MLS1004SEF-Week 6 Lecture Notes on Medical Microbiology and Virology II - 20 Feb 2024 PDF

Lecture 6 Introduction to Medical Microbiology and Virology II MLS 1004SEF Introduction to Medical Laboratory Science and Laboratory Instrumentation II By Dr. Andy YY CHEUNG [email protected] Book references Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology. Brooks, G...

Lecture 6 Introduction to Medical Microbiology and Virology II MLS 1004SEF Introduction to Medical Laboratory Science and Laboratory Instrumentation II By Dr. Andy YY CHEUNG [email protected] Book references Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology. Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies. Mahon, C. R., & Lehman, D. C., (2019). Textbook of diagnostic microbiology. Elsevier. The Five “I’s in microbiology Inoculation: to introduce microbes into a culture media so that it reproduces there Isolation: to obtain pure culture Incubation: to grow microbes under proper conditions Inspection: to observe characteristics of microbes Identification: to ID organism to exact species level Inoculation: to introduce microbes into a culture media so that it reproduces there    Inoculation : Introduction of microbes into sterile culture medium Culture media (singular = medium) : contains nutrients for microbial growth Culture: to cultivate microbes in/on culture medium   Sterile: No living microbes “Aseptic technique”: to prevent contamination Factors affecting bacterial growth  Chemical Requirements  CHONPS sources        Carbon Hydrogen Oxygen Nitrogen Phosphorus Sulfur   Trace Elements Water     Temperature pH Osmotic Pressure Oxygen Physical Requirements Growth Media Bacteria and other microbes have particular requirements for growth In order to successfully grow bacteria in lab, we must provide an environment suitable for growth Media = mixtures of nutrients that the microbes need to live Media also provide a surface and the necessary moisture and pH to support microbial growth How is media made? When lab personnel make media, they measure out a quantity of dry powdered nutrient media, add water and check the pH They pour the media into bottles, cap it and autoclave The autoclave exposes the media to high temperature (121°C) and pressure (15 psi) for 20 minutes Once the media is autoclaved it is considered sterile (all life forms killed) Biological and physical indicators of sterilization Different types of culture media I. Based on their consistency a) solid medium – contains 1 - 3 % agar - Colony morphology can be observed b) liquid medium – no agar e.g. Blood culture, enrichment broth c) semi solid medium– 0.2 – 0.5 % agar e.g. SIM (Sulfide Indole Motility medium) Sulfide Indole Motility Medium Selenite F Broth – for the isolation of Salmonella & Shigella Agar Plated media       Complex polysaccharide No nutritive value Used as solidifying agent for solid or semi-solid culture media in Petri plates, slants, and tubes (or called agar deep) Generally not metabolized by microbes Liquefies at 85°C or above Solidifies at 32 - 40°C Agar deep Broth in tube (no agar) Slants Pouring a plate Watch Videos: https://www.youtube.com/watch?v=gxamN 2CpmX4 https://www.youtube.com/watch?v= oe7AY0QTcD4 Slant Tubes The medium has been allowed to solidify at an angle in order to get a flat inoculating surface After autoclaving the media (in tube) for 20 minutes, the tubes are placed in a slanted position to allow the agar to solidify These tubes are called slants Microorganisms grow on the surface of agar plates and slants Different types of culture media II. Based on the constituents/ ingredients a) simple / basal medium contain carbon and nitrogen sources contain ions (calcium, magnesium, potassium, sodium, and phosphate) essential for cell survival and growth e.g. peptone water b) complex medium non-synthetic: chemical composition is not specifically defined; They have added ingredients e.g. extracts and digests of yeasts, meat, or plants that provide special nutrients e.g. nutrient broth/agar, tryptic soy broth/agar, and brain heart infusion broth/agar Different types of culture media II. Based on the constituents/ ingredients c) synthetic or defined medium Media prepared from pure chemical substances its exact composition is known d) Special media Enriched media Enrichment media Selective media Differential media Enriched media prepared by adding additional substances like blood, serum, or egg yolk in the basal medium used to grow fastidious microorganisms as they require additional nutrients and growth-promoting substances. e. g. Blood agar, Chocolate agar Blood agar Chocolate agar Enrichment media favor the growth of a particular microorganism over undesirable commensal or contaminating microbes to isolate pathogens from a mixed culture Are usually liquid media The growth of a broad spectrum of Vibrio sp. is promoted by peptones, a sodium chloride concentration of 10 g/liter, and a high pH of 8.5 alkaline peptone water Sodium selenite helps suppress the growth of competitive organisms and enhances the recovery of Salmonella by inhibiting the normal gut flora present in the sample Selenite F Broth Selective media contain certain ingredients which can inhibit the growth of unwanted organisms and allow the growth of target organisms allow the selection of one or more types of microorganisms e.g. MacConkey agar MacConkey agar Differential media also known as indicator media contain compounds that allow groups of microorganisms to be visually distinguished by the appearance of the colony or the surrounding media, usually on the basis of some biochemical difference between the two groups MacConkey agar Blood agar Blood agar Non-selective, differential media Contains 5-10% mammalian blood (usually sheep or horse) Used to detect hemolytic activity Beta hemolysis: hemolysin lyse the blood cells completely, producing a clear area around the colony Alpha hemolysis: Incomplete hemolysis produces a greenish discoloration around the colony, caused by hydrogen peroxide produced by the bacterium, oxidizing hemoglobin producing the green oxidized derivative met-hemoglobin Gamma hemolysis: no effect on the red cells left: no lactose fermentation MacConkey agar right: lactose fermentation MacConkey’s media is both selective & differential 1. Selective: bile salts and crystal violet inhibits the growth of Grampositive bacteria; only Gram-negative bacteria grows well; 2. Differential: neutral red (pH-sensitive dye) and lactose (type of sugar) have been added to media Bacteria that use lactose for food (lactose fermenters), produce acidic metabolites that trigger the pH sensitive dye to turn pink lactose fermenting bacteria will grow in bright pink colonies while non-lactose fermenters will be colorless and clear Viral culture 1. Laboratory animals 2. Embryonated chicken egg 3. Cell / tissue culture -> requires living cells/tissues for growth intracellular bacteria ( L ) Image source: https://www.pipette.com/Culture-Tubes-Test-Tubes ( R ) Image source: https://www.slideshare.net/vivekaiden/cultivation-of-virus-120166509 Different types of culture media III. Based on oxygen requirement - Aerobic media - Anaerobic media Robertson’s cooked meat medium contains pieces of fat-free minced cooked meat of ox heart Before inoculation, medium is boiled to make it oxygen-free [After inoculation, it is covered with a layer of sterile liquid paraffin oil to prevent the entry of oxygen into the medium] Reducing agents: sulfhyrdyl (-SH) groups of muscle protein glutathione in beef heart Thioglycolate medium- sodium thioglycolate to chemically combine with dissolved oxygen to deplete the oxygen in the media (A) Facultative Anaerobe, grows both aerobically and anaerobically, growth is seen throughout the tube (B) Obligate Anaerobe, incapable of growth in the presence of oxygen, growth is seen approximately 1/4 to 1/2 of the way from the top of the tube (C) Facultative Anaerobe, grows both aerobically and anaerobically, growth is seen throughout the tube (D) Microaerophile, requires oxygen but at concentrations below atmosphere, grows just below the surface of the media but not at the top (E) Obligate Aerobe: oxygen is required for growth, growth at the top of the tube only Labeling plates All Petri plates should be labeled and stored in the following manner: 1. Make certain that all plates are labeled on the bottom half (i.e. the portion of the Petri plate that contains the media) 2. Include the following: a. Your initials or identifying mark b. Date (mm/dd/yy) c. Type of specimen / Code # / letter Labeling plates 3. All plates are incubated in the "upside down" position “Upside down” means that ½ of the Petri plate with media faces up The empty ½ of the Petri plate is down Plates will be incubated at 37° C for 24 hrs, then stored at room temperature until next week, when you will observe for results Agar plates are stored upside down to prevent condensation Labeling tube and specimens Inoculation: to introduce microbes into a culture media so that it reproduces there Broth/Liquid Culture Stroke culture Stab culture Lawn culture (for susceptibility testing) Streak plate method (for isolation too) Pour plate method (for isolation and bacterial enumeration too) Spread plate method (for isolation and bacterial enumeration too) Platinum / Nichrome loops Inoculation https://www.youtube.com/watch?v=bRadiLXkqoU https://www.youtube.com/watch?v=Osgj53nou6I Broth/Liquid Culture Liquid culture in a tube, bottle or flask may be inoculated by touching with a charged loop or with a pipette or with a syringe Stroke culture It is made in tubes containing agar slopes (slant) It is used for providing a pure growth of bacterium Stab culture It is prepared by puncturing with charged long straight wire (loop) Stab culture is employed mainly for anaerobe stock cultures or motility test Lawn (or carpet) culture Lawn cultures are prepared by flooding the surface or plate with suspension of bacteria It provides uniform surface growth of bacteria It is useful for antibiotic sensitivity test Antibiotic sensitivity test A pure bacterial culture is suspended in saline, its turbidity is standardized, and it is swabbed uniformly across an agar plate Using aseptic technique, saline suspension of a specific organism is collected with a sterile swab The swab is then streaked across a Mueller– Hinton agar plate to form a bacterial lawn To obtain uniform growth, the agar plate is streaked with the swab in one direction, rotated 60° and streaked again, rotated another 60° and streaked again Antibiotic susceptibility testing Isolation: to obtain pure culture In the clinical laboratory it is necessary to isolate bacteria in pure culture : obtain sufficient growth of bacteria for demonstration their properties such as study of morphological, biochemical, antigenic and pathogenic properties, bacteriophage and bacteriocin susceptibility; determine its sensitivity to antibiotics Colony – macroscopically visible collection of millions of bacteria originating from a single bacterial cell -> pure culture In microbiology, colony-forming unit (CFU) is a unit which estimates the number of microbial cells (bacteria and fungi) in a sample that are viable, able to multiply via binary fission under the controlled conditions Isolation: Streak-plating method Streaking is a technique used in microbiology for the isolation of single colonies of microorganisms, either from a mixed species or from the same species Four Quadrant Streak procedure https://www.youtube.com/watch?v=FutAgWDymAM Isolation: Streak-plating method a rapid qualitative isolation technique is based on the principle of dilution The main criterion of isolation is to obtain a reduced number of colonies In this technique, a loopful of culture is spread on an agar plate to get individual cells far apart enough from each other The streaking method gradually dilutes the inoculum such that the bacterial cells can give rise to individual colonies Quadrant Streaking also known as a four-quadrant streak the most common method of streaking where the petri dish is divided into four quadrants and then inoculated A loopful of inoculum is taken and streaked such that the first quadrant contains the highest concentration of the inoculum, followed by the second quadrant, third quadrant, and fourth quadrant the inoculation loop is sterilized after streaking in every quadrant By the time the fourth quadrant is streaked, the inoculum is diluted enough to give rise to individual colonies Streak plate technique Streak plate technique Streak plate technique Streak plate technique Other streaking techniques Dr Ekta, Microbiology Isolation: Pour Plating (not commonly used in routine practice) https://www.youtube.com/watch?v=0X39eeZbS98 Isolation: Pour-plating method Isolation: Pour-plating method 1 ml of appropriately diluted inoculum is added to 9 ml of molten agar and poured on petri-dish Colonies appear through out the depth of medium Used to estimate viable count, recommended method for obligate anaerobic bacteria Following the incubation, the numbers of isolated colonies are counted Counts above 300 are considered Too Numerous To Count (TNTC) because it is impossible to tell whether colonies are separated Plates with less than 30 colonies do not have a statistically significant number of colonies Thus, the sample must be serially diluted to obtain at least one plate with 30 – 300 CFU/mL (suitable colony counting) It is generally desirable to make duplicate or triplicate plantings of each dilution and to average the resulting counts Bacteria enumeration: Pour-plating method Spread Plate Technique for Colony Counting (not commonly used in routine practice) https://www.youtube.com/watch?v=gwPZhsyMI9M Isolation: Spread-plating method Bacteria enumeration: Spread-plating method = 87 x 105 / 0.1 = 8.7 x 107 CFU/mL Pour plating vs spread plating Incubation: to grow microbes under proper conditions Factors affecting microorganism growth: Oxygen requirements: Obligate / facultative aerobes / anaerobes; microaerophilic Carbon dioxide (CO2) pH Osmotic pressure Optimum temperature for growth and bacterial growth curve When optical density (OD) is directly proportional to CFU/mL Bacterial Growth Curve 4 phases Microorganism, incubation temperatures and examination timepoints Microorganism Incubate at Examine at Bacteria 35 ± 2°C 24 to 48 hours Yeast 35 ± 2°C 24 to 48 hours Mold 25°C 25°C and Dimorphic fungi 35 ± 2°C Up to 7 days 24 to 48 hours and Up to 7 days Categories of Oxygen Requirement Aerobe – utilizes oxygen and can detoxify it obligate aerobe - cannot grow without oxygen facultative anaerobe – utilizes oxygen but can also grow in its absence microaerophilic – requires only a small amount of oxygen Anaerobe – does not utilize oxygen obligate anaerobe - lacks the enzymes to detoxify oxygen so cannot survive in an oxygen environment aerotolerance anaerobes – do no utilize oxygen but can survive and grow in its presence Categories of Oxygen Requirement Carbon dioxide Requirement All microbes require some carbon dioxide in their metabolism Capnophilic – grows best at higher CO2 tensions than normally present in the atmosphere Bacteria Aerobes Anaerobes Obligate aerobes Obligate anaerobes Facultative anaerobes Aerotolerant anaerobes Gas requirements for growth Grow in ambient air, which contains 21% oxygen and small amount of (0.03%) of carbon dioxide Does not require oxygen for growth Have absolute requirement for oxygen in order to grow Grow only under condition of high reducing intensity and for which oxygen is toxic Are capable of growth under both aerobic and anaerobic conditions Are anaerobic bacteria that are not killed by exposure to oxygen Require increased concentration of carbon dioxide (5% to 10%) and approximately 15% oxygen Grow under reduced oxygen (5% to 10%) and increased carbon dioxide (8% to Microaerophiles 10%); Higher oxygen tensions may be inhibitory to them Capnophiles O2 CO2 21% 0.03% not not required affected not required affected not toxic affected not not affected affected not not affected affected ↓15% ↑5-10% ↓5-10% ↑8-10% Incubation- anaerobic  Reducing media ◦ Heated to drive off O2 ◦ Contain chemicals (thioglycollate or oxyrase) that combine O2 thioglycollate broth Anaerobic jar, candle jar and anaerobic bag McIntosh and Filde's anaerobic jar Gas-pak Anaerobic jar https://www.youtube.com/watch?v=aFDYx-7ceS8 Anaerobic jar Gas-pak is a method used in the production of an anaerobic environment These are commercially available, disposable sachets containing a dry powder or pellets, which, when mixed with water and kept in an appropriately sized airtight jar, produce an atmosphere free of oxygen gas The medium, the gas-pak sachet (opened and with water added) and an indicator are placed in an air-tight gas jar which is incubated at the desired temperature These chemicals react with water to produce hydrogen and carbon dioxide Hydrogen and oxygen reacting on Palladium catalyst and combine to form water simpler technique than the McIntosh and Filde's anaerobic jar where one needs to pump gases in and out Anaerobic chamber Anaerobic chamber https://www.youtube.com/watch?v=9ghSOnjD3Ls https://www.youtube.com/watch?v=wMTgCGWQ77c Anaerobic chamber an anaerobic incubation system provides oxygen free atmosphere for inoculating culture media and for incubation fitted with airtight rubber gloves to insert hands for working with specimens contains catalyst, desiccant, hydrogen gas, carbon dioxide gas, nitrogen gas and an indicator Indicators of anaerobic incubation Chemical indicators Resazurin: pink (with oxygen) -> colorless (anaerobic) methylene blue dye: blue (with oxygen) -> colorless (anaerobic) Biological indicators Pseudomonas aeruginosa - strict aerobe Clostridium tetani - strict anaerobe Incubation Culture tubes should be stored upright in plastic rack Petri plates should be incubated upside-down (lid on the bottom ) Inspection: to observe characteristics of microbes Inspection: Observation and Description Colony Morphology Microscopic examination (grams stain) Lecture 6 Introduction to Medical Microbiology and Virology II -supplementary slides MLS 1004SEF Introduction to Medical Laboratory Science and Laboratory Instrumentation II By Dr. Andy YY CHEUNG [email protected] LABORATORY SAFETY Human pathogens in clinical samples (NOT just in medical microbiology laboratory) Laboratory procedures are often potentially hazardous if proper technique is not followed, and laboratory-acquired infections can occur Among the common hazards that might expose laboratory personnel to the risk of infection are the following: (1) aerosols— generated by homogenization of infected tissues, centrifugation, ultrasonic vibration, or broken glassware; (2) ingestion— from mouth pipetting, eating or smoking in the laboratory, or inadequate washing of hands; (3) skin penetration—from needle sticks, broken glassware, hand contamination by leaking containers, handling of infected tissues, or animal bites; and (4) splashes into the eye Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies. LABORATORY SAFETY Good biosafety practices include the following: (1) training in and use of aseptic techniques; (2) interdiction of mouth pipetting; (3) no eating, drinking, or smoking in the laboratory; (4) use of personal protective equipment (eg, coats, gloves, masks) not to be worn outside the laboratory; (5) sterilization of experimental wastes; (6) use of biosafety hoods; and (7) immunization if relevant vaccines are available. Additional precautions and special containment facilities (Biosafety Level 4) are necessary when personnel are working with high-risk agents such as the filoviruses and rabies virus Brooks, G. F., Carroll, K. C., Butel, J. S., Morse, S. A., & Mietzner, T. A. (2013). Jawetz, Melnick & Adelberg’s Medical Microbiology 26th Ed., by The McGraw-Hill Companies. Personal Protective Equipment (PPE) Disposable gloves Lab coats Safety glasses/goggles Face shields Masks WHO: Good Microbiological Practices & Procedures -Personal Protective Equipment (PPE) https://www.youtube.com/watch?v=Cuw8fqhwDZA&t Biohazard label Ref: Safety in the Clinical Microbiology Laboratory Summary of biosafety levels (BSLs) for infectious agents Biosafety Level 1 (BSL 1): No known pathogenic potential for immunocompetent individuals. Typical examples include Bacillus subtilis. Most undergraduate laboratory courses operate under BSL 1 precautions. Precautions include adherence to standard laboratory techniques. Biosafety Level 2 (BSL 2): Level 1 practices plus laboratory coats, protective gloves, limited access, decontamination of all infectious waste, and biohazard warning signs. Apparatus includes partial containment equipment (such as classes I and II biological safety cabinets) when procedures may lead to the production of infectious aerosols. This category includes the most common microorganisms associated with laboratory-acquired infections, including HBV, HIV, Staphylococcus,and enteric pathogens such as Salmonella and Shigella. Biosafety Level 3 (BSL 3): Level 2 procedures plus special laboratory clothing and controlled access are recommended for handling clinical material suspected of containing Mycobacterium tuberculosis, Brucella, Coccidioides immitis, Rickettsia, and specific viruses such as arbovirus. The air movement must be carefully controlled to contain the infectious materials. Biosafety Level 4 (BSL 4): Level 3 practices plus entrance through a separate room in which street clothing is changed and replaced with laboratory clothing. Maximum containment includes the use of a class II biological safety cabinet and the decontamination of all personnel and materials before leaving the area. This level is primarily used in research facilities and includes a limited number of exotic viruses including filovirus and arenavirus. Biosafety Cabinets come in three classes, depending on the protection to personnel, specimen, and environment Class I Biosafety Cabinet Features: Front opening Exhaust Plenum Exhaust HEPA filter Sash Uses: In an animal lab for cage dumping Aerate cultures that potentially produce aerosols It is only employed in low- and moderate-risk microbiological studies. Fermenters and centrifuge housing Class II Type A1 Biosafety Cabinet Features: Front opening Sash Exhaust HEPA filter Rear plenum Supply HEPA filter Blower Class II Type A2 Biosafety Cabinet Features: The front opening Sash Exhaust HEPA filter Supply HEPA filter Positive pressure plenum Negative pressure plenum Class II Type B1 Biosafety Cabinet Features: The front opening Sash Exhaust HEPA filter Supply plenum Supply HEPA filter Blower The negative pressure exhaust plenum Class II Type B2 Biosafety Cabinet Features: The front opening Sash Exhaust HEPA filter Supply plenum Supply HEPA filter Blower The negative pressure exhaust plenum Container Supply Blower Filter screen Class II Type C1 Biosafety Cabinet Features: When in recirculating mode, they can function as type A cabinets, and when in exhausting mode, as type B cabinets. Comparison between different types of Class II BSCs Definitions Antisepsis: Use of chemical agents on skin or other living tissue to inhibit or eliminate microbes; no sporicidal action is implied Disinfection: Use of physical procedures or chemical agents to destroy most microbial forms; bacterial spores and other relatively resistant organisms (e.g., mycobacteria, viruses, fungi) may remain viable; disinfectants are subdivided into high-, intermediate-, and low-level agents Germicide: Chemical agent capable of killing microbes; includes virucide, bactericide, sporicide, tuberculocide, and fungicide High-level disinfectant: A germicide that kills all microbial pathogens except large numbers of bacterial spores Intermediate-level disinfectant: A germicide that kills all microbial pathogens except bacterial endospores Low-level disinfectant: A germicide that kills most vegetative bacteria and lipid-enveloped and medium-size viruses Sterilization: Use of physical procedures or chemical agents to destroy all microbial forms, including bacterial spores Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Sterilization Sterilization is the total destruction of all microbes, including the more resilient forms such as bacterial spores, mycobacteria, nonenveloped (non-lipid) viruses, and fungi This can be accomplished using physical, gas vapor, or chemical sterilants Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Sterilization Saturated steam under pressure is a widely used, inexpensive, nontoxic, and reliable method of sterilization. Three parameters are critical: the time of exposure to steam, temperature, and amount of moisture. The most commonly used sterilization cycle is use of saturated steam heated at 121°C for 15 minutes. Maintaining the proper temperature is critical because a drop of 1.7°C increases the needed exposure time by 48%. If no moisture is present, then the temperature must reach 160°C. Dry heat sterilization requires prolonged exposure times and damages many instruments, so it is not currently recommended. Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Sterilization Ethylene oxide gas is used to sterilize temperature- or pressuresensitive items Treatment is generally for 4 hours, and sterilized items must be aerated for an additional 12 hours to eliminate the toxic gas before the items are used Although ethylene oxide is highly efficient, strict regulations limit its use, because it is flammable, explosive, and carcinogenic to laboratory animals For these reasons, ethylene oxide sterilization is avoided if acceptable alternatives are available Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Sterilization Hydrogen peroxide vapors are effective sterilants because of the oxidizing nature of the gas This sterilant is used for the sterilization of instruments A variation is plasma gas sterilization, in which hydrogen peroxide is vaporized, and then reactive free radicals are produced with either microwave-frequency or radio-frequency energy Because this is an efficient sterilizing method that does not produce toxic byproducts, plasma gas sterilization has replaced many of the applications for ethylene oxide However, it cannot be used with materials that absorb hydrogen peroxide or react with it Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Sterilization Two chemical sterilants have also been used: peracetic acid and glutaraldehyde Peracetic acid, an oxidizing agent, has excellent activity, and the end products (i.e., acetic acid and oxygen) are nontoxic In contrast, safety is a concern with glutaraldehyde, and care must be used when handling this chemical Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Disinfection Microbes are also destroyed by disinfection procedures, although more resilient organisms can survive Unfortunately, the terms disinfection and sterilization are casually interchanged and can result in some confusion This occurs because disinfection processes have been categorized as high level, intermediate level, and low level High-level disinfection can generally approach sterilization in effectiveness, whereas spore forms can survive intermediate-level disinfection, and many microbes can remain viable when exposed to low-level disinfection Even the classification of disinfectants by their level of activity is misleading. The effectiveness of these procedures is influenced by the nature of the item to be disinfected, number and resilience of the contaminating organisms, amount of organic material present (which can inactivate the disinfectant), type and concentration of disinfectant, and duration and temperature of exposure Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Disinfection High-level disinfectants are used for items involved with invasive procedures that cannot withstand sterilization procedures (e.g., certain types of endoscopes and surgical instruments with plastic or other components that cannot be autoclaved) Disinfection of these and other items is most effective if cleaning the surface to remove organic matter precedes treatment Examples of high-level disinfectants include treatment with moist heat and use of liquids such as glutaraldehyde, hydrogen peroxide, peracetic acid, and chlorine compounds Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Disinfection Intermediate-level disinfectants (i.e., alcohols, iodophor compounds, phenolic compounds) are used to clean surfaces or instruments where contamination with bacterial spores and other highly resilient organisms is unlikely These have been referred to as semicritical instruments and devices and include flexible fiberoptic endoscopes, laryngoscopes, vaginal specula, anesthesia breathing circuits, and other items Low-level disinfectants (i.e., quaternary ammonium compounds) are used to treat noncritical instruments and devices, such as blood pressure cuffs, electrocardiogram electrodes, and stethoscopes Although these items come into contact with patients, they do not penetrate through mucosal surfaces or into sterile tissues Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Disinfection The level of disinfectants used for environmental surfaces is determined by the relative risk these surfaces pose as a reservoir for pathogenic organisms For example, a higher level of disinfectant should be used to clean the surface of instruments contaminated with blood than that used to clean surfaces that are “dirty,” such as floors, sinks, and countertops The exception to this rule is if a particular surface has been implicated in a nosocomial infection, such as a bathroom contaminated with Clostridium difficile (spore-forming anaerobic bacterium) or a sink contaminated with Pseudomonas aeruginosa In these cases, a disinfectant with appropriate activity against the implicated pathogen should be selected Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Antisepsis Antiseptic agents are used to reduce the number of microbes on skin surfaces. These compounds are selected for their safety and efficacy Alcohols have excellent activity against all groups of organisms except spores and are nontoxic, although they tend to dry the skin surface because they remove lipids They also do not have residual activity and are inactivated by organic matter Thus the surface of the skin should be cleaned before alcohol is applied Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Antisepsis Iodophors are also excellent skin antiseptic agents, having a range of activity similar to that of alcohols They are slightly more toxic to the skin than is alcohol, have limited residual activity, and are inactivated by organic matter Iodophors and iodine preparations are frequently used with alcohols for disinfecting the skin surface Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Antisepsis Chlorhexidine has broad antimicrobial activity, although it kills organisms at a much slower rate than alcohol Its activity persists, although organic material and high pH levels decrease its effectiveness The activity of parachlorometaxylenol is limited primarily to gram-positive bacteria Because it is nontoxic and has residual activity, it has been used in hand washing products Triclosan is active against bacteria but not against many other organisms It is a common antiseptic agent in deodorant soaps and some toothpaste products Murray, P. R., Rosenthal, K. S., & Pfaller, M. A. (2016). Medical microbiology 8th edition. Germicidal Properties of Disinfectants and Antiseptic Agents Autoclaves (1) and Hot Air Oven (2) 1 2 Biological and chemical indicator Hierarchy of surface disinfection Virusolve+ is highly effective as a cleaner and disinfectant against bacteria, mycobacteria, fungi, viruses and spores. Ijaz, M. K., Nims, R. W., Zhou, S. S., Whitehead, K., Srinivasan, V., Kapes, T.,... & McKinney, J. (2021). Microbicidal actives with virucidal efficacy against SARS-CoV-2 and other beta-and alphacoronaviruses and implications for future emerging coronaviruses and other enveloped viruses. Scientific Reports, 11(1), 5626. BSC-Fumigation BSC fumigation is performed with a disinfectant in a gas or vapor state The term “gas” refers to a chemical that is a stable gas at room temperature A “vapor” refers to a chemical that is stable as a liquid at room temperature and converted either to a gas or microscopic droplets prior to its release into the cabinet This chemical then penetrates all internal surfaces within the BSC, including through the HEPA filters The chemical is typically one capable of killing bacterial endospores, which among bacteria, viruses, fungi, algae, and protozoa, are considered the most resistant to chemical disinfection There are four underlying needs for fumigating a BSC: 1. To ensure safety to service personnel who need to access potentially biologically contaminated areas of the BSC, e.g., for HEPA filter changes or motor replacement. 2. To help prevent contamination of samples being processed inside a BSC, e.g., changeover of pharmaceutical lot production run. 3. To prevent outbreak of highly infectious agents that could harm laboratory personnel, before non-routine service or as a part of a regular contamination control regimen, e.g., BSL-3 or -4 facilities. 4. Fumigation should also be performed prior to relocation of the biological safety cabinet in order to protect the movers and reduce the potential release of biohazardous material during the transportation process. BSC-Fumigation The three most commonly used chemicals in BSC fumigation are formaldehyde (CH2O), chlorine dioxide (ClO2), and hydrogen peroxide (H2O2) Health and Safety, Effectiveness, Material Compatibility, Ease of Use (refer to the pdf attachment) BSC-Fumigation Careers in Microbiology Medical Microbiology Clinical Microbiology Public Health Food microbiology Environmental microbiology Where do Microbiologists work? Hospitals, Clinical laboratories, Government laboratories Health Authority Healthcare industry Pharmaceutical industry Food safety laboratories Food production specialist Environmental consulting companies Universities