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Practical Medical Microbiology

Practical Medical Microbiology. Lab # 7 Laboratory identification of Gram +ve rods, and acid fast stained bacteria. Gram +ve Rods. Non spore forming G+ve rods Corynebacterium diphtheriae. Culture medium: Loffler's serum medium Biochemical test: Elek's test

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Practical Medical Microbiology

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  1. Practical Medical Microbiology Lab # 7 Laboratory identification of Gram +ve rods, andacid fast stained bacteria

  2. Gram +ve Rods

  3. Non spore forming G+ve rods Corynebacterium diphtheriae • Culture medium: Loffler's serum medium • Biochemical test: Elek's test • Microscopic examination

  4. MICROSCOPIC EXAMINATION OF C.dIPHTHERIAE • Name of stain: Gram stain • Gram reaction: Gram +ve • Shape: Rods, non sporulated • ramified aggregations • "Chinese characters” having • clubbed ends • Size: Large

  5. Culture media for Corynebacterium diphtheriae Name of the medium • Loffler's serum medium Type of the medium • Enriched • Horse serum • + nutrient broth (3:1) • + 1% glucose Constituents Uses • Cultivation of Corynebacterium diphtheriae

  6. Elek’s test for c.diphtheriae • To differentiate between toxigenic & • non toxigenic strains of C. diphtheriae • In a plate: • Agar +20%horse serum • C .diphtheriae producing toxin (Ag) forms complex with antitoxin(Abs) in the filter paper ppt line

  7. Elek’s test for c.diphtheriae

  8. Gram +ve Rods • Pathogenic • Bacillus anthracis • Non pathogenic • Bacillus subtilis

  9. Spore forming G+ve rods Culture medium Tests

  10. Spore forming gram +ve rods Bacillus spp.

  11. Microscopic examination of Bacillus spp. • Name of stain: Gram stain • Gram reaction: Gram +ve • Shape: rods • Size: large • Arrangement: chains

  12. Tests for identification of bacillus spp. • Gelatin is • liquefied • by m.o. • producing • gelatinase. • In test tube: • N. broth • +gelatin • (12-15%) • Test for • gelatinase • Producing • m.o e.g. • Bacillus spp.

  13. Tests for identification of bacillus spp. Gelatinase +ve growth Gelatinase –ve growth

  14. Tests for identification of bacillus spp. • Nutrient agar+ 1% soluble starch • Test for m.o producing amylase e.g. Bacillus spp. • Starch + I2 blue color • Starch • amylase • Glucose I2 No blue color • Amylase hydrolyze • starch forming • clear zone around • the growth. • Reagent • Iodine

  15. Tests for identification of bacillus spp. Amylase –ve growth e.gE.coli Amylase +vegrowth e.gBacillus spp

  16. Tests for identification of bacillus spp. • Nutrient agar+ 1% casein (milk protein) • Test for m.o. producing caseinase e.g. • Bacillus, Staphylococcus • Casein around the growth is hydrolyzed by caseinase transparent zone

  17. Tests for identification of bacillus spp. Caseinase +ve growth Caseinase –ve growth

  18. Spore forming gram +ve rods Clostridium tetani

  19. Microscopic examination of clostridium tetani • Name of stain: • Gram stain • Gram reaction: • Gram +ve • Shape: Rods • Arrangement: • Resemble tennis • rackets or • drumsticks

  20. Growth Requirement for clostridium tetani Anaerobic jar

  21. Tests for identification of clostridium spp. • Skimmed milk • M.O. with • proteolytic activity: • utilizes casein giving • alkaline end products + short polypeptide (blue) • M.O. with • saccharolyticactivity: • utilizes lactose producing acid (red) & gas (stormy clot) • To differentiate • between • Clostridium spp.: • C. tetani • (proteolytic) • C. Perfringens • (saccharolytic) Indicator • Litmus

  22. Tests for identification of clostridium spp. Clostridium perfringens Clostridium tetani

  23. Test for identification of clostridium perfringens • Test for m.o. producing lecithinase e.g. C. perfringens • Lecithin • Lecithinase • Diacyl glycerol + • free fatty acids • Insoluble opaque • zone around • the growth • Nutrient agar + 10% egg yolk suspension

  24. Test for identification of clostridium perfringens White PPT of free fatty acids

  25. Acid fast bacteria Acid fast bacteria Mycobacterium spp. • Microscopic examination • Culture medium: LowensteinJensen medium • Test: Niacin test

  26. Microscopy • Name of stain: • Acid fast stain • Shape: Rods • Arrangement: • Single or bundles • Size: Small to medium • Non spore forming • Non motile

  27. Culture medium for mycobacterium spp.

  28. Culture medium for mycobacterium spp. Lowenstein Jensen plain medium

  29. Test for mycobacterium tuberculosis • Dubes &Davis • medium • To differentiate • Mycobacterium • tuberculosis • human strain • (niacin producer) • from bovine • strain (-ve) • M.o. producing • niacin (nicotinic • acid) forms • yellow azodye with • the indicator Indicator • Cyanogen • bromide • + aniline

  30. Test for mycobacterium tuberculosis -ve Niacin +ve Niacin

  31. Required Spots for lab#7

  32. Spots Corynebacterium diphtheriae Loffler's serum medium Potential Questions • Name of the spot/medium • Mention the type of the medium • What’re the components of the medium? • What’s the importance of the medium? • Mention the name of the m.o

  33. Spots Corynebacterium diphtheriae Elek’s test Potential Questions • Mention the name of the spot/test • What’s the principle of the test? • What’s the importance of the test? • Explain the result observed

  34. Spots Bacillus spp. Microscopic examination Potential Questions Name of the stain Type of the stain Name of the microorganism Items of the scheme

  35. Spots Bacillus spp. Gelatinase test Gelatinase +ve growth Gelatinase –ve growth Potential Questions • Mention the name of the spot/test • What’s the principle of the test? • What’s the importance of the test? • Describe the result observed

  36. Spots Bacillus spp. a)E.coli b)Bacillus spp Amylase test a) b) Potential Questions • Mention the name of the spot/test • What’s the principle of the test? • Mention the name of the reagent used • What are the components of the medium? • What’s the use of the test? • Mention the name of the m.o giving the +ve result and mention other test for that m.o • Explain the result observed

  37. Spots Bacillus spp. Caseinase test Caseinase –ve growth Caseinase +ve growth Potential Questions • Mention the name of the spot/test • What’re the constituents of the medium? • What’s the principle of the test? • What’s the importance of the test? • Explain the result demonstrated • Mention the name of the m.o

  38. Spots Clostridium spp. Litmus milk Potential Questions • Mention the name of the spot/test • What’s the principle of the test? • Mention the name of Indicator used • What’s the importance of the test? • Explain the result demonstrated • and give an example of m.o for each result A B Clostridium perfringens Clostridium tetani

  39. Spots Clostridium spp Lecithinase test Potential Questions • Mention the name of • the spot/test • What’s the principle of the test? • Mention the components • of the medium • Mention the name of the m.o • Explain the result observed

  40. Spots Mycobacterium spp. Microscopic examination Potential Questions Name of the stain Type of the stain Name of the microorganism Items of the scheme

  41. Spots Mycobacterium spp. Lowenstein Jensen medium Potential Questions • Mention the name of • the spot/medium • What’s the type of the medium? • Mention the components • of the medium • Explain the principle of the medium

  42. Spots Mycobacterium spp. Niacin test Potential Questions • Mention the name of • the spot/test • What’s the principle of the test? • Mention the name of the indicator • Explain the importance of the test • Mention the name of m.o for • each result -ve Niacin +ve Niacin

  43. Thank YOU Next lab: Other Gram negative rods.

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