT= D85= D85= log N1-logN2 log 109-log106 9- 6 Example 1: • calculate the D value for a bacterial suspension of 109 cfu/ml that was subjected to 85˚C for 15 minutes at which point its density was reduced to 106 cfu/ml. Δt: 15 minutes N1: 109cfu/ml N2: 106cfu/ml T= 85˚ C D85= 5 minutes

14. Δt Δt Δt DT= 2= 2= log N1-logN2 log 108-log100 8- 0 Example 2: • the D90 value for a bacterium is 2 minutes. If starting culture has 108 cfu/ml, how long should this suspension be kept at 90C to kill the entire population? Δt: ? minutes N1: 108cfu/ml N2: 1 cfu/ml T= 90˚ C Δt= 16 minutes

15. 106 105 104 DB.subtilis DE.coli 100oC 103 The D value: an index for sensitivity to thermal killing • Which one is more sensitive to heat killing at 100˚C? • Bacillus subtilis or E.coli? • At 100C the time required to reduce Bacillus population is longer than that required for E.coli # Bacteria Time

16. 106 105 # Bacteria D100 D110 D120 104 120oC 100oC 110oC 103 Time The D value is temperature dependent D value decreases as the temperature increases ie. there is less time required to reduce the population by one log at higher temperatures

17. ΔT Z = log D1-logD2 Kinetics of thermal reduction: the Z value • Z value • increase in temperature required to reduce D by 1/10 (one log reduction) 100 10 D value (min) ΔT: Temperature change D1: initial D value D2: secondary D value 1 log 1 Z =10˚C 100 105 110 120 115 Temperature (T)

18. Kinetics of thermal reduction: the Z value • by having D values for different temperatures • one can seek for altering the sterilization protocol to fit to the industrial setting • One question would be: how much the temperature can increase to reduce the D value to a given length • This would provide a pragmatic approach in setting up SOPs in industrial settings

19. Δt Δt D111= 2 = log1012-log100 12- 0 The use of Z value • Example: • A food processing company produces canned meat. Prevention of Clostridium botulinum spores from growing is important. The D121 for botulinum spores is 0.2 minutes and the Z value is 10˚C. the company wants to sterilize the canned food at 111˚C. what should be the length of sterilization if they consider to kill 1012 spores per can content. since every10˚C decrease in treatment causes 10-fold increase in D value then: D111= D121x10 ie. D111= 0.2x10 = 2 minutes using, Δt= 2x12= 24 minutes They should heat treat their product at 111˚C for 24 minutes.

20. Problem : try this on your own The Z value for a microorganism is 2oC. it takes 54 minutes at 75oC to reduce the population from 109 to 106. At what temperature should the microorganism be treated to achieve the same result in 10.8 sec. Answer=800C

21. Low Temperatures • Freezing • stops microbial reproduction due to lack of liquid water • some microorganisms killed by ice crystal • disruption of cell membranes • Refrigeration • slows microbial growth and reproduction • Does not prevent psychrophilic microorganisms

22. Filtration • Porous material with 0.1-0.45 um pore size • reduces microbial population or sterilizes solutions of heat-sensitive materials by removing microorganisms • also used to reduce microbial populations in air

23. Filtration Bacillus megaterium Trapped on a nylon Membrane with 0.2 um pore size Enterococcus faecalis Trapped on a polycarbonate Membrane with 0.4 um pore size

24. Filtering air • surgical masks • cotton plugs on culture vessels • high-efficiency particulate air(HEPA)filters • used in laminar flow biological safety cabinets

25. Ultraviolet (UV) Radiation • limited to surface sterilization because it does not penetrate glass, dirt films, water, and other substances • has been used for water treatment • Kills by inducing massive number of mutations • How about escaping mutants?

26. Ionizing Radiation • Gamma radiation from cobalt 60 is used • penetrates deep into objects • destroys bacterial endospores; not always effective against viruses • used for sterilization of antibiotics, hormones, sutures, plastic disposable supplies, and food

27. Chemical Control Agents -Disinfectants and Antiseptics

28. Phenolics • commonly used as laboratory and hospital disinfectants (2%) • act by denaturing proteins and disrupting cell membranes • tuberculocidal, effective in presence of organic material, and long lasting • disagreeable odor and can cause skin irritation

29. Alcohols • bactericidal, fungicidal, but not sporicidal • Effective if diluted to 70% in water (95% is much less active) • inactivate some enveloped viruses • denature proteins and possibly dissolve membrane lipids

30. Halogens - Iodine • skin antiseptic • oxidizes cell constituents and iodinates proteins • at high concentrations may kill spores • skin damage, staining, and allergies can be a problem

31. Halogens - Chlorine • oxidizes cell constituents • important in disinfection of water supplies and swimming pools, used in dairy and food industries, effective household disinfectant • destroys vegetative bacteria and fungi, but not spores • can react with organic matter to form carcinogenic compounds

32. Quaternary Ammonium Compounds • detergentsthat have antimicrobial activity and are effective disinfectants • organic molecules with hydrophilic and hydrophobic ends • cationic detergents are effective disinfectants • kill most bacteria, but not Mycobacterium tuberculosis or endospores • safe and easy to use, but inactivated by hard water and soap

33. Aldehydes • highly reactive molecules that cross link proteins • sporicidal and can be used as chemical sterilants • combine with and inactivate nucleic acids and proteins

34. Sterilizing Gases • used to sterilize heat-sensitive materials • EtO penetrates plastic packages • Toxic, needs to be aerated • microbicidal and sporicidal • combine with and inactivate proteins • BPL used for sterilizing vaccines • Decomposes after use, but is carcinogenic

35. Evaluation of antimicrobial efficiency: Phenol coefficient 5 Minutes TEST 10 Minutes 5 Minutes Phenol 10 Minutes

36. Evaluation of antimicrobial efficiency calculation: The reciprocal of the lowest concentration of the test material that prevents the microorganism from growing over 10 minutes exposure but not at 5 minutes relative to that of phenol is considered as phenol coefficient of the test compound. In this example: PC= 320/160 PC= 2