Disinfection & Sterilization PDF

DISINFECTION & STERILIZATION Control of Microbes is important because:  Microorganisms possess the ability to cause illness, which may sometimes lead to death.  Microorganisms are also able to cause spoilage of food, as well as personal possessions.  In order to effectiv...

DISINFECTION & STERILIZATION Control of Microbes is important because:  Microorganisms possess the ability to cause illness, which may sometimes lead to death.  Microorganisms are also able to cause spoilage of food, as well as personal possessions.  In order to effectively control microorganisms, we need to know how a particular treatment affects a population of microbial cells.  It is important to know whether a particular treatment will attempt to kill all the cells, or just slow their growth, without killing them. Important terms  Sterilization destroys all microbial life  Disinfection (Sanitizing)- treatment that reduces the number of pathogens to a level at which they pose no threat of disease  Antiseptics kill microorganisms on living tissue Important terms  Microbiocidal treatments are those that kill all microorganisms  Microbiostatic treatments are those treatments that inhibit microbial growth. When the treatment is withdrawn, cells begin to grow again. Example, Refrigeration Physical treatments used to control microorganisms 1. Heat ◦ Simple, inexpensive, effective for materials that are not heat-sensitive. ◦ Heat may be in the form of dry heat (open flames, ovens) or moist heat (boiling, autoclaving). ◦ Autoclaving is very effective at destroying resistant endospores. Dry Heat a) Hot air ovenn-1600c –1800c for 1 hour (dry heat sterilization) b) Flaming - (direct exposure) c) Red heat- holding inoculating loops etc d) incineration- destruction by burning. e) Infra-Red Radiation-Infra-Red rays are directed from an electrically heated element onto the objects to be sterilized. Hot air sterilizer Moist Heat  Boiling, steam and steam under pressure  Uses temperatures of  1) < 1000c,  2) 1000c,  3) > 1000c Autoclave  Saturated steam under pressure  Temp.0c of 1210c for 15 minutes at 15 lbs pressure ◦ Destroys micro-organisms including spores ◦ Widely used for sterilization of surgical supplies and bacteriological culture media 2. Cold ◦ Microbiostatic treatment. Low temperatures often do not kill microorganisms, but instead slow their growth. ◦ eg. Refrigeration slows the growth of most species of microorganisms, thereby preserving foods. ◦ Most psychrophiles do not pose health risks. An exception is Listeria monocytogenes. 3. Radiation ◦ UV radiation of wavelengths 10nm to 400 nm is lethal to most microorganisms because it damages their DNA. ◦ Germicidal lamps that emit UV are commonly used to kill microorganisms on surfaces 4. Filtration ◦ Microorganisms other than viruses may be removed from liquids by filtration. ◦ Filtration is suitable for removing cellular organisms from media, vitamin solutions and antibiotic solutions that are heat-sensitive. 5. Drying ◦ Means removal of water. ◦ Done either by evaporation (commonly used in food industry) or by freeze- drying, where products are first frozen, then vacuum dried. ◦ Freeze-dried products include proteins and various blood products. 6. Osmotic strength ◦ High concentrations of salt or sugar solutions are often used to preserve foods. ◦ High salt and sugar concentrations act to decrease the amount of water available to microbes. ◦ High osmotic strength solutions of salt or sugar cause cells to plasmolyse. Chemical treatments used to control microorganisms Important terms:  Chemicals, including antibiotics that are used to treat disease are called chemotherapeutic agents.  Chemicals that kill microorganisms are called germicides.  Chemicals that inhibit microbial growth are called germistats  Germicides used on inanimate objects are called disinfectants  Germicides applied to living tissue are called antiseptics Testing germicides  Germicides may be tested by comparing their effectiveness to phenol (a traditional germicide).  The phenol coefficient is determined by adding a test organism to a series of dilutions of the disinfectant;  The phenol coefficient is the number obtained by dividing the greatest dilution of the disinfectant killing the test organism by the greatest dilution of phenol showing the same result.  The effectiveness of germicides may also be tested against various microorganisms.  This may be carried out using the disc diffusion method. Disk diffusion method  This involves the use of paper discs impregnated with test germicide and placed on a plate with the test organisms.  After incubation, there may be a clear zone free of bacteria around the disc called the zone of inhibition.  The size of the zone of inhibition is a useful index of the bacterium’s sensitivity to the germicide. Disk diffusion method  An example of a plate from a disc diffusion study.  Note the varying sizes of the inhibitory zone present around the different disks.  Each disk is impregnated with a different antibiotic agent.  Interpretation of the test requires comparison of the measured zone diameter with the accepted cutoff values for each antibiotic/organism pair.  http://www.uphs.upenn.edu/bugdrug/antibiot ic_manual/bk.html Classes of germicides There are six major classes of germicides: 1. Phenols & phenolics 2. Alcohols 3. Halogens & Hydrogen peroxide 4. Heavy metals 5. Surfactants 6. Alkylating agents 1. Phenols & phenolics ◦ These kill microbes by denaturing vital cellular proteins, including enzymes. ◦ Phenolics act on lipids, such as the cytoplasmic membrane. An example is Paracresol, brand name Lysol 2. Alcohols ◦ Kill by disrupting lipids in the cell membrane and by denaturing proteins. ◦ Used to disinfect sites on the skin (eg. Ethanol, isopropanol). ◦ Alcohols are not effective against endospores. 3. Halogens & Hydrogen peroxide ◦ Oxidizing agents. ◦ Iodine is used as antiseptic, chlorine as a disinfectant and is an additive in bleach, swimming pools & water treatment facilities. ◦ Hydrogen peroxide is used as an antiseptic for cleaning wounds. 4. Heavy metals ◦ Salts of heavy metals react with the sulphydryl groups of proteins, in so doing inactivating them, and killing the cell. ◦ Mercurochrome and silver nitrate are used as antiseptics. 5. Surfactants ◦ These are compounds (such as soaps) that have hydrophobic and hydrophilic parts. ◦ They act to emulsify oily materials making them easier to wash away with water. ◦ Frequent hand washing removes surface microbes and so controls the spread of infections. 6. Alkylating agents ◦ Compounds that attach short chains of Carbon atoms to proteins & inactivate them. ◦ Examples are glutaraldehyde and formaldehyde. Antimicrobial chemotherapy  Chemicals used to control microbial growth with in a host, i.e. to treat infection 1. Antibiotics- active against bacteria 2. Antivirals- active againts viruses 3. Antimycotics/ Antifungals- active against fungi 4. Antihelminthics- active against helminths 5. Antiprotozoals- active against protozoa Some of the Main classes of antibiotics  Penicillin- bactericidal, broad-spectrum, act well on Gram positive bacteria, minimal side effects  Cephalosporins- similar in structure to penicillins  Sulphonamides- interfere with folic acid synthesis, broad-spectrum, especially acive against Gram-negative bacteria, bacteriostatic  Tetracyclines- broad-spectrum, block protein synthesis  Quinolones- block DNA replication, broad-spectrum. Aseptic Techniques  In order to carry out experiments with microbes they need to be pure and readily available.  This means providing suitable conditions for their growth and multiplication in the laboratory.  Bacteria and fungi are the most commonly cultured microorganisms using laboratory media.  Viruses must be cultured in living host cells; these are called tissue cultures. STERILIZATION May be carried out using: 1. Moist heat ◦ Involves exposure to steam and quite often pressure. ◦ This is done using an autoclave, which is similar to a pressure cooker ◦ It maintains high temperature and pressure for an extended period, killing any microorganisms present. (121ºC, 15psi) ◦ Growth medium is usually autoclaved. 2. Dry heat, which involves exposure to a flame- done for inoculating needles. 3. Filtration, where microbial cells may be removed from liquids using filters. Filter sterilization is used for heat-sensitive substances such as vitamins and antibiotics. 4. Chemicals such as alcohol and bleach are used to sterilize laboratory surfaces, never for media as they are toxic to microbial growth. Growth media  Most experiments are done with a pure culture, that is, a culture consisting of one type of organism, grown from one cell.  Since pure cultures rarely exist in nature, they must be artificially cultivated.  In order to obtain a pure culture, all the materials used to obtain the pure culture must be sterilized.  This includes flasks, petri dishes, test- tubes, pipettes, inoculating needles and medium.  The medium used to culture a micro- organism depends greatly on its nutritional requirements: Defined medium  Defined medium is prepared from pure chemicals, so that the composition is known. Complex medium  Complex media is made from extracts of natural materials, such as beef, yeasts or blood, so that the exact composition is not known.  A liquid complex medium is called a broth. Growth medium may contain agar, which solidifies it. Selective medium  Selective media favour the growth of only particular microorganisms.  Selective media is used to isolate favoured species in a complex mixture of other microorganisms. Differential media  Differential media are used to isolate microorganisms by the appearance of their colonies. Streptococcus sp. on Blood agar plate  Aside from selecting the appropriate medium, microorganisms need the correct temperature and pH in order to thrive.  Different microorganisms respond differently to oxygen and so this requirement should also be met. Isolation of a microorganism (pure cultures)  Involves separating a single cell from all the others and providing it with nutrients in order for it to survive.  In order to isolate a single cell, the population must be diluted or reduced.  This is done in one of three ways:  Streak plate- Microorganisms are streaked onto media so that fewer and fewer organisms are distributed.  Pour plate- diluted sample of the microorganism is added to the media and poured into a petri dish to encourage the growth of single colonies.  Spread plate- diluted sample is poured onto medium in a Petri dish and spread evenly using a sterile rod.  After culturing, microorganisms may be examined under the microscope.  In order to better view them, they may be stained.  Most stains are only effective after the organism has been killed and attached to the microscope slide (fixed). References  Brock Biology of Microorganisms- Madigan, Martinko & Parker  Medical Microbiology- Jawetz, Melnick & Adelberg  Prescott, Harley & Klein’s Microbiology- Willey, Sherwood & Woolverton