Microbial Control Methods: Understanding Sterilization and Disinfection

1. Control of Microorganisms by Physical and Chemical Agents

2. What is the meaning of sterilization? • It is the complete destruction or removal of all living organism including viruses, bacteria, fungi and their spores. • Cleaning : is a process intended to remove foreign material, like dust, dirt, organic materials including microorganism. • Disinfection is the destruction of infectious organism( not including bacterial spores) by using chemical ( disinfectants and antiseptics. It results in reduction of the contaminating organism. • -Disinfectant are toxic and irritant material and are used for control of microorganism ( on the floors for example) • - Antiseptic are less irritant and less toxic and used for disinfection of body surfaces ( living tissues). • Preservation: is the prevention of multiplication of microorganism in the formulated products including pharmaceuticals and foods.

3. Figure 8.1-Microbial Control Methods

4. Definition of Frequently Used Terms in Microbial control methods • Sterilization • destruction or removal of all viable organisms from an object or habitat. • Disinfection • killing, inhibition, or removal of pathogenic organisms that may cause disease:substantial reduction of total population. • Disinfectants • agents, usually chemical, used for disinfection; not necessary kills viable spores • Sanitization • reduction of microbial population to levels deemed safe (based on public health standards) • Antisepsis • prevention of infection or sepsis. of living tissue by microorganisms using antiseptics. • antiseptics • chemical agents that kill or inhibit growth of microorganisms when applied to tissue-should not be toxic as disinfectants to kill host tissues.

5. Antimicrobial Agents • agents that kill microorganisms or inhibit their growth e;g Chemotherapy- chemical agent to kill or inhibit growth of microorganisms within host tissues. • -cidal agents to kill • -static agents to inhibit growth • cide • suffix indicating that agent kills • germicide • kills pathogens and many nonpathogens but not necessarily endospores • include bactericides, fungicides, algicides, and viricides • -static • suffix indicating that agent inhibits growth • include bacteriostatic and fungistatic

6. The Pattern of Microbial Death A microbial population is not killed instantly population death usually occurs exponentially as growth rate. microorganisms were previously considered to be dead when they did not reproduce in conditions that normally supported their reproduction however we now know that organisms can be in a viable but nonculturable (VBNC) condition . once they recover they may regain the ability to reproduce and cause infection. Decimal Reduction Time (D): D is the time required to kill 90% of the microorganisms or spores in a sample under specified conditions. Microorganisms is defined as dead when they don’t grow or reproduce when inoculated in culture medium.

7. Conditions Influencing the Effectiveness of Antimicrobial Agent Activity Population size: • larger populations take longer to kill than smaller populations Population composition: • microorganisms differ markedly in their sensitivity to antimicrobial agents. Bacterial spores are much more resistant to Microbial agents e.g Mycobacterium tuberculosis. Concentration or intensity of an antimicrobial agent: • usually higher concentrations or intensities kill more rapidly • relationship is not linear Duration of exposure: longer exposure to antimicrobial agents more organisms killed. Temperature: • higher temperatures usually increase amount of killing Local environment: • many factors (e.g., pH, viscosity and concentration of organic matter) can profoundly impact effectiveness • organisms in biofilms are physiologically altered and less susceptible to many antimicrobial agents. Organic matter in biofilms protects biofilm microorganisms.

8. The Use of Physical Methods in Control Heat Low temperatures Filtration Radiation

9. Moist Heat Sterilization must be carried out above 100oC which requires saturated steam under pressure Moist heat destroys viruses, bacteria and fungi carried out using an autoclave (Fig8.3) also known as Steam Sterilizer effective against all types of microorganisms including spores degrades nucleic acids, denatures proteins, and disrupts membranes

10. The Autoclave or Steam Sterilizer Figure 8.3-Autoclave Steam Sterilizer

11. Table 8.2

12. MeasuringHeat-Killing Efficiency Thermal death time (TDT) • shortest time needed to kill all microorganisms in a suspension at a specific temperature and under defined conditions Decimal reduction time (D or D value) • time required to kill 90% of microorganisms or spores in a sample at a specific temperature .

13. Pasteurization • controlled heating at temperatures well below boiling- Louis Pasteur • used for milk, beer and other beverages • process does not sterilize but does kill pathogens present and slow spoilage by reducing the total load of organisms present. Dry Heat Sterilization • less effective than moist heat sterilization, requiring higher temperatures and longer exposure times • items subjected to 160-170oC for 2 to 3 hours • oxidizes cell constituents and denatures proteins. Filtration • reduces microbial population or sterilizes solutions of heat-sensitive materials by removing microorganisms • also used to reduce microbial populations in air

14. Filtering Liquids Depth filters • thick fibrous or granular materials bonded in thick layers that remove microorganisms by physical screening(size), entrapment, and/or adsorption to the surface of the filter materials. • Solution contain microorganisms are sucked under vacuum • membrane filters • porous membranes with defined pore sizes that remove microorganisms primarily by physical screening. This has replaced Depth Filters.

15. Figure 8.5-Membrane Filter Sterilisation

16. Figure 8.6-Membrane Filter

17. Filtering Air • surgical masks used in hospitals and Labs • cotton plugs on culture vessels • high-efficiency particulate air (HEPA) filters used in laminar flow biological safety cabinets (remove 99.97% of particles) Figure 8.7 (a)-Laminar Floor

18. Ultraviolet (UV) Radiation • UV (260nm) quite lethal is limited to surface sterilization because it does not penetrate glass, dirt films, water, and other substances. • UV prevent replication and transcription of Microbial DNA. • has been used for water treatment Figure 7.9

19. Ionizing Radiation • Excellent sterilization agent e.g Gamma radiation • penetrates deep into objects • destroys bacterial endospores; not always effective against viruses • used for sterilization and pasteurization of antibiotics, hormones, sutures, plastic disposable supplies, and food

20. Figure 8.8-Sterilization with Ionization; Radiation machine which uses Cobalt 60 as a Gamma radiation to sterilize fruits, veg, fish, meat, etc..

21. Chemical Control Agents -Disinfectants and Antiseptics Phenolics commonly used as laboratory and hospital disinfectants 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 Alcohols bactericidal, fungicidal, but not sporicidal inactivate some viruses denature proteins and possibly dissolve membrane lipids Halogens any of five elements: fluorine, chlorine, bromine, iodine, and astatine iodine and chlorine are important antimicrobial agents

22. 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 • iodophore • iodine complexed with organic carrier 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

23. Heavy Metals • e.g., ions of mercury, silver, arsenic, zinc, and copper • effective but usually toxic • combine with and inactivate proteins; may also precipitate proteins Ammonium Compounds • These are detergents that have antimicrobial activity and are effective disinfectants • organic molecules (cleansing agents) with hydrophilic and hydrophobic ends for food utensils, small instruments and skin antiseptics. • Because of its positively charged nitrogen, cationic detergents/ammonium compound, are effective disinfectants. • They disrupt microbial membrane , may denature protein. • kill most bacteria, but not Mycobacterium tuberculosis or endospores • safe and easy to use, but inactivated by hard water and soap • E.gBenzalkonium chloride and Cetylpyridinium Aldehydes • highly reactive molecules • sporicidal and can be used as chemical sterilants • combine with and inactivate nucleic acids and proteins • E.gFormal dehydeandglutaraldehyde

24. Sterilizing Gases used to sterilize heat-sensitive materials such as disposable petri dishes, syringes, heart lung machine components, sutures, catheters microbicidal and sporicidal combine with and inactivate proteins E.g Ethylene oxide gas (EtO)

25. Chemical in common use are : • 1-Alcolhol: kill vegetative bacteria only e.g. 75% ( absolute alcohol is not effective becuase water is important for the denaturation of bacterial protein). It is used for skin disinfection. • 2- Phenol 2% used as a disinfectant in microbial laboratories. • 3- ethylene oxide gas ; used for the disinfection f plastic and rubber articles. • 4-halogenes: kill vegetative bacteria, spores and viruses. • Iodine: used in alcohol for skin antisepsis ( e.g. betadine). • 5- Glutaraldehyde 2 % used for the decontamination of endoscopes. • 6-hydrogen peroxide used in wound cleaning. • 7-formaldehyde gas used as a disinfectant for fumigation of areas contaminated by infection agents, for rubber, leather, shoes, books. • 8- Quaternary ammonium salts; cetavlon ( soap) • 9- Heavy metals ( e.g. mercury and silver) they cause denaturation of enzymes and other essential proteins.`

26. Figure 8.11-Ethylene Oxide Sterilizer

27. Chemotherapeutic Agents chemicals that can be used internally to kill or inhibit the growth of microbes within host cells (covered later in book) their selective toxicity allows them to target the microbe without harming the host most are antibiotics, chemicals synthesized by microbes that are effective in controlling the growth of bacteria

28. CHEMOTHERAPEUTIC AGENTS • Definitions: • Antimicrobial agents are substances that inhibit or kill the growth of microorganisms. Antimicrobial agents that are produced by microorganisms are naturally occurring chemotherapeutics and were defined by Waxman as antibiotics. • Examples of naturally occurring chemotherapeutic agents are : penicillins and cephalosporins, chloroamphinicol, tetracyclines, macrolids, nystatin and griseofulvin. • Examples of synthetics chemotherapeutic agents are: sulphonamides, nitrofuran, isonictinc acid deriivatives, diaminopyrimidines, quinolones and imidazoles. • The chemical substance that used as a chemotherapeutic agent must be a selective toxic. By means toxic for the microorganism and not toxic to the host. • Antibiotics: An antibiotic is a product produced by a microorganism or a similar substance produced wholly or partially by chemical synthesis, which in low concentrations, inhibits the growth of other microorganisms. • The minimum inhibitory concentration (MIC): is the lowest concentration of an antibiotic that inhibit the growth of an organism. • Bactericidal antibiotics: are those antibiotics that destroy bacteria, like penicillins, cephalosporins. • Bacteriostatic antibiotics: are those inhibit the multiplication of the bacterial cells. Like tetracyclines and chloroamphenicol

30. Susceptibility Tests 1. Broth dilution - MIC test 2. Agar dilution - MIC test

31. 2 ug/ml 32 ug/ml 4 ug/ml 8 ug/ml 16 ug/ml Sub-culture to agar medium MIC = 8 ug/ml MBC = 16 ug/ml Minimal Inhibitory Concentration (MIC) vs. Minimal Bactericidal Concentration (MBC) 1 ug/ml

32. Susceptibility Tests • Agar diffusion

34. Antibiotic Mechanisms of Action Alteration of Cell Membrane Polymyxins Bacitracin Neomycin Transcription Translation Translation

35. THANKS