Specimen Cultivation

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 totalnumber 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 •  CO2 • 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 O2 • Sodium thioglycolate broth: • Thioglycolic acid - reducing agent to create anaerobic atmosphere deeper in tube • Resazurin - oxygen-reduction indicator; in presence of O2, 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 C14O2production

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 35-370 C, optimum growth temperature for most human pathogens • Fungi often grown room temperature • Many fungi dimorphic growth: • Yeast at 370 C • Mold at RT • Candida albicans different growth: • Yeast at RT • Mold at 370C, in the presence of serum (Germ tube test)

40. Culture Incubation: Atmosphere • Most pathogenic bacteria grow best in 2-10% CO2 • Clinical Micro Labs routinely use 5% CO2 incubators • Some bacteria require 5-10% CO2 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 CO2 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 CO2 and H2 • H2 combines with O2 in presence of catalyst (palladium): 2H2 + O22H2O • Thus O2 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