3rd Year Bacteriology Lecture Notes PDF

Bacteriology Lecture- 3rd Year (1st Term) Made by: Pumicpic, Grazia Lorres A. (BSMT-MED223; NU-MOA) INTRODUCTION TO MICROBIOLOGY, INFECTION MICROBES CONTROL AND LABORATORY SAFETY, AND QUALITY Pathogens CONTROL...

Bacteriology Lecture- 3rd Year (1st Term) Made by: Pumicpic, Grazia Lorres A. (BSMT-MED223; NU-MOA) INTRODUCTION TO MICROBIOLOGY, INFECTION MICROBES CONTROL AND LABORATORY SAFETY, AND QUALITY Pathogens CONTROL - Microbes that cause disease Lesson 1 Midterm - Also known as infectious agents - Characteristics: Microbiology - 3% of known microbes - Study of living organisms of microscopic size - Distinct shapes - STUDY OF MICROBES - Some produce toxin - Is the study of living organisms of microscopic size. - Spread easily - Microbiology is the study of microbes. Individual - Live everywhere microbes car be observed only with the use of - Causes infection and disease various types of microscopes. - Medical microbiology is the subdivision concerned Nonpathogens with the causative agents of infectious disease of - Microbes that do not cause disease man, the response of the host to infection and - Ex. Saprophytes - live on dead matter various methods of diagnosis, treatment and - Characteristics: prevention. - Harmless We learn this to know the causative agents - Can be beneficial of any infectious diseases and how is it - Many live on surface of skin being prevented an - 70% of all bacteria - The various categories of microbes include viruses, bacteria, archaea, protozoa, and certain types of NOTE: there are some bacteria that affects humans as algae and fungi. themselves and some affects humans by the toxins they secrete also called as “Microbial intoxication” Individual microbes can be observed only with the use of - Ex. Shigella- this has a toxin that can affect humans various types of microscopes. Acellular microbes Medical Microbiology - Also called as infectious particles - Subdivision concerned with the causative agents of Ex. Virus and prions infectious disease of man, the response of the host to - For a virus to survive, it needs to replicate inside the infection and various methods of diagnosis, cell from the host. treatment, and prevention. Keywords: it needs to be INSIDE of a HOST “Normal Flora” Cellular microbes - Normal residents of our inner biomes Ex. bacteria, all archaea, some algae, all protozoa, and some - Can be opportunistic. fungi - They live normally inside the body but once - Also called cellular microorganisms the immune system is low, they strike or - Divided into two: Prokaryotes and eukaryotes harm the host. Categories of Microbes: 1. Viruses 2. Bacteria 3. Archaea 4. Protozoa 5. Certain types of algae and fungi? The two categories of diseases caused by pathogens  Bacteriology Lecture- 3rd Year (1st Term) Made by: Pumicpic, Grazia Lorres A. (BSMT-MED223; NU-MOA) - Pathogens cause two major types of diseases: Clostridium Toxins A and Glycosylating Diarrhea Yes - Infectious diseases difficile B toxins - a type of disease in which a pathogen Clostridium Perfringens Adenylate Diarrhea No colonizes a person's body. perfringens enterotoxin cyclase - Ex. Neisseria, Mycobacteria, Streptococcus, Perfringolysin Pore-forming Unknown; No Staphylococcus O toxin may be involved in - Microbial intoxications - a type of disease in which a pathogen produces toxin that once ingested by the History of Microbiology: EARLIEST KNOWN INFECTIOUS host can cause an intoxication. DISEASES - The earliest known account of a "pestilence" Toxin - poisonous molecules produced by microorganisms occurred in Egypt about 3180 BC. This may represent capable of causing harm to a host (Forbes, 2020). the first recorded epidemic, although words like pestilence and plague were used without definition in early writings. - Around 1900 BC, near the end of the Trojan War, the Greek army was decimated by an epidemic of what is thought to have been bubonic plague. The bacteria that cause the plague was Yersinia pestis which was used as a bioterrorism - The Ebers papyrus, describing epidemic fevers, was discovered in a tomb in Thebes, Egypt; it was written around 1500 BC. - A disease thought to be smallpox occurred in China around 1122 BC. - Epidemics of plague occurred in Rome in 790, 710, and 640 BC and in Greece around 430 BC. Organism Toxin name Toxin type Clinical Routinely significanc detected History of Microbiology e for - Aside from already mentioned: definitive diagnosis - There are early accounts of rabies, anthrax, dysentery, smallpox, ergotism, botulism, Bacillus Edema toxin Adenylate Edema and Yes anthracis (ET) plus cyclase skin measles, typhoid fever, typhus fever, protective necrosis antigen (PA) diphtheria, and syphilis. - Syphilis -It made its first appearance in Europe in Lethal toxin Metalloprote Yes 1493. Many people believe that syphilis was (LT) plus PA ase carried to Europe by Native Americans who were Bacteroides Bacteroides Metalloprote Unknown; No brought to Portugal by Christopher Columbus. fragilis fragilis ase implicated enterotoxin in diarrhea Christopher Columbus Bordetella Pertussis toxin ADP-ribosyla Tracheobron Yes pertussis (PT) tion chitis - First to describe Syphilis Adenylate Adenylate No cyclase toxin cyclase (ACT) Clostridium Botulinum Metalloprote Muscle Yes botulinum neurotoxin ase paralysis, (BoNT) botulism Bacteriology Lecture- 3rd Year (1st Term) Made by: Pumicpic, Grazia Lorres A. (BSMT-MED223; NU-MOA) - Regarded as the "FATHER OF BACTERIOLOGY AND PROTOZOOLOGY" - He used the term "ANIMALCULES" or the tiny living and moving cells seen under the microscope - Termed “ANIMALCULES” from the “invisible creatures” described by Lucretius and Girolamo Fracastoro - First true microbiologist - FATHER OF BACTERIOLOGY AND PROTOZOOLOGY LUCRETIUS (98-55 B.C) and GIROLAMO FRACASTORO (1478-1553) - They suggested that disease were caused by "invisible creatures" Aristotle GIROLAMO FRACASTORO - Created the Theory of Spontaneous Generation - Italian, wrote poems about STDs (Syphilis) (Abiogenesis) "infections result from tiny self- multiplying bodies that can be spread by direct/indirect contact" - Abiogenesis = From non-living things - It states that life arises from non-living material if it contain "pneuma" (vital heat). - The theory suggests that organisms do not descend from other organism or from a parent and only require that certain condition in their environment be fulfilled in order for creation to occur. - Living organisms arise from non-living organisms in combination with energy (earth, water, air, vital heat etc.) First one who brought syphilis to Europe - Native Americans First to describe Syphilis - Christopher Columbus ANTONIE VAN LEEUWENHOEK (1632-1723) - He is considered as the "first true microbiologist" - He is the first person to observe and accurately describe living microorganisms (rain water, pond, lake, etc.) Francesco Redi Bacteriology Lecture- 3rd Year (1st Term) Made by: Pumicpic, Grazia Lorres A. (BSMT-MED223; NU-MOA) - Experiments with maggots, in which it was revealed that its offspring are flies - demonstrated that maggots were the offspring of flies, not products of spontaneous generation GRADUATE ATTRIBUTES 1. Leadership and Teamwork 2. Responsible Citizenship 3. Innovation, Creative, and critical thinking 4. Academic and Professional Competence 5. Effective Communication 6. Whole person character John Needham 7. Life and Career skill orientation - Experiments with a clean broth that is exposed on 8. Technological literacy air, wherein it became turbid (became blurry). - No fly dropped on his broth so he believes that there is microbes that arose in spontaneous generation. - microbes arose spontaneously in broth from a "life force." LOUIS PASTEUR (1822-1895) - exposure of a broth to air was not introducing a “life force” to the broth but rather airborne microorganisms - X Life force - ✓airborne bacterial contamination - He boiled bacteria to sterilize it - “oni vivum ex vivo” : “Life only comes from life” - :Life is a germ, and a germ is life” - THEORY OF BIOGENESIS (making new living things) - Re resolved the issue on spontaneous generation Lazzaro Spallanzani with a series of ingenious and persuasive experience - Heated but sealed flasks remain clear, without any - He demonstrated that microorganisms are present in signs of spontaneous growth, unless the flask were air and can contaminate sterilized solution but air subsequently opened to the air itself does not create microorganism Bacteriology Lecture- 3rd Year (1st Term) Made by: Pumicpic, Grazia Lorres A. (BSMT-MED223; NU-MOA) - He proposed reuse of heat to kill microorganism - Introduce the smallpox vaccine through cowpox (aseptic technique) present contaminated from inoculation (from milkmaid of infection cow) unwanted organism NOTE: Virulation - China; if open scab/wound would be - Developed vaccine for bacterial anthrax and viral infected; experiment on smallpox rabies LOUIS PASTEUR (1882-1895) PIERRE PAUL EMILE ROUX RUDOLF VIRCHOW (1821-1902) (1853-1933) - Proposed the theory of biogenesis in which states - PAsteur used the term “vaccine” for an attenuated that living cells can arise only from pre-existing culture (reduce virulence). They made a series of living cells experiments to produce an attenuated strains of bacteria IGNAZ SEMMELWEIS (1816-1865) NOTE: First erodicated disease using a vaccine = smallpox - He demonstrated that routine hand washing was Attenuated = weakened virulence prevent the spread of disease(puerperal fever) Ex. Sinovac (attenuated vaccine for COVID) - Father of Hand washing MODERN THERAPY “MAGIC BULLET” JOSEPH LISTER (1827-1912) - THEORY OF ANTISEPSIS SELMAN WAXMAN (1888-1973) - Demonstrated system of antiseptic injury in britain - Discovered streptomycin and neomycin antibiotics (antiseptic and preventive medicine (phenol)) - Streptomycin = Streptomyces griseus - Pioneered and promote handwashing before and - Neomycin = Streptomyces fradiae after operation/surgery, wearing gloves, sterilizing surgical instrument and other use of phenol as an ALEXANDER FLEMMING (1881-1955) antimicrobial agent for surgical wound dressing - Accidentally discovered penicillin (penicillium notatum) from a fungus ROBERT KOCH - Discovered lysozyme - First to show proof that bacteria cause disease (Koch’s postulates) PAUL EHRLICH (1854-1915) - Discovered: Bacillus anthracis (1876) mycobacterium - Discovered salvarsan (arsphenamine) for the tuberculosis (1882) which is a PTB causative agent treatment of syphilis Notes: PTB: pulmonary tuberculosis/Koch disease - Culture bacteria on boiled potatoes, gelatin, meat ELIE METCHNIKOFF (1845-1915) extract and protein (ingredients of culture medium) - First to describe the immune system cell and the - He developed a culture media for observing bacterial process of phagocytosis growth isolated from the human body JOHN TYNDALL (1820-1893) KOCH POSTULATES - Showed that dust carry agents that could 1. Microorganism must be found in diseased but not contaminate a sterile broth healthy individual 2. Microorganism must be cultured through diseased FERDINAND COHN (1828-1898) 3. Inoculation a healthy individual with the cultured - Discovered that there are bacteria that could microorganism withstand a series of heating and boiling because of 4. The microorganism must be reisolated front he heat resistance structure known as endospores inoculated diseased individual and matched to the original microorganism FANNY HESSE - Suggested the use of Agar, solidifying agent, in the IMMUNOLOGY (ADVENT OF VACCINATION) proportion of culture media EDWARD JENNER (1749-1823) JULIUS RICHARD PETRI (1852-1921) Bacteriology Lecture- 3rd Year (1st Term) Made by: Pumicpic, Grazia Lorres A. (BSMT-MED223; NU-MOA) - Developed the petri dish ROBERT HOOKE (ENGLISH MAN 1665) - Observing a thin slice of cork reported to be world that like smallest structural units “little boxes or cell” - Cell theory - Cell = little boxes = smallest structural unit ANTONIE VAN LEEUWENHOEK (DUTCH MERCHANT, AMATEUR SCIENTIST) - Van Leeuwenhoek microscope - First to observe microorganisms - “Father of Ancient BActeriology and Scientific Microscope” - Science of Bacteriology another factor microscopy” - Debunked Spontaneous generation theory EUKARYOTES (eukaryotic cells) MICROBIAL TAXONOMY - True nucleus of eukaryotes (Gr. Karyon nucleus) - Area of biological science that comprises 3 distinct - Membrane bound organelles; the microtubules and but highly interrelated disciplines the microfilaments form a complex intracellular ✓ Classification structure. ✓ Nomenclature (naming) - Flagella and cilia - complex multi structured ✓ Identification structure - Provides a consistent means to classify names and - Haploid 23 gametes identify organisms. This consistency allows - Diploid contains full set of genes biologists worldwide to use a common label for - Derived from each gamete (egg and sperm cell) organisms studies within the multitude of biologic - Almost entirely in diploid structure disciplines (ailey’s and Scott) PROKARYOTES CLASSIFICATION - DNA is not separated - It is an organization of microorganisms that have - No nuclear membrane similar morphology, physiologic, genetic traits into - Relatively small specific groups of taxa. - Only 1 chromosome - DNA in a circle with a length of mm FORMAL LEVEL OF BACTERIAL HIERARCHICAL - Relatively small in size (1um in diameter) absence of CLASSIFICATION nuclear hormones. Part of almost all bacteria is circle with a length of 1 mm NUCLEOID - Seen in electron M. PLASMIDS - Transfer information CLASSIFICATION 1. Family - group that contain multiple genera - Organisms with common attribute Bacteriology Lecture- 3rd Year (1st Term) Made by: Pumicpic, Grazia Lorres A. (BSMT-MED223; NU-MOA) - Adding suffix -aceae ; and root name - Ex. family - streptococcaceae; Genus- Rule: streptococcus - Genus always capitalize first letter, species small - EXCEPT: Enterobacteriaceae(names after letters enteric bacteria) - Capital first letter of genus then period, then full 2. Genus species name (ex. S. aureus) - Contains different species with several - Informal designation to name group of organisms, important features in common either capitalization - Ex. Streptococcus escherichia - Printed in italic 3. Species - Underlined in script - Most basic of taxonomic groups - Collection of bacterial strains Identification - Is the most basic of the taxonomic group - Process by which microorganism is identified and can be defined as a collection of - Process of key features are delineated bacteria. That share common physillic and - Methods: genetic features and are different notably 1. Genotype - genetic makeup, genes, nucleic from after microbial sp. acids and DNA Base component ratio - Ex. streptococcus pneumoniae; Escherichia 2. Phenotypic - based on the feature beyond coli - Diarrhea the genetic level and include both readily observable characteristics and those that SUBSPECIES may require analytic procedure 4. Serotype - Macro-micro (inoculated) - based on serologic differences - Anaerobic - aerobic - Ex. E. coli (>700 serotypes) - Biochemical testing results 5. Biotype - Based on biochemical differences - Ex. Treponema pallidum subsp. Pallidum PHENOTYPIC AND GENOTYPIC CHARACTERISTICS (can cause syphilis) GRAM POSITIVE: Purple stain color GRAM NEGATIVE: Pink/Red INFECTION CONTROL AND LABORATORY SAFETY LABORATORY BIOSAFETY NOMENCLATURE - “Protecting people from dangerous pathogens” - naming of microorganism because of - Prevents unintentional exposure to harmful ICNB(International code of nomenclature of pathogens bacteria) and BC (Bacteriological code) - Protects user Bacteriology Lecture- 3rd Year (1st Term) Made by: Pumicpic, Grazia Lorres A. (BSMT-MED223; NU-MOA) - Those outside the laboratory - primary health service, diagnostic services, - The environment research - GMT, protective clothing biohazard sign WHO: Laboratory has safety and security components - open bench plus BSL c. Biosafety level 3 CONTAINMENT LABORATORY BIOSECURITY - negative airflow directional - Protect pathogens from dangerous people: theft, - special diagnostic services misuse, diversion, etc. - special clothing, controlled access d. Biosafety level 4 MAXIMUM CONTAINMENT PRINCIPLE OF BIOSAFETY - airlock entry 1. Practice and Procedure - dangerous pathogen. a. Standard practice - astronaut umit, only hands are put in b. Special practice and consideration - WHO container programs - 2. Safety Equipment CDC BIOSAFETY LEVEL - Primary containment barrier BSL 4 - Ebola, Smallpox (highly lethal) - Minimize exposure to hazard BSL 3 - HIV, H1N1 Flu, Yersinia, TB, SARS, Rabies, - PPE West Nile virus, Rickets, the plague - BSC BSL 2 - Most Chlamydiae, Hepatitis A -C, Influenza A, - covered / ventilated animal cage system Lyme disease, salmonella, Mumps, measles - Engineering controls and equipment BSL 1 - RESEARCH 3. Facility Design and Construction - Secondary barrier/engineering controls RESEARCH BSL 1-4 PRACTICES OF SAFETY EQUIPMENT - Contributed to work protection - Protect outside laboratory BIOLOGICAL SAFETY CABINET (BSC) 4. Increasing Levels of Protection - Downflow airpasses hrough ULPA/HEPA filter and - Biosafety Level 1-4 create an ISO class 3 work zone Class 1 Biosafety Cabinet BIOHAZARD SYMBOL - represent chain of infection - protects operator and environment from exposure to biohazards 1. Agents - cause of infection - does not prevent sample being handled from being 2. Host - get infected exposed 3. Source - where pathogen originate - allow room (unsterilized) air to pass through 4. Transmission - MOT - absolute circulated air and exhausted since prone to contamination CHAIN OF INFECTION Germs -> where germs live -> how germs gets out -> Germs Class 2 Type A2 get around -> How germs get in -> sick person - most common class 2 cabinet - 30% air exhausted, 70% recirculated RISK GROUPS CLASSIFICATION - must have positively-pressurized contaminated 1. Biosafety levels (1-4) plenum - Level of biocontainment to isolate - with type A1 dangerous biological agents - Each level has own containment through: Class 2 Type B1 Laboratory practices, safety equipment, and - Common plenum, 70% air exhausted, 30% facility construction recirculated eliminates recirculation with dedicated a. Biosafety Level 1 - BASIC exhaust feature - Basic training research; open branch work - exhaust air in class 2 B cabinet is discharged b. Biosafety level 2 -BASIC Bacteriology Lecture- 3rd Year (1st Term) Made by: Pumicpic, Grazia Lorres A. (BSMT-MED223; NU-MOA) Class 2 Type B2 SHARPS - medical devices that cut or go into the skin - suitable for work with toxic chemicals ❖ *H4 patient = patient with HIV - safest of all class 2 biosafety cabinet (intheory) ❖ * Do not recap using hands ❖ HIV, HBV, HCV = Discuss that can be transmitted Class 3 biological safety through needlestick injury HIV = 0.3% Infectious HBV = 6-3% HCV = 1.8% INFECTION PREVENTION AND CONTROL (IPC) TRANSMISSION - Variety of measures practiced by health care - based precautions Supplement Standard Precautions personnel to prevent spread, transmission and with known or Suspected Colonization of infection acquisition and spread of infections - Patient to PAtient AIRBORNE - Patient to Staff Member - Fitted Respirator (N95), placed in negative pressure - Staff Member to Patient room - Staff to Staff *DROPLET - regular medical mask HIERARCHY OF CONTROLS CONTACT - - Elimination - most effective - Substitution SHARES CONTAINER = puncture - proof container physical - Engineering controls methods of sterilization - Administrative Control 1.) Incineration - PPE - least effective 2.) Moist heat 3.) Chemicals GENERAL STRATEGIES OF IPC 4.) Dry heat 1. Hand Hygiene 5.) Filtration - any action of hand cleaning 6.) Ionizing radiation 2. Hand rub - hand not visible soiled INCINERATION - using alcohol-based sanitizer/antiseptic gel - literally burned to asher 3. Handwashing - 870-980 °C - visibly soiled - safest method to ensure no microbe left - using gloves or going to bathroom - Ex. Priors and infective chem - 2 HAPPY BIRTHDAY (15-20 mins) 5 MOMENTS FOR HAND HYGIENE (research) DRY HEAT STEPS OF ON OUTBREAK INVESTIGATION (research) - sterilize glassware - 1.5 to 3 hours ; 100 - 180°C (100°C 15 min) 1. Verity diagnosis other heat methods: Boiling Autoclave (121.6°C, 15 min at 15 2. Confirm outbreak psi)" 3. Find additional carer 4. Characterize carer PARTEINIZATION (63℃, 30 min) 5. Form hypothesis OVER DRY HEAT (100-180°C, 1.5-3 hours) 6. Clest hypo theoro Flash method (72°C, 15 seconds) 7. Institute control measure 8. Evaluate effectiveness MOIST HEAT 9. Communicate findings - sterilize biohazard co trash and heat - table objects Bacteriology Lecture- 3rd Year (1st Term) Made by: Pumicpic, Grazia Lorres A. (BSMT-MED223; NU-MOA) - autoclave is used for this IQPC (INDIVIDUALIZED QUALITY CONTROL PROGRAM) - gravity - displacement autoclave must access: specimen, test system, reagent, environment, ? QUALITY PROGRAM - implement and monitor quality system *Basic elements of a QC program (a-k) *specimen collection and transport Laboratory - responsible for providing written politico and - 2 Most temp common: 121 °C and 132°C procedures that ensure positive identification and optimum - fastest and simplest physical method of Sterilization integrity of a patient's specimen. - Biologic waste (30 mino 121°C/ 4 min 132°C) - Infectious med waste (132°C for 30-60 min) Critical info: name, hospital or lab no PRIONS ordering physician, receiving microbial therapy, suspected - require much more extensive sterilization agent or syndrome, autoclave at 134°c for 1 mins (prevacuum) immunization history (if applicable), travel history when “At 132°C for 1 hour (gravity displacement) ” certain microorgan or parantes are suspected Immerse in l N sodium hydroxide for 1 hour, rinse in water, important to know what antibiotic autoclave 121°C in gravity do or 134°c in prevacuum sterilizer patient takes to know if they are susceptible/resistant to for 1 hour or testing immerse in l N sodium hydroxide for I hour, heat in gravity d. *collection Instruction Retail (Research) [12 items] at 121°C for 30 mins, clean and subject to routine equipment * examples of unacceptable specimens [8 items] 4 OPTIONS FOR STERILIZATION (Elsevier, 2018) (Principles Culture media extraction = extract blood both arms and practices of Infectious Disease) If both army infected by culture: undergoing sepsis QNS quantity not sufficient for testing → REJECT FILTRATION Methods or choice for antibiotic solutions, toxic chemicals, *Standard operating Procedure Manual (SOPM) Is define- radioisotopes, vaccines and carbohydrates, etc Personnel - sufficient for volume and complexity of the work IONIZING RADIATION performed. - Sterilizing disposables > Primary at least 4 MT Most common chemical sterilant (chemical sterilant) *Reference Laboratories - Ethylene oxide (ETO) - used to heat-resistant object *patient reports - review checking specimen workup QUALITY CONTROL - internal activities - only released to authorized personnel QUALITY ASSURANCE - external activities Ex. 2nd day initial report, 5th day final result *positive patient outcomes reduced length of Stay - clinicians should be notified about “panic reduced cost of stay values(immediate call doctor)"/ potentially life reduced turnaround time threatening values appropriate microbial therapy. - all patient records should be maintained for at least 2 years. Proficiency Testing Bacteriology Lecture- 3rd Year (1st Term) Made by: Pumicpic, Grazia Lorres A. (BSMT-MED223; NU-MOA) - read guidelines of RITM guidelines for microbiology - lab maintain average score of 80% to retain license strains used in Quality control of Routine Antimicrobial susceptibility Tests (Table 10-1 Rodriguez) performance checks - Instrumento "Antimicrobial susceptibility tests” - user - prepared and nonexempt, commercially prepared media Bacteriology - QC forms should contain:? -non-fastidious (rapidly growing) can be saved up to 1 year on trypticase soy agar (TOA) Expiration dates - 1 month for agar plates, 6 months for tubed media -long-term storage -frozen, non-fastidious every year * Instruments requiring quality control. - fastidious 1. Thermometer Calibration - every 3 years 2. Check the thermometer periodically. -stock isolates 3. Check presence of gas daily - freeze in 10% sodium milk 4. Easily read if permanently immersed in glycol - trypticase soy broth (TAB) ↳ prevent room fluctuation?. if door is opened 5. Temps for thermometer? Mycobacteriology 6. Carbon Dioxide incubator -acid- fast bacilli - glycerol 7. centrifuge 8. Tachometer Quality Assurance Program - preanalytical, analytical, port - analytical Representative sample test for sterility At least 5?? Of any lot tested for a batch of 100 or fewer units maximum of 10 units are tested in a large batch Batch - any one Shipment of a product with the some lot number if a separate department reject if diff shipment even if some lot no Antimicrobial susceptibility tests ensure precision and accuracy of supplies and microbiologists performing test Reference strains MIC (broth dilution) - testing Kirby - Bauer - agar diffusion testing susceptibility test system tested with use (usually daily) for 20 consecutive days weekly ou testing (outside reference range during 20-day testing period) Kirby Bauer = has zone of inhibition for each antimicrobial tested