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CLINICAL BACTERIOLOGY(Lecture) 2 Categories of Disease Caused by INTRODUCTION TO MICROBIOLOGY Pathogens Pathogens cause two major types of dise...

CLINICAL BACTERIOLOGY(Lecture) 2 Categories of Disease Caused by INTRODUCTION TO MICROBIOLOGY Pathogens Pathogens cause two major types of diseases: MICROBIOLOGY - Is the study of living organisms of Infectious Diseases microscopic size. - a type of disease in which a pathogen colonizes a person's body. - Microbiology is the study of microbes. Individual microbes can be observed only with Microbial Intoxications the use of various types of microscopes. - a type of disease in which a pathogen produces toxin that once ingested by the - The various categories of microbes include host can cause an intoxication. viruses, bacteria, archaea, protozoa, and certain types of algae and fungi. av Microbes Microbes that cause disease are known as pathogens. Those that do not cause disease are called nonpathogens. esPathogenic Nonpathogenic - Cause infection - Harmless History of Microbiology and disease - Can be beneficial A. Earliest Known Infectious Diseases - Distinct shapes an - Many live on - Some produce surface of skin - The earliest known account of a "pestilence" toxins occurred in Egypt about 3180 BC. This may - Saprophytes live represent the first recorded epidemic, although - Spread easily on dead matter words like pestilence and plague were used without definition in early writings. - Live everywhere - 70% of all bacteria - Around 1900 BC, near the end of the Trojan War, sh Categories of Microbes the Greek army was decimated by an epidemic of what is thought to have been bubonic plague. The two major categories: - The Ebers papyrus, describing epidemic fevers, 1. Acellular Microbes was discovered in a tomb in Thebes, Egypt; it was - also called infectious particles. written around 1500 BC. 2. Cellular Microbes - A disease thought to be smallpox occurred in - also called microorganisms China around 1122 BC. - Epidemics of plague occurred in Rome in 790, 710, and 640 BC and in Greece around 430 BC. B. Christopher Columbus E. Antonie Van Leeuwenhoek (1632-1723) - Aside from those already mentioned: there are - He is considered as the "first true early accounts of rabies, anthrax, dysentery, microbiologist”. smallpox, ergotism, botulism, measles, typhoid fever, typhus fever, diphtheria, and syphilis. - He is the first person to observe and accurately av describe living microorganisms (rain water, pond, - Syphilis - It made its first appearance in lake, etc.) Europe in 1493. Many people believe that syphilis was carried to Europe by Native - Regarded as the "FATHER OF BACTERIOLOGY Americans who were brought to Portugal by AND PROTOZOOLOGY" Christopher Columbus. - He used the term "ANIMALCULES" or the tiny es C. Lucretius (98-55 B.C) Fracastoro (1478-1553) and Girolamo living and moving cells seen under the microscope. F. Aristotle - Theory Of Spontaneous Generation (Abiogenesis) an - It states that life arises from non-living material if it contains "pneuma" (vital heat). - The theory suggests that organisms do not descend from other organisms or from a parent - They suggested that diseases were caused by and only require that certain condition in their "invisible creatures". environment be fulfilled in order for creation to sh occur. D. Girolamo Fracastoro - Living organisms arise from non-living organisms in combination with energy (earth, water, air, vital heat etc.). G. Francesco Redi - Italian, wrote poems about STDS (Syphilis) "infections result from tiny self- multiplying bodies that can be spread by direct/indirect contact". - demonstrated that maggots were the o spring BIOGENESIS of flies, not products of spontaneous generation. - He resolved the issue on Spontaneous H. John Needham Generation with a series of ingenious and persuasive experiments. - He demonstrated that microorganisms are present in the air and can contaminate sterile solutions, but air itself does not create microbe. - He proposed the use of heat in killing microorganisms (ASEPTIC TECHNIQUE) or a method used in preventing contamination by unwanted organisms. - microbes arose spontaneously in broth from a "life force." - PASTEUR'S CONTRIBUTION TO MICROBIAL av SCIENCE I. Lazzaro Spallanzani He disproved the theory of spontaneous generation. He developed the vaccine against anthrax (1881) and rabies (1885) He improved the wine-making process (fermentation and pasteurization) es K. Rudolf Virchow (1821-1902) an - Heated but sealed flasks remained clear, without any signs of spontaneous growth, unless the flasks were subsequently opened to the air. J. Louis Pasteur (1822-1895) - Proposed the theory of biogenesis, which states that living cells can arise only from sh pre-existing living cells. Theory of Antisepsis L. Ignaz Semmelweis (1816-1865) - suggested that the exposure of a broth to air was not introducing a "life force" to the broth but rather airborne microorganisms. he boiled broth to sterilize it. - Pasteur articulated "Omne vivum ex vivo" ("Life only comes from life") "...life is a germ and a germ is life". - He demonstrated that routine handwashing can prevent the spread of diseases (puerperal 1. The microorganism must be found in diseased fever). but not healthy individuals M. Joseph Lister (1827-1912) 2. The microorganism must be cultured from the diseased individual 3. Inoculation of a healthy individual with the cultured microorganism 4. The microorganism must be re-isolated from the inoculated, diseased individual and match to the original microorganism Immunology (Advent of Vaccination) - He introduced the system of antiseptic surgery av in Britain (antiseptic and preventive medicine O. Edward Jenner (1749-1823) pioneer) (phenol). - He pioneered promoting handwashing before and after operation, the wearing of gloves, sterilizing surgical instruments, and the use of phenol as an antimicrobial agent for surgical es wound dressing. N. Robert Koch (1843-1910) - He introduced the smallpox vaccination through cowpox inoculation. an P. Louis Pasteur and Pierre Paul Emile Roux sh - He was the first to show irrefutable proof that bacteria cause diseases. - He discovered: Bacillus anthracis (1876), Mycobacterium tuberculosis (1882). - Pasteur used the term "vaccine" for an - He was the first to cultivate bacteria on boiled attenuated culture (reduced virulence) They potatoes, gelatin, meat extract and protein made a series of experiments to produce (ingredients of culture medium) attenuated strains of bacteria. - He developed a culture media for observing bacterial growth isolated from the human body. KOCH’S POSTULATES Four (4) criteria to assess if a microorganism can cause a disease (Segre, 2013):  Modern Therapy: “Magic Bullet” - He discovered that there are bacteria that could withstand a series of heating and boiling Q. Selman Waksman (1888-1973) because of heat-resistant structures known as endospores. U. Fanny Hesse (1850-1934) - He discovered the streptomycin and neomycin antibiotics. - She suggested the use of Agar, a solidifying av R. Alexander Flemming (1881-1955) agent, in the preparation of culture media. V. Julius Richard Petri (1852-1921) es - He accidentally discovered the antibiotic Penicillin (Penicillium notatum) (fungi). - He developed the petri dish. an - He Discovered the lysozyme. W. Robert Hooke, ENGLISH-MAN (1665) S. Paul Ehrlich (1854-1915) sh - Observe thin slices of cork. - He discovered salvarsan (arsphenamine) for - Proposed cell theory the treatment of syphilis. T. Ferdinand Cohn (1828-1898)  Microbial Taxonomy Distinguishing Characteristics: TAXONOMY - Relatively small in size (1µm in - is an area of biologic science that comprises diameter) three distinct but highly interrelated disciplines: ○ Classification - Absence of nuclear membrane ○ Nomenclature (naming) ○ Identification - The DNA of almost all bacteria Classification is a circle with a length of 1mm. It is the organization of microorganisms that have - Most prokaryotes have only similar morphologic, physiologic, and genetic traits single chromosome into specific groups or taxa. - The specialized region of cell The formal levels of bacterial hierarchical containing DNA is termed as classification in successively smaller taxa or av NUCLEOID and can be subsets are as follows visualized by EM (electron microscope) - Have the capacity to exchange small packets of genetic information (this information is carried on PLASMIDS) es Family - encompasses a group of organisms that may contain multiple genera and consists of organisms with a common attribute. an - The name of a family is formed by adding the su x -aceae to the root name of one of the group's genera, called the type Classification genus Eukaryotes Distinguishing characteristic: Genus - Contains di erent species that sh - The "true nucleus" of have several important features eukaryotes (from Gr karyon in common. "nucleus") is only one of their Species - (abbreviated as sp., singular, or distinguishing features. spp., plural) is the most basic of the taxonomic groups and can - The membrane-bound be defined as a collection of organelles, the microtubules, and bacterial strains that share the microfilaments form a common physiologic and genetic complex intracellular structure. features and di er notably from other microbial species. - Flagella and cilia -complex multi-stranded structures. Subspecies - Serotype: - based on serologic Prokaryotes - No membrane bound di erences organelles ○ Eg. E. coli (>700 serotypes)  - based on features beyond the genetic level and include both readily observable - Genotype: - based on characteristics and those that may require biochemical di erences ○ Eg. extensive analytic procedures to be detected. Treponema pallidum subsp. ➔ Macroscopic and microscopic pallidum morphology (inoculated) ➔ Staining characteristics(gram staining, any staining technique) Nomenclature ➔ Nutritional requirements (an/aerobic organism, needs oxygen, doesn't need Naming of microorganisms according to additional vitamins, etc. for growth) established rules and guidelines set forth by the: ➔ Biochemical Testing (results) International Code of Nomenclature of Bacteria (ICNB) Phenotypic the Bacteriological Code (BC) Macroscopic The microbial growth av Genus and Species morphology patterns on artificial media as observed when RULES: inspected with the unaided eye. 1. Genus designation – first letter is always Examples include the size, capitalized, texture, and pigmentation of bacterial colonies es 2. Species designation – first letter is always lower case Microscopic morphology The size, intracellular shape, inclusions, collular appendages, and 3. Printed in italics arrangement of cells when observed with the aid of 4. Underlined in script microscopic an magnification. 5. The name may be abbreviated by using the upper case form of the first letter of the genus Staining The ability of an organism characteristics to reproducibly stain a designation followed by a period (.) and the full particular color with the species name, which is never abbreviated application of specific dyes and reagents. 6. Informal designation may be used to label a particular group of organisms. These Staining in used in sh designations are not capitalized or italicized. conjunction with microscopic morphology for bacterial identification. Identification For example, the Gram stain for bacteria is a Process by which a microorganism's key critical criterion for features are delineated. di erential identification. Environmental The ability of an organium IDENTIFICATION METHODS: requirements to grow at various temperatures, in the A. Genotypic Characteristics presence of oxygen and - organism's genetic make-up, including the other gases, at various pH nature organism's genes and constituent nucleic levels, or in the presence acids of other ions and salts, ➔ DNA base composition ratio such as NaCl Nutritional The ability of an organism B. Phenotypic Characteristics requirements to utilize various carbon  and nitrogen sources as Nucleic acid (DNA The order of bases along a nutritional substrates and ribonucleic strand of DNA or RNA is when grown under specific acid [RNA]) base known as the base environmental conditionis sequence sequence. The extent to characteristics, which sequences are Resistance profiles The exhibition of a including those homologous (similar) characteristic inherent determined by between two resistance to specific hybridization microorganisms can be antibiotics, heavy metals, assays determined directly or or toxins indirectly by various molecular methods. The Antigenic The profiles of degree of similarity in the properties microorganisms sequences may be a established by various measure of the degree of serologic and immunologic organism relatedness, methods to determine specifically, the ribosomal relatedness among RNA (RNA) sequences that various microbial groups. remain stable in av comparison to the genome Subcellular Molecular constituents of as a whole properties the call that are typical of a particular taxon, or organism group, as Role of Taxonomy in Diagnostic established by various Microbiology analytic methods. Some examples include cell wall - Establishes and maintains records of key es components, components of the cell membrane, and enzymatic content of the characteristics microorganisms of clinically relevant microbial cell - Facilitates communication among Chemotaxonomic The chemical constituents technologists, microbiologists, physicians, and properties of the cell, such as the scientists by assigning universal names to an structure of teichoic acids, clinically relevant microorganisms. This is fatty acid analysis, and essential for: protein profiles, as determined by analytical Establishing an association of particular methods diseases or syndromes with specific microorganisms Epidemiology and tracking outbreaks sh Genotypic Accumulating knowledge regarding the Deoxyribonucleic DNA comprises four bases management and outcome of diseases acid (DNA) base (guanine, cytosine, associated with specific composition ratio adenine, and thymine). The extent to which the microorganisms DNA from two organisms is made up of cytosine and Establishing patterns of resistance to guanine (i.e., G+ C antimicrobial agents and recognition of content) relative to their changing microbial resistance patterns total base content can be used as an indicator of relatedness or lack Understanding the mechanisms of thereof. For example, an antimicrobial resistance and detecting organism with a G + C new resistance mechanisms exhibited content of 50% is not by microorganisms closely related to an organism with a G + C Recognizing new and emerging content of 25%, pathogenic microorganisms  Recognizing changes in the types of ➔ Aware of potential hazard infections or diseases caused by characteristic microorganisms ➔ Trained and proficient in techniques Revising and updating available technologies for the development of ➔ Supervisors responsible for: new methods to optimize the detection appropriate laboratory and identification of infectious agents facilities, personnel and and the detection of resistance to training antiinfective agents (microbial, viral, fungal, and parasitic) b. Special practices and considerations -WHO programs Developing new antiinfective therapies (microbial, viral, fungal, and parasitic 2. SAFETY EQUIPMENT ➔ Primary containment barrier av INFECTION CONTROL AND ➔ Minimize exposure to hazard -prevent LABORATORY SAFETY contact/contain aerosols According to WHO: ➔ Engineering controls/equipment - The risk associated with biological materials in the laboratory has a ➔ PPE-gown, gloves, respirator, face es safety and a security component LABORATORY BIOSAFETY ➔ shield booties BSC - Containment principles, technologies, and practices implemented to prevent unintentional ➔ Covered or ventilated animal cage exposure to pathogens and toxins, or their systems unintentional release. an 3. FACILITY DESIGN AND CONSTRUCTION "PROTECTING PEOPLE FROM DANGEROUS ➔ Secondary barrier/engineering PATHOGENS" controls ➔ Protect the users. ➔ Contributes to worker protection ➔ Protect those outside the labs ➔ Protect the environment ➔ Protects outside the laboratory sh Ex-bldg. and lab design, LABORATORY BIOSECURITY ventilation, autoclaves, cage, wash - Institutional and personal security measures facilities 4 designed to prevent the loss, theft, misuse, diversion, or intentional release of pathogens 4. INCREASING LEVELS OF and toxins. PROTECTION-BIOSAFETY LEVELS 1-4 "PROTECTING PATHOGENS FROM Biohazard Symbol DANGEROUS PEOPLE” Principles of Biosafety 1. PRACTICE AND PROCEDURES a. Standard practices ➔ Most important concept/strict adherence  risk that can be readily transmitted from one individual to another. E ective There are four circles within the symbol, treatment and signifying the chain of infection. preventive 1. Agent measures are not usually available 2. Source Biosafety Levels 3. Host Level of the biocontainment precautions 4. Transmission: The means of transmission, required to isolate dangerous biological agents mostly direct or indirect. Some routes of in an enclosed facility. transmission include air, insect, direct contact and contaminated surfaces. Combination of laboratory practices and av procedures, safety equipment (primary barriers and laboratory facilities (secondary Risk Risk Level Description Group barriers) (based on the risk groups of microorganism). 1 No or low A microorganism individual that is unlikely to specific containment controls that are and cause human or required for the following (CDC): 2 es community risk Moderate animal disease A pathogen that Laboratory practices Safety equipment Facility construction individual can cause human or risk, low animal disease but community is unlikely to be a risk serious hazard to an the environment. Laboratory exposure may cause serious infection but e ective treatment and preventive measures are sh available 3 High A pathogen that individual usually causes risk, low serious human or community animal disease but CDC Biosafety Levels risk does not ordinarily spread from fone BSL 4 Dangerous and Ebola infected individual exotic, posing a to another. E ective high risk of Smallpox treatment and aerosol-transmitt preventive ed infections. (no BSL 4 in the measures are Infections caused Philippines available by these microbes are 4 High A pathogen that frequently fatal individual usually causes and without and serious human or treatment or community animal disease and  vaccines Work can be performed on an open table or bench BSL 3 Microbes there HIV, H1N1 Flu, can be either Yersinia pestis PPE (Personal Protective Equipment) needed indigenous or (The Plague), exotic, and they Tuberculosis, can cause serious SARS, Rabies, or potentially West Nile Virus, SAFETY EQUIPMENT lethal disease Rickets through Special containment devices or equipment, respiratory such as BSCs - not generally required transmission Protective laboratory coats or uniforms are BSL 2 Microbes Most recommended potential hazard chlamydiae, to personnel and Hepatitis A-C, Wear gloves to protect hands from exposure the environment. Influenza A, to hazardous materials Includes bacteria Lyme Disease, av and viruses that Salmonella, STANDARD MICROBIOLOGICAL cause only mild Mumps, Measles PRACTICES disease to humans, or are Hand washing practices di cult to contract via Eating, drinking, smoking, handling contact aerosol in a lab lenses applying cosmetics, and storing setting. (most food-not permitted in laboratory es BSL 1 laboratories Not known to Canine hepatitis, Food must be stored outside the laboratory area in cabinets or refrigerators designated consistently non- pathogenic and used for this purpose cause disease in Escherichia coli, healthy adult and Mouth pipetting is prohibited humans, and of non-infectious an minimal potential bacteria Policies for safe handling of sharps must be hazard implemented. tolaboratory personnel and environment BIOSAFETY LEVEL 2 - Designed for laboratories that deal with BIOSAFETY LEVEL 1 indigenous moderate-risk agents present in the community. sh - For undergraduate and secondary educational training and teaching laboratories. - Practices, equipment and facility design applicable to clinical, diagnostic, teaching - Microbes not known consistently to cause laboratories disease in healthy adults and present minimal potential hazard to lab and environment - Microbes that possess moderate hazards to laboratorians. - Open bench-No containment Nonpathogenic strain of Escherichia - Accidental percutaneous or mucous coli, Bacillus subtilis membrane exposures, or ingestion of infectious materials BSL-1 PRACTICES Hepatitis virus, HIV, Salmonella enterica spp Standard microbiological practices are followed BSL-2 PRACTICES  Standard Microbiological Practices (Same as controlled BSL-1) SAFETY EQUIPMENT Access to laboratory is restricted when working is being conducted All procedures must be conducted within a BSC (preferably Class II or Class III), or other physical containment devices. SAFETY EQUIPMENT Use protective laboratory clothing with a solid-front, such as tie-back or Properly maintained BSCs, personal wrap-around gowns, scrub suits. protective equipment (gloves, goggles, mask, face shield) and/or physical Eye and face protection (goggles, mask, containment devices must be used face shield or other splash guard) especially during: Procedures with potential for Gloves must be worn. creating infectious aerosols or splashes are conducted PPE with respirators av High concentrations or large volumes of infectious agents are Biosafety Cabinets used Sink with eyewash STANDARD MICROBIOLOGICAL PRACTICES Exhaust air-Not recirculated Autoclave/Decontamination proper es Self-closing doors with automatic locking Self-Closing doors STANDARD MICROBIOLOGICAL PRACTICES Sink with eyewash apparatus readily available Same as BSL-1 & 2 A sign incorporating Method for decontaminating all laboratory Universal biohazard symbol wastes should be available in the facility, an Laboratory's biosafety level preferably within the laboratory Supervisor's name, Telephone number Example: Autoclave, Chemical disinfection, Required procedures for entering incineration) BIOSAFETY LEVEL 3 BIOSAFETY LEVEL 4 - Applicable to clinical, diagnostic, teaching, - For working with dangerous and exotic sh research agents that pose a high individual risk of life-threatening disease that may be - Serious/potentially lethal disease through transmitted via the aerosol route, for which respiratory transmission there are no available treatment or vaccines. - Primary hazards-relate to autoinoculation, - Highest level of biological safety ingestion and exposure to infectious aerosols Mycobacterium tuberculosis, Coxiella - Dangerous and exotic microbes burnetiid, St. Louis encephalitis Example: EBOLA, Marburg Viruses, Crimean-Congo hemorrhagic fever BSL-3 PRACTICES BSL-4 PRACTICES Laboratorians - under medical surveillance and receive immunization Same with BSL-3 Access to laboratory is restricted and Change clothes before entering  Shower upon exiting pressure back to the positive plenum, and it will Decontaminate all materials before exiting not leak out Class III BSC In the A2 cabinet, about 70% of air from the Separate building for laboratory positive plenum is recirculated as downflow, Vacuum lines and decontamination and the remaining 30% is systems discharged to the lab through the exhaust filter Biological Safety Cabinet (BSC) Class II Type B1 Has a common plenum from which 70% of air is Mainly used for handling pathogenic biological exhausted, and 30% samples or for applications that require a sterile re-circulated to the work area as the downflow work zone. av This cabinet also has a A biological safety cabinet creates inflow and dedicated exhaust feature downflow of air that provides operator that eliminates protection. re-circulation when work is performed towards the Air that contains the infectious material is back within the interior of the cabinet. sterilized, either by heat, ultraviolet light, or, es most commonly, by passage through a HEPA filter that removes most particles larger than 0.3 mm in diameter. Toxic chemicals employed as an adjunct to microbiological processes should only be allowed if ➔ The downflow air passes through an they do not interfere with ULPA/HЕРА asses through filter and work when re- circulated in the downflow creates an ISO Class 3 work zone to an protect samples from the risk of cross- The exhaust air in class contamination IIB cabinets is discharged ➔ The air exhausted also passes through outside the building. A an ULPA/HEPA filter prior to release to class IIB cabinet is protect the environment. selected if radioisotopes, toxic chemicals, or carcinogens will be used. Class II Type A2 Is the most common Class sh II cabinet Class II Type B2 Are suitable for work with toxic chemicals employed It has a plenum from as an adjunct to which 30% of air is microbiological processes exhausted, and 70% under all circumstances re-circulated to the work since no re- circulation area as the downflow occurs. Both the Class II Type A1 In theory, Type B2 and Type A2 must have biological safety cabinets the positively-pressurized may be considered as the contaminated plenum to safest of all Class II BSCs be surrounded by since the total exhaust negative pressure. In case feature acts as a fail-safe there is a leakage on the in the event that the positive plenum, the downflow and / or leaking aerosol will be exhaust HEPA filtration pulled by the negative systems cease to function  - Illumination is adequate for all laboratory normally. activities. However, Class II Type B2 biosafety cabinets require - Storage space is adequate large laboratory spaces due to their installation Issues to Consider system and will require elaborate ducting works After reading the patient's case history, Class III biological Suitable for work with consider safety microbiological agents assigned to biosafety The role of the laboratory worker and levels 1, 2, 3 and 4. They microbiology laboratory in an infection are frequently specified prevention and control program for work involving the most lethal biological hazards The surveillance of health care-associated infections (HAls) and av Work is performed the required laboratory support through glove ports in the front of the cabinet. The information needed in an outbreak investigation During routine operation, negative pressure relative to the ambient The role of the laboratorian as an environment is educator in infection prevention and es maintained within the biosafety cabinet. This provides an additional control Bioterrorism and emerging pathogens fail-safe mechanism in case physical containment INFECTION PREVENTION AND is compromised. On all CONTROL Class III BSCs, a supply of an HEPA filtered air provides product protection and WHAT IS INFECTION PREVENTION AND prevents cross CONTROL (IPC)? contamination of samples - Variety of measures practiced by health care personnel to prevent Exhaust air is usually spread, transmission and acquisition HEPA filtered and and spread of infection. incinerated. Alternatively, double HEPA filtration sh with two filters in series Goals of Infection Prevention and may be utilized Control Laboratories Facilities in BSL-2 IPC is a universal discipline with relevance to all aspects of healthcare. - Laboratory should be designed in such a way that it can be easily cleaned. Assures that HCWs understand how pathogens can be transmitted in their - Laboratory contains a sink for washing. work environment. - Laboratory tops are impervious to water but Apply current scientifically accepted resistant to acids, alkalis and organic solvents. infection control principles appropriately. - An autoclave to decontaminate infectious material is available.  Minimize opportunity for transmission Personal Protective Equipment of pathogens to patients and healthcare workers Eye Protection Goggles - used as eye protectors from Hierarchy of Safety and Health Controls flying particles, dust, wind, chemical fumes or other external hazards Respiratory Mask Protection - a covering for the face that prevents droplets from the mouth and nose from spreading in the air. Respirator - masks are a form of respiratory protection PPE that protects the wearer from av inhaling harmful airborne particles, gases or vapors that are present in the - The National Institute for Occupational environment. Safety and Health (NIOSH) defines five rungs of the Hierarchy of Controls: Gloves Gloves protect and comfort 1. Elimination hands against cold or heat, damage by friction, abrasion es 2. Substitution 3. Engineering controls 4. Administrative controls Isolation or chemicals, and disease Hospital gowns worn by 5. Personal protective equipment. Gown/Coveralls medical professionals as personal protective - The hierarchy is arranged beginning with the equipment (PPE) in order to most e ective controls and proceeds to the provide a barrier between an patient and professional least e ective Shoe cover Shoe covers are important as General Strategies they help maintain a sanitary environment by eliminating tracked-in dirt and microbes and they protect the wearer from accidental spills and body fluids. sh HAND HYGIENE Hair Covering Surgical caps are a part of - Hand Rub medical protective clothing ➔ When hands are not visibly and should prevent germs soiled using alcohol-based from the hair or scalp of antiseptic gel or sanitizer. surgical personnel from contaminating the operating - Hand wash area. ➔ When hands are visibly soiled ➔ After using gloves or going to Face Protection Face Shield the bathroom - that protects the mucous membranes of the eyes, nose and mouth during patient-care procedures.  Donning and Do ng of PPE PROPER DISPOSAL Remember: 1. Approved sharps containers must be placed in Sequence of Donning Sequence of areas where sharps may be utilized of PPE Do ng of PPE 1. Gown 1. Gloves 2. Containers must be labeled with the date that waste accumulation begins 2. Mask or respirator 2. Face shield or goggles 3. Sharps containers must be properly closed and 3. Goggles or face sealed prior to disposal. shield 3. Gown DISEASES TRANSMITTED THROUGH NEEDLE 4. Gloves 4. Mask or respirator STICK INJURY HIV (0.3%) Safe Injection Practices HBV (6-30%) av HCV (1.8%) Intended to prevent transmission of infectious diseases during phlebotomy. SHARPS Transmission - Based Precaution - Do not uncover or unwrap the sharps until time of usage Supplement standard precautions in patients with es - Keep sharp pointed away from yourself and others known or suspected colonization of infection of highly transmissible or epidemiologically important pathogens. - Never recap or bend Airborne Droplet Contact - Keep fingers away from the tip an Tuberculosis, Pneumonia, Scabies, Measles, Influenza, Poliomyelitis, - If reusable sharp, place inside a secure Varicella Diphtheria, Congenital and closed container after usage COVID-19 Ruella ○ Use tongs, forceps or one-handed Filtered scoop to recap Respirator Regular Use (N95 Mask) Medical Mask gown/apron - Never hand a sharp to someone else or put and gloves for Patient must Distance at any procedure sh in on a tray for someone to pick up. be placed in a least 3 feet involving negative apart. Educate contact with a Remember: pressure room about cough patient or Sharps containers should be placed where (Ideally) etiquette. May their sharps are used be placed in a immediate private room environment. Containers should have date when waste or cohorting MAy be accumulation began labeled placed in a private room Containers have to be properly closed and or cohorting. sealed before disposal Waste Management DO NOT RECAP! If it is essential that a needle be recapped due to Collection, transport, processing or disposal, the nature of your work, the use of a mechanical managing and monitoring of waste materia device (e.g., tongs, forceps, or the one-handed scoop method) must be used. Purpose: ORANGE RADIOACTIVE/ ○ Reduce hazardous nature waste NUCLEAR WASTES ○ Reduce volume of waste ○ Prevent misuse or abuse of waste lodine 125 (1-125) ○ Ensure occupational safety and health lodine 131 (1131) All wastes generated by the hospital shall be H3-Thymidine segregated and disposed properly in accordance to the DENR-DOH Administrative Order No. 02 Cesium 137 Biodegradable, non-biodegradable and infectious Chromium 51 wastes shall be placed in green, black and yellow bags respectively. Technetium 99m RED SHARPS & BLACK NON-INFECTIOUS PRESSURIZED DRY WASTES CONTAINERS av Paper & Paper Needles & syringes Products Scalpel blades Bottles Glass vials - tuberculin es Packaging materials / insulin GREEN NON-INFECTIOUS Stylet WET WASTES Capillary tubes Leftover food Ampules Used cooking oil Blood evacuation an Fish entrails, scale & tubes fins Pipette slides / cover Fruits & vegetable slips peelings Rotten fruits & Aluminum cover vegetables Blood lancets YELLOW INFECTIOUS & sh PATHOLOGICAL Empty aerosol cans WASTES Rusty pins, nails, clips Foreign bodies & screws removed from any body parts Broken glasses Used gloves Microbial Growth Control Used tubing-IV, nebulizer STERILIZATION AND DISINFECTION - May be accomplished by Physical or chemical Used test strips means Used urine bags Sterilization - All forms of microbial life are killed Used mask/face mask ○ Targets endospores/bacterial spores - most important structure present in bacteria being targeted by sterilization.  Physical Agents Chemical Agents Sanitation Use of chemical agents on food-handling equipment Sunlight Agents that can to meet public health damage cell standards Drying membrane ○ Surface-active Minimizes chances of Heat disinfectants disease transmission ○ Dry heat ○ Phenolic ○ Moist heat compounds Factors that Organic load ○ Alcohols limit success or ○ Total number of Filtration degree of organisms present Agents that damage sterilization, (microbial Radiation proteins disinfection, or load/bioburden) ○ Acids and decontaminatio ○ Organisms and other Ultrasonic and Sonic alkalines n in a health contaminating materials vibrations ○ Alcohols care setting (blood and body fluids) Type of organisms present av Agents that can modify functional groups of proteins Concentration and and nucleic acid exposure time to the ○ Heavy metals germicide ○ Oxidizing agents ○ Dyes Physical and chemical ○ Alkylating nature of the surface agents es Microbial Growth Control Temperature, pH, humidity Presence of a biofilm (microorganisms living together) Disinfection Pathogenic organism, but not all microorganisms are destroyed an ○ Targets vegetative state Antisepsis Destruction of vegetative state on living tissues ○ Targets vegetative state sh Disinfectant Chemical agent applied to inanimate objects ○ Sodium hypochlorite & Lysol Antiseptic Chemical agent applied to living tissues ○ Hydrogen peroxide ○ Betadine ○ Antiseptic mouthwash Decontaminatio Removes pathogenic n microorganisms Degerming Mechanical removal of most microbes in a limited area ○ Skin around puncture site  5. Spores BACTERIAL PHYSIOLOGICAL IDENTIFICATION - Highly refractile resting cells that are highly durable and dehydrated with thick walls Prokaryotic Cell Components A. Cytoplasmic Structures B. Cell Envelope Structures C. Surface Polymers or Appendages Gram Stain Schae er-F B. Cell Envelope av I. Plasma Membrane (Cell Membrane) - A phospholipid bilayer with embedded proteins that envelop the cytoplasm es A. Cytoplasmic Structures 1. Nuclear Area (Nucleoid) - Single circular chromosome attached to a an mesosome II. Cell Wall (Peptidoglycan or murein layer) - Maintains the shape of the cell and 2. Plasmids protects cell from osmotic pressure - Repeating disaccharide attached by - Small circular, dsDNA molecule polypeptide - Antibiotic resistance sh 3. Ribosomes - Site of protein synthesis - Consists of RNA and protein 4. Inclusions Metachromatic Granules – represents reserves of polyphosphates used in the synthesis of ATP Polysaccharide Granules – consist of III. Periplasmic Space glycogen and starch granules - Between cell membrane and cell wall - Consist of gel like matrix containing nutrient binding nutrients Contains enzymes for degrading and detoxifying macromolecules.  IV. Outer Membrane C. Surface Polymers and Appendages - Cells initial barrier (certain antibiotics and evasion of phagocytes) I. Glycocalyx ( Slime Layer and Capsule) - Composed of Lipopolysaccharides - General Substances that surround cells (LPS), lipoproteins and phospholipids - Gelatinous polymer of polysaccharide, - Contains Porins polypeptide, or both Water-filled structures that control the passage of a. Capsule - substance is organized and nutrients and solutes. firmly attached to cell wall b. Slime Layer - unorganized and loosely attached to the cell wall. Functions: - Prevents phagocytosis av - Attachment to various surfaces in its natural environment - B. anthracis es - S. pneumoniae - K. pneumoniae CELL ENVELOPE CHARACTERISTICS GRAM GRAM POSITIVE NEGATIVE an Peptidoglycan Layer Thick Thin (single (multilayer) layer) II. Flagella Teichoic acids Present in many Absent - Exterior protein filaments that rotate Periplasmic Space Absent Present and cause bacteria to be motile - Four arrangements of flagella : Outer Membrane Absent Present sh a. Monotrichous – a single polar LPS Content Virtually none High flagellum Lipid & Lipoprotein Low High (due to outer b. Amphitrichous – two or more flagella membrane) at one pole of the cell c. Lophotrichous – single/tuft of flagella at each end of the cell. d. Peritrichous – flagella distributed over the entire cell. III. Fimbriae and Pili BACTERIAL METABOLISM - Hairlike appendages that are shorter, straighter and thinner than flagella. Various biochemical pathways exist for a. Fimbriae (sing. Fimbria) substrate breakdown in the microbial world, – also called common pili, can occur at the and the particular pathway used determines poles or can be evenly distributed from few to the end product and final pH of the medium. several hundred. Diagnostic Schemes Function: for adherence of cells to one another and to environmental 1. Utilization of various substrates as a carbon surfaces. source b. Pili (pilus) 2. Production of specific end products from – also called sex (conjugation) pili, are hollow various substrates protein tubes, longer than fimbria and number av is 1 or 2 per cell 3. Production of an acid or alkaline pH in the test medium Function: join bacterial cell in preparation of DNA transfer from one Fermentation Respiration cell to another. Anaerobic process E cient energy carried out by both generating process in es obligate and facultative anaerobes which molecular oxygen is the final electron acceptor - End products: lactate, - Obligate aerobes and Fimbrae Pili ethanol, butyrate, facultative anaerobes acetoin an IV. Axial Filaments (Endoflagella) - Used for MRVP - Bundles of fibrils anchored at one end of spirochete and spiral around the cell. Fermentation Respiration - The rotation of filaments propels the spirochete in a spiral motion. Definition The energy The cellular production process that process that produces sh occurs mainly energy when in the oxygen is microorganism present s when the oxygen is not present Presence of Occurs in the Occurs in the Oxygen absence of presence of oxygen oxygen. Even can occur in the absence of oxygen as well. Energy 2 ATP 36 ATP Molecules Products Ethanol CO2  Organic acids H2O Carbohydrate Utilization and Lactose Fermentation CO2 Energy Ability of microorganism to use various Types Ethanol Aerobic sugars for growth in an integral part of most fermentation respiration, diagnostic identification schemes. and lactic acid anaerobic fermentation respiration Fermentation of sugar is usually detected by Organisms Microbes, Higher acid production and a concomitant change of mainly by organisms color. Due to pH indicator present in the culture yeast and medium bacteria For Enterobacteriaceae: determination of Glucose to Pyruvic Acid the m.o. ability to ferment lactose nonfermenters, late lactose fermenters av Embden-Meyerhof-Parnas (EMP) Glycolytic Pathway Pentose Phosphate Pathway Entner-Doudoro Pathway Anaerobic Utilization of Pyruvic Acid es 1. Alcoholic fermentation - The major end product is ethanol. Used by yeast 2. Homolactic fermentation an - The end product is almost exclusively lactic acid. - Streptococcus spp., Lactobacillus spp. 3. Heterolactic fermentation - The end product is propionic acid. - Carried by Propionibacterium acnes sh 4. Mixed acid fermentation - lactic, acetic, succinic, formic acids. - Escherichia spp., Salmonella spp., Shigella spp. 5. Butanediol fermentation - The end products are acetoin 2,3-butanediol. - Krebsiella spp., Enterobacter spp., Serratia spp.  Colony BACTERIAL GROWTH CURVE COLONIES Stages of Bacterial Growth Curve – groups of bacteria forming on solid media as a result of division of one or a few organisms. Generation time of bacteria in a culture varies according to their cellular properties TYPES OF COLONIES It takes 20 minutes for fast growing 1. S or smooth colonies bacterium (E. coli) or 24 hours for slow growing - Uniform texture & homogeneity, easily bacteria (M. tb) emulsified in NSS, virulent organisms 1. Lag Phase or Period Rejuvenescence 2. M or mucoid colonies - Associated with capsulated and - Period when there is no cell division, start of virulent microorganism, slimy/watery biosynthesis, adjustment phase to a new av environment 3. R or rough colonies - Granulated in appearance, hard to 2. Log or Exponential Phase emulsify in NSS - Period when microorganism are actively Other Info: growing and dividing, phase where microorganism are utilized in physiological, es biochemical, and antimicrobial testing 3. Stationary/Plateau Phase - Balance between cell division and dying organisms, but number of viable organisms are an constant 4. Death or Decline Phase - Period of cessation of bacterial growth as the number of dead cells exceeds the living microorganisms. sh OPACITY Automated Methods for ID The bacterial colony also varies in optical Patterns of microbial growth are compared properties. Based on the opacity, the bacterial with the test organism using a computer colonies are of following types – software 1. Opaque – Non transparent (cannot see Growth is detected through colorimetric, through it) fluorometric, or turbidimetric analysis 2. Translucent – Semi transparent (partially Used for various m.o. can be seen) Principle: Pure bacterial isolate during 3. Transparent – Can clearly see through it incubation at 35C for 16-24hrs utilized the carbohydrate in the reagents, thus producing 4. Iridescent – Shows di erent color in light acid or alkaline end products as early as 2 reflection hours. av BACTERIAL IDENTIFICATION BD Phoenix System, Vitek System Conventional Methods Microscopy Biochemical characterization serves as the Fundamental method used for both the fundamental source of information for ID of detection and characterization of bacteria es most bacteria after microscopy. Microscope – vital in magnifying m.o. Motility test – wet mount and hanging drop preparation Remember: o Unit of measurement in determining Staining – gram staining, acid-fast staining bacterial size is micrometer (um) an structural staining o Ocular micrometer is located within Manual biochemical test – carbohydrate the eyepiece fermentation (LIA), catalase and coagulase tests o Refractive index – measure of the relative velocity at which light passes Use of routing and selective media through a material or specimen sh Analytical Profile Index o Total magnification – product of the lens and the objectives that are used Consists of plastic strips and microtubes that contain dehydrated biochemical substrates Types of Microscopes Commonly used for gram (-) enteric bacteria 1. Light Microscope - visible light passes Same principle with biochemical manual tubed through the specimen then through the lenses method that reflect light Principle: Biochemical substrates (pHbased a. Brightfield microscope – most commonly substates) are inoculated with pure culture used microscope, forms a dark image against suspended in sterile physiologic saline and a brighter background, can distinguish incubate for 18-24 hours, at 35C, some between 2 dots that are 0.2 um apart reagents may be added after incubation. b. Phase contrast microscope – permits detailed examination of internal structures in - Faster result in terms of bacterial living organisms, fungi, staining not included identification (in mins.) c. Fluorescence microscope – involves - Components: Ionization Phase and the TOF excitation of fluorochromes (dye) using light Phase d. Darkfield microscope – uses a dark field - Procedure: bacterial isolate is ionized by condenser that blocks light, used to observe transferring it from the culture plate to a metal spirochetes plate, where it is treated with matrix solution until it forms a crystallized microbial protein or 2. Electron Microscope matrix lattice which is then analyzed using - Utilizes electrons instead of light MALDI-TOF instrument. - Has electromagnetic fields instead of lenses - This method creates unique mass spectral to form images fingerprints that are compared to a massive av database of mass spectra - Presence of built-in camera to capture images of cells in black-and-white transmission Molecular Diagnosis a. Transmission Electron Microscope (TEM) - Considered the most important method for Allows visualization of the internal structures of microbial identification cells Resolving power: 0.2 nm, magnification: es 150, 000x to 10M x Used to examine very thin specimens and m.o. - Useful in confirming the taxonomy of the emerging and re-emerging pathogens b. Scanning Electron Microscope (SEM) Scans Polymerase Chain Reaction (Nucleic the surface of the cells or specimens Resolving Acid Amplification Assay) power: 200 nm, magnification: 20x to 10,000x an Specimen is positioned at the bottom of the - Most commonly used amplification technique column in molecular diagnosis. 3. Digital Microscopy - Increases the nucleic acid of the test sample or target microorganism from a very small - A technology that utilizes the science of amount to a million copies staining like the gram reaction - Utilized for rapid detection of nucleic acid in sh - Capture cellular images through web-based biological samples, a very significant tool in the interface diagnosis of the etiologies of diseases. - Uses an automated microscope and the Types of PCR interface to present images on screen or computer monitor 1. Conventional PCR Matrix-Assisted Laser Desorption - Principle: The DNA sequence is amplified Ionization-Time-of Flight Mass utilizing a Taq polymerase Spectrometry - Clinical/Diagnostic Application: Microbial - Excellent tool for the rapid identification of a detection, phylogenetic study, gene analysis, wide range of pathogenic species therapeutic cancer detection, therapeutic management, and DNA profiling and cloning - Utilized for species, subspecies, and strain identification of bacteria. 2. Real-time PCR STAINING TECHNIQUES - Known as quantitative PCR STAINING - Quantifies the target nucleic acid after each replication cycle in the same PCR equipment To observe and appreciate the appearance utilizing commercially available fluorescence detecting thermocyclers. To di erentiate one organism from another - Uses fluorescent dyes to mark specific DNA To help in the identification of organisms and their special structures - Measure the amplified product and determine the number of copies of target substance Classification: present in the original specimen. - Basic - Acidic - Advantages: combines amplification and av product detection at one time, used for Types of Stains multiple sample analysis, reduces Simple Stains cross-contamination with the amplified Indirect, Relief, or Negative Stains production, and lowers turn-around time. Special Stains – Capsular Stain, Cell Wall Stain, Metachromatic Granules, Types of RT-PCR Flagellar Di erential Stain es a. Nested PCR (Single-tube Nested PCR or STNPCR): Gram Stain - E ective for divergent nucleic acid samples such those isolated from formalin-fixed, para n-embedded tissue an b. Reverse-Transcription PCR (RT-PCR): - Transcribes RNA ‘reversely’ into complementary DNA by way of the reverse transcriptase enzyme, hence, transcription is the di erential phase compared to the conventional PCR sh c. Real-time RT-PCR/Reverse Transcription Quantitative PCR - Useful for measuring the abundance of certain RNAs to determine the gene expression. - Gold standard method for the detection of Reasons why Gram (+) becomes (-) SARS-CoV-2 a. Over-decolorization d. Multiplex PCR b. Smear too thick - Allows simultaneous amplification of multiple gene segments rather than separate test runs c. Improper washing between steps for each.  d. Too old culture Result: e. Impure or mixed culture Reasons why Gram (-) becomes (+) a. Under-decolorization b. Smear too thick General Rules for Gram Staining All cocci are gram positive except: - Neisseria, Other Staining Methods Branhamella, Moraxella, and Velionella. 1. LANA (L-alanine 4-nitroanilide) All bacilli are gram negative except: - av Mycobacteria, Clostridium, Corynebacterium, 2. 3% KOH method Bacillus, Erysipelothrix, Listeria, Lactobacillus. ○ To a drop of #% KOH, emulsify a loopful of bacterial colonies Higher forms of organisms (Actinomyces, Streptomyces, yeasts, and molds) are gram ○ Loop is gently pulled out positive. ○ Stir continuously for 60 seconds with es All spiral organisms are reported as gram negative. a wooden stick Acid-Fast Stain - All bacteria are non-acid fast except the an Mycobacteria group. - One does not belong to the Mycobacteria group but is slightly acid fast: Nocardia asteroides - Acid Fast organisms (AFO) are very hard to stain due to the presence of unsaponifiable sh wax called mycolic acid or hydroxymethoxy acid. - In staining, the mycolic acid is temporarily removed through the steaming process. PURPOSE ZIEHL-NEELSEN KINYOUN’S Primary Carbol Fuchsin Carbol Fuchsin Stain Mordant Heat Phenol, Tergitol Decolorizer 3% acid alcohol 3% acid alcohol Counterstai Methylene Blue Malachite n green  - Dacron or rayon polyester swabs are Wk.3 SPECIMEN COLLECTION, advisable HANDLING, AND PROCESSING - Swab collection system is available that provides Fundamentals: transport media and protects the specimen from dying. 1. If possible, collect the specimen in the acute phase of the infection and before antibiotics are administered. 2. Select the correct anatomic site for collection of the specimen. 3. Collect the specimen using the proper technique and supplies with minimal contamination from normal biota. - A wound is not an appropriate specimen source 4. Collect the appropriate quantity of specimen. when the exact anatomic site is not provided. av 5. Package the specimen in a container designed to - Before the specimen is collected, the area should be maintain the viability of the organisms and avoid cleansed to eliminate as much of the commensal hazards that result from the leakage. flora as possible 6. Label the specimen accurately with the specific - The specimen should be collected by needle anatomic site and the patient information. aspiration whenever possible, rather than by swab from the advancing margin of the lesion es 7. Transport the sample to the laboratory promptly or make provisions to store the specimen in an environment that will not degrade the suspected - Aspirated material should be placed into a sterile tube or transport vial and not "squirted" onto a swab. organism. Selection of common clinical specimens for 8. Notify the laboratory in advance if unusual bacterial culture. pathogens or agents of bioterrorism are suspected. an Things to consider in Specimen Collection: Anatomic site Appropriate Inappropriate Lower Freshly Saliva, - Site of collection respiratory expectorated oropharyngeal - Materials used for collection tract mucus secretions, sinus - Containers to be used and drainage - State of the patient