Control of Microorganisms by Physical and Chemical Agents

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.

Figure 8.1-Microbial Control Methods

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) . 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

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.

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 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 relationship is not linear 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.

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

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

The Autoclave or Steam Sterilizer Figure 8.3-Autoclave Steam Sterilizer

Table 8.2

Measuring Heat-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 .

Dry Heat Sterilization 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

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.

Figure 8.5-Membrane Filter Sterilisation

Figure 8.6-Membrane Filter

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

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

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

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

Chemical Control Agents- Disinfectants and Antiseptics Phenolics Commonly used as laboratory and hospital disinfectants Act by denaturing prptein 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 Halogens - Chlorine 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 iodophor 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 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.g Benzalkonium chloride and Cetylpyridinium 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

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) : 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

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

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, 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. 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

Antibiotic combinations—Combinations of antibiotics that may be used (1) to broaden the antibacterial spectrum for empiric therapy or the treatment of polymicrobial infections, (2) to prevent the emergence of resistant organisms during therapy, and (3) to achieve a synergistic killing effect. Antibiotic synergism—Combinations of two antibiotics that have enhanced bactericidal activity when tested together compared with the activity of each antibiotic. Antibiotic antagonism—Combination of antibiotics in which the activity of one antibiotic interferes With the activity of the other (e.g., the sum of the activity is less than the activity of the individual drugs). Beta-lactamase—An enzyme that hydrolyzes the beta-lactam ring in the beta-lactam class of antibiotics, thus inactivating the antibiotic. The enzymes specific for penicillins and cephalosporins are the penicillinases and cephalosporinases, respectively. 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).

Susceptibility Tests 1. Broth dilution - MIC test

Minimal Inhibitory Concentration (MIC) vs Minimal Inhibitory Concentration (MIC) vs. Minimal Bactericidal Concentration (MBC)

Susceptibility Tests 3.Agar diffusion

Antibiotic Mechanisms of Action

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