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Practical Medical Microbiology

Practical Medical Microbiology. Lab # 4 Laboratory Identification of Gram Negative Rods ( Enterobacteriaceae ). Classification of GRAM NEGATIVE RODS. Oxidase negative Enterobacteriaceae. Oxidase positive Vibrio spp. Enterobacteriaceae. General Characteristics: N on- spore forming.

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Practical Medical Microbiology

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  1. Practical Medical Microbiology Lab # 4 Laboratory Identification of Gram Negative Rods (Enterobacteriaceae )

  2. Classification of GRAM NEGATIVE RODS Oxidase negative Enterobacteriaceae Oxidase positive Vibrio spp.

  3. Enterobacteriaceae General Characteristics: • Non- spore forming. • Motile (except Klebsiella& Shigella are non-motile). • Ferment glucose with acid or acid + gas production. • Oxidase negative. • Reduce nitrates to nitrite.

  4. Microscopic examination OF GRAM NEGATIVE RODS Morphological Description: • Type of stain: Gram stain • Gram reaction: Gram -ve • Color: Pink • Shape: Rods • Size: Small • Arrangement: Single

  5. Classification of ENTEROBACTERIACEAE • According to fermentation of lactose on MacConkey’s Agar Medium:

  6. Cultural Characteristics • Fermentation of lactose on MacConkey’s Agar Medium: Pink colonies of Lactose fermentors e.g. E.coli Yellow colonies of Lactose non-fermentors e.g. Salmonella sp. Plain medium

  7. Cultural Characteristics MacConkey’s agar medium Selective Differential Lactose • Contains crystal violet&bile salts • Inhibit the growth of all M.Os. except • Enterobacteriaceae Lactose non- fermentors Lactose fermentors Oxidative decarboxylation of amino acids Fermentation of lactose Lactic acid Alkaline end products Neutral red indicator Yellow colonies Pink colonies

  8. Cultural Characteristics Uses: • Isolationof Enterobacteriaceae from clinical Specimens. • Differentiation between lactose fermentors & lactose non-fermentors.

  9. Cultural Characteristics • Growth of lactose fermentors on Eosin Methylene Blue Agar Medium (EMB): Green metallic sheen of typical coliforms e.g.E.coli Plain medium

  10. Cultural Characteristics • Growth of lactose fermentors on Eosin Methylene Blue Agar Medium (EMB): Pink colonies of atypical coliforms e.g. Klebsiella spp. Plain medium

  11. Cultural Characteristics Eosin Methylene Blue Agar medium Selective Differential Lactose • Contains eosin & methylene blue • Inhibit all m.o. except • Enterobacteriaceae Typical coliforms Atypical coliforms Fermentation of lactose Large amount of lactic acid Moderate amount of lactic acid Precipitation of eosin No precipitation of eosin Green metallic sheen Pink colonies

  12. Cultural Characteristics Uses: • Isolationof Enterobacteriaceaefrom clinical specimens. • Differentiationbetween typical coliforms as E. coli and atypical coliforms as Klebsiella spp.

  13. Biochemical reactions and confirmatory tests • Nitrate reduction test. • Carbohydrate fermentation test. • IMViC tests.

  14. 1- Nitrate reduction test 1- Partial reduction Nutrient broth + KNO3 + M.O. Nitrate Reductase NO3 NO2 Sulphanilic acid + α-Naphthyl amine Red azodye(+ve result)

  15. 1- Nitrate reduction test Nutrient broth + KNO3 + M.O. 2- Complete reduction Nitrate Reductase NO3 N2 Sulphanilic acid + α-Naphthyl amine No red azodye(Persistent yellow color) (False negative result) Zn Dust (reducing agent) Yellow color (No red azodye) (False negative result)

  16. 1- Nitrate reduction test Nutrient broth + KNO3 + M.O. 3- No reduction No Nitrate Reductase NO3 Sulphanilic acid + α-Naphthyl amine Zn Dust (Reducing agent) Red azodye (True negative result) No red azodye (yellow color) NO3 NO2

  17. 1- Nitrate reduction test N.B. Persistence of the yellow color after addition of Zn dust means complete reduction of NO3 by nitrate reductase (false negative result) while appearance of the red azodyeafter addition of the Zn dust means no reduction (true negative result). Uses: • Test for M.O. producing nitrate reductase e.g. Enterobacteriaceae. +ve Result -ve Result

  18. 2- carbohydrate fermentation test Peptone water + Glucose Peptone water + Lactose Uses:Identification of M.O. by determination of type of sugars utilized by each. e.g. E. coli ferment glucose with production of acid & gas. Shigella E.coli Salmonella +ve gas (In derham tube) ̶ ve gas ̶ ve gas +ve acid (Red) +ve acid (Red) ̶ ve acid (Yellow) • Neutral red

  19. 3- IMVic Tests 1- Indole test • Uses: Totest ability of M.O. to produce tryptophanase thatconvert tryptophan to indole. Tryptone broth + M.O. Negative indole test Positive indole test Tryptophanase Tryptophan Indole Pink in the organic layer Kovac’s reagent • Kovac’s reagent =Alc. p-dimethyl- Aminobenzaldehyde & conc.HCl Indole

  20. 3- imviCtestS 2- Methyl red test Methyl red-VogesProskauer broth (MR-VP) (Glucose PO4 medium) • Uses: To test ability of m.o. to utilize glucose and to convert glucose to acid. M.O. Acid (pH<4.4) Glucose Methyl red (indicator)

  21. 3- imviCtestS 3- Voges-Proskauer test Methyl red-VogesProskauer broth (MR-VP) (Glucose PO4 medium) • Uses: To test ability of m.o. to utilize glucose and to convert glucose to acetoin. M.O. Acetyl methyl carbinol (Acetoin) (pH=6) Negative Positive Glucose Barritt’s reagent (Alc. α̶naphthol + KOH) (indicator)

  22. 3- imviCtestS • N.B.Voges-Proskauer test must be negative if methyl red test is positive and vice versa.

  23. 3- imviCtestS 4- Citrate utilization test • Uses: Test the ability of m.o. to utilize citrate as a sole carbon source e.g. Klebsiella & Enterobacter sp. Simmons citrate agar medium + M.O. Citrate NaCO3 alkaline Bromothymol blue (Indicator)

  24. Results of IMViCtests for coliforms Indole MR VP Citrate + + - -

  25. Results of IMViCtests for coliforms Indole MR VP Citrate + + - -

  26. Results of IMViC tests for coliforms Indole MR VP Citrate + - + -

  27. Required Spots for lab#4

  28. Spots Potential Questions • Name of the stain • Type of the stain • Name of the microorganism(Gram –ve rods) • Items of the scheme Microscopic examination

  29. Spots MacConkey’s Agar Medium A Potential Questions • Mention type of medium? • What is the name of indicator used? • What is the principle of media action? • What is the use of the medium? • What is the name of the microorganism in (A) & in (B)? B

  30. Spots Eosin Methylene Blue agar medium A Potential Questions • Mention type of medium • What is the principle of media action? • What is the name of indicator used? • What is the use of the medium? • What is the name of the microorganism in (A)& (B)? B

  31. Spots Nitrate reduction test A B Potential Questions • What is the principle of the test? • What is the name of the reagent used? • What is the use of the test? • What is the name of the microorganism in (A) & (B)? • What is the result in (A) & (B)?

  32. Spots Carbohydrate fermentation test A B C Potential Questions • What is the principle of the test? • What is the result of the test in tube (A) , tube (B) and tube (C)? • What is the use of the test? • What is the name of the microorganism?

  33. Spots IMViC tests Indole test A B Potential Questions • What is the principle of the test? • What is the composition of the medium? • What is the name of the reagent used? • What is the use of the test? • What is the result of the test in (A) & (B)

  34. Spots IMViC test A B Citrate Utilization test Potential Questions • What is the principle of the test? • What is the composition of the medium?? • What is the name of the reagent used? • What is the use of the test? • What is the result of the test in (A) & (B?

  35. Spots IMViC test for E.coli Potential Questions • What is the name of the organism giving this result? • What is the result?

  36. SPOTS IMViC test for Klebsiella & Enterobacter: Potential Questions • What is the name of the organism giving this result? • What is the result?

  37. spots Potential Questions • What is the name of the organism giving this result? • What is the result?

  38. spots Potential Questions • What is the name of the organism giving this result? • What is the result?

  39. Thank YOU Next lab: Enterobacteriaceae 2

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