1. Chapter 43
Basic Microbiology

2. The Medical Assistant’s Role in the Microbiology Laboratory
Preparing cultures
Allow bacteria to grow at least 12 hours before examining culture
Sensitivity identifies which antibiotics will kill microorganism causing infection

3. The Medical Assistant’s Role in the Microbiology Laboratory
Use exact technique to avoid laboratory error
Use sterile equipment
Send culture to laboratory in reasonable amount of time

4. The Medical Assistant’s Role in the Microbiology Laboratory
Identification of organisms done successfully within 24–72 hours

5. Microbiology Bacteria are found naturally in the body Normal flora
Always present and help with immune system
Pathogens cause disease

6. Microbiology Classification
Taxonomy deals with classification of living organisms
Carolus von Linnaeus devised current classification system
No universal agreement on one system

7. Microbiology Classification Kingdoms Plants Animals Protists
Prokaryotes (lower protists)
Eukaryotes (higher protists)

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

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

10. Microbiology Cell structure Basic bacterial cell >>
DNA (deoxyribonucleic acid)

11. Equipment Autoclave Used to sterilize equipment
Not used with presterilized and disposable equipment

12. Equipment
Microscope
Used to view organisms that cannot be seen with naked eye
Prepared slides used

13. Equipment
Safety hood
Aerosols can be released into air when culturing and are potentially dangerous if inhaled
Use of hood is mandatory when performing culture on specimen with potential aerosol
Used to minimize odors

14. Equipment Incubator Has constant temperature of 35–37°C
Grows aerobic or anaerobic organisms
When culturing, set up some cultures in oxygenated environment as well as oxygen-reduced environment

15. 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

16. Equipment Inoculating equipment Inoculating loop Inoculating needle
Stab culture used for certain biochemical tests used for identification

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

18. Equipment Refrigerator Used to store materials Temperature of 2–8°C
Never store food or drink or medication with specimens

19. Safety When Handling Microbiology Specimens
Personal protector when handling microbiology specimens
Wear PPE at all times
Remove when leaving for the day
Buttoned laboratory coat or apron, safety goggles, and gloves

20. Safety When Handling Microbiology Specimens
Personal protector when handling microbiology specimens
Use of hood or shield
Never eat, drink, smoke, or put objects into mouth
Do not touch contact lenses or apply makeup
Wash hands frequently

21. 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

22. Safety When Handling Microbiology Specimens
Specimen handling
Check for leaks and contamination on containers
Wear gloves
Use appropriate container
Handle all specimens as if contaminated

23. Safety When Handling Microbiology Specimens
Disposal of waste and spills
Biohazard symbol
Separation of biohazardous wastes
Disinfect spills with 10 percent bleach solution

24. Quality Control
All equipment with temperature controls should be monitored daily
Microscopes should be cleaned and kept dust-free
Media of all types should not be used past shelf life
Should be stored at proper temperatures
Checked for growth with known organisms for quality control

25. Quality Control
Procedure manual with all standard operating procedures written down should be updated periodically
Many microbiology laboratories subscribe to associations that periodically send unknown samples to be set up and identified

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

27. Collection Procedures
Common microbiology specimen sites
Place in appropriate container
Bring to laboratory
Rejecting specimens

28. 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

29. Collection Procedures
Factors determining successful isolation of causative pathogens
Specimen labeled properly
Specimen brought to the laboratory in a minimal amount of time
Specimen collected before administration of antibiotics
Specimen inoculated onto proper media and placed in correct atmosphere to ensure growth

30. Specific Collection Requirements
Urine
Collecting a clean-catch specimen
Use of catheterization
Nose
Nasal-pharyngeal swab collects specimen
Place swab in sterile tube for transport to laboratory

31. Specific Collection Requirements
Throat
Use sterile tongue depressor to hold patient’s tongue down
Avoid swabbing sides of mouth and tongue

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

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

34. Specific Collection Requirements
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35. Specific Collection Requirements
Stool
Ova and parasites
Bacterial cultures
Non-sterile containers
Contamination of urine

36. Specific Collection Requirements
Cerebrospinal fluid (CSF)
Lumbar puncture
Fluid dispersed in several departments of clinical laboratory
Use of incubator
Refrigeration can kill meningitis-causing bacteria

37. Specific Collection Requirements
Blood
Development of septicemia
Collection of cultures
Variety of collection devices available

38. Bacterial Shapes
Cocci
Bacilli
Spirilla

39. Microscopic Examination of Bacteria
Dyes
Derived from coal tar
Acidic dyes carry a negative ion
Basic dyes carry a positive ion
Methylene blue binds to DNA and RNA of cell

40. Microscopic Examination of Bacteria
Simple stain
Uses single stain on fixed slide for given period of time
Shows structure and arrangement of bacterial cell
Takes no more than 3 minutes to stain
Gives little information other than size and morphological arrangement

41. Microscopic Examination of Bacteria
Differential stain
A common differential stain is the gram stain
Use of decolorizer and counterstain
Developed in 1884 by Dr. Hans Christian Gram
Differentiates bacteria by gram stain ability of being negative or positive
Use of gentian or crystal violet reagents

42. 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

43. 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

44. Microscopic Examination of Bacteria
Special techniques
Used when flagella, spore, capsule, or nuclei of cells are present
Tests without staining
Wet slide preparation
Hanging drop

45. 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 percent KOH and coverslip
KOH clears debris

46. Microscopic Examination of Bacteria
Potassium hydroxide (KOH) preparation
Set slide at room temperature for one-half hour before examination for debris settlement
Use of phase or dark-field microscope
Dispose of properly (live organisms)
Direct microscopic examination of culture and infectious bacteria

47. Culture Media Inoculate material on proper medium for growth
Reliability of results
Fastidious bacteria need specialized medium to grow
Aerobic bacteria grow only in oxygen

48. Culture Media Common bacteria and growth requirements Transport media
Can be solid, liquid, or semisolid substance

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

50. Culture Media Agar Broth tubes store semisolid media Solid media
Poured in petri dish or tubes
Broth tubes store semisolid media

51. Culture Media Media classification
Basic
Differential
Selective
Enriched
Common specimens, suspected pathogens, media recommendations

52. Microbiology Culture Inoculating the media
Roll swab onto upper quadrant of agar plate
Use loop to inoculate sputum or liquid
Spread flamed loop or needle back and forth in sweeping motion to dilute bacteria

53. Microbiology Culture Inoculating the media
After inoculating agar plate, turn upside down and place in proper environment for growth
Broth tube inoculation
Deep inoculation/slant

54. 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

55. Microbiology Culture Primary culture
Read after media incubated 24–48 hours
Observe for gross colony characteristics
Size
Shape
Color
Elevation

56. Microbiology Culture Primary culture
Observe for gross colony characteristics
Density
Consistency
Hemolytic versus nonhemolytic
Odor
Pigment production
Use of bright, direct light

57. Microbiology Culture Subculture
More than one pathogen grows in culture
Separation of pathogenic bacteria from normal flora
Use of inoculation loop or needle
Pick suspicious pathogen and streak onto media
Allow to grow for 24 hours

58. Identification Systems
Streptococcus screening
Rapid identification important
Many rapid test kits available
Latex agglutination test based on antigen and antibody agglutination

59. Identification Systems
Streptococcus screening
Rules for accurate screening
Use correct swab in taking throat cultures
Always run positive and negative control along with actual test
Read and understand directions before starting test

60. Identification Systems
Streptococcus screening
Rules for accurate screening
Never use outdated kits and materials
Observe all safety guidelines

61. Sensitivity Testing
Often ordered on pathogenic organisms recovered from culturing process

62. Parasitology Becoming more common
Geographical area determines types of parasites seen
Examination methods

63. Parasitology Specimen collection
Use of wide-mouth containers with tight lid to prevent leakage
Strictly follow laboratory procedure
Label specimen correctly
Follow standard precautions and OSHA guidelines

64. Parasitology Common parasites Enterobius vermicularis Diagnosis of
Pinworms
Nematode
Diagnosis of

65. Parasitology Common parasites Trichomonas vaginalis
Found five times more often in women than in men
Transmitted sexually
Recovery of trichomonad

66. Mycology Extensive field
Candida has several species that cause yeast infections in body
Dermatophytes