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MICROBIOLOGY LAB TESTS

MICROBIOLOGY LAB TESTS. The following microbiology tests will be performed in lab this semester. Gram stain Motility EMB ( Eosin Methylene blue agar) MacConkey agar Carbohydrate fermentation ( glucose, lactose, sucrose) Gelatine liquefaction test Mannitol salt Indol

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MICROBIOLOGY LAB TESTS

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  1. MICROBIOLOGY LAB TESTS

  2. The following microbiology tests will be performed in lab this semester • Gram stain • Motility • EMB ( Eosin Methylene blue agar) • MacConkey agar • Carbohydrate fermentation ( glucose, lactose, sucrose) • Gelatine liquefaction test • Mannitol salt • Indol • SIM ( indol, motility,sulfur reduction)

  3. IMViC tests (Indole ,Methyl Red,Voges-Proskauer (VP) test,Citrate Test) • Triple sugar Iron Agar • Skim Milk agar • Catalase

  4. Gram stain • Purpose: The Gram stain is the most commonly used differential stain for determining cell morphology. • The gram stain divides most bacteria into two main groups based on the amount of the sugar: peptidoglycan present in the bacteria cell wall.

  5. Gram stainProcedure • Procedure: • 1. clean your slide • Put ½ drop of water on the slide • Using a bacteria loop add bacteria to the slide • Let slide air dry • Heat fix the slide • Add crystal violet to slide and let sit for 2 minutes. • Wash off under water • Add iodine to slide and let sit for 2 minutes

  6. Procedure continue: • Wash off under water • THIS IS THE MOST IMPORTANT STEP!! Holding slide at an angle, carefully add ethyl alcohol for 8 seconds • After 8 seconds are up, wash off alcohol under water. • Add safranin red for at least 2 minutes • Wash off and blot dry • Examine under microscope

  7. Gram Positive bacteria • Gram positive bacteria stain purple and have a thick layer of peptidoglycan in the cell wall

  8. Gram Negative bacteria • Gram negative bacteria stain pink and have a thin layer of peptidoglycan in the cell wall

  9. Purpose: “The movement of bacteria is detected by use of a medium with low agar concentration. The medium is inoculated with an inoculating needle Reading the test: An organism that is motile will grow out into the surrounding medium away from the stab line. Motility

  10. Test Tube Results Test tube 1: Motile Test Tube 2:Non-motile Test Tube 3：Motile Motility

  11. Motility Test

  12. EMB ( Eosin Methylene blue agar) Purpose: This agar a is selective and differential medium used for isolation and differentiation among Enterobacteriaceae.

  13. EMB ( Eosin Methylene blue agar) EMB is selective for gram negative bacteria meaning only gram negative bacteria will grow on this medium.

  14. EMB ( Eosin Methylene blue agar) • EMB also contains lactose. • Organisms which do not ferment lactose will have colorless colonies • Organisms which do ferment lactose will have pink colonies. • E. coli develops a metallic green sheet of colonies

  15. EMB with E. coli

  16. Organisms which do ferment lactose will have pink colonies. • .

  17. Organisms which do ferment lactose will have pink colonies

  18. MacConkey agar Purpose: MacConkey is both selective and differential used to differentiate between gram negative and gram positive bacteria.

  19. MacConkey agar MacConkey is selective for gram negative bacteria meaning only gram negative bacteria will grow on this medium.

  20. MacConkey agar • MacConkey also contains lactose. • Organisms which do not ferment lactose will have colorless colonies • Organisms which do ferment lactose will have pink colonies

  21. Organisms which do not ferment lactose will have colorless colonies

  22. Organisms which do ferment lactose will have pink colonies

  23. MacConkey Agar Left: Shigella - result Right: Escherichia + result

  24. A B • Answers: • A 3. B • 2. A 4. B

  25. Carbohydrate fermentation( glucose, lactose, sucrose) • Purpose: This test is used to determine the ability of an organism to ferment various simple carbohydrates ( sugars) • A small tube ( Durham tube) is inverted and place in each larger tube of liquid medium • The inverted tube is able to trap any gas produced. • The indicator, phenol red will turn yellow below a pH of 6.8 9 showing fermentation) and darker pinkish-red above pH 7.4 ( showing no fermentation)

  26. Carbohydrate fermentation( glucose, lactose, sucrose) • Reading the Test: • Acid: (yellow): Positive for metabolizing sugar + ( a color change has happened) • Acid, Gas: ( yellow plus gas bubble): Positive for metabolizing sugar and gas production (+) ( color change plus bubbles) • Negative: NO color change

  27. Carbohydrate fermentation( glucose, lactose, sucrose)

  28. Carbohydrate Fermentation Tests Glucose w/ Durham Fermentation Tube

  29. Carbohydrate Fermentation Tests Sucrose Fermentation

  30. Gelatine liquefaction test • Purpose: This test is used to determine the ability of the bacteria to produce gelatinases that digest and liquefy gelatin. • Reading Test: • Once the tubes are inoculated with the bacteria and incubated, they will cause the gelatin to melt. • In order to determine whether or not the reaction has taken place, the incubated tubes must be placed on ice until the control tube is once again solid.

  31. Positive Test Negative Test Gelatine liquefaction test

  32. Mannitol salt • Purpose: Mannitol salt agar is both selective and differential. • Principle: Mannitol salt helps determine two characteristics of bacteria, whether they are salt tolerant or not, and whether they are able to ferment mannitol

  33. Mannitol salt used to id Staphlococcus bacteria • Mannitol salt agar is a selective and differential primary culture medium useful in recovery and identification of staphylococci from specimens containing mixed flora. • The high salt concentration (7.5%) inhibits most gram-negative and gram-positive bacteria except Staphylococcus species

  34. The Staphylococcus aureus ferments mannitol and turns the medium yellow.  The Serratia marcescens does not grow because of the high salt content

  35. Streptococcus agalactiae does not grow on MSA because of the high salt content. Staphylococcus epidermidis grows but does not ferment mannitol.

  36. Indol • Purpose: The indole test is used to identify bacteria capable of producing indole. • Reading the Test: • When Kovac’s solution is added to the inoculated tube, a red layer will form when indol is present = positive test. • No red layer = negative test

  37. IndolWith the Kovac’s reagent added: • Positive Test • Negative Test

  38. SIM Test(Indol, motility, sulfur reduction) • Purpose: The SIM test performs three different tests: motility, indole and sulfur reduction. • Motility and indole have been mentioned earlier • Sulfur reduction: This test is used to identify those bacteria capable of reducing sulfur.

  39. Black precipitate indicates a positive test (for H2S production) No precipitate means a negative test SIM Test(, sulfur reduction portion)

  40. IMViC tests • The IMViC tests are a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. • A coliform is a gram negative, aerobic or facultative aerobic rod which produces gas from lactose within 48 hours. • The presence of some coliforms indicate fecal contamination.

  41. These four tests include • Indole test ( discussed earlier) • Methyl Red test • Voges-Proskauer (VP) test * • Citrate Test

  42. Indole Test broth with tryptophan IMViC Series

  43. Methyl Red test and Voges-Proskauer (VP) test * • These tests both use the same broth for bacterial growth. • The broth is called MRVP broth. • After growth, the broth is separated into two different tubes, one for the Methyl Red (MR) test and one for the Voges-Proskauer (VP) test.

  44. The pH indicator Methyl Red is added to one tube and a red color appears at pH's lower than 4.2, and indicated positive test. Methyl Red

  45. Methyl Red Test IMViC Series

  46. VP test • The reagents used for the VP test are Barritt's A (alpha-napthol) and Barritt's B (potassium hydroxide). • When these reagents are added to a broth in which acetyl methyl carbinol is present, they turn a pink-burgundy color (a positive VP test). • This color may take 20 to 30 minutes to develop. E. coli does not produce acetyl methyl carbinol,

  47. Voges-Proskauer Test  MRVP broth IMViC Series Leave uncapped for 15-20 minutes……..

  48. . VP test

  49. Citrate Test This test uses Simmon's citrate agar to determine the ability of a microorganism to use citrate as its sole carbon source.

  50. Simmons Citrate Test • The citrate agar is green before inoculation, and turns blue as a positive test indicator

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