Control of Microorganisms by Physical and Chemical 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.

What is the meaning of sterilization?

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

• 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.

Definition of Frequently Used Terms in Microbial control methods

• 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

Antimicrobial Agents

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.

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.

Conditions Influencing the Effectiveness of Antimicrobial Agent Activity

Heat Low temperatures Filtration Radiation

The Use of Physical Methods in Control

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

Moist Heat Sterilization

The Autoclave or Steam Sterilizer

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 temperature .

Measuring Heat-Killing Efficiency

Pasteurizationcontrolled heating at temperatures well below boiling- Louis Pasteurused for milk, beer and other beveragesprocess does not sterilize but does kill pathogens present and slow spoilage by reducing the total load of organisms present.

Dry Heat Sterilizationless effective than moist heat sterilization, requiring higher temperatures and longer exposure times items subjected to 160-170oC for 2 to 3 hoursoxidizes cell constituents and denatures proteins.

Filtrationreduces microbial population or sterilizes solutions of heat-sensitive materials by removing microorganismsalso 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.

Filtering Liquids

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

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

Ionizing Radiation

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

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

Chemical Control Agents -Disinfectants and Antiseptics

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

Heavy Metalse.g., ions of mercury, silver, arsenic, zinc, and coppereffective but usually toxiccombine 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 sterilantscombine 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)

Sterilizing Gases

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.`

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

Chemotherapeutic Agents

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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

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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).

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 aret he penicillinases and cephalosporinases, respectively.

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Susceptibility Tests

1. Broth dilution - MIC test

2. Agar dilution - MIC test

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1 ug/ml

MIC = 8 ug/mlMBC = 16 ug/ml

Minimal Inhibitory Concentration (MIC)vs.

Minimal Bactericidal Concentration (MBC)

32 ug/ml 16 ug/ml 8 ug/ml 4 ug/ml 2 ug/ml

Sub-culture to agar medium

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Susceptibility Tests

3. Agar diffusion

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Antibiotic Mechanisms of Action

Transcription

Translation

Translation

Alteration of Cell Membrane Polymyxins Bacitracin Neomycin

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