© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Chapter 43 Basic Microbiology

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. The Medical Assistant’s Role in the Microbiology Laboratory Obtain and test specimens Prepare slides and cultures Allow cultures to grow at least 12 hours before examining for identification Sensitivity identifies which antibiotic(s) will kill microorganism causing infection

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. The Medical Assistant’s Role in the Microbiology Laboratory Normal flora: natural bacteria Pathogen: disease causative microorganism Use technique to avoid laboratory error Use sterile supplies Deliver culture to laboratory in reasonable amount of time

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. The Medical Assistant’s Role in the Microbiology Laboratory Identification of organisms done successfully within 24 to 72 hours Many test kits available

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microbiology Classification –Taxonomy deals with classification of living organisms –Kingdoms: plants, animals, protists –Lower protists, or prokaryotes (blue-green algae and bacteria) –Higher protists, or eukaryotes (protozoa, algae, fungi)

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microbiology Nomenclature –System for naming bacteria –Genus First name; capitalized –Species Second name; not capitalized

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microbiology Nomenclature –Bacteriologists and microbiologists –Parasitology –Virology –Mycology –Reference laboratory –Report certain types of bacteria and yeasts to Department of Public Health

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microbiology Cell structure –DNA (deoxyribonucleic acid) –Lower protist Basic bacterial cell >>

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Equipment Autoclave –Used to sterilize equipment –Many laboratories no longer use autoclaves because of use of presterilized and disposable equipment

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Equipment Microscope –Used to view organisms that cannot be seen with naked eye on prepared slide –Delicate instrument –Cared for properly as stated by manufacturer

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Equipment Safety hood –Aerosols can be released into air when culturing –Potentially dangerous if inhaled –Use of hood mandatory when performing culture on specimen with potential aerosol –Used to minimize odors

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Equipment Incubator –Constant temperature of 35–37°C –Grows aerobic or anaerobic organisms –Temperature requirements must be met for adequate growth

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Equipment Anaerobic equipment –Absence of oxygen to grow anaerobic bacteria –Use of candle jar –Gas pack jar –Specimens sent to reference laboratories –Gram stain used to observe gross morphological features of bacteria

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Equipment Inoculating equipment –Loop used to inoculate organisms –Needle used when performing stab culture Inoculating loop>> Inoculating needle>>

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Equipment Incinerator –Quickest method of sterilization –Electrical incinerator or Bunsen burner Media –Host of substances –Used to foster growth of bacteria

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Equipment Refrigerator –Used to store materials –Temperature of 2–8°C –Never store food or drink with specimens, kits, media

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Safety When Handling Microbiology Specimens Personal protective equipment –PPE worn at all times –Laboratory coat or apron, safety goggles, gloves –May need to work behind shield or use safety hood –Never eat, smoke, drink, or put objects into mouth –Do not touch contact lenses or apply makeup –Wash hands

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Safety When Handling Microbiology Specimens Work area –Use strong germicide before and after daily use or immediately after spills –Dust-free and clean at all times –Uncluttered –Avoid body burns or files

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Safety When Handling Microbiology Specimens Specimen handling –Look for leaks and contamination on containers –Wear gloves –Use appropriate container –Handle all specimens as if contaminated

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Safety When Handling Microbiology Specimens Disposal of waste and spills –Separation of biohazardous wastes (red bags) –Disinfect spills with 5% phenol or 10% bleach solution <<Biohazard symbol

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Quality Control All equipment with temperature controls monitored daily Microscopes cleaned and kept dust-free Before use, expiration date checked on all testing kits

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Quality Control Media of all types should not be used past shelf life and should be stored at proper temperatures Check specific list of bacteria to use on various media to test for growth Laboratory manual updated periodically

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Quality Control All chemicals or reagents with Material Safety Data Sheets (MSDS) should be available to reference Document all quality control testing in proper laboratory logs

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Collection Procedures Check to see if culture was: –Collected properly –Delivered within a reasonable period of time –Collected in sufficient quantity

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Collection Procedures All specimens taken from site of infection Place in appropriate container Deliver to laboratory Rejecting specimens

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Collection Procedures Factors determining successful isolation of causative pathogens –Proper collection from infection site –Collection of specimen during infection period –Sufficient amount of specimen –Appropriate specimen container –Appropriate transport medium

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Collection Procedures Factors determining successful isolation of causative pathogens –Specimen labeled properly –Specimen delivered to laboratory in minimal amount of time –Specimen collected before administration of antibiotics –Specimen inoculated onto proper media and placed in correct atmosphere to ensure growth

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Specific Collection Requirements for Cultures Urine –Obtaining clean-catch specimen –Use of catheterization Nose –Nasal-pharyngeal swab collects specimen –Place swab in sterile tube for transport to laboratory

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Specific Collection Requirements for Cultures Throat –Specimens taken using culturette –Use sterile tongue depressor to hold patient’s tongue down –Avoid swabbing sides of mouth and tongue –Take specimen directly from affected area

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Specific Collection Requirements for Cultures Wound –Use of sterile needle or swab to aspirate pus-filled fluid from wound –Use of anaerobic transport medium

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Specific Collection Requirements for Cultures Sputum –Patient coughs deeply and expectorates into sterile container –Should be morning specimen –Use of special sterile container

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Specific Collection Requirements for Cultures Stool –Ova and parasites –Bacterial cultures –Keep at between room and body temperature –Non-sterile containers

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Specific Collection Requirements for Cultures Cerebrospinal fluid (CSF) –Lumbar puncture –Fluid dispersed in several departments of clinical laboratory –Use of incubator –Refrigeration can kill meningitis-causing bacteria –STAT order for processing

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Specific Collection Requirements for Cultures Blood –Development of septicemia –Cultures collected by same means as regular blood collection –Variety of collection devices available

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Foodborne Illnesses Bacterial infections and parasites 48 million cases of “food poisoning” each year contracted from: –Raw or undercooked meats and seafood –Contaminated food handlers –Unwashed produce

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Foodborne Illnesses Symptoms: mild to severe diarrhea, nausea, vomiting, abdominal pain, fever, headaches, flu-like symptoms Diagnostics: stool cultures, blood tests, following the symptoms Treatments: antibacterial medications and treating symptoms

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Bacterial Shapes Cocci Bacilli Spirilla

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microscopic Examination of Bacteria Dyes (stains) –Derived from coal tar –Basic dyes carry a positive ion –Acidic dyes carry a negative ion

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microscopic Examination of Bacteria Simple stain –Uses single stain on fixed slide for given period of time –Shows arrangement and structure of bacterial cell –Takes no more than 3 minutes to stain –Does not give much information

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microscopic Examination of Bacteria Differential stain –Stain result varies –Common differential stain is Gram stain –Developed in 1884 by Dr. Hans Christian Gram –Differentiates bacteria by Gram stain ability of being negative or positive –Retain or lose color through decolorization –Use of gentian or crystal violet reagents

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microscopic Examination of Bacteria Differential stain –Identifies Gram-positive and Gram-negative bacteria Staphylococcus Streptococcus E. coli Proteus –Morphological arrangement, shape, and Gram- stain characteristic help identify bacteria

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microscopic Examination of Bacteria Acid-fast stain –Specific stain –Allows microscopic examination of acid-fast mycobacteria –Use of heat or powerful dye –Ziehl-Neelsen stain –Kinyoun stain

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microscopic Examination of Bacteria Special techniques –Used when flagella, spore, capsule, or nuclei of cells present –Organisms in living state, without staining –Wet-mount preparation –Hanging drop preparation

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microscopic Examination of Bacteria Potassium hydroxide (KOH) preparation –Used for study of fungi and spores –Fragments of human hair, skin, or nails placed on slide with drop of 10% KOH and coverslip –KOH clears debris

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microscopic Examination of Bacteria Potassium hydroxide (KOH) preparation –Set slide at room temperature for a half hour before examination for debris settlement –Direct examination of specimens –Use of phase or dark-field microscope –Dispose of properly (live organisms)

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Culture Media Inoculate material on proper culture medium for growth and identification Reliability of results Aerobic bacteria grow only in oxygen Anaerobic bacteria live and grow in absence of oxygen

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Culture Media Transport media for reference laboratory Media solid, liquid, or semisolid substance Agar: solid form of media Semisolid media: made by adding less agar Liquid media: in broth form stored in tubes

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Culture Media Media nutrients to support growth of bacteria –Vitamins –Sugar –Salt –Minerals –Amino acids –Addition of special products

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Culture Media Media classification –Basic –Differential –Selective –Enriched Check known organisms for quality control and for contaminants

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microbiology Culture Inoculating the media –Roll swab onto upper quadrant of agar plate –Use loop to inoculate sputum or liquid –Inoculum spread back and forth in sweeping motion with flamed loop or needle

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microbiology Culture Inoculating the media –After inoculating agar plate, turn upside down and place in proper environment for growth –Liquid broths and agar slant tubes have screw caps

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microbiology Culture Other types of streaking –Lawn streak used to place organism over entire area of agar plate for sensitivity testing –Colony count used to plate urine cultures –Laboratories have slightly different ways of performing basic streaks

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Microbiology Culture Primary culture –After media incubated for 24 to 48 hours, initial or primary culture read Subculture –More than one pathogen grows in culture –To identify each organism, separate bacteria to other media –Separate pathogenic bacteria from normal flora

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Rapid Identification Systems Streptococcus screening –Rapid strep testing –Many rapid test kits available –Rapid identification important –Latex agglutination test based on antigen and antibody agglutination

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Rapid Identification Systems Streptococcus screening –Read and understand directions before starting test –Never use outdated materials –Observe all safety guidelines and precautions –Use correct swab in taking throat cultures –Always run positive and negative control along with actual test

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Sensitivity Testing Often ordered on pathogenic organisms recovered from culturing process Culture and Sensitivity (C&S) Culture plate showing antibiotic discs on bacteria>>

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Parasitology Parasitic infections becoming more common Different geographic areas have different types of parasites Common methods of fecal specimen examination for parasitic identification is direct wet-mount slide

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Parasitology Specimen collection –Wide-mouth containers with tight lid to prevent leakage –Container in biohazard transport bag to avoid contamination –Laboratory user’s manual for specific instructions –Strictly follow laboratory procedure –Label specimen correctly

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Parasitology Common parasites –Giardiasis lamblia Spread through fecal to oral route –Enterobius vermicularis Nematode (round worm) Pinworms To diagnose: adult worm or ova has to be located in specimen

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Parasitology Common parasites –Trichomonas vaginalis Found five times more often in women than in men Transmitted sexually Recovery of trichomonad

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Parasitology Common parasites –Hookworm Infection through hair follicles, pores, open wounds/sores Poor hygiene and warm moist climates Concern is anemia Diagnostic tests: stool cultures and CBC

© 2014 Cengage Learning. All Rights Reserved. May not be scanned, copied or duplicated, or posted to a publicly accessible website, in whole or in part. Mycology Extensive field Candida has several species that cause yeast infections in body Dermatophytes cause infections on hair, skin, nails