1. Diagnostic Testing in the Microbiology Laboratory Jane Wong Public Health Microbiologist September 30, 2003 jwong@ucbcidp.org

2. Topics Some basic principles of microbiology testing A crash course in microbiology Follow a specimen through the lab Laboratory staffing issues

3. Media and Culture Media: Nutrients (agar, pH indicators, proteins and carbohydrates) used to grow organisms outside of their natural habitats Culture: The propagation of microorganisms using various media

4. Direct and Indirect Testing Direct: Demonstration of the presence of an infectious agent –Culture –Microscopy –Molecular methods such as PCR Indirect: Demonstration of presence of antibodies to a particular infectious agent –Serology

5. Sterile versus Non-sterile Body Sites Sterile body sites: –These sites normally do not contain any bacteria, so any bacteria found there are significant Blood Spinal fluid Non-sterile body sites: –These sites are open to the external environment and normally contain bacteria Throat Feces

6. Specimens from Sterile Sites Any organism growing in a normally sterile site is significant Identify it

7. Specimens from Non-Sterile Sites Only look for specific pathogens Physician will order test for a specific organism, or group of organisms Other “normal flora” bacteria will be present, but are not be identified

8. Sensitivity The fraction of those with the disease correctly identified as positive by the test. Isolation and identification of a known pathogenic organism may not be a very sensitive test –If the organism is present, it may not be found 100% of the time There can be false negatives

9. Specificity The fraction of those without the disease correctly identified as negative by the test. Isolation and identification of a known pathogenic organism is a very specific test –If the organism is not present in the specimen it will not be found

10. Documentation Specimen is logged in upon arrival in laboratory All tests and results are recorded and initialed by microbiologist All media and reagents are batch tested with positive and negative controls All equipment is checked at least once a day to be sure it is operating within predetermined parameters

11. Specimen Appropriateness Collection Transport to lab Inoculation of media Culture and isolation Confirmation Report

12. Appropriate Specimen From relevant body site Adequate amount Quality

13. Collection No contamination Appropriate equipment Good instructions to patient

14. Transport to Laboratory Safe packaging Good labeling Temperature

15. Inoculation of Media Use appropriate culture media –What kind of specimen is it? –What test did the physician request?

16. Culture media Used to grow bacteria Can be used to: –Enrich the numbers of bacteria –Select for certain bacteria and suppress others –Differentiate among different kinds of bacteria

17. Microbiological Culture Media

18. Isolation of Individual Bacteria Specimen is “streaked”, using a sterile loop, onto solid media. The agar plates (media) are incubated at appropriate temperature and atmosphere –Often at 35º C. –Often at 5% CO 2 –Usually first examined after 24 hours

19. “Streaking a Plate”

20. Growth of Colonies Bacterial Colony –Result of one bacterium being isolated from others during “streaking procedure” –That bacterium grows in numbers exponentially –Many bacteria have a generation time of 20 minutes –2 72 organisms in one colony after 24 hours!

21. Classical bacterial identification can only be performed on pure cultures of bacteria (ideally, all descendants from one bacterial cell)

22. Mixed Culture of Soil Organisms Containing Bacillus anthracis

23. Colony “Picking” Sterile needle or loop is touched to surface of colony and transferred to fresh, sterile media Incubation for another 24 hours

24. Colonies of Bacteria in Pure Culture

25. Pure Culture of Francisella tularensis Colonies After 72 hours Growth

26. Pure Culture of Yersinia pestis Colonies on Blood Agar After 48 hours of Growth

27. Yersinia pestis Colonial Morphology Viewed With Transmitted Light

28. Confirmation Now we have a pure culture of bacteria Testing is now done to confirm the identification of the bacteria culture –Stains –Biochemical tests –Serological tests (using known antibodies) –Molecular tests (nucleic acid probes)

29. Gram Stain of Streptococcus sp.

30. Yersinia pestis Gram stain

31. Gram stain of Brucella sp.

32. B. anthracis Gram stain showing spores

33. Gram stain of B. anthracis from broth culture

34. Examples of Biochemical Tests Left: API 50 Test Above: Antimicrobial Sensitivity Test

35. Yersinia pestis E-Test (Antimicrobial Sensitivity Test)

36. Nitrate and Urea Reactions

37. Reactions on MacConkey Agar

38. Triple Sugar Iron (TSI) Test

39. Case Study Patient arrives in emergency room with f ever (temperature greater than 100 degrees F). The fever is accompanied by chills or night sweats. Flu-like symptoms. Non-productive cough, chest discomfort, shortness of breath, fatigue, muscle aches

40. Patient Admitted to Hospital Blood cultures ordered Blood drawn and immediately placed in blood culture bottles

41. Blood Bottles Incubated Bottles are automatically tested every 10 minutes. Positive results are tagged for quick processing. Negative bottles can be batch-scanned out of the system and unloaded at the end of protocol.

42. 18 Hours of Incubation Blood culture incubator signals that there is growth in one of the bottles. It is removed and a Gram stain is performed

44. Microbiologist Suspects Bacillus anthracis Reports results so far to supervisor Streaks a fresh blood agar plate and incubates it May perform wet mount test with India Ink to see “capsule” around individual bacteria Inoculates media to observe motility

45. Bacillus anthracis India Ink Preparation

46. Growth on a Blood Agar Plate (Petri Dish) After 18-24 Hours

47. Gram stain of B. anthracis from broth culture

48. Motility B. anthracis is non-motile. Other Bacillus species are motile.

49. Laboratory Cannot Rule Out Bacillus anthracis Refers the culture to a reference laboratory that is part of the Laboratory Response Network (LRN)

50. Report Final report goes to physician The validity of this report is dependent upon: –Appropriateness of specimen –Proper collection and adequacy of specimen –Appropriate transport to lab –Use of media of known quality –Culture and isolation by knowledgeable personnel using equipment known to be operating correctly –Confirmation by tests of known quality –Results interpreted and reported by professional staff –No transcription or computer errors

51. Molecular Tests Biotechnology has given diagnostic laboratories very powerful tools – for rapid detection and identification of human pathogens –for strain typing for epidemiological investigations

52. The Flip Side! Biotechnology companies attract recent college graduates –Majors in biology and allied fields –Salaries usually higher than clinical or government public health labs offer –Appeal to public service only goes so far! Result: public health and clinical laboratories have trouble recruiting and retaining laboratory personnel.

53. Other Factors in Personnel Shortage Training opportunities have been drastically reduced Pay is not competitive Much of the work force is approaching retirement age

54. Licensing Applications/Year For Clinical Laboratory Scientist Certification