Assisting in Microbiology and Immunology Chapter 55 Copyright

Learning Objectives Define, spell, and pronounce the terms listed in the vocabulary. Apply critical thinking skills in performing patient assessment and care. Cite the protocols for the collection, transport, and processing of specimens. Identify the elements needed for microbial growth. Compare bacteria with viruses. Describe the characteristics of common viral diseases. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 2

Learning Objectives Describe the bacterial structures used in identification. Compare bacteria with fungi, parasites, and protozoa. Describe various bacterial morphologies. Explain the characteristics of common diseases caused by bacteria. Describe the unusual characteristics of chlamydia, rickettsia, and mycoplasma. Identify the characteristics of common diseases caused by fungi as well as protozoan and parasitic diseases. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 3

Learning Objectives Perform patient education on the collection of a stool specimen for ova and parasite testing. Describe the equipment needed in a microbiology laboratory. List the different growth media used for culturing. Describe the preparation of a bacterial smear. Perform the procedure for the inoculation of a blood agar plate. Perform a urine culture. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 4

Learning Objectives Perform a screening urine culture test. Prepare a direct smear or culture smear for staining. Compare and contrast the throat culture for Streptococcus pyogenes with the rapid strep test. Perform a rapid strep test. Describe three microbiology tests that use a rapid identification technique. Describe the method used for antimicrobial susceptibility testing. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 5

Learning Objectives Explain how pinworm testing is done and when it must be performed. Perform a cellulose tape collection for pinworms. Discuss the purpose of immunology testing. Describe three rapid immunology tests that could be done in the POL. Perform the Mono-test for mononucleosis. Discuss legal and ethical issues in laboratory testing. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 6

Microbiology The medical assistant needs to understand the role of microorganisms. Ø Responsibilities include preventing nosocomial infections and assisting with infection control The main objective of microbiology procedures is to identify the organisms responsible for illness so that the physician can properly treat the patient. Microbiology procedures may be performed in the POL or in the microbiology department of a medical referral laboratory. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 7

Specimen Collection and Transport Specimen collection and handling are among the most critical considerations in patient care because any results the laboratory generates are directly dependent on the quality of the specimen and its condition on arrival in the laboratory. When collecting specimens for microbiological analysis, the medical assistant should ask two questions: “In what ways can I prevent extraneous microorganisms from contaminating this sample? ” “What can I do to prevent myself from becoming infected while I collect this sample? ” Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 8

Specimen Collection and Transport: Table 55 -1 Microorganisms are living organisms and must be provided with conditions that permit their survival but do not permit their multiplication. If microorganisms are allowed to multiply after their collection, the culture results will not reflect the true disease state. Transport media Ø Stuart medium or Amies medium The medical assistant must strictly follow the directions. Transport the specimen directly after collection or by courier. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 9

Containers for Transport Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of

Collection Devices From De la Maza LM, Pezzlo MT, Baron EJ: Color atlas of diagnostic microbiology, St Louis, 1997, Mosby. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 11

Critical Thinking Application Aaron Mitchell, age 9 years, was brought into the clinic this morning at 9 o’clock with scabbing sores on his upper lip. Dr. Chowdry suspects impetigo and orders a wound culture. How will Anna collect this culture? What device might she use? How should she store this specimen until the courier, who does not come until 3: 00 pm, arrives? Anna knows that impetigo is highly contagious. How can she protect herself from becoming infected? Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 12

Classification of Microorganisms Bacteria, fungi, protozoa Parasitic worms Viruses Once inside a host cell they use the host cell’s structures to multiply; obligate intracellular parasites Ø Table 55 -2 Ø Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 13

Naming of Microorganisms Binomial system of nomenclature Assigns two names: the first is the genus and the second is the species Ø genus name begins with a capital letter, species name with a lowercase letter Ø Neisseria gonorrhoeae Ø The genus name of the organism may be represented by a single letter after the organism’s full genus and species name is written once in a report. For example, it is common to refer to Escherichia coli as E. coli Ø Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 14

Critical Thinking Application While preparing to collect the specimen from Aaron, Anna receives a telephone call from Bio. Stat. Lab, the referral laboratory the clinic uses. The results from Ms. Tina Walker’s urine culture and from Mr. Robert Livore’s abscess culture are complete. Anna listens carefully to the technician from the referral laboratory regarding Mr. Livore’s abscess culture and jots down “staphylococcus” on the reporting form. She pauses when the technician stops and asks what species of “staph. ” Why is this important? The technician also reports Ms. Walker’s test results, indicating that the organism causing her urinary tract infection was identiﬁed as Escherichia coli. How could E. coli have infected the urinary tract? Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 15

Typical Pathogenic Bacteria are single-celled prokaryotic organisms that reproduce by binary fission. Often classified by their shape, staining characteristics, and the environmental conditions in which they thrive. Three types of cell wall structures based on reactions in specialized stains: gram positive, gram negative, acid fast. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 16

Bacterial Shapes Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier

Critical Thinking Application Anna knows that impetigo is caused by Staphylococcus aureus. Without using a microscope, she knows what the organism will look like. How does she know? Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 18

Bacterial Oxygen Requirements Aerobes – require oxygen to live Anaerobes – die in the presence of oxygen Facultative anaerobes – flexible concerning oxygen requirements and, although they are anaerobes, can survive in the presence of oxygen Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 19

Bacterial Physical Structures Flagella – thin, long structures that aid in propulsion Capsules – thick gelatinous coats surrounding the cell wall Endospores – intracellular structures that allow the cell to remain viable when environmental conditions are not favorable Tables 55 -3 to 55 -5 list some important infectious diseases caused by typical pathogenic bacteria Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 20

Unusual Pathogenic Bacteria Viruses range between 10 and 100 nanometers (nm); typical pathogenic bacteria are between 1000 and 5000 nm; these are tiny bacteria that fall between the size range of viruses and typical pathogenic bacteria Chlamydia – tiny bacteria; require host cells for growth Mycoplasma – no PG in cell wall but are not obligate parasites like rickettsia and chlamydia Rickettsiae – tiny gram-negative bacteria that are transmitted by blood-sucking arthropods; cannot multiply outside of a host cell Table 55 -6 Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 21

Fungi: Table 55 -7 Mycology – study of fungi and diseases they cause Fungi are eukaryotes, larger than bacteria Present in soil, air, and water Few cause disease Transmission – direct contact with infected persons, prolonged exposure to a moist environment, inhalation of contaminated dust or soil Treatment – resistant to antibiotics used for bacterial infections; treated with drugs active against their unusual cell walls Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 22

Parasites Parasitology – study of all parasitic organisms that live on or in the human body Transmitted by ingestion during the infective stage, direct penetration of the skin, inoculation by a vector Identified in urine, sputum, or tissue fluid or through tissue biopsy Helminths – worms; live on or in living organisms and nourish themselves at the expense of the host organism Protozoa – single-celled parasitic eukaryotes ranging in size from microscopic to macroscopic; present in moist environments and bodies of water; transmitted through contaminated feces, food, and drink Table 55 -8 Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 23

Parasites: Roundworms and Whipworms From Stepp CA, Woods MA: Laboratory procedures for medical office personnel, Philadelphia, 1998, Saunders. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 24

The Microbiology Laboratory Most laboratories have: Ø Refrigerator, autoclave, safety cabinet, microscope, and incubator Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 25

Inoculating Equipment Inoculating loops and needles Copyright © 2011, 2007, 2003, 1999 by Saunders,

Incineration Equipment Fastest way to sterilize reusable equipment Used to heat-fix smears Some laboratories use a Bunsen burner connected to a natural gas supply but most use an electric incinerator because of the reduced ﬁre hazard Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 27

Incineration Equipment (cont’d) From Stepp CA, Woods MA: Laboratory procedures for medical office personnel, Philadelphia, 1998, Saunders. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 28

Culture Media All-purpose or nutritive – supports growth of a wide variety of bacteria; will not support fastidious bacteria Selective – supports growth of one type of organism while inhibiting growth of others Differential – contains chemicals or dyes that alter the appearance of certain bacterial types; used in biochemical testing Enriched – contains complex organic materials that certain fastidious species must have in order to multiply; blood agar, needed for the growth of Streptococcus pyogenes, is made by adding sterile sheep blood to an all-purpose medium; used to cultivate pathogens Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 29

Culture Media Solid, liquid, or semisolid From Stepp CA, Woods MA: Laboratory procedures for medical office personnel, Philadelphia, 1998, Saunders. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 30

Inoculation of Media Quadrant streak – done with a loop to spread sample thinly over agar medium in several directions; separates bacteria so they grow in individual colonies Lawn or spread streak – used when assessing antibiotic effectiveness or counting colonies; spread sample with a swab or loop continuously over the entire plate Plates are incubated in an inverted position so condensation that accumulates on underside of lid does not fall down onto the growth; temperature and conditions of incubation depend on specimen source and suspected pathogens Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 31

Identification of Pathogens in the Microbiology Laboratory Culture is incubated 18– 24 hours; examined for evidence of pathogens Suspicious colonies are subcultured onto appropriate medium to isolate them in pure culture Throat culture – Streptococcus pyogenes, also known as group A beta-hemolytic Streptococcus, causes septic sore throat; grown on sheep blood agar plate; lysis indicates beta-hemolytic “strep” Ø Urine culture – count number of bacteria colonies present in a sample; each colony that grows on the plate represents 1000 colony-forming units per milliliter Ø Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 32

Beta Hemolysis Around Strep Colonies From Stepp CA, Woods MA: Laboratory procedures for medical office personnel, Philadelphia, 1998, Saunders. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 33

Critical Thinking Application The technician from the referral laboratory indicated that Ms. Walker had a urinary tract infection. Anna recorded the results “>100, 000 cfu/ml” on the patient's record. What does this number mean? Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 34

Differential Staining Ø Gram’s stain – Gram stain differentiates bacteria into two categories according to cell wall thickness; sequence of primary dyes applied to slide; dyes are taken up differently according to the chemical composition of the cell walls • Gram-positive – stain purple • Gram-negative – stain pink or red Ø Acid-fast stain – differentiates bacteria into two categories based on presence or absence of a waxy lipid in the cell wall; is used in identiﬁcation of Mycobacterium species Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 35

Gram-Positive and Gram-Negative Cocci Gram stain From De la Maza LM, Pezzlo MT, Baron EJ: Color atlas of diagnostic microbiology, St Louis, 1997, Mosby. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 36

Identification of Pathogens in the Microbiology Laboratory Biochemical testing Ø Performed to identify the genus and species Rapid identification methods Designed to give the physician a positive indication of the problem Ø Rapid strep testing Ø Influenza A and B testing Ø RSV testing Ø Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 37

Rapid Identification Tests From Stepp CA, Woods MA: Laboratory procedures for medical office personnel, Philadelphia, 1998, Saunders. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 38

Antimicrobial Susceptibility Testing “Culture and sensitivity” test – to determine the appropriate antibiotic Culture the organisms from a patient sample and test to determine the organism’s susceptibility to certain antibiotics The physician’s decision for antimicrobial agents is based on numerous factors. Ø Test results, physical examination, and the knowledge of the patient Asepsis must be strictly observed to ensure safety and good results; the organism must be isolated in pure culture before the test Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 39

Kirby-Bauer Antimicrobial Susceptibility Test Inoculate sterile water with pure bacterial culture Spread suspension in lawn pattern on agar medium Disks containing an antimicrobic agent are placed on agar After incubation, the zone of inhibition (area of no growth) around each disk is measured in millimeters and compared with values provided by the manufacturer of the disks Three determinations: Ø S – susceptible; antibiotic effective against organism Ø R – organism is resistant to antibiotic Ø I – intermediate – additional testing must be performed to determine the dosage of antimicrobial needed for therapeutic treatment Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 40

Antimicrobial Susceptibility Testing Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of

Critical Thinking Application Anna has recorded the results of Ms. Walker’s urine culture and notes that 10 antimicrobial agents had been tested, but the Escherichia coli was susceptible to only ﬁve of them. How will Dr. Ling determine which of these ﬁve antibiotics would be best for Ms. Walker? Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 42

Pinworm Testing: Procedure 55 -7 Testing for pinworms Specimens best collected at night or early before bowel movement, urination, or bathing. Ø Petroleum jelly paraffin swabs used to collect eggs. Ø Parent may also be instructed to bring child to office. Ø Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 43

Immunology Testing Provides information about past or present infections with bacteria or viruses and certain cancers Demonstrates the reaction between antigen and antibody Amount of antibody at any given time can be measured with serological testing and is referred to as the titer Agglutination and precipitation – antibody/antigen molecules clump together (agglutinate); they precipitate to the bottom of a test tube Immunochromatographic assay – when antigen-antibody reaction occurs a color change is visible Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 44

Immunology Testing The medical assistant may perform CLIA-waived tests for infectious mononucleosis, H. pylori, HIV, and Lyme disease. Mononucleosis testing – CBC and serologic tests CBC shows increased number of lymphocytes that appear atypical on the differential examination Ø Antibodies react with surface antigens of horse erythrocytes, causing agglutination Ø Procedure 55 -8 Ø Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 45

Critical Thinking Application Tiffany Warhola, a seventh grade student, visits Dr. Chowdry complaining of extreme fatigue and sore throat. Dr. Chowdry orders a rapid strep test and a mononucleosis test. What sample will Anna need for the rapid strep test? What sample will she need for the mononucleosis test? Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 46

Patient Education List of teaching topics to help educate patients An explanation of patient’s type of infection Ø How infection spreads Ø Normal barriers to infection Ø Risk factor Ø Patient preparations, collection Ø Hand washing, proper storage and cleaning Ø Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 47

Reportable Diseases Certain infectious diseases must be reported to the CDC or local board of health. Each state legislature determines how data are reported as well as what diseases must be reported. National data published weekly by the CDC in the MMWR – the Morbidity and Mortality Weekly Report. Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved. 48