Dr. Rasheeda Hamid Abdalla Assistant Professor

 What is the meaning of sterilization?  Definition of Frequently Used Terms in Microbial control methods  Antimicrobial Agents  Conditions Influencing the Effectiveness of Antimicrobial Agent Activity  The Use of Physical Methods in Control of Microorganisms  Chemical Control Agents- Disinfectants And Antiseptics  Chemotherapeutic Agents

 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.

Figure 8.1-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 :Destruction of most microorganisms ( whether or not pathogenic) on wounds, clothing, or hard surfaces, through the use of chemicals or heat  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.

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,fungistatic,algistatic and virustatic

 A microbial population is not killed instantly  population death usually occurs exponentially as growth rate.  microorganisms considered to be dead when they did not reproduce in conditions that normally supported their reproduction  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-value): Is the time required at a given temperature to kill 90% of the exposed microorganisms  Microorganisms is defined as dead when they don’t grow or reproduce when inoculated in culture medium.

 Population size: larger populations take longer time to be killed than smaller populations  Population composition: microorganisms differ markedly in their sensitivity to antimicrobial agents e.g. Mycobacterium tuberculosis. Bacterial spores are much more resistant to Microbial agents  Concentration or intensity of an antimicrobial agent: usually higher concentrations or intensities kill more rapidly

 Duration of exposure: the longer exposure to antimicrobial agents the 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.

 Heat - Moist Heat Sterilization -Dry Heat Sterilization  Low temperatures  Filtration  Radiation

 Must be carried out above 100 o C 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

Figure 8.3-Autoclave Steam Sterilizer

Table 8.2

 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 temperat ure.

 Dry Heat Sterilization  Less effective than moist heat sterilization, requiring higher temperatures and longer exposure times  Items subjected to o C for 2 to 3 hours  Oxidizes cell constituents and denatures proteins.  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.

 Reduces microbial population or sterilizes solutions of heat- sensitive materials by removing microorganisms  Also used to reduce microbial populations in air

 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.

Figure 8.5-Membrane Filter Sterilisation

Figure 8.6-Membrane Filter

 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

 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. Figure 7.9

 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

 Phenolics  Commonly used as laboratory and hospital disinfectants  Act by denaturing protein and disrupting of cell membrane  Tuberculosidal, effective in presence of organic material, and long lasting disagreeable odor and can cause skin irritation  Alcohol  Bactericidal, Fungicidal, but not sporicidal  Inactive 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

 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  Is a preparation containing iodine complexed with a solubilizing agent  Diluted iodophore is often used to sanitize equipment and bottles

 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

 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  Disrupting plasma membranes, they allow cytoplasmic constituents to leak out of the cell  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.g Benzalkonium chloride and Cetylpyridinium chloride  Aldehydes  Highly reactive molecules  Sporicidal and can be used as chemical sterilants  Combine with and inactivate nucleic acids and proteins  E.g Formal dehyde and glutaraldehyde

 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) : gas is commonly used to sterilize objects that are sensitive to temperatures greater than 60 °C and / or radiation such as plastics. -Ethylene oxide treatment is generally carried out between 30°C and 60°C with relative humidity above 30% and a gas concentration between 200 and 800 mg/l

 Chemical in common use are : 1-Alcolhol: kill vegetative bacteria only e.g. 75% ( absolute alcohol is not effective because 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 of 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 infectious agents 8- Quaternary ammonium salts 9- Heavy metals ( e.g. mercury and silver) they cause denaturation of enzymes and other essential proteins.`

Figure 8.11-Ethylene Oxide Sterilizer

 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

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, chloroamphenicol, tetracyclines, macrolids, nystatin and griseofulvin.  Examples of synthetics chemotherapeutic agents are: sulphonamides, nitrofuran, isonictinc acid derivatives, 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.  The minimum bactericidal concentration (MBC) :is the lowest concentration of an antibacterial agent required to kill a particular bacterium  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

Antibacterial spectrum—Range of activity of an antimicrobial against bacteria. A broad-spectrum antibacterial drug can inhibit a wide variety of gram-positive and gram-negative bacteria, whereas a narrow-spectrum drug is active only against a limited variety of bacteria.  Bacteriostatic activity—The level of antimicrobial activity that inhibits the growth of an organism. This is determined in vitro by testing a standardized concentration of organisms against a series of antimicrobial dilutions. The lowest concentration that inhibits the growth of the organism is referred to as the minimum inhibitory concentration (MIC).  Bactericidal activity—The level of antimicrobial activity that kills the test organism. This is determined in vitro by exposing a standardized concentration of organisms to a series of antimicrobial dilutions. The lowest concentration that kills 99.9% of the population is referred to as the minimum bactericidal concentration (MBC).

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