Microbial control

1. Microbial control Dr. Bhavesh Patel (M.Sc., Ph.D.) Principal V.P. and R.P.T.P. Science College Vallabh Vidyanagar, Gujarat (bhavesh1968@rediffmail.com)

2. Microbial control What is control ? • Restricting the growth and normal activities of microorganisms.

3. Microbial control Why control ? • To prevent transmission of diseases. • To prevent contamination at various levels. • To prevent spoilage of materials.

4. Microbial control How control ? • By killing the microorganisms (cidal) • By inhibiting the microorganisms (static) • By removing the microorganisms

5. Microbial control Mechanism of antimicrobial action • Affecting the cell wall • Affecting the cell membrane • Interfering with protein structure and function • Affecting nucleic acids

6. Microbial control Factors affecting antimicrobial activities • Type and state of microorganisms • Initial microbial load • Concentration or dose of agent • Time of exposure • Environmental conditions

7. Microbial control - Practical Concern • Does the application require sterilization ? • Is the item to be reused ? • Can the item withstand heat, pressure, radiation or chemical ? • Will the agent penetrate to the necessary extent ? • Is the method cost and labor efficient and is it safe ?

8. Microbial control - Some useful terms • Sterilization :killing or removal of all life form i.e. free from life. • Disinfection :surface removal of organisms. • Sanitation :reduction in number of organism. • Antiseptic :agent that prevent sepsis. • Pasteurization :killing of organism without affecting quality of material.

9. Kinetics of heat sterilization • Death from heating is an exponential (first order) function and occurs more rapidly as the temp raised. • Thus if we practically wants to sterilize a microbial population , it will take longer at low temp than at higher temp. • It is thus required to adjust time and temp to achieve sterilization for each specific set of conditions. • The nature of heat is also important – moist heat is more penetrating than dry heat.

10. Kinetics of heat sterilization

11. Kinetics of heat sterilization Death rate-Decimal reduction time (D-value) • It is the time in minute to kill 90% of the cells in a population. OR • It is the time to reduce the population by one decimal Death rate- Thermal Death Point (TDP) • Lowest temperature required to kill all the microorganisms in the liquid suspension in 10 min. Death rate- Thermal Death Time (TDT) • Minimum time required to kill all microorganisms in liquid suspension at given temperature

12. Physical control • Heat (low temp, high temp) • Desiccation • Osmotic pressure • Radiation(ionizing & non ionizing) • Filtration(porcelain, barkfeld, sintered glass)

13. Moist heat Boiling (100oC) Tyndalization (100oC,3days) Autoclave (15 lbs/sq.in.,121oC-20min.) Pasteurization(LTHT-63oC,30min.,HTST-72oC,15sec.) Dry heat Incineration(>500oC) Hot air oven (160oC,2hrs. or 170oC,1 h.) Flaming (>500oC) High temperature

14. Moist vs Dry heat Temp(oC) Time(min) Moist heat 121 15 125 10 134 3 Dry heat 121 600 140 180 160 120 170 60

15. Autoclave

16. Low temp.(Static) Freezing (below 4oC) Chilling (4-6oC) High temp.(Cidal) Moist heat (>100oC) Dry heat (>100oC) Heat

17. Control by drying - vacuum, heat etc. Susceptibility to dessication varies widely: Neisseria gonnorrhea: Only survives about one hour. Mycobacterium tuberculosis: May survive several months. Viruses are fairly resistant to dessication. Clostridium spp. and Bacillus spp.: May survive decades. Desiccation

18. Depth filters-particulate material (porcelain, glass, earth, asbestos) Membrane filters (cellulose acetate, polycarbonate, etc.) different pore sizes; 0.2 or 0.4 micrometer remove bacteria HEPA (high-efficiency particulate air) filters used for hospitals, laminar flow hoods in laboratories, being marketed or home use to reduce allergens Filtration

19. Laminar air flow Membrane filtration Filtration

20. Osmotic pressure • High sugar and salt conc. Can be used to restrict the growth (Bacteriostatic). • Many microbes can tolerate sugar conc. Up to 10% (saccharophilic or saccharolytic). • Some can tolerate salt conc. Up to 30% (halophiles).

21. Radiation

22. Radiation Radiation: Three types of radiation kill microbes: 1. Ionizing Radiation: Gamma rays, X rays, electron beams, or higher energy rays. Have short wavelengths (less than 1 nanometer). Dislodge electrons from atoms and form ions. Cause mutations in DNA and produce peroxides. Used to sterilize pharmaceuticals and disposable medical supplies. Food industry is interested in using ionizing radiation. Disadvantages: Penetrates human tissues. May cause genetic mutations in humans.

23. Radiation 2. Ultraviolet light (Non ionizing Radiation): Wavelength is longer than 1 nanometer. Damages DNA by producing thymine dimers, which cause mutations. Used to disinfect operating rooms, nurseries, cafeterias. Disadvantages: Damages skin, eyes. Doesn’t penetrate paper, glass, and cloth. 3.Microwave Radiation: Wavelength ranges from 1 mm to 1m Heat is absorbed by water molecules. May kill vegetative cells in moist foods. Bacterial endospores, which do not contain water, are not damaged by microwave radiation.

24. Radiation

25. Chemical Agents • Phenolics • Alcohols • Halogens • Heavy metals • Quaternary Ammonium Compounds • Aldehydes • Sterilizing Gases

26. Chemical Agents: Phenolics • Aromatic organic compounds with attached -OH • Denature protein & disrupt membranes • Phenol, orthocresol, orthophenylphenol, hexachlorophene • Commonly used as disinfectants (e.g. “Lysol”); are tuberculocidal, effective in presence of organic matter, remain on surfaces long after application • Disagreeable odor & skin irritation; hexachlorophene once used as an antiseptic but its use is limited as it causes brain damage

27. Chemical Agents: Alcohols • Ethanol; isopropanol; used at concentrations between 70 – 95% • Denature proteins; disrupt membranes • Kills vegetative cells of bacteria & fungi but not spores • Used in disinfecting surfaces; thermometers; “ethanol-flaming” technique used to sterilize glass plate spreaders or dissecting instruments at the lab bench

28. Chemical Agents: Halogens • Act as oxidizing agents; oxidize proteins & other cellular components • Chlorine compounds • Used in disinfecting municipal water supplies (as sodium hypochlorite, calcium hypochlorite, or chlorine gas) • Sodium Hypochlorite (Chlorine Bleach) used at 10 - 20% dilution as bench top disinfectant • Halazone tablets (parasulfone dichloro amido benzoic acid) used by campers to disinfect water for drinking

29. Chemical Agents: Halogens • Iodine Compounds • Tincture of iodine (iodine solution in alcohol) • Potassium iodide in aqueous solution • Iodophors: Iodine complexed to an organic carrier; e.g. Wescodyne, Betadyne • Used as antiseptics for cleansing skin surfaces and wounds

30. Chemical Agents: Heavy Metals • Mercury, silver, zinc, arsenic, copper ions • Form precipitates with cell proteins • At one time were frequently used medically as antiseptics but much of their use has been replaced by less toxic alternatives • Examples: 1% silver nitrate was used as opthalmic drops in newborn infants to prevent gonorrhea; has been replaced by erythromycin or other antibiotics; copper sulfate used as algicide in swimming pools

31. Chemical Agents: QuaternaryAmmonium Compounds • Quaternary ammonium compounds are cationic detergents • Amphipathic molecules that act as emulsifying agents • Denature proteins and disrupt membranes • Used as disinfectants and skin antiseptics • Examples: cetylpyridinium chloride, benzalkonium chloride

32. Chemical Agents: Aldehydes • Formaldehyde and gluteraldehyde • React chemically with nucleic acid and protein, inactivating them • Aqueous solutions can be used as disinfectants

33. Chemical Agents:Sterilizing Gases • Ethylene oxide (EtO) • Used to sterilize heat-sensitive equipment and plastic ware • Explosive; supplied as a 10 – 20% mixture with either CO2 or dichlorofluoromethane • Its use requires a special EtO sterilizer to carefully control sterilization conditions as well as extensive ventilation after sterilation because of toxicity of EtO • Much of the commercial use of EtO (for example, plastic petri dishes) has in recent years been replaced by gamma irradiation

34. Chemical Agents:Sterilizing Gases • Betapropiolactone (BPL) • In its liquid form has been used to sterilize vaccines and sera • Decomposes after several hours and is not as difficult to eliminate as EtO, but it doesn’t penetrate as well as EtO and may also be carcinogenic • Has not been used as extensively as EtO • Vapor-phase hydrogen peroxide • Has been used recently to decontaminate biological safety cabinets