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CHAPTER 20 MEDICAL MICROBIOLOGY PRETEST True or False 1. Microbiology is the scientific study of microorganisms and their activities. 2. A disease that can be spread from one person to another is known as an infectious disease. 3. Droplet infection is the transfer of pathogens from a fine spray emitted from a person already infected with the disease. 4. Streptococci are round bacteria that grow in pairs. 5. Chickenpox is caused by a virus. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 2 PRETEST, CONT. True or False 6. The course adjustment on a microscope is used to obtain precise focusing of an object. 7. The purpose of transport media is to provide nutrients for the multiplication of the specimen. 8. A throat specimen should be collected from the tonsillar area and posterior pharynx. 9. A wet mount is used to examine microorganisms in the living state. 10. A smear is material spread on a slide for microscopic examination. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 3 Content Outline Introduction to Microbiology 1. Microbiology: the scientific study of microorganisms and their activities a. Microorganisms: Tiny living plants or animals that cannot be viewed with the naked eye 2. Anton van Leeuwenhoek (1632-1723) a. Designed a magnifying glass strong enough for viewing microorganisms • Precursor of modern microscope b. First to observe and describe protozoa and bacteria Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 4 Introduction to Microbiology, cont. 3. Microbiology: Deals with unicellular (onecelled) microscopic organisms 4. All life processes of a microorganism are performed by one cell including: a. Ingestion of food substances and their use for energy b. Growth c. Reproduction d. Excretion Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 5 Introduction to Microbiology, cont. 5. Microorganisms are ubiquitous a. Means they are found everywhere: • Air • Plants • Food • Animals • Water • Humans • Soil Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 6 Introduction to Microbiology, cont. 6. Only a small number of microorganisms are pathogenic (disease-producing) 7. Pathogen infects host: produces symptoms of that disease a. Assists physician in diagnosing that disease Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 7 Introduction to Microbiology, cont. 8. Microbiologic tests: a. Also help physician identify pathogen causing disease 9. Identification of pathogen: leads to proper treatment 10.Most microbiologic tests: performed at outside laboratory a. MA often responsible for collecting the specimen Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 8 Normal Flora 1. Every individual has normal flora: 2. Normal flora: Harmless microorganisms that normally reside in many parts of the body but do not cause disease a. Examples of body areas with an abundant normal flora • Surface of skin • Mucous membranes of gastrointestinal (GI) tract • Respiratory tract • Genitourinary tract Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 9 The Normal Flora, cont. 3. Normal flora may be beneficial a. Example: • Normal flora in intestinal tract: – Feed on potentially harmful microorganisms – Synthesize vitamin K (needed for blood clotting) From Mahon CR, Manuselis G, Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. 10 Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. The Normal Flora, cont. 4. Sometimes normal flora can become pathogenic and cause disease a. May occur during lowered body resistance Courtesy Cathy Bissonette Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 11 Infection 1. Infection: invasion of the body by pathogenic microorganisms a. If body conditions are favorable • Pathogen grows and multiplies • Results in an infectious disease – Infectious disease: a disease caused by a pathogen that produces harmful effects on its host Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 12 Infection, cont. 2. Not all invading pathogens are able to cause disease 3. Body attempts to stop invasion a. Uses second line of defense mechanisms: • Inflammation: a protective response of the body to the entrance of foreign matter. – The purpose of inflammation is to destroy invading microorganisms and to repair injured tissue • Phagocytosis by white blood cells (WBCs) • Production of antibodies Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 13 Infection, cont. 4. First line of defense: works to prevent entrance of pathogens into body a. Examples: • Skin • Mucous membranes • Mucus and cilia in nose Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 14 Infection, cont. • Coughing and sneezing • Tears and sweat • Acid nature of urine and vaginal secretions • Hydrochloric acid secreted by stomach Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 15 Infection, cont. 5. If body defenses are successful: pathogens are destroyed a. Individual does not get infectious disease 6. If body defenses are overcome by pathogen a. Individual gets infectious disease Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 16 Infection, cont. 7. Many infectious diseases are contagious a. Contagious: capable of being transmitted directly or indirectly from one person to another • Droplet infection: inhalation of pathogens from a fine spray emitted by person infected with disease • When infected individual exhales (breathing, talking, coughing, sneezing) – Pathogens are dispersed on minute liquid particles • Infected individual: should cover mouth when coughing and sneezing Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 17 Stages of an Infectious Disease 1. Infection: invasion and multiplication of pathogenic microorganisms in the body 2. Incubation period: interval of time between invasion by a pathogen and appearance of first symptoms of disease a. May range from a few days to several months b. Pathogen is growing and multiplying during this time Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 18 Stages of an Infectious Disease, cont. 3. Prodromal period: first symptoms appear a. Examples: • Headache • Feeling of illness From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 19 Stages of an Infectious Disease, cont. 4. Acute period: disease is at its peak and symptoms fully developed a. Example: fever 5. Decline period: symptoms begin to subside 6. Convalescent period: patient regains strength and returns to good health Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 20 Microorganisms and Disease 1. Groups of microorganisms known to contain species capable of causing human disease: a. Bacteria b. Viruses c. Protozoa d. Fungi (yeast) e. Animal parasites Courtesy Cathy Bissonette. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 21 Bacteria 1. Microscopic single-celled organisms 2. Of 1700 species known to dwell in humans: only about 100 produce human disease 3. Antibiotics: help in controlling bacterial infections a. Not effective against viral infections 4. Classified according to shape Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 22 Classification of Bacteria by Shape Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 23 Staphylococci 5. Cocci: round shape a. Staphylococci • Round bacteria that grow in grapelike clusters From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 24 Staphylococci, cont. • S. epidermidis: normally present on skin surface and mucous membranes of the mouth, nose, throat, intestines – Usually nonpathogenic – Break in the skin can allow invasion 1) Example: cut 2) Results in a mild infection Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 25 Staphylococci, cont. • S. aureus: associated with pathologic conditions - Boils - Abscesses - Carbuncles - - Pimples Staphylococcal food poisoning - Impetigo - Wound infections Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 26 Staphylococci, cont. • Staphylococcal infections usually cause pus formation (suppuration) – Termed: pyogenic infections From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 27 Streptococci b. Streptococci • Round bacteria that grow in chains From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 28 Streptococci, cont. • Before antibiotics: major cause of human death • Diseases caused by streptococci: - Streptococcal sore throat - Scarlet fever - Rheumatic fever - Pneumonia - Puerperal sepsis - Erysipelas - Carbuncles - Impetigo From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 29 Diplococci c. Diplococci • Round bacteria that grow in pairs • Diseases caused by diplococci: – Pneumonia – Gonorrhea – Meningitis From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 30 Bacilli 6. Bacilli: rod shaped bacteria a. Found in soil and air Courtesy Cathy Bissonette. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 31 Bacilli, cont. b. Some are able to form spores • Spore: a hard, thick-walled capsule formed by some bacteria that contains only the essential parts of the protoplasms of the bacterial cell • Enables them to resist adverse conditions – Heat – Disinfectants Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 32 Bacilli, cont. • Diseases caused by bacilli: – Botulism – Tetanus – Gas gangrene – Gastroenteritis caused by Salmonella food poisoning – Typhoid fever – Pertussis – Bacillary dysentery – Diphtheria – Tuberculosis Courtesy Cathy Bissonette. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 33 Bacilli, cont. c. Escherichia coli (E. coli): a species of bacillus • Normal flora of the large intestine From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 34 Bacilli, cont. • Normally harmless • May cause a urinary tract infection (UTI) if enters urinary tract due to: – Lowered resistance – Poor hygienic practices From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 35 Spirilla 7. Spirilla: spiral or curved shaped bacteria a. Treponema pallidum b. Causative agent of syphilis c. Cannot be grown in culture media • Diagnosis of syphilis made through serologic tests Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 36 Spirilla, cont. b. Vibrio cholerae • Causative agent of cholera – Immunization and proper sanitation: have eliminated cholera in United States Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 37 Viruses 1. Smallest living organisms 2. Electron microscope must be used to view them 3. Can infect: plants, animals, humans 4. Use nutrients inside host cells a. For metabolic and reproductive needs Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 38 Viruses, cont. 5. Diseases caused by viruses: a. Influenza b. Chickenpox c. Rubeola (measles) d. Rubella (German measles) e. Mumps Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 39 Viruses, cont. f. Poliomyelitis g. Smallpox h. Rabies i. Herpes simplex j. Herpes zoster Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 40 Viruses, cont. k. Yellow fever l. Hepatitis m. Majority of diseases in upper respiratory tract • Example: Common cold Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 41 What Would You Do? What Would You Not Do? Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 42 What Would You Do? What Would You Not Do? Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 43 Microscope 1. Compound microscope: used most often in medical office a. Consists of two-lens system 2. Components: a. Support system b. Optical system Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 44 Parts of a Microscope Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 45 Support System 1. Frame a. Supports microscope b. Consists of: • Base: for support • Arm: – For carrying it – To support magnifying and adjusting systems Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 46 Support System, cont. 2. Stage a. Flat, horizontal platform b. Slide is placed on stage Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 47 Support System, cont. c. Has small round opening in center • Permits light from below to pass through the object being viewed and up into lenses d. Standard microscope: has metal clips to hold slide in place • Slide must be moved by hand Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 48 Support System, cont. e. Mechanical stage: allows movement of slide in vertical or horizontal position using adjustment knobs • Needed for: – Differential white blood cell count – Inspection of gram-stained smear Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 49 Support System, cont. 3. Light source a. Located at the base b. Consists of: built-in illuminator • Switch for turning on and off c. Light directed to condenser above • Then through object to be viewed Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 50 Support System, cont. 4. Condenser a. Compound microscopes have lens system between light source and object • Known as substage condenser Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 51 Support System, cont. b. Popular type: Abbe's condenser • Consists of two lenses • Collects and concentrates light rays and directs them up – Brings light rays to a focus on the object so it is well-illuminated Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 52 Support System, cont. 5. Diaphragm a. Controls the amount of light focused on an object b. Amount of light controlled by iris diaphragm c. Located beneath or inside condenser d. Consists of: • Series of horizontally arranged interlocking plates • With central opening (aperture) Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 53 Support System, cont. e. Diaphragm lever: increases or decreases aperture • Increases or decreases light admitted Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 54 Support System, cont. f. As magnification increases: more light is needed • Example: Oil immersion requires increased light intensity g. Low-power objective • Do not need as much light Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 55 Support System, cont. h. Degree of illumination: also influenced by density of the object • Stained structures: require more light than unstained specimens 6. Adjustment knobs a. Coarse adjustment: to obtain an approximate focus quickly b. Fine adjustment: to obtain precise focusing Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 56 Support System, cont. c. Adjustment knobs: -May be mounted as two separate knobs -May be placed together Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 57 Optical System 1. Compound microscopes have two-lens magnification system a. Magnification: The ratio of the apparent size of an object viewed through the microscope to the actual size of the object 2. The Eyepiece a. First lens system b. Also called ocular lens Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 58 Optical System, cont. c. Located at top of body tube d. Marked 10×(magnifies 10 times) e. Monocular microscope: has one eyepiece f. Binocular microscope: has two eyepieces Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 59 Optical System, cont. 3. Objective Lenses a. Second lens system b. Three objective lenses: located on a revolving nosepiece c. Each objective has a different degree of magnification: engraved on metal shaft • Low-power objective: 10× • High-power objective: 40× • Oil-immersion objective: 100× Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 60 Optical System, cont. d. May have color-coded rings to identify objective • Example: – Low-power objective: yellow ring – High-power objective: blue ring – Oil-immersion objective: white ring Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 61 Optical System, cont. e. Metal shafts of objectives: differ in length • Low-power objective: shortest length • High-power objective: length between low and oil objectives • Oil-immersion objective: longest length Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 62 Optical System, cont. f. Total magnification: multiply the ocular magnification by the objective magnification: • Low-power total magnification: -100× (10×10) • High-power total magnification: -400× (10×40) • Oil-immersion magnification: -1000× (10×100) Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 63 Optical System, cont. 4. Focus a. Barrel focus: body tube or barrel moves while stage remains stationary b. Stage focus: stage moves while body tube remains stationary Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 64 Optical System, cont. 5. Low and High Power a. Low-power objective used for: • Initial focusing and light adjustment • Initial observation and scanning requirements b. High-power objective used for: • A more thorough study of the slide Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 65 Optical System, cont. c. Working distance: distance between tip of lens and slide • High-power objective: working distance is short • Care must be taken when using high-power objective – To prevent it from striking and breaking slide – To prevent damaging the lens Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 66 Optical System, cont. d. Most compound microscopes are parafocal • Parafocal: once focused on low power the nosepiece can be rotated to high power and focused with fine adjustment Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 67 Optical System, cont. 6. Oil Immersion a. Provides highest magnification b. Used to view very small structures or the detail of structures • Microorganisms • Blood cells Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 68 Optical System, cont. c. Oil immersion has a very short working distance • Lens nearly rests on slide Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 69 Optical System, cont. d. Must use immersion oil • Does not dry out when exposed to air for a long time e. Drop of oil placed on slide Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 70 Optical System, cont. • Oil resides between objective and slide • Provides path for light to travel between slide and lens • Prevents scattering of light rays – Permits clear viewing of small structures Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 71 Optical System, cont. f. Oil immersion used for: • Differential white blood counts • Examination of gramstained smears Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 72 Care of the Microscope 1. Carry with two hands: one on arm and one under base a. Place down gently: prevents jarring it • Could damage delicate parts Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 73 Care of the Microscope, cont. 2. Do not touch the lenses: prevents fingerprints a. Avoid wearing mascara: difficult to remove from lens 3. When not in use: keep covered with plastic dust cover a. Store in case or cupboard b. Rotate nosepiece to low objective • Position as close as possible to stage Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 74 Care of the Microscope, cont. 4. Clean by washing enameled surfaces with soap and water a. Dry thoroughly with a soft cloth 5. Clean stage after each use: a. Wipe stage with gauze or tissue b. If immersion oil gets on stage: • Remove with gauze moistened with xylene Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 75 Care of the Microscope, cont. 6. Ocular, objectives and condenser a. Consist of hand-ground optical lenses • Must keep spotlessly clean – Use dry lens paper Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 76 Care of the Microscope, cont. • Optical glass: softer than ordinary glass – Do not use tissue or gauze to clean: scratches the lens • If lenses are especially dirty – Clean with commercial lens cleaner or xylene – Dry and polish with lens paper Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 77 Clean with lens paper: Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 78 Care of the Microscope, cont. 7. Light source: keep free of dust, lint, and dirt a. Periodically polish with lens paper Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 79 Care of the Microscope, cont. 8. Microscope that is malfunctioning a. Have repaired by qualified service person • Do not try to fix yourself – May result in further damage Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 80 Microbiologic Specimen Collection 1. Purpose: a. To identify pathogen causing the disease • Assists in the diagnosis – Example: Obtaining a urine specimen to assist in diagnosis of a urinary tract infection 2. Specimen: a small sample or part taken from the body to show the nature of the whole Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 81 Microbiologic Specimen Collection, cont. 3. MA often responsible for collecting microbiologic specimens a. Examples: throat, nose, and wound specimens 4. MA may assists physician in collecting specimens a. Examples: vagina, cervix, urethra, rectum Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 82 Microbiologic Specimen Collection, cont. 5. Sterile swab used to collect specimen a. Swab: small piece of cotton wrapped around the end of a slender wooden or plastic stick • Passed across body surface or opening – To obtain a specimen for microbiologic analysis Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 83 Sterile Swab Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 84 Microbiologic Specimen Collection, cont. 6. To prevent inaccurate test results: a. Use good techniques of medical and surgical asepsis b. Do not contaminate with extraneous microorganisms • Extraneous microorganisms: undesirable microorganisms that can enter the specimen in various ways. – They grow and multiply – Can obscure and prevent identification of pathogens that might be present Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 85 Microbiologic Specimen Collection, cont. c. To prevent contamination of a specimen with extraneous microorganisms: • Supplies used to collect specimen must be sterile – Examples: Swabs and specimen containers Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 86 Microbiologic Specimen Collection, cont. • Do not allow swab to touch areas surrounding collection site – Example: Throat specimen: Do not allow swab to touch inside of mouth Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 87 Microbiologic Specimen Collection, cont. 7. Follow OSHA Bloodborne Pathogens Standard when performing microbiologic procedures: a. Wear gloves b. No eating, drinking, smoking, or applying makeup • Pathogens can be transmitted through hand-tomouth contact c. Do not lick labels for specimen containers d. Cover breaks in skin with a bandage Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 88 Microbiologic Specimen Collection, cont. e. If accidentally touch specimen: • Wash area immediately with soap and water f. If specimen comes in contact with worktable: • Immediately clean with soap and water followed by disinfectant (e.g., phenol) g. At end of day: • Clean worktable with disinfectant Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 89 Microbiologic Specimen Collection, cont. 8. After collection: place specimen in proper container a. Securely fasten lid b. Label with: • Patient's name • Date • Source of specimen • Initials Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 90 Handling and Transporting Microbiologic Specimens 1. Delay in processing specimen: a. May cause death of pathogens b. Overgrowth of specimen by normal flora (usually collected along with pathogen) 2. If specimen analyzed in office: a. Examine under microscope or culture immediately Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 91 Handling and Transporting Microbiologic Specimens, cont. 3. If specimen transported to outside laboratory by courier a. Place in transport medium (e.g., Starswab II, Culturette) • Prevents drying of specimen • Preserves specimen in its original state b. Outside laboratory: provides instructions on care and handling of specimen Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 92 Handling and Transporting Microbiologic Specimens, cont. c. Specimen must be accompanied by laboratory request which includes: • Physician's name and address • Patient's name, age, and gender • Date and time of collection • Type of microbiologic examination requested Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 93 Handling and Transporting Microbiologic Specimens, cont. • Source of specimen – Examples: throat, wound, urine • Physician's clinical diagnosis – Clinical diagnosis: a tentative diagnosis of a patient's condition obtained through the evaluation of the health history and the physical examination without the benefit of laboratory or diagnostic tests Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 94 Handling and Transporting Microbiologic Specimens, cont. • If patient is receiving antibiotic treatment – Antibiotics suppress growth of bacteria – Could cause false-negative results Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 95 Handling and Transporting Microbiologic Specimens, cont. 4. Wound Specimens a. Must be collected from within wound (rather than from surface of wound) • To obtain accurate and reliable test results b. Usually two swabs used Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 96 Handling and Transporting Microbiologic Specimens, cont. c. Swab inserted into area of wound that contains the most drainage d. Swab gently rotated from side to side • To completely absorb microorganisms present e. Swab placed in specimen container f. Process repeated with second swab Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 97 Collection and Transport System 1. Types available a. Culturette b. Starswab II Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 98 Collection and Transport System, cont. 2. Consist of sterile swab and plastic tube that contains a transport medium a. Comes packaged in peel-apart envelope b. Should be stored at room temperature Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 99 Collection and Transport System, cont. 3. Procedure for use a. Complete laboratory request form b. Sanitize hands c. Apply gloves Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 100 Collection and Transport System, cont. d. Check expiration date e. Peel open envelope f. Remove cap from the collection tube g. Remove cap/swab unit from envelope h. Collect specimen using aseptic technique • Do not allow swab to touch any other area Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 101 Collection and Transport System, cont. i. Insert swab into collection tube j. Push cap/swab as far as it will go to completely immerse in transport medium k. Remove gloves and sanitize hands Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 102 Collection and Transport System, cont. l. Label tube m. Place in biohazard specimen transport bag • Place laboratory request in outside pocket n. Chart procedure o. Transport to laboratory to laboratory within 24 hours Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 103 What Would You Do? What Would You Not Do? Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 104 What Would You Do? What Would You Not Do? Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 105 Cultures 1. Once a microbiologic specimen is collected a. Examined to determine type of microorganisms present 2. Most specimens: only contain a small number of pathogens a. Must induce pathogens to grow and multiply Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 106 Cultures, cont. 3. Culture medium: a mixture of nutrients on which microorganisms are grown in the laboratory a. Must contain growth requirements: • Presence or absence of oxygen • Proper nutrition • Temperature • pH • Moisture Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 107 Cultures, cont. b. Solid culture medium • Blood agar most commonly used – Sheep's blood added to agar 1) Contains nutrients to grow bacteria From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 108 Cultures, cont. c. Liquid culture medium • Referred to as broth • Usually contained in a tube • Example: nutrient broth Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 109 Cultures, cont. 4. Culture media: must be stored in refrigerator a. Warm to room temperature before use b. Do not use cold culture medium • Results in death of microorganisms Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 110 Cultures, cont. 5. Petri plate: frequently used to hold solid culture medium a. Consists of shallow circular dish with a cover (made of glass or clear plastic) b. Microorganisms cultured on surface of medium in plate c. Allows examination of culture • Prevents microorganisms from entering or escaping Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 111 Cultures, cont. d. Culture: a mass of microorganisms growing in a laboratory culture medium From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 112 Cultures, cont. 6. Most offices use commercially prepared culture media in disposable plastic Petri plates a. Plates are packaged in plastic bag • Prevents medium from drying out b. Stored in refrigerator with medium side facing up • Prevents condensation on surface of medium Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 113 Cultures, cont. c. Expiration date: check before using • Do not use plates that are: – Expired – Dried out – Contaminated Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 114 Cultures, cont. 7. Inoculation of solid culture medium in Petri plate a. Specimen swab is lightly rolled across surface of medium (streaking) • Only remove Petri plate cover during streaking of specimen – Prevents contamination with extraneous microorganisms b. Culture is incubated for 24 to 48 hours Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 115 Cultures, cont. 8. Most specimens for analysis contain a mixture of microorganisms a. Mixed culture: contains two or more types of microorganisms • Suspected pathogen must be separated to analyze b. Pure culture: contains only one type of microorganism • Tests to identify pathogen are performed on pure culture Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 116 Cultures, cont. 9. Viruses cannot be grown by the above method a. Are identified by: • Culturing on living tissue • Serologic tests Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 117 Streptococcus Testing 1. Streptococcal sore throat (streptococcal pharyngitis) a. Primarily affects children and young adults b. Causative agent: group A beta-hemolytic streptococcus Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 118 Streptococcus Testing, cont. 2. Potentially serious condition a. Small percentage of patients develop a poststreptococcal sequela • Sequela: a morbid secondary condition that occurs as a result of a less serious primary infection – Example: rheumatic fever b. Early diagnosis and treatment of “strep throat” with antibiotics is important Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 119 Rapid Streptococcus Tests 1. Detects group A streptrococci from a throat swab Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 120 Rapid Streptococcus Tests, cont. a. Requires only 4 to 10 minutes to process • Diagnosis can be made while patient is at office – If streptococci present: antibiotics prescribed Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 121 Rapid Streptococcus Tests, cont. 2. Direct antigen identification test: most frequently used a. Particles sensitized to streptococcal antibody are combined with specimen (antigen) b. Produces a color change if streptococcal is present (through antigen-antibody reaction) c. Test includes a control • To ensure results are accurate and reliable Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 122 Rapid Streptococcus Tests, cont. 3. Includes a control to verify the results are accurate and reliable 4. Advantage a. Provides physician with immediate results Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 123 Rapid Streptococcus Tests, cont. 5. Examples of tests a. Q Test Strep b. Clearview Strep A c. Quick Vue In-Line Strep A Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 124 Hemolytic Reaction and Bacitracin Susceptibility 1. Streptococci classified into three types based on their hemolytic properties: a. Alpha b. Beta: produces streptolysin (exotoxin) that hemolyzes red blood cells (RBCs) c. Gamma Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 125 Hemolytic Reaction and Bacitracin Susceptibility, cont. 2. Further divided according to their antigenic properties a. Into 15 subgroups designated by letters A through O • Group A streptococci: susceptible to the antibiotic bacitracin – Are destroyed by it Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 126 Hemolytic Reaction and Bacitracin Susceptibility, cont. 3. Test uses these hemolytic and antigenic properties to interpret test results a. Causative agent of strep throat: • Group A beta-hemolytic streptococcus Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 127 Hemolytic Reaction and Bacitracin Susceptibility, cont. 4. Testing procedure: a. Throat specimen inoculated on blood agar medium b. Bacitracin disc placed on surface of medium c. Medium incubated for 18 to 24 hours Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 128 Hemolytic Reaction and Bacitracin Susceptibility, cont. c. Plate examined for hemolytic and antigenic reactions d. Positive reaction for Group A betahemolytic streptococcus • Hemolytic reaction: clear wide colorless zone around bacterial colonies – Streptolysin from beta streptococci has hemolyzed RBCs • Antigenic reaction: clear zone of inhibition around the bacitracin disk – Group A streptococci are susceptible to and destroyed by bacitracin Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 129 Bacitracin Susceptibility Test From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 130 Sensitivity Testing 1. Determines susceptibility of pathogenic bacteria to antibiotics a. Allows physician to determine best antibiotic to use to treat patient's condition 2. Must be performed on a pure culture Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 131 Sensitivity Testing, cont. 3. Disk-diffusion method a. Most common method b. Use disks impregnated with various antibiotics • Dropped on surface of solid culture medium in Petri plate inoculated with pathogen Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 132 Sensitivity Testing, cont. c. Culture is incubated • Allows antibiotics to diffuse into culture medium d. If pathogen is susceptible to an antibiotic: e. Clear zone without bacterial growth around disk • Indicates antibiotic is effective against pathogen Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 133 Sensitivity Testing, cont. e. If pathogen is resistant to an antibiotic • No clear zone around disk – Indicates antibiotic was unable to kill pathogen Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 134 Sensitivity Testing From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 135 What Would You Do? What Would You Not Do? Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 136 What Would You Do? What Would You Not Do? Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 137 Microscopic Examination of Microorganisms 1. Can be examined in fixed or in living state 2. Examining microorganisms in a fixed state a. Most microorganisms are examined this way b. Preparation of a smear through heat fixation: followed by staining • Example: Gram staining • Easier to examine when stained Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 138 Microscopic Examination of Microorganisms, cont. 3. Examining microorganisms in a living state a. Allows visualization of movement b. Helpful in identification of certain motile microorganisms • Example: Trichomonas vaginalis Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 139 Microscopic Examination of Microorganisms, cont. c. Microorganisms suspended in liquid medium • Microorganisms are free to move about d. Wet mount: most common method used Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 140 Wet Mount Method 1. MA prepare slide 2. Drop of fluid containing organism: placed on glass slide a. Covered with coverslip b. Coverslip ringed with petroleum jelly • Provides seal – Reduces rate of evaporation – Evaporation could lead to drying out of slide and death of specimen Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 141 Wet Mount Method, cont. 1. Slide placed under microscope 2. Examined under high power by physician 3. Slide discarded in biohazard sharps container Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 142 Wet Mount Method Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 143 Smears 1. Smear: material spread on a slide for microscopic examination Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 144 Smears, cont. 2. Prepared: a. Directly from specimen swab b. Specimen first grown on culture medium 3. Most smears must be stained before viewing under microscope Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 145 Smears, cont. 4. Smears can be prepared and examined immediately a. Helpful when time is a factor 5. Provides a preliminary clue a. While more time-consuming tests are being performed Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 146 Gram Stain 1. Used in combination with other tests: to diagnose and treat infectious diseases 2. Bacteria in a smear are colorless a. Makes them difficult to identify b. Staining allows for direct viewing of bacteria • Size • Shape • Growth patterns Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 147 Gram Stain, cont. 3. Discovered by Christian Gram in 1883 a. Differentiates bacteria on basis of their color reactions to various stains Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 148 Gram Stain, cont. 4. Gram-positive bacteria: exhibit a purple color a. When treated with purple crystal violet dye: gram-positive bacteria permanently retain this dye Courtesy Cathy Bissonette. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 149 Gram Stain, cont. 5. Gram-negative bacteria: exhibit pink or red color a. Gram-negative bacteria are unable to retain purple dye: are counterstained with a red dye 6. Staining characteristics of bacteria: From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. a. Caused by differences in the chemical composition of bacterial cell walls Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 150 Gram Stain, cont. 7. Infectious diseases caused by grampositive bacteria: a. Streptococcal sore throat b. Scarlet fever c. Rheumatic fever d. Diphtheria Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 151 Gram Stain, cont. e. Lobar pneumonia f. Tetanus g. Botulism Courtesy Cathy Bissonette. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 152 Gram Stain, cont. 8. Infectious diseases caused by gramnegative bacteria: a. Whooping cough b. Gonorrhea c. Meningitis From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 153 Gram Stain, cont. d. Bacillary dysentery e. Cholera f. Typhoid fever g. Plague From Mahon CR, Manuselis G Jr: Textbook of diagnostic microbiology, ed 2, Philadelphia, 2000, Saunders. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 154 Gram Stain, cont. 9. Categories of gram stained bacteria: a. Gram-positive rods b. Gram-negative rods c. Gram-positive cocci d. Gram-negative cocci • Example: gonorrhea caused by a gram-negative diplococcus Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 155 Prevention and Control of Infectious Diseases 1. Practice good techniques of medical asepsis. 2. Obtain proper nutrition and rest. 3. Use good hygienic measures. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 156 Prevention and Control of Infectious Diseases, cont. 4. Infected individuals should contact their physicians to ensure early diagnosis and treatment. 5. MA responsible for educating community members a. Practices that reduce transmission of pathogens b. Practices that control and prevent infectious diseases Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 157 POSTTEST True or False 1. Microorganisms that reside in the body but do not cause disease are known as transient flora. 2. The invasion of the body by a pathogenic microorganism is known as infection. 3. The interval of time between the invasion by a pathogen and the first symptoms of disease is known as the prodromal period. 4. Staphylococcal infections usually result in pus formation. 5. E. coli normally reside in the urinary tract. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 158 POSTTEST, CONT. True or False 6. The high-power objective has a magnification of 40x. 7. Examination of urine sediment requires the use of the oil immersion objective. 8. A mixed culture contains two or more types of microorganisms. 9. The purpose of sensitivity testing is to identify the type of microorganism present. 10. When viewed under a microscope gram-positive bacteria appear pink or red. Elsevier items and derived items © 2008 by Saunders, an imprint of Elsevier Inc. 159