Physical Methods of Microbial Control PDF

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Methods in Microbial Control...

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OUTLINE Definition of important terms Physical methods of microbial control Evaluation of methods of of microbial control

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STERILIZATION Killing or removal of all forms (including endospores) of microbial life in a material or an object

COMMERCIAL STERILIZATION Killing of microorganisms and endospores of public health importance with heat  Public health importance – those that can cause infection in human or can cause outbreak / common infection in the body  It is achieved and required to prevent pathogenic microorganisms from proliferating in food products in normal storage and handling conditions. 

HISTORY

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IGNAZ SEMMELWEIS “Hand disinfection” Gynecologist First who demonstrated that handwasing techniques could actually reduce the occurrence of a specific infection in Europe. It was documented as “puerperal fever” aka childbed fever Puerperal fever type of postpartum infection that is common in mothers who just gave birth type of bacterial infection that can occur in the uterine tract of the women who just gave birth or those who underwent medical abortion. He used calcium hypochlorite/Ca(OCl)2 or “chlorinated lime” Once he started implemented the hand washing technique using chlorinated lime to medical practitioners who is working in that hospital – they noticed a drastic reduction of these puerperal fever.

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JOSEPH LISTER Father of Antiseptic Surgery He introduced the antiseptic process which dramatically decreased the deaths not only from childbirth but also in surgeries. He used “liquefied phenol” – formerly known as carbolic acid o Became one of the widely used antiseptic especially in medical practice

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DISINFECTION Reducing the number of pathogenic microorganisms to the point where they no longer cause disease Most of the time, it applies to nonliving things or inanimate object

ANTISEPSIS Removal of pathogens from living tissue

DEGERMING Mechanical removal of microbes from a limited area There is a movement that is used to sweep away microorganisms It doesn‟t necessarily kill all the time but it helps in facilitating the removal because of the mechanical action.

SANITIZATION Use of chemical agent on food handling equipment to meet public health standards and minimize chances of disease transmission

DEFINITION OF TERMS

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SEPSIS microbial contamination the presence of significant amount of microorganisms in a surface or in body HOSPITAL o life-threatening condition caused by a body‟s response towards an infection – usually a severe infection. o It can be deadly if undiagnosed and uncontrolled o Usually requires high-end

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-STATIC AGENT An agent that inhibits growth of certain microorganisms, but does not necessarily kill them More applicable for chemical means of microbial control Static agents are most commonly applicable to bacteria and fungi Eg., bacteriostatic, fungistatic

-CIDAL AGENT Agent that kills microorganisms

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ASEPSIS Absence of significant contamination

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NORMAL BACTERIAL GROWTH CURVE

NORMAL GROWTH CURVE It starts from the lag phase then it will result this introduction to a suitable medium that will result to exponential growth phase known as the log phase. Later on, the number of cells living and dying are equal – resulting to the stationary phase. Then later on, resources become scarce and limited – they start fighting for these resources until such time that the medium can no longer support the growth of microorganism – we call it the death phase.

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–CIDAL AGENT IS INTRODUCED Will result to this bacterial growth phase curve During the lag phase upon the introduction of –cidal agent (something that will kill microbial cells) - it will result to a drastic reduction of microbial growth. STATIC AGENT IS INTRODUCED A static agent will only decrease the exponential growth because you inhibited the growth but did not necessarily kill them that easily. It is by the time that the resources or the metabolism already peaks and later on slows down for microorganisms that they start dying naturally. Inhibited the log phase without drastically reducing the number of cells that are still viable. Inhibit growth and cells still die naturally PHYSICAL METHODS OF MICROBIAL CONTROL

Factors which Influence the Effectiveness of Antimicrobial Treatment

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time it take to eliminate the population As time passes by, the population decreases or the population load will be reduced drastically – because as time passes by, there is a higher contact between the microbial cells and method of microbial control ; resulting to their death.

2) Type of Microbes  Endospores are very difficult to destroy  Vegetative pathogens have varying susceptibility to different microbial controls  3) Environmental Influence  Some compounds present in the surrounding (tissue, surface that you are treating, area that you would like to disinfect) may alter efficacy of antimicrobials  This is more applicable for chemical methods of microbial control  Example of compounds: o Biological fluids like blood, feces, saliva o Inorganic compounds 4) Time of Exposure  Some methods of microbial control are time-dependent  Eg., chemical anti-microbials, radiation treatment – they are more effective at longer time

PHYSICAL METHODS

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01 HEAT Most commonly used method of microbial control

1) Number of Microbes  It takes more effort to kill more microbial cells  The more microbes present, the more

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Because of the presence of water, it allows the penetration of heat in a more efficient manner sterilization is achieved at a shorter period of time (quicker) EXAMPLES:

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Boiling o Heat samples up to 100 °C or more at sea level o Kills vegetative forms of bacterial pathogens, almost all viruses, fungi and their spores within 10 minutes or less o Commonly applied in the household

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Autoclaving o Uses hot steam under pressure which kills all organisms and endospores o Steam is created at 121 °C (greater than the boiling point, at this point steam/water vapor are created) at 15 psi for 15 minutes o Take note: autoclaving does not only take the use of the equipment for 15 minutes, because if you actually put your samples inside an autoclave and you start the machine/equipment for sterilization – you will not immediately reach the temperature, pressure, as well as the time required. Which means that you only start counting when you reach 121 °C and their pressure 15 psi – that‟s the only time that you‟ll start timing and making sure that that condition (that temp and pressure) is maintained for at least 15 minutes- and that‟s the only time that you can stop the autoclaving.

TYPES

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DRY HEAT Kills through oxidation effects resulting to denaturation of microbial proteins and enzymes Example:

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Direct flaming o instrument/surface is exposed to direct/open flame o This is utilized with the use of Bunsen burner or an alcohol lamp o This is done for inoculating loops, needles, forceps, mouth of test tube, petri plates

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Incineration o achieved through burning the samples that are contaminated with the highly infectious microorganism to ashes o used for medical supplies/wastes that are treated as infectious wastes to prevent contaminating other waste products that are in the landfill o achieved through high temperature

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Hot air sterilization o requires equipment which is hot air sterilizer o samples are exposed for a long period of time usually at least two hours o temperature up to 170 degrees Celsius o applicable if you want to dry your apparatus while sterilizing them

When you stop the heating process, you can‟t open the machine immediately because it entails lots of pressure inside the chamber which will prevent you from opening it abruptly o o

Hot Air Sterilization Incineration Direct Flaming

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MOIST HEAT involves water (H2O) it involves a specific types of denaturation known as coagulation coagulates proteins and penetrates cells better than dry heat does o if there is coagulation, there is inherent loss in solubility which results to clumping or aggregation of

Autoclaving may take more or less an hour or more This type of moist heat sterilization is widely used in sterilization not only of lab instruments, equipment‟s, culture media – but also used in sterilization of a lot of our pharmaceutical products such as parenteral solutions.

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the proteins moist heat is deemed superior to dry heat because you only need a shorter period of time in order to penetrate the cell better.

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Pasteurization o Named in honor of Louis Pasteur o Process in which liquid foods are treated with mild heat, usually to less than 100 °C (212 °F), to eliminate pathogens and extend shelf life o As temperature is increased, much less time will be needed to kill the same number of microbes. But for pasteurization, it focuses on the use of milder heat but at a longer period of heating. o Classic Method of Pasteurization - Heating milk to 65 °C for 30 minutes - High Temperature Short Time Pasteurization (HTST) - Milk is exposed to 72 °C for 15 seconds - Ultra High Temperature Pasteurization (UHT) - Aka Ultra Heat Treated (UHT) Most common; found in a lot of liquid food products/dairy products - Milk is treated at 140 °C for only 3 seconds and then cooled very quickly in a vacuum chamber - EXAMPLE: Nestle

extremely low temperature, sometimes even less than 10 °C and that results to immediate freezing of the outside structures of the cell. And with that, if the outside cell structures are already frozen, the cold temperatures can no longer easily penetrate the internal structures – resulting to simply inhibition/slowing of metabolic rate o

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03 FILTRATION Removal of microbes by passage of a liquid or gas through a screen-like material with small pores Eg., HEPA filters and Membrane Filters

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04 LOW TEMPERATURE It can be either in the form of: 

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Refrigeration o Utilize temperature that is a little bit higher o 0 to 7 °C or USP Cold storage (2-8°C) o Reduces metabolic rate of most microbes - Reduction of metabolic rate or inhibition of metabolism; without this metabolic processes, they cannot proliferate, they cannot grow in number – but it does not necessarily result to killing. - You are just slowing down/reducing their metabolic rates but they are still viable microbial cells

Freezing o Less than 0°C o Sometimes, to really make sure that microorganisms in the sample are killed, -2°C is utilized 2 FORMS OF FREEZING

Slow freezing - Use of 0 to -2°C - More harmful/more lethal form of freezing because with this type of freezing, you allow really cold temperatures to penetrate the internal structures of microorganisms - When you want to make sure that no microorganisms will grow and that present microorganisms are to be killed – you have to utilize slow freezing

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Membrane Filter For liquids Used to remove bacteria Useful for removing microbial contaminants from various type of heatsensitive samples Pore size o 0.2 microns (micrometers) o smaller than the average size of a bacterium o bacterium size – 1 mcm a lot of antibiotic solution, vitamins, as well as parenteral products are subjected to membrane filtration after they are formulated because a lot of drug products are heat sensitive...