Clinical Microbiology Chapter 4, 5, 6, 7 PDF

- BIOFILMS microorganisms living together in communities, CHAPTER 4: CONTROL OF also provide protection to the MICROORGANISM microorga...

- BIOFILMS microorganisms living together in communities, CHAPTER 4: CONTROL OF also provide protection to the MICROORGANISM microorganisms against chemical and physical means of destruction. STERILIZATION - ref ers to the destruction of all - PRIONS are naked pieces of f orms of lif e, including bacterial protein, similar to a virus but spores. without the nucleic acid. thought to be the agents that cause a DISINFECTION number of degenerative - ref ers to a process that diseases of the nervous system eliminates a def ined scope of (transmissible spongiform microorganisms, including some encephalopathy—mad cow spores. Physical or chemical disease, Creutzfeldt - Jakob methods may be used, but most disease). disinf ectants are chemical agents applied to inanimate NUMBER OF ORGANISMS objects. - microbial load (bioburden) - If the number of organisms is ANTISEPTIC plotted against the time they are - A substance applied to the skin exposed to the killing agent f or the purpose of eliminating or (exposure time) logarithmically, reducing the number of bacteria the result is a straight line. The present. death curve is logarithmic. - Antiseptics do not kill spores, and cannot be used as disinf ectants. CONCENTRATION OF DISINFECTING AGENT FIGURE 4-1: Dif f erent types of organisms and - Proper concentrations of their resistance to killing agents. disinf ecting agents ensure the inactivation of target organisms FACTORS THAT INFLUENCE THE DEGREE OF and promote saf e and cost- ef f ective practices. KILLING PRESENCE OF ORGANIC MATERIAL TYPES OF ORGANISMS - Organic material, such as blood, - Cell walls of mycobacteria are mucus, and pus, af f ects killing rich in lipids, which may account activity by inactivating the f or their resistance to chemical disinf ecting agent. and environmental mental - Bleach (sodium hypochlorite) stresses, particularly is easily inactivated by organic desiccation. material. - viruses containing lipid-rich envelopes are more susceptible NATURE OF SURFACE TO BE to the ef f ects of detergents and DISINFECTED wetting agents. - Certain medical instruments are - The organisms known today to manuf actured of biomaterials be the most resistant to the that exclude the use of certain actions of heat, chemicals, and disinf ection or sterilization radiation are prions. methods because of possible damage to the instruments.  - For example, endoscopic materials are most likely to instruments are readily damaged produce inf ection if contaminated by the heat generated in an and require sterilization. autoclave. SEMICRITICAL MATERIAL TEMPERATURE - contact with mucous - Disinf ectant is room temperature membranes, they require high- (20° to 22°C). Their activity is level disinf ection agents. generally increased to some degree by an increase in NONCRITICAL MATERIALS temperature and decreased by a - require intermediate-level to low- decrease in temperature. level disinf ection bef ore contact with intact skin. pH - The pH of the material to be High-level disinf ectants have activity against disinf ected or sterilized can bacterial endospores, whereas intermediate- af f ect the activity of the level disinf ectants have tuberculocidal activity disinf ecting or sterilizing agent. but not sporicidal activity. BIOFILMS PHYSICAL METHODS - Considered as a community of HEAT bacteria or other - MOIST HEAT or heat under microorganisms. steam pressure, is the agent - Catheter and pipes used in autoclaves. Putting - To disinf ect materials that may steam under 1 atm of pressure, have a biof ilm present, the or 15 psi, achieves a concentration of the disinf ectant temperature of 121°C. At this may need to be increased, the temperature, all microorganisms contact time may need to be (except f or prions) and their increased, or both. endospores are destroyed within approximately 15 minutes of COMPATIBILITY OF DISINFECTANTS exposure. - Some disinf ectants may inactivate other disinf ectants. - DRY HEAT may also be used as - For example, the use of bleach a sterilizing agent, although it and a quaternary ammonium requires much longer exposure compound together may negate times and higher temperatures the activity of both disinf ectants. than moist heat. METHODS OF DISINFECTION AND - BOILING AND STERILIZATION PASTEURIZATION are methods that achieve disinf ection but not sterilization; these methods do E. H. Spaulding categorized medical materials not eliminate spores. into three device classifications: 1. Critical materials BOILING (100°C) kills 2. Semicritical materials most microorganisms in 3. Noncritical material approximately 10 CRITICAL MATERIAL minutes. - Invade sterile tissues or enter the vascular system. Thes e PASTEURIZATION, used mostly in the food  industry, eliminates CHEMOSTERILIZER f ood-borne pathogens - All disinf ectants are regulated by and organisms the U.S. Environmental responsible f or food Protection Agency (EPA). spoilage. It is perf ormed Agents that are classif ied as at 63°C for 30 minutes. sterilants are regulated by the U.S. Food and Drug FILTRATION Administration (FDA) when they - Most bacteria, yeasts, and molds are to be used to sterilize devices are retained by pore sizes of 0.45 that will come in contact with and 0.80 µm; however, this pore patients. Chemical agents exert size may allow passage of their killing effect by the Pseudomonas-like organisms, following mechanisms: and theref ore a 0.22-µm size is Reaction with available f or critical sterilizing components of the (e.g., parenteral solutions). cytoplasmic membrane Membranes with pore sizes of Denaturation of cellular 0.01 µm are capable of retaining proteins small viruses. Reaction with the thiol (– - HEPA FILTERS are able to SH) groups of enzymes remove microorganisms larger Damage of RNA and than 0.3 µm and are used in DNA laboratory hoods and in rooms of immunocompromised patients. DISINFECTANTS VS. ANTISEPTICS - GERM THEORY of disease was TABLE 4-2: Control of Microorganisms Using one of the most important Heat contributions by microbiologists Method. to the general welf are of the worldwide population. The RADIATION medical community gradually - Two Forms: Ionizing and grew aware of the problem of Nonionizing nosocomial (hospital-acquired) inf ections and the need to - IONIZING RADIATION e f orm of practice asepsis to prevent the gamma rays or electron beams, contamination of wounds, is of short wavelength and high dressings, and surgical energy. This method of instruments. sterilization is used by the medical f ield f or the sterilization - The germ theory of disease also of disposable supplies such as contributed to the development syringes, catheters, and gloves. of antimicrobial chemotherapeutics. - NON IONIZING RADIATI ON radiation in the f orm of ultraviolet - Ignaz Semmelweis (1816– rays is of long wavelength and 1865) and Joseph Lister (1827– low energy. 1912) are considered to be important pioneers f or the TABLE 4-3: Chemical Agents Commonly Used promotion of asepsis. as Disinf ectants and Antiseptic - Ignatz Semmelweis CHEMICAL METHODS 1816-1865, Demonstrated routine  hand washing to prevent minutes and sporicidal in 3 to 10 the spread of disease. hours. Died with inf ection of Streptococcus HALOGENS pyogenes IODOPHORS - Joseph Lister - can be used as a disinf ectant in Academic surgeon one of two forms: tincture or benef ited by reading iodophor. Pasteur’s works about - TINCTURES are alcohol and bacteria as causes of iodine solutions, used mainly as inf ection bef ore he antiseptics. ventured into studies of - IODOPHOR (Povidone-iodine antisepsis. ~ Betadine) is a combination of Introduced handwashing iodine and a neutral polymer in 1867 and the use of carrier that increases the phenol as antimicrobial solubility of the agent. Iodophors agent f or surgical wound may be used as antiseptics or dressing to British disinf ectants, depending on the surgery. concentration of f ree iodine. Listerian Technique - This combination allows the slow was approved in the release of iodine. United States at the 1st of f icial meeting of the CHLORINE AND CHLORINE COMPOUNDS American Surgical Association in 1883, 20 CHLORINE AND CHLORINE years af ter Semmelweis’ COMPOUNDS initial publications. - Some of the oldest and most commonly used disinf ectants. ALDEHYDES - Form of hypochlorite, such as the liquid sodium hypochlorite FORMALDEHYDE (household bleach) and solid - An aldehyde generally used as calcium hypochlorite. formalin, a 37% aqueous - A solution containing 0.5% to 1% solution or f ormaldehyde gas. sodium hypochlorite is - It is not recommended that generally used f or disinf ecting. f ormaldehyde in any f orm be Such solutions are generally used as a disinf ectant or sterilant stable f or no longer than 30 days on a routine basis. with 50% of the original concentration of chlorine GLUTARALDEHYDE dissipating by 30 days. - A saturated f ive-carbon dialdehyde that has broad- DETERGENT: QUATERNARY spectrum activity and rapid killing AMMONIUM COMPOUNDS action and remains active in the - Derived by substitution of the presence of organic matter. f our-valence ammonium ion with - Extremely susceptible to pH alkyl halides. changes because it is active only - Their action is mediated through in an alkaline environment. disruption of the cellular - When used as a 2% solution, it is membrane, resulting in leakage germicidal in approximately 10 of cell contents.  - Certain bacteria, particularly Heavy metal disinf ectants are slowly gram-negative bacteria such as bactericidal; their action is primarily Pseudomonas aeruginosa, are bacteriostatic. intrinsically resistant to quaternary ammonium PERACETIC ACID compounds. - used in a gaseous f orm as a sterilant primarily in the PHENOLICS pharmaceutical and medical device manuf acturing industries. - Phenolics are molecules of phenol (carbolic acid) that have been HYDROGEN PEROXIDE AND chemically substituted, typically by PERACETIC ACID halogens or alkyl, phenyl, or benzyl - The major advantage to the use groups. of the combination of H2O2 and - Fairly broad spectrum of activity but are peracetic acid over each of its not sporicidal. individual components is a shorter contact time. CHLORHEXIDINE GLUCONATE (CHG) - Used f or more than 30 years in hospital setting GENERAL LABORATORY SAFETY - severe skin reactions may occur Hazardous Waste in inf ants younger than 2 Chemical Saf ety months. Fire saf ety Storage of compressed gases HEXACHLOROPHENE Electrical Saf ety - Primarily ef f ective against gram- Miscellaneous Saf ety Considerations positive bacteria. Saf ety Training - Chlorinated bisphenol that interrupts bacterial electron BOX 4-2: Classif ication of Inf ective transport, inhibits membrane- Microorganisms by Risk Group. bound enzymes at low concentrations, and ruptures BIOLOGICAL SAFETY CABINETS bacterial membranes at high (BSCs) concentrations. - Biological saf ety cabinets (BSCs) are a f orm of engineering CHLOROXYLENOL control that is used throughout - parachlorometaxylenol [PCMX] the microbiology laboratory. - PCMX at concentrations of 0.5% - These hoods are a type of to 4% acts by microbial cell wall containment barrier that protects disruption and enzyme the worker f rom the aerosolized inactivation. transmission of organisms. TRICLOSAN Biosafety Level 1. - A diphenyl ether that disrupts the - Are not known to cause disease cell wall. consistently in healthy adults. - The reaction time is - Minimal threat to laboratory intermediate, and the personnel and environment. persistence is excellent. Biosafety Level 2. HEAVY METALS - Inf ectious agents that require BSL-2 containment and practices are agents that pose a  moderate potential hazard f or the Incubators employees and the environment. Heating blocks Water baths Biosafety Level 3. Ref rigerators - BSL-3 containment and practices are required for Freezers inf ectious agents that are either indigenous or exotic. These EQUIPMENT QUALITY CONTROL agents have the potential for - Equipment used in the clinical aerosol transmission, and microbiology laboratory must be diseases with these agents may tested f or proper perf ormance at have serious lethal intervals appropriate f or each consequences. piece. This process may involve checking the percentage of CO2 Biosafety Level 4. in an incubator daily or - BSL-4 containment and measuring the rpm of a practices are required when centrif uge twice a year. working with agents that are - A preventive maintenanc e dangerous and exotic. Thes e program must be established as agents have a high risk of an additional control measure. causing lif e-threatening inf ections, can be transmitted by MEDIA QUALITY CONTROL aerosols, or have an unknown - Moisture: Plates should be f ree risk of transmission. As in all of moisture bef ore use but should microbiology practices, specific never show signs of drying training is required. Laboratory around the edges. personnel must receive thorough - Sterility: Plates should be f ree of training in the handling of these contaminants. dangerous agents, and they - Breakage: Petri dishes should must be trained in how to use the not be cracked or broken. containment barriers that are in - Appearance: Blood-based place f or protection. plates should not show signs of hemolysis, and any other plate FIGURE 4-7: Hazardous material classif ication that deviates f rom the normal symbol. (Courtesy Lab Saf ety Supply, Inc., color should not be used. Janesville, WI. REAGENT QUALITY CONTROL - Reagents should be tested on each day of use with both CHAPTER 5: PERFORMANCE positive and negative controls. Reagents that are documented IMPROVEMENT IN THE MICROBIOLOGY to have consistent and LABORATORY dependable results may be tested less f requently. Some reagents may be tested more GENERAL GUIDELINES FOR ESTABLISHING than once a day. - For the Gram stain, known QUALITY CONTROL gram-positive bacteria (Staphylococcus aureus ATCC TEMPERATURE 25923) and known gram- Daily temperature checks are required on all negative bacteria (Escherichi a temperature dependent equipment: coli ATCC25922) are recommended.  - QC in microbiology are: Physician’s name, All stains address, and phone Bacitracin number β-Lactamase Specif ic anatomic site Catalase Date and time of Coagulase specimen collection Gelatin Clinical diagnosis or relevant patient history Germ tube solution Hippurate Antimicrobial agents (if patient is receiving any) Kovacs reagent Name of individual Nitrate transcribing orders ANTIMICROBIAL SUSCEPTIBILITY QUALITY CONTROL PRESERVATION, STORAGE, AND TRANSPORT - The CLSI provides guidelines for OF SPECIMEN control of susceptibility testing. The recommended control Specimen transport is another essential organisms are specif ic strains component of the preanalytical process f rom the American Type of microbiology testing. Culture Collection. The primary goal in the transportation of specimens to the laboratory is to maintain the specimen as near to its original state CHAPTER 6: SPECIMEN COLLECTION AND as possible with minimal deterioration PROCESSING and to prevent risk to the specimen handler. Specimens should be transported to the TABLE 6-1: Specimen Collection Guideline laboratory ideally within 30 minutes of collection, pref erably within 2 hours. LABELING AND REQUISITION - Proper identification of each SPECIMEN STORAGE specimen includes a label f irmly attached to the container with the The individual responsible f or storing the f ollowing inf ormation: specimen needs to be inf ormed as to the Name best storage environment f or each Identif ication number specimen type. Some specimens, such Room number as urine, stool, sputum, swabs (not for Physician anaerobes), f oreign devices such as Culture site catheters, and viral specimens can be Date of collection maintained at ref rigerator temperature (4°C) f or 24 hours. Time of collection Body f luids, genital specimens, and ear and eye swabs. If cerebrospinal fluid is - The requisition form should not processed immediately, it can be provide the f ollowing inf ormation: stored in a 35°C incubator f or 6 hours. Patient’s name Patient’s age (or date of birth) and gender PRESERVATIVES Patient’s room number or location  Two specimen types in which The quantity of the specimen is preservatives can be used are urine and inadequate to perf orm all tests stool. requested. Boric acid is used in commercial The specimen transport time is more than products to maintain accurate urine 2 hours and the specimen has not been colony counts. preserved. Stool specimens f or bacterial culture The specimen is received in a f ixative that are not transported immediately to such as f ormalin; stools f or O & P the laboratory can be ref rigerated; if the examinations are an exception. delay is longer than 2 hours, the An anaerobic culture is requested on a specimen can be added to Cary-Blair specimen in which anaerobes are transport media. indigenous. Stools for Clostridium difficile toxin Microbiology processing of a particular assay should be collected without a specimen results in questionable data preservative and can be ref rigerated; if (e.g., Foley catheter tip). the delay is expected to be longer than 48 The specimen is dried up. hours, the specimen should be f rozen at More than one specimen f rom the same − 70°C. source was submitted f rom the same patient on the same day; blood cultures are an exception. ANTICOAGULANTS One swab was submitted with multiple used to prevent clotting of specimens, requests f or various organisms. including blood, bone marrow, and Gram stain of expectorated sputum synovial fluid. reveals f ewer than 25 white blood cells Sodium polyanethol sulf onate (SPS) is (WBCs) and more than 10 epithelial cells the most common anticoagulant used for per low-power f ield and mixed bacterial microbiology specimens. The f lora. concentration of SPS must not exceed 0.025% (wt/vol) because some MACROSCOPIC OBSERVATION Neisseria spp. and certain anaerobes are inhibited by higher concentrations. Notations f rom the macroscopic observation should include SPECIMEN PRIORITY the f ollowing: TABLE 6-3: Levels of Specimen Prioritization Swab or aspirate Stool consistency (f ormed or liquid) Blood or mucus present UNACCEPTABLE SPECIMENS AND SPECIMEN Volume of specimen Fluid—clear or cloud REJECTIONS The f ollowing situations are examples of suboptimal specimens that must be rejected: MICROSCOPIC OBSERVATION The inf ormation on the requisition does Microscopic observation serves several not match the inf ormation on the purposes: specimen label. If the patient name or 1) It can be used to determine the quality of source does not match, the specimen the specimen. Sputum specimens that should be collected again. represent saliva rather than lower There is no patient identif ication on the respiratory tract secretions can be specimen container. determined by the quantitation of WBCs The specimen is not submitted in the or epithelial cells. Similar Assessment appropriate transport container or the and determination can be made in container is leaking. samples collected f rom a wound site. Absence of WBCs may indicate that the  sample may not have been taken f rom SMEARS FROM THICK LIQUIDS OR the actual site of inf ection. SEMISOLIDS 2) It can give the microbiology technologist - Swabs also can be used as the and the physician an indication of the tool f or preparation of smears inf ectious process involved. Gram stain f rom thick liquid or semisolid of a sputum specimen revealing WBCs specimens such as feces. and gram-positive diplococci is indicative SMEARS FROM THICK, GRANULAR, of Streptococcus pneumoniae. OR MUCOID MATERIALS 3) The routine culture workup can be guided - Opaque material must be thinly by the results of the smear. The spread so that a monolayer of technologist can correlate the bacterial material is deposited in some isolates with the types detected in the areas. It is most desirable to smear; this may alert the technologist to have both thick and thin areas. the presence of additional organisms not - A better presentation of granules yet growing, such as anaerobes. is possible if granules or grains 4) It can dictate the need f or nonroutine or are “fished” f rom the additional testing. The presence of f ungal surrounding materials and elements in a specimen f or bacterial crushed on a separate slide culture would alert the technologist to using the technique. notif y the physician to request a f ungus culture. INCUBATION Most bacteria cultures are incubated at 35°C to 37°C. Aerobes grow in ambient air, whereas anaerobes cannot grow in the presence of oxygen and require an anaerobi c atmosphere. CHAPTER 7: MICROSCOPIC EXAMINATION OF MATERIALS FROM INFECTED SITES PREPARATION OF SAMPLES Samples f or routine bright-f ield microscopy are prepared in a manner that f acilitates adequate examination within a reasonable time. For smears, specimens should be examined grossly to determine the best approach (Table 7.1). Both thick, but not opaque, and thin (monolayer) smear areas should be produced by the smear process chosen. SMEARS FROM SWABS - Smears should not be prepared f rom a swab af ter it has been used to inoculate culture media.