ST220 Concorde Career College MICROBIOLOGY Microbiology Objectives Define the term microbiology. Provide a timeline of events leading up to the current understanding of microbiology. List and identify the parts of a microscope and understand its use in a laboratory setting. Microbiology Microbiology – Study of microorganisms Microorganisms – Microscopic organism (plant or animal) Microscopic – Minute; visible only with the aid of a microscope Microbiology Microscope An optical instrument for making an enlarged image of an object too small to be seen by the naked eye Consists of a lens, or a combination of lenses Often provides light enhancement May be monocular or binocular Microbiology Microscope Original van Leeuwenhoek microscope Microbiology Microscope Operating microscope used for neurosurgical procedures Microbiology Microbiology Timeline Robert Hooke 1635-1703 Cell theory – Proposed the idea that all living things are composed of cells after viewing slices of cork through a microscope in the year 1665. Note: Hooke coined the term cell which is from the Latin word cella meaning "storeroom" or "small container" Robert Hooke Microbiology Microbiology Timeline Anton van Leeuwenhoek 1632-1723 First to observe bacteria with a microscope Made the first accurate drawings of bacteria and protozoa Anton van Leeuwenhoek Microbiology Microbiology Timeline Francesco Redi 1626-1697 Disproved the theory of abiogenesis or spontaneous generation (it was thought that new living cells could spontaneously arise from nonliving material) Francesco Redi Microbiology Microbiology Timeline Louis Pasteur 1822-1895 Proved the theory of biogenesis (new living cells can only arise from preexisting living cells) Developed the “germ theory of disease” Promoted hand washing and a clean environment to reduce the spread of disease in hospitals Developed several vaccines (anthrax, cholera, rabies) Developed "pasteurization," a method of destroying harmful microbes in perishable food products using heat, without destroying the food Louis Pasteur Microbiology Microbiology Timeline Edward Jenner 1749-1823 Developed smallpox vaccination Father of Immunology Edward Jenner Microbiology Microbiology Timeline Ignaz Semmelweis 1818-1865 Correlated frequent handwashing in the obstetrics ward with lower rates of infection (puerperal fever) Note: the term puerpera refers to a woman who has just given birth Ignaz Semmelweis Microbiology Microbiology Timeline Joseph Lister 1827-1912 Recognized the significance of Pasteur’s findings and established the first principles of asepsis and the practice of sterile technique Used carbolic acid (phenol) to treat surgical wounds and dressings Wore clean gown and gloves for each surgical procedure Boiled instruments between surgical procedures Joseph Lister Microbiology Microbiology Timeline Robert Koch 1843-1910 Proved Pasteur’s germ theory Developed pure culture techniques still used today Robert Koch Microbiology Microbiology Timeline Hans Gram 1853-1938 Developed staining methods (Gram’s stain) utilized to identify various species of bacteria Hans Gram Microbiology Organism Identification Staining – Involves application of a colored dye (called a stain) to an organism to enhance visualization and allow identification of a specific organism. Gram Stain Acid-fast Stain Microbiology Organism Identification Gram Stain Organism is stained with blue/purple dye Weak iodine solution is added to promote colorfastness Slide is washed with alcohol If blue/purple dye remains the organism is Gram positive If blue/purple dye is removed the organism is Gram negative and is then stained with pink/red dye to enhance visibility Positive or Negative? Anthrax rods in CSF Pseudomonas aeruginosa Microbiology Organism Identification Acid-fast Stain Organism is stained with red dye Smear is treated with acid Most organisms quickly loose the red color when treated with acid Those that remain colored (e.g., Mycobacterium tuberculosis) are called acid-fast Acid-fast Stain Mycobacteria paratuberculosis in bovine ileum Microbiology Organism Identification Culturing – Growing cells in enriched media Fermentation – Action of organism on sugars Observation – Reaction to test chemicals Inoculation – Injection and observation of test animals Immunology – Antigen-antibody reaction Culture Microbiology Microscope Types Compound Light Microscope Dark Field Microscope Phase Contrast Microscope Fluorescence Microscope Electron Microscope Microbiology Compound Light Microscope Also called “bright field” Two lens system First lens located in the objective near the specimen Second lens in the eyepiece Light source Microbiology Dark Field Microscope Similar to bright field microscope – contains a light condenser with an opaque disc that blocks direct light; allowing peripheral light to enter Utilized when microbes cannot be stained Effective for viewing motility when microbes are suspended in liquid (wet mount) Dark Field Microbiology Phase Contrast Microscope Light condenser contains ring shaped diaphragm that allows light of various brightness (phases) to pass through – highlighting the internal structures of the cell Phase Contrast Samples Microbiology Fluorescence Microscope Utilized ultraviolet light to visualize fluorescent microbes or those stained with fluorochromatic dye Especially useful in identification of rabies and syphilis Fluorescence Microscope Microbiology Electron Microscope Utilizes a beam of electrons in place of light which improves resolution Utilizes an electromagnetic lens in place of a glass lens which focuses the electron beam onto the specimen Scanning electron microscope is pictured Electron Microscope Pollen Microbiology Microscope Components Eyepiece Body tube Arm Revolving nosepiece Objectives Stage with clips Focus adjustment knobs Diaphragm Light source Base Microbiology Microbiology Microscope – Care and Handling When carrying the microscope hold the arm with one hand and support the base with the other Never touch the lenses Use only lens paper to clean the lenses When finished with the microscope Rotate the nosepiece to the low power objective Lower the nosepiece down to the stage Secure the cord Replace the dust cover Microbiology Objectives List and identify the components of a cell and describe the function of each. List and define fluid movement concepts. List the major classifications of organisms. Microbiology Cell Theory Anton von Leeuwenhoek Observed pond water under microscope Noted organisms that he called “animalcules” Principles of modern cell theory Cells are the smallest complete living things (basic unit of organization of all organisms) All organisms are composed of one or more cells in which all life processes occur Cells arise only from preexisting cells through the process of cell division. All existing cells are descendants of the first cells formed in history. Microbiology Cell Structure Cell membrane Cytoplasm Nucleus Nuclear membrane Nucleoplasm Chromatin Nucleolus Mitochondria Lysosomes ER – endoplasmic reticulum (rough, smooth) Golgi apparatus Ribosomes Microbiology Cell Membrane Also called the plasma membrane or plasmalemma Consists of a double phospholipid layer containing proteins Phospholipids allow free passage of water molecules through the cell membrane via osmosis (hydrophilic/hydrophobic) Some proteins allow passage of molecules and ions via transport channels or by active transport Other proteins act as receptor sites and identity markers Phospholipid Bilayer Microbiology Cytoplasm Main constituent is water that contains chemical compounds (e.g., mineral salts) in solution and organic compounds in colloidal suspension Also contains storage granules, fat droplets, and vacuoles (area within the cytoplasm surrounded by a membrane filled with a watery mixture of nutrients or waste products Cytoplasm Microbiology Nucleus Control center of the cell Surrounded by a membrane Contains genetic material Microbiology Nuclear Membrane Porous double nuclear membrane that allows passage of materials (e.g., messenger RNA) to the cytoplasm Inner layer surrounds the nucleoplasm Outer layer connects with the ER Nuclear Membrane Microbiology Nucleoplasm Fluid portion of the nucleus Microbiology Chromatin Genetic material contained within the nucleoplasm Darkly stained threads of nucleic acids Duplicates, shortens, and thickens during cell division and becomes visible as chromosomes Microbiology Nucleolus Spherical particle within the nucleoplasm Produces ribosomes Microbiology Mitochondria Powerhouse of the cell Composed of two membranes Outer membrane forms capsular shape Inner membrane folds into itself to increase surface area (folds are called cristae) Aerobic phase of cellular respiration occurs in the mitochondria Mitochondrion Microbiology Lysosomes Small structures in the cytoplasm containing powerful digestive enzymes and surrounded by a membrane Perform three important functions 1. Work with food vacuoles to digest stored food 2. Maintain and repair organelles (building blocks of protoplasmic structures) 3. Destruction of old or weakened cells Lysosomes Microbiology Endoplasmic Reticulum (rough, smooth) Complex system of membranes that make up channels called cisternae Connects the outer nuclear membrane with the cell membrane All cells have rough ER Rough ER has attached ribosomes that synthesize protein Some cells have smooth ER Smooth ER transports fat or synthesizes sex hormones Endoplasmic Reticulum Microbiology Golgi Apparatus Collection of flat saclike cisternae Store compounds secreted by the cell Aid in synthesis of necessary substances (e.g., carbohydrates) Golgi Apparatus Microbiology Ribosomes Small granules distributes throughout the cytoplasm that are attached to the ER Site of protein synthesis Ribosomes on Rough ER Microbiology Fluid Movement Concepts Diffusion Osmosis Active Transport Filtration Microbiology Diffusion Movement of molecules through a medium from an area of high concentration to an area of low concentration. Microbiology Osmosis Movement of a solvent (e.g., water) through a semipermeable membrane from an area of high concentration to an area of low concentration Osmosis Microbiology Active Transport Movement of substances (e.g., sugars, amino acids, large proteins, fats) through the cell wall from an area of lower concentration to an area of higher concentration using energy in the form of ATP (adenosine triphosphate) Active Transport Microbiology Filtration Passage of a liquid or gas through a permeable membrane to remove particulate matter Microbiology Movement of Items Other Than Water Endocytosis Exocytosis Microbiology Endocytosis Particles are taken into the cell (e.g., bacteria, nutrients) Three types of endocytosis Phagocytosis Pinocytosis Receptor-mediated endocytosis Microbiology Phagocytosis Movement of other organisms or fragments of organic matter into the cell Macrophage Phagocytosis Microbiology Pinocytosis Movement of fluids (other than water) into the cell Pinocytosis Microbiology Receptor-Mediated Phagocytosis Receptors on the cell surface detect specific molecules and allow rapid movement of the molecule into the cell Microbiology Exocytosis Movement of particles (e.g., hormones, neurotransmitters, digestive enzymes) out of the cell Via secretory granules originating in the Golgi apparatus Platelet Exocytosis Microbiology Major Classifications of Organisms (Nomenclature) Kingdom (plantae, animalia, fungi, protists, monera) Phylum Class Order Family Genus Species Classification Microbiology Specific Classes of Microorganisms Algae Protozoa Fungi (yeasts, mushrooms, molds) Bacteria Rickettsiae Viruses Prions Microbiology Objectives Define the term algae, list its characteristics, and explain its relationship to humans. Define the term protozoa, list its characteristics, and explain its relationship to humans. Microbiology Algae (Definition and Characteristics) Protist Kingdom (simplest organisms) Eukaryotic (nucleus surrounded by a membrane) Photosynthetic (contains chlorophyl – manufactures carbohydrates and oxygen by combining CO2 and H2O using sunlight) Nonparasitic Lacks roots, stems, leaves, and flowers May be microscopic (unicellular) or massive (multicellular) Algae Microbiology Algae (Relationship to Humans) Ecological balance Indication of ecological imbalance Food source Blue-green algae – Cyanobacteria responsible for epidemic diarrhea Microbiology Protozoa (Definition and Characteristics) Protista Kingdom (simplest organisms) Unicellular – animal like microorganisms Saprophytes (lives on dead or decaying matter) Microbiology Ciliated Protozoa Microbiology Flagellated Protozoa Microbiology Protozoa (Relationship to humans) Common protozoa infections Malaria (Plasmodium vivax, P. malariae) Gastroenteritis (Entamoeba histolytica, Giardia lamblia) Leishmaniasis – inflammation of the skin or viscera (Leishmania species) Sleeping sickness (Trypanosoma brucei gambiense) Vaginal infections (Trichomonas vaginalis) Trypanosoma brucei Microbiology Malaria (Plasmodium malariae) Microbiology Protozoa Spread of infection (opportunistic – most commonly affects immunosuppressed individuals) Fecal-oral contamination Ingestion of contaminated food or water Vector Mosquito Human sexual contact Microbiology Trichomonas vaginalis Microbiology Objective Define the term fungus (yeasts, mushrooms, and molds), list the characteristics of each, and explain the relationship of each to humans. Define the term helminth, list its characteristics, and explain its relationship to humans. Microbiology Fungi (Definition and Characteristics) Protist Kingdom Eukaryotic Chemoheterotrophic (uses chemicals as energy source) Requires an external carbon source Lack chlorophyll Saprophytic or parasitic Reproduction occurs either by budding (sexual) or spore formation (asexual) Microbiology Fungi (Definition and Characteristics - continued) Grow in irregular masses Devoid of roots, stems, and leaves May be unicellular or filamentous (threadlike in structure) Includes yeasts, mushrooms, and molds Mycology is the study of fungi Mycoses are fungal diseases Microbiology Yeast (baking, brewing) Saccharomyces cerevisiae Microbiology Yeast (esophageal candida albicans) Microbiology Mushroom Microbiology Mold Microbiology Onychomycosis Microbiology Fungus (Relationship to humans) Benefits Molds – decompose organic matter/produce antibiotics Mushrooms – edible Yeasts – fermentation/leavened bread/normal body flora Microbiology Fungus (Relationship to humans) Mycoses – Typically opportunistic in humans Immune deficiency Immunosuppression Corticosteroid use Chemotherapy Antibiotic therapy Comorbid condition (e.g., diabetes) Microbiology Fungus (Relationship to humans) Mycoses (examples) Aspergillosis – air conditioning contaminant that commonly affects the respiratory tract Zygomycosis – contaminant found in soil and on fruit and plants Candida (yeast) – normal mucosal flora Cryptococcus – enters through the respiratory tract and can spread to other internal organs including the nervous system (cryptococcal meningitis) Microbiology Helminths (Definition and Characteristics) Worms (includes eggs and larvae) Parasitic (referred to an infestation rather than infection) Microbiology Helminths (Relationship to humans) Roundworms Ascaris Prevalent in larval form in Asia Prevalent in egg form in the Southern US Enter the digestive system via contaminated food Infests lungs and intestines Microbiology Human Intestinal Roundworm (Ascaris Lumbricoides) Microbiology Helminths (Relationship to humans) Roundworms Trichina Encapsulated worms found in host’s muscle (e.g., pork) Undercooked pork is ingested Digestive juices dissolved by new host Worms mature and infest new host’s muscle Microbiology Trichina Microbiology Helminths (Relationship to humans) Roundworms Filariasis (caused by a small threadlike worm) Common in tropical and subtropical areas Transmitted by biting insects (e.g., flies, mosquitos) May infest cardiovascular system (e.g., heartworm) May infest lymphatic system (e.g., elephantiasis) FILARIASIS Microbiology Elephantiasis Microbiology Heartworm Microbiology Helminths (Relationship to humans) Pinworms Common type Enterobius vermicularis (seat worm) Especially prevalent in children Initially ingested Female exits anus to lay eggs, anal eggs cause itching, eggs are reintroduced to the GI tract via the fingers, re-infestation occurs Microbiology Enterobius vermicularis Microbiology Helminths (Relationship to humans) Hookworms Small larvae live in soil Larvae penetrate intact skin of the bare foot Transported to the intestine by the blood Leech blood from the host’s intestinal mucosa causing anemia Microbiology Hookworm Microbiology Helminths (Relationship to humans) Flatworms Tapeworm Ingested in improperly cooked meat (e.g., beef, pork, and fish) May grow to the length of the intestinal tract (up to 50 feet) Microbiology Human Intestinal Flatworm Tapeworm (Taenia Pisiformis) Tapeworm Microbiology Helminths (Relationship to humans) Flatworms Flukes Leaf shaped flatworms Invade the blood, lungs, liver, and intestine Microbiology Fluke Microbiology Objectives Define the term bacteria, list its characteristics, and explain its relationship to humans. Microbiology Bacteria Unicellular, prokaryotic (lacks a true nucleus) organisms that usually multiply by cell division May be capable of producing spores (resistant form that can tolerate adverse conditions) Compose the largest group of pathogens Microbiology Bacteria May be: Aerobic Motile (flagella) Free-living Saprophytic Pathogenic - Anaerobic - Nonmotile - Commensal - Parasitic - Beneficial Microbiology Bacterial Classifications Bacilli – rod shaped cells Straight Slender Cigar shaped with tapered ends Common bacillary diseases: Tetanus Diphtheria Tuberculosis Typhoid fever Microbiology Clostridium tetani Microbiology Tetanus Anaerobic, Gram positive rod Found in soil, dust, and manure Enters through a break in the skin Affects the CNS causes tetany Symptoms appear 3-21 days following exposure Usually fatal by asphyxiation Vaccine available Microbiology Other common clostridium diseases Botulism – Clostridium botulinum Food poisoning – Clostridium perfringens Antibiotic-associated colitis – Clostridium difficile Necrotizing enteritis – Clostridium perfringens Uterine and wound infections – Clostridium perfringens and others Microbiology Corynebacterium diphtheriae Microbiology Diphtheria Acute bacterial disease Enters the respiratory tract Commonly affects the throat (tonsils), nose, and skin/may also affect the pericardium/swollen lymph nodes/produces a membrane Usually fatal by asphyxiation Symptoms appear 1-6 days following exposure Vaccine available Microbiology Mycobacterium tuberculosis Microbiology Tuberculosis Usually attacks the lungs, but may affect any body tissue Diagnosed with skin test or chest X-ray Enters the respiratory tract Respiratory symptoms include: Cough that lasts longer than 2 weeks Pain in the chest Coughing up blood or sputum No vaccine/treatment lengthy Microbiology Bacterial Classifications Cocci – spherical cells Round (characteristic arrangements) Diplococci – pairs Gonorrhea Meningitis Streptococci – chains Strep throat Staphylococci - clusters Microbiology Neisseria gonorrhoeae Microbiology Gonorrhea Gram negative diplococci STD Purulent penile or cervical discharge Frequent painful urination Ectopic pregnancy Sterility Epididymitis Microbiology Gonorrhea Glans penis Microbiology Gonorrhea Vaginal Microbiology Gonorrhea Transmitted to the neonatal eye during childbirth Microbiology Neisseria Meningitidis Microbiology Bacterial Meningitis Bacterial normal flora in upper aerodigestive tract High fever Headache Neck stiffness Photosensitivity Microbiology Streptococcus pyogenes Microbiology Strep Throat Fever Pain Redness Swelling Microbiology Necrotizing Fascitis Also caused by streptococcus pyogenes Microbiology staphylococcus aureus Microbiology Staph Infection Carpal tunnel release – 1 week postop Patient complaining of worsening throbbing pain in the hand and pain with any movement of the fingers Microbiology Carpal tunnel filled with purulent matter caused by a s. aureus deep wound infection Microbiology Infection spread into the forearm through the flexor tendon compartments as well as up into the small finger flexor tendon sheath. She was treated with repeated radical debridement, intravenous antibiotics, and delayed wound closure. Her infection was cured, but she was left with stiffness of the small finger. Microbiology Staphylococcus aureus Gram positive cocci Normal skin flora Opportunistic pathogen Major cause of SSI Microbiology Common minor infections caused by s. aureus Folliculitis Boils Sties Impetigo Superficial abscesses Microbiology Common severe infections caused by s. aureus Food poisoning TSS Pneumonia Osteomyelitis Lactation mastitis Endocarditis Bacteremia SSI Rheumatic and scarlet fevers Microbiology Bacterial Classifications Vibrio – comma shaped rods Spirilla – corkscrew shaped rods Spirochetes – corkscrew shaped rods capable of waving and twisting motions Microbiology Vibrio cholerae Cholera Found in drinking water contaminated with feces Severe diarrhea Death can occur within hours of onset of symptoms Microbiology Spirilla Commonly found in fresh water streams Causes diarrhea – poses a danger to wilderness campers One cell can multiply to over a million cells in less than three hours (exponentially) Microbiology Spirochete Treponema pallidum (syphilis) Microbiology Syphilis – Treponema pallidum STD – Four stages Primary syphilis – transmitted from primary sore (chancre) of affected individual to the mucous membranes (vaginal, anal, oral) of another Microbiology Secondary syphilis Rash appears 3-6 weeks after initial chancre (usually on palms of hands or soles of feet) Transmission may occur from ANY physical contact during this phase Microbiology Latent syphilis Noncontagious stage Tertiary syphilis Can damage any organ system Causes mental illness, blindness, neurologic disorders, heart disease, and death Microbiology Objective Define the term rickettsia, list its characteristics, and explain its relationship to humans. Microbiology Rickettsia Distinct bacterial group Discovered by American pathologist Harold Taylor Ricketts Gram negative bacilli (rods) or cocci (spheres) Obligate intracellular parasites Pleomorphic (occur in more than one morphologic form – able to mutate) Common feature – spread by arthropod vectors Microbiology Rickettsia prowazekii Causes typhus Louse-borne Microbiology Rickettsia rickettsii Causes Rocky Mountain Spotted Fever Spread by ticks, dogs, rodents Microbiology Objective Define the term virus, list its characteristics, and explain its relationship to humans. Microbiology Virus A virus is a small microorganism (smaller than bacteria) that cannot replicate unless it is within a living cell (obligate intracellular parasite). Most viruses are pathogenic with the exception of bacteriocidal viruses (bacteriophages). Viruses are NOT affected by antibiotics. All viruses are capable of mutation. Microbiology Virus – Main components Capsid – Outer protein shell that encloses the genetic material Core – Certain viruses also contain a protein layer underneath the capsid that also surrounds the genetic material Genetic material – DNA or RNA (“usually” not both) Envelope - Some viruses have an additional outer covering called an “envelope” that may appear to have spike-like projections Virus Anatomy Microbiology Viruses can affect: Plants Animals Bacteria All viruses have an affinity for a particular host. For example a human cannot contract a plant virus. Microbiology Classification – Viruses are classified by the disease that they cause. Examples: Common cold Polio Chickenpox Smallpox Measles Hepatitis AIDS Microbiology Common Cold Virus (rhinovirus) Microbiology Measles http://www.mayoclinic.com/health/measles/DS00331 Microbiology Poliomyelitis http://www.who.int/topics/poliomyelitis/en/ Microbiology Hepatitis B (affected liver) http://www.emedicinehealth.com/hepatitis_b/article_em.htm Microbiology Chickenpox (varicella) http://emedicine.medscape.com/article/969773-overview Microbiology Shingles (Herpes Zoster) https://health.google.com/health/ref/Shingles Microbiology HIV (Human immunodeficiency virus – causes AIDS) http://www.cdc.gov/hiv/topics/basic/ HIV Attacking a White Blood Cell Microbiology Smallpox Virus (Variola Virus) http://www.who.int/mediacentre/factsheets/smallpox/en/ Smallpox Victim Smallpox Victim Microbiology Objective Define the term prion, list its characteristics, and explain its relationship to humans. Microbiology Prion (pronounced “pree-on”) The word prion represents the term proteinaceous infectious particle Prions were first identified in 1982 by Stanley Prusiner of UCSF ALL known prion diseases are FATAL because the immune system does not recognize proteins as foreign and protection does not develop Microbiology Proteinaceous Particle (Infectious) Microbiology Prion Simple protein Much smaller than a virus Unique because it lacks a genome - (All other known infectious agents contain genetic material) Microbiology Prion Protein particles exist in two forms. The normal, an innocuous (harmless) protein called PrPc can change its shape to a harmful, diseasecausing form called PrPSc The abnormal, conversion from PrPc to PrPSc then proceeds via a chain reaction Several PrPSc proteins form long filamentous aggregates that gradually damage neuronal tissue PrPSc is VERY resistant to high temperatures, UV-irradiation, and strong degradative enzymes Microbiology Prion Prion diseases affect animals and humans Microbiology Sheep Scrapie First described in the 18th century http://www.aphis.usda.gov/animal_health/videos/scrapie-narrated.mpg Microbiology Cows Bovine Spongiform Encephalopathy – BSE (Mad Cow Disease) http://www.5min.com/Video/Risks-of-Mad-Cow-Disease-173033501 http://www.fda.gov/animalveterinary/guidancec omplianceenforcement/complianceenforcement /bovinespongiformencephalopathy/default.htm Microbiology Deer Chronic Wasting Disease (CWD) http://www.cwd-info.org/index.php/fuseaction/about.main Microbiology Humans Creutzfeldt-Jacob Disease (CJD) Gross Appearance First reported in the 1920s http://www.mayoclinic.com/health/creutzfeldt-jakob-disease/DS00531 Microbiology Humans Creutzfeldt-Jacob Disease (CJD) Cross-Section Microbiology Humans Creutzfeldt-Jacob Disease (CJD) Micro - Histology Microbiology Humans Kuru Appeared in New Guinea in the early 1900s Recently identified as a prion disease Ritual mortuary cannibalism caused fatal epidemic Disease irradicated when cannibalism was terminated Microbiology How are prion diseases acquired? Theories include: Genetic transmission Spontaneous mutation of the proteinaceous particle Consumption of infected meat (including cannibalism) Transplantation/injection (e.g., dura mater, cornea, growth hormone) Contact with contaminated surgical instrumentation Microbiology Recommended Practices Incinerate items used on known spongiform encephalopathy patients Quarantine items until disease is ruled out Staff education Develop policies, procedures, and guidelines for suspected cases Microbiology Modified Sterilization Protocol (when incineration is NOT an option) Steam Sterilization (previously cleaned items) 272º F for 18 minutes or 250º F for 1 hour OR Soak 1 hour in 5,000 ppm sodium hypochlorite (NaOH) or 1 molar (M) sodium hydroxide THEN rinse, clean, and steam sterilize as described above