1. Specimen Cultivation How do you grow these bugs?

2. Clinical Microbiology Diagnosis  “Gold Standard” – culture isolation and identification of viable pathogen  Future trend - rapid, non growth dependent detection of MO  Microbial antigen (specific antibody, i.e. ELISA)  Microbial nucleic acid - sensitive, need 1 copy, amplification of DNA, i.e. PCR; RT-PCR (RNA reverse transcribe to DNA)

3. Diagnosis: Require Pure Culture  Identify MO by biochemical methods  Antimicrobial sensitivity testing  In the clinical lab - streak original sample out on primary isolation media to get isolated colonies

4. MO Isolated  Each isolated colony pure culture, progeny of single cell  Each bacteria forms a characteristic colony: shape, size, color, texture, adherence to medium  Colonial characteristics often used as first step in identification of bacteria

5. Bacterial Colonial Morphology

6. Culture Media  Artificial media on which bacteria and fungi grown  Some bacteria never successfully grown on artificial lab media  Treponema pallidum – grown in testes of rabbits  Mycobacterium leprae – grown in armadillo or foot-pad of mice

7. Obligate Intracellular Parasite  Some bacteria – Rickettsia, Chlamydia  All viruses (non-cellular)  Cultured in living host - whole organism or tissue cell culture  Identify virus grown in tissue cell culture by:  Electron microscope - see virus in cell  Cytopathic effects (damage or change in cell)  Inclusion body (viral products)  Syncytia formation (cell fusion)  Rounding up of cell (death)

8. Cytopathic Effect: Inclusion Body (Rabies virus)

9. Cytopathic Effect: Syncytia Formation (Herpesvirus)

10. Cytopathic Effect: Cell Rounding (Poliovirus)

11. Diagnosis: Obligate Intracellular Parasite  Growth time consuming and expensive  Detect MO by immunological methods  Direct assay - antigens of MO, usually in tissues (use specific antibody as a reagent)  Indirect assay - host immunological response (antibody) against MO (use specific microbial antigen as a regent)

12. Diagnosis: Antibody Assay  Serology – diagnose infection by assaying for antibodies in patient’s serum against causative agent:  Take both acute (patient most ill) and convalescent (patient recovering) phase specimen  One looks for a 4-fold rise in antibody titer between acute and convalescent specimens  ELISA – Enzyme linked immunosorbent assay:  Use microbial antigen (specificity)  Enzyme detection (sensitivity)  Rapid testing

13. Diagnosis: Parasites  Not easily grown on artificial media or in living hosts  Detection based on visual microscopic identification of:  Parasite (trophozoite, free-living stage)  Ova or cyst stage of parasite

14. Entamoeba histolytica (ameba): Trophozoite

15. Entamoeba coli (ameba): Cyst

16. Taenia solium (tapeworm): Scolex

17. Taenia solium (tapeworm): Proglottids

18. Enterobius vermicularis (roundworm): Ova

19. Ascaris lumbricoides (roundworm): Ova

20. Culture – When?  Specimen plated immediately  Delay may result in:  Loss of fastidious or anaerobic MOs  Overgrowth by normal flora - change of total number and relative number of MOs

21. Culture Media  Diagnostic labs vary in choice of routine plating media used for growing different types of specimens  Take into account what pathogens anticipated in specimen  Also consider:  Growth requirements   CO 2  Temperature requirements (RT, 37ºC)

22. General Purpose Culture Plating Media  Supports growth of most common pathogens, non-selective  Permits isolation and differentiation of wide variety of bacteria  Differences: colony size, shape, color, texture, adherence to culture media

23. Nutrient Agar Plate

24. Columbia Blood Agar (CBA) Plate: Differential  Allows differentiation based on bacteria hemolysin that destroy red blood cells in the agar  Alpha (  ) hemolysis - incomplete hemolysis and appears as green halo surrounding the colony  Beta (  ) hemolysis - complete hemolysis and appears as clear area surrounding the colony  Gamma (  ) hemolysis - no hemolysis

25. Chocolate Agar (Choc) Plate  Essentially same as blood agar, except RBCs lysed  Releases hemin and NAD for fastidious MOs  Gives medium chocolate brown color  CBA and Choc termed “enriched media” because of blood nutrients in media

26. Selective Media  Special nutrients that support growth of certain pathogens and/or inhibitors that suppress growth of competing NF  Columbia Blood agar with antibiotics (Columbia CNA) – select for G(+):  Sheep blood  Antibiotics Colistin & Nalidixic Acid  Why does it inhibit G(–) bacteria?  MacConkey, Salmonella-Shigella, Hektoen Enteric agar – select for G(-)  Bile salts to inhibit MOs  Why does it inhibit G(+) ?

27. Selective/Differential Media  Selective media is also usually differential  Addition of a carbohydrate (CHO) and a pH indicator differentiate MO that ferment the CHO and those that do not  Mannitol Salt Agar (MSA):  Selective - 7.5% NaCl to suppress MOs not halophilic  Differential - Mannitol (CHO) and pH indicator phenol red

28. MSA Plate  MO grows on media ferments mannitol, acid is produced and lowers pH  At low pH, phenol red = yellow  MO that ferments mannitol turn media yellow

29. MacConkey Agar (Mac) Plate  Selective - Crystal violet and bile salts inhibit G(+) bacteria, fungi  Differential – Lactose, pH indicator neutral red (red or pink at acid pH)  Mac plates example of enteric agar plates which facilitate isolation and differentiation of enteric pathogens

30. Mac Agar Plate  MO able to grow on media and ferment lactose produce pink colonies (acid pH) i.e. E. coli  MO that grow and don’t ferment lactose produce colorless colonies (neutral pH) i.e. Salmonella, Shigella

31. Reducing Media  Used for cultivating anaerobes  Contains compounds that chemically combine with dissolved oxygen in media to deplete O 2  Sodium thioglycolate broth:  Thioglycolic acid - reducing agent to create anaerobic atmosphere deeper in tube  Resazurin - oxygen-reduction indicator; in presence of O 2, resazurin = pink

32. Growth of MOs in Thioglycolate Broth

33. Enrichment Media  To prevent missing bacteria present in small number  Usually liquid, provides nutrients and environmental conditions favor growth of one type MO while unsuitable for others  Enrich stool culture for enteric pathogens found in low numbers relative to NF:  Gram negative broth - bile & citrate salts inhibit G(+); mannitol enrich for Salmonella, Shigella)  Tetrathionate broth - bile salts, thiosulfate, tetrathionate inhibit most G(+) & G(-) rods, except Salmonella  Selenite broth - selenite inhibits G(-) rods, enterococci; allows recovery of Salmonella, Shigella

34. Blood Culture  Collected specimen inoculated into blood culture media directly at bedside of patient  Two bottles of liquid media inoculated:  Aerobic growth - Tryptic Soy Broth (TSB)  Anaerobic growth – Thioglycolate Broth  Bottles examined for turbidity, 7-14 days  If turbidity develops, some media removed for Gram staining and subculture onto solid media

35. Blood Culture  Blood culture may routinely be Gram stain and subculture at specific intervals (24 hrs,48 hrs, etc.) even in absence of turbidity  Bactec machines automatically detect growth in blood cultures by radioactive  C 14 O 2 production

36. Quantitative Culture  Often done on urine specimens  A known volume of specimen plated on agar medium via calibrated loop and number of colonies counted  Caution – this represents number of bacteria present at time of plating  For clean catch urine specimen: >100,000 colonies/ml considered significant and indicative of disease  For bladder or kidney specimen >10,000 colonies/ml considered significant and indicative of disease. Why?

37. Quantitative Urine Culture Counts

38. Culture: Unusual MO  Some rarely encountered pathogens need special media and/or procedure for isolation  If physician suspects one of these MO, must notify lab so appropriate media prepared and proper precautions taken, if necessary  Brucella  Bordetella  Legionella

39. Culture Incubation: Temperature  Inoculated media incubated at 35-37 0 C, optimum growth temperature for most human pathogens  Fungi often grown room temperature  Many fungi dimorphic growth:  Yeast at 37 0 C  Mold at RT  Candida albicans different growth:  Yeast at RT  Mold at 37 0 C, in the presence of serum (Germ tube test)

40. Culture Incubation: Atmosphere  Most pathogenic bacteria grow best in 2-10% CO 2  Clinical Micro Labs routinely use 5% CO 2 incubators  Some bacteria require 5-10% CO 2 in order to grow or to grow well (Neisseria, Streptococcus, Haemophilus)  In the lab, we will grow these MOs in a “candle jar” to provide higher CO 2 needed for growth

41. Anaerobic Culture  Reducing media may be used  Plates may be incubated in special jar or pouch in oxygen free atmosphere (nitrogen gas)  In an anaerobic jar, oxygen free atmosphere generated by chemical reaction

42. Anaerobe Jar  Envelopes of sodium bicarbonate and borohydride placed in jar and water added  Chemical reaction generates CO 2 and H 2  H 2 combines with O 2 in presence of catalyst (palladium): 2H 2 + O 2  2H 2 O  Thus O 2 removed  Indicator strip – methylene blue:  Colorless in absence of oxygen  Blue in presence of oxygen

43. Culture Incubation: Time  Most routine cultures: 16-18 hrs (overnight), before report negative  CSF and blood cultures: one week, before report negative  Wound cultures: 48 hrs, before report negative  Fungal cultures: 3-4 weeks, before report negative  Mycobacterium : 6-10 weeks, before report negative

44. Class Assignment  Textbook Reading: –Chapter 7 Microscopic Examination of Infected Materials –Chapter 8 Use of Colonial Morphology for the Presumptive Identification of Microorganisms  Key Terms  Learning Assessment Questions