1. Chapter 43 Basic Microbiology

2. 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

3. 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

4. The Medical Assistant’s Role in the Microbiology Laboratory • Identification of organisms done successfully within 24 to 72 hours • Many test kits available

5. 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)

6. Microbiology • Nomenclature • System for naming bacteria • Genus • First name; capitalized • Species • Second name; not capitalized

7. Microbiology • Nomenclature • Bacteriologists and microbiologists • Parasitology • Virology • Mycology • Reference laboratory • Report certain types of bacteria and yeasts to Department of Public Health

8. Microbiology • Cell structure • DNA (deoxyribonucleic acid) • Lower protist Basic bacterial cell >>

9. Equipment • Autoclave • Used to sterilize equipment • Many laboratories no longer use autoclaves because of use of presterilized and disposable equipment

10. 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

11. 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

12. Equipment • Incubator • Constant temperature of 35–37°C • Grows aerobic or anaerobic organisms • Temperature requirements must be met for adequate growth

13. 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

14. Equipment • Inoculating equipment • Loop used to inoculate organisms • Needle used when performing stab culture Inoculating loop>> Inoculating needle>>

15. Equipment • Incinerator • Quickest method of sterilization • Electrical incinerator or Bunsen burner • Media • Host of substances • Used to foster growth of bacteria

16. Equipment • Refrigerator • Used to store materials • Temperature of 2–8°C • Never store food or drink with specimens, kits, media

17. Safety When HandlingMicrobiology 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

18. Safety When HandlingMicrobiology 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

19. Safety When HandlingMicrobiology Specimens • Specimen handling • Look for leaks and contamination on containers • Wear gloves • Use appropriate container • Handle all specimens as if contaminated

20. Safety When HandlingMicrobiology Specimens • Disposal of waste and spills • Separation of biohazardous wastes (red bags) • Disinfect spills with 5% phenol or 10% bleach solution <<Biohazard symbol

21. Quality Control • All equipment with temperature controls monitored daily • Microscopes cleaned and kept dust-free • Before use, expiration date checked on all testing kits

22. 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

23. 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

24. Collection Procedures • Check to see if culture was: • Collected properly • Delivered within a reasonable period of time • Collected in sufficient quantity

25. Collection Procedures • All specimens taken from site of infection • Place in appropriate container • Deliver to laboratory • Rejecting specimens

26. 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

27. 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

28. 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

29. Specific Collection Requirementsfor 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

30. Specific Collection Requirements for Cultures • Wound • Use of sterile needle or swab to aspirate pus-filled fluid from wound • Use of anaerobic transport medium

31. Specific Collection Requirements for Cultures • Sputum • Patient coughs deeply and expectorates into sterile container • Should be morning specimen • Use of special sterile container

32. Specific Collection Requirements for Cultures • Stool • Ova and parasites • Bacterial cultures • Keep at between room and body temperature • Non-sterile containers

33. 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

34. Specific Collection Requirements for Cultures • Blood • Development of septicemia • Cultures collected by same means as regular blood collection • Variety of collection devices available

35. 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

36. 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

37. Bacterial Shapes Bacilli Cocci Spirilla

38. Microscopic Examination of Bacteria • Dyes (stains) • Derived from coal tar • Basic dyes carry a positive ion • Acidic dyes carry a negative ion

39. 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

40. 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

41. 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

42. 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

43. 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

44. 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

45. 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)

46. 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

47. 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

48. Culture Media • Media nutrients to support growth of bacteria • Vitamins • Sugar • Salt • Minerals • Amino acids • Addition of special products

49. Culture Media • Media classification • Basic • Differential • Selective • Enriched • Check known organisms for quality control and for contaminants

50. 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