Laboratory Biochemical Assays A Quick & Concise Review
Parts of the Microscope <ul><li>Coarse Focus </li></ul><ul><li>Fine Focus </li></ul><ul><li>Stage </li></ul><ul><li>Conden...
Negative Staining <ul><li>Natural size & shape  </li></ul><ul><li>No heat fix; prevents distortion </li></ul><ul><li>Congo...
Negative Stain <ul><li>Bacteria will be clear cells  </li></ul><ul><li>Background will be stained dark or navy blue </li><...
Gram Stain <ul><li>Hallmark staining procedure </li></ul><ul><li>Gram positive vs. gram negative </li></ul><ul><li>Crystal...
Gram Stain <ul><li>Purple are gram +  </li></ul><ul><li>Pink are gram - </li></ul>
Acid Fast Stain <ul><li>Mycobacterium  ssp. </li></ul><ul><li>Thick & waxy, lipoidal cell wall </li></ul><ul><li>Carbol fu...
Acid Fast Stain <ul><li>Acid fast cells will be pink </li></ul><ul><li>Non-acid fast cells will be green </li></ul>
Spore Stain <ul><li>Bacillus  &  Clostridium  ssp. </li></ul><ul><li>Malachite green w/ heat </li></ul><ul><li>Safranin </...
Spore Stain <ul><li>Spores are green  </li></ul><ul><li>Vegetative cells are pink </li></ul>
Capsule Stain <ul><li>Klebsiella  ssp. </li></ul><ul><li>No heat fixing </li></ul><ul><li>Crystal violet </li></ul><ul><li...
Capsule Stain <ul><li>Capsule will appear as a light blue, or clear halo around the bacterial cell </li></ul>
Differential & Selective Media <ul><li>Selective:  </li></ul><ul><li>Isolate specific groups of bacteria </li></ul><ul><li...
Differential & Selective Media <ul><li>Differential: </li></ul><ul><li>Distinguish between morphologically& biochemically ...
Mannitol Salt Agar (MSA) <ul><li>High salt (7.5%) </li></ul><ul><li>Phenol red </li></ul><ul><li>Mannitol </li></ul>
Mannitol Salt Agar (MSA) <ul><li>Halophiles </li></ul><ul><li>Mannitol fermentation </li></ul><ul><li>Useful in isolating ...
SA on MSA
Blood Agar <ul><li>Fastidious organisms </li></ul><ul><li>Hemolytic properties </li></ul>
Blood Agar <ul><li>Gamma hemolysis – no hemolysis (SA) </li></ul><ul><li>Alpha hemolysis – incomplete lysis; greenish halo...
SP on Blood Agar
MacConkey Agar <ul><li>Crystal Violet </li></ul><ul><li>Bile Salts </li></ul><ul><li>Lactose </li></ul><ul><li>Neutral Red...
MacConkey Agar <ul><li>Inhibits gram positive organisms </li></ul><ul><li>Ability to ferment lactose </li></ul><ul><li>EC ...
EA & EC on MacConkey Agar
Eosin Methylene Blue Agar (EMB) <ul><li>Lactose vs. non-lactose fermenters </li></ul><ul><li>EC vs. EA </li></ul>
Eosin Methylene Blue Agar (EMB) <ul><li>EC has a metallic green sheen to it’s growth </li></ul><ul><li>EA has a fish-eye a...
EA vs. EC on EMB Agar
Starch Hydrolysis <ul><li>Exoenzymes amylase & maltase </li></ul><ul><li>Breakdown starch in the agar </li></ul>
Starch Hydrolysis <ul><li>Plate turns blue-black after the addition of iodine? </li></ul><ul><li>Plate shows a clear zone ...
Starch Hydrolysis
Gelatin Tube <ul><li>Exoenzyme gelatinase </li></ul><ul><li>Protein hydrolysis </li></ul>
Gelatin Tube <ul><li>Gelatin is liquified? </li></ul><ul><li>How to confirm? </li></ul><ul><li>If it remains a liquid? </l...
Carbohydrate Fermentation <ul><li>Can bacteria ferment sugars? </li></ul><ul><li>Acid? </li></ul><ul><li>Acid & Gas? </li>...
Carbohydrate Fermentation <ul><li>Yellow tube? </li></ul><ul><li>Bubble in the Durham tube? </li></ul><ul><li>Red or pink ...
Carbohydrate Fermentation
Triple Sugar Iron (TSI) <ul><li>Enterobacteriaceae </li></ul><ul><li>Glucose (0.1%) </li></ul><ul><li>Alkaline slant/acid ...
Triple Sugar Iron (TSI)
IMViC Test <ul><li>I ndole </li></ul><ul><li>M ethyl Red </li></ul><ul><li>V oges-Proskauer </li></ul><ul><li>C itrate Uti...
Indole <ul><li>Tryptophanase degrades tryptophan </li></ul><ul><li>Indole is a waste product of the reaction </li></ul>
Indole <ul><li>Kovacs reagent </li></ul><ul><li>Cherry-red reagent layer if indole is present  </li></ul>
Indole
Methyl Red <ul><li>Ferment glucose in the MRVP broth </li></ul><ul><li>Large quantities of acid end-products </li></ul>
Methyl Red <ul><li>Methyl Red indicator used to detect pH changes (reacts at a pH~4) </li></ul><ul><li>Red color in the br...
Voges-Proskauer <ul><li>Glucose fermentation </li></ul><ul><li>Acid end-products </li></ul><ul><li>Conversion of these aci...
Voges-Proskauer <ul><li>Barrits Reagents A & B </li></ul><ul><li>Detects acetoin, a non-acidic end-product </li></ul><ul><...
MRVP Test
Citrate Utilization <ul><li>Citrate as a sole carbon source </li></ul><ul><li>Simmons citrate agar slant </li></ul><ul><li...
Citrate Utilization <ul><li>Blue slant – positive test </li></ul><ul><li>Green slant – negative test </li></ul>
Citrate Utilization
Litmus Milk <ul><li>Skim milk </li></ul><ul><li>Lactose </li></ul><ul><li>Milk proteins – casein, lacto-albumin </li></ul>...
Litmus Milk <ul><li>Lactose fermentation - pink </li></ul><ul><li>Litmus reduction - white </li></ul><ul><li>Gas formation...
Litmus Milk
Litmus Milk
Nitrate Reduction Test <ul><li>Nitrates to Nitrites (NO 3  to NO 2 ) </li></ul><ul><li>Anaerobic Process </li></ul><ul><li...
Nitrate Reduction Test <ul><li>Mix solutions A & B </li></ul><ul><li>Red color develops? </li></ul><ul><li>No color develo...
Nitrate Test
Catalase <ul><li>Degrade H2O2 using catalase </li></ul><ul><li>Phenomenon of aerobic bacteria </li></ul><ul><li>TSA </li><...
Catalase <ul><li>Bubbles? </li></ul><ul><li>No bubbles? </li></ul>
Catalase
Oxidase <ul><li>Cytochrome oxidase C activity </li></ul><ul><li>Phenomenon of aerobic respiration </li></ul><ul><li>TSA pl...
Oxidase <ul><li>Black color develops? </li></ul><ul><li>No color or pink coloration? </li></ul>
Oxidase, at the bottom
Upcoming SlideShare
Loading in...5
×

Laboratory Biochemical Assays Mcb1010 L

1,366
-1

Published on

0 Comments
1 Like
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total Views
1,366
On Slideshare
0
From Embeds
0
Number of Embeds
0
Actions
Shares
0
Downloads
33
Comments
0
Likes
1
Embeds 0
No embeds

No notes for slide

Laboratory Biochemical Assays Mcb1010 L

  1. 1. Laboratory Biochemical Assays A Quick & Concise Review
  2. 2. Parts of the Microscope <ul><li>Coarse Focus </li></ul><ul><li>Fine Focus </li></ul><ul><li>Stage </li></ul><ul><li>Condenser </li></ul><ul><li>Objectives </li></ul><ul><li>Oculars </li></ul><ul><li>Light Source </li></ul>
  3. 3. Negative Staining <ul><li>Natural size & shape </li></ul><ul><li>No heat fix; prevents distortion </li></ul><ul><li>Congo Red </li></ul><ul><li>HCl </li></ul>
  4. 4. Negative Stain <ul><li>Bacteria will be clear cells </li></ul><ul><li>Background will be stained dark or navy blue </li></ul>
  5. 5. Gram Stain <ul><li>Hallmark staining procedure </li></ul><ul><li>Gram positive vs. gram negative </li></ul><ul><li>Crystal Violet </li></ul><ul><li>Grams Iodine </li></ul><ul><li>95% ethanol </li></ul><ul><li>Safranin </li></ul>
  6. 6. Gram Stain <ul><li>Purple are gram + </li></ul><ul><li>Pink are gram - </li></ul>
  7. 7. Acid Fast Stain <ul><li>Mycobacterium ssp. </li></ul><ul><li>Thick & waxy, lipoidal cell wall </li></ul><ul><li>Carbol fuschin </li></ul><ul><li>Acid alcohol </li></ul><ul><li>Brilliant green </li></ul>
  8. 8. Acid Fast Stain <ul><li>Acid fast cells will be pink </li></ul><ul><li>Non-acid fast cells will be green </li></ul>
  9. 9. Spore Stain <ul><li>Bacillus & Clostridium ssp. </li></ul><ul><li>Malachite green w/ heat </li></ul><ul><li>Safranin </li></ul>
  10. 10. Spore Stain <ul><li>Spores are green </li></ul><ul><li>Vegetative cells are pink </li></ul>
  11. 11. Capsule Stain <ul><li>Klebsiella ssp. </li></ul><ul><li>No heat fixing </li></ul><ul><li>Crystal violet </li></ul><ul><li>Copper sulfate </li></ul>
  12. 12. Capsule Stain <ul><li>Capsule will appear as a light blue, or clear halo around the bacterial cell </li></ul>
  13. 13. Differential & Selective Media <ul><li>Selective: </li></ul><ul><li>Isolate specific groups of bacteria </li></ul><ul><li>Chemical substances inhibit the growth of one type of bacteria </li></ul><ul><li>Permit the growth of another </li></ul>
  14. 14. Differential & Selective Media <ul><li>Differential: </li></ul><ul><li>Distinguish between morphologically& biochemically related groups of bacteria </li></ul><ul><li>Produce characteristic change in the surrounding media or their appearance on the media </li></ul>
  15. 15. Mannitol Salt Agar (MSA) <ul><li>High salt (7.5%) </li></ul><ul><li>Phenol red </li></ul><ul><li>Mannitol </li></ul>
  16. 16. Mannitol Salt Agar (MSA) <ul><li>Halophiles </li></ul><ul><li>Mannitol fermentation </li></ul><ul><li>Useful in isolating SA </li></ul>
  17. 17. SA on MSA
  18. 18. Blood Agar <ul><li>Fastidious organisms </li></ul><ul><li>Hemolytic properties </li></ul>
  19. 19. Blood Agar <ul><li>Gamma hemolysis – no hemolysis (SA) </li></ul><ul><li>Alpha hemolysis – incomplete lysis; greenish halo </li></ul><ul><li>Beta hemolysis – complete hemolysis; clear halo (SP) </li></ul>
  20. 20. SP on Blood Agar
  21. 21. MacConkey Agar <ul><li>Crystal Violet </li></ul><ul><li>Bile Salts </li></ul><ul><li>Lactose </li></ul><ul><li>Neutral Red </li></ul>
  22. 22. MacConkey Agar <ul><li>Inhibits gram positive organisms </li></ul><ul><li>Ability to ferment lactose </li></ul><ul><li>EC vs EA </li></ul><ul><li>Salmonella vs. Shigella </li></ul>
  23. 23. EA & EC on MacConkey Agar
  24. 24. Eosin Methylene Blue Agar (EMB) <ul><li>Lactose vs. non-lactose fermenters </li></ul><ul><li>EC vs. EA </li></ul>
  25. 25. Eosin Methylene Blue Agar (EMB) <ul><li>EC has a metallic green sheen to it’s growth </li></ul><ul><li>EA has a fish-eye appearance </li></ul>
  26. 26. EA vs. EC on EMB Agar
  27. 27. Starch Hydrolysis <ul><li>Exoenzymes amylase & maltase </li></ul><ul><li>Breakdown starch in the agar </li></ul>
  28. 28. Starch Hydrolysis <ul><li>Plate turns blue-black after the addition of iodine? </li></ul><ul><li>Plate shows a clear zone around the bacterial colony after the addition of iodine? </li></ul>
  29. 29. Starch Hydrolysis
  30. 30. Gelatin Tube <ul><li>Exoenzyme gelatinase </li></ul><ul><li>Protein hydrolysis </li></ul>
  31. 31. Gelatin Tube <ul><li>Gelatin is liquified? </li></ul><ul><li>How to confirm? </li></ul><ul><li>If it remains a liquid? </li></ul><ul><li>If it re-solidifys? </li></ul>
  32. 32. Carbohydrate Fermentation <ul><li>Can bacteria ferment sugars? </li></ul><ul><li>Acid? </li></ul><ul><li>Acid & Gas? </li></ul><ul><li>3 tubes, lactose, dextrose and sucrose </li></ul><ul><li>Phenol red </li></ul><ul><li>Durham Tube </li></ul>
  33. 33. Carbohydrate Fermentation <ul><li>Yellow tube? </li></ul><ul><li>Bubble in the Durham tube? </li></ul><ul><li>Red or pink tube? What happened? </li></ul>
  34. 34. Carbohydrate Fermentation
  35. 35. Triple Sugar Iron (TSI) <ul><li>Enterobacteriaceae </li></ul><ul><li>Glucose (0.1%) </li></ul><ul><li>Alkaline slant/acid butt </li></ul><ul><li>Acid slant/acid butt </li></ul><ul><li>Alkaline slant/alkaline butt </li></ul><ul><li>Blackening of the butt </li></ul><ul><li>Read the tube when? </li></ul>
  36. 36. Triple Sugar Iron (TSI)
  37. 37. IMViC Test <ul><li>I ndole </li></ul><ul><li>M ethyl Red </li></ul><ul><li>V oges-Proskauer </li></ul><ul><li>C itrate Utilization </li></ul>
  38. 38. Indole <ul><li>Tryptophanase degrades tryptophan </li></ul><ul><li>Indole is a waste product of the reaction </li></ul>
  39. 39. Indole <ul><li>Kovacs reagent </li></ul><ul><li>Cherry-red reagent layer if indole is present </li></ul>
  40. 40. Indole
  41. 41. Methyl Red <ul><li>Ferment glucose in the MRVP broth </li></ul><ul><li>Large quantities of acid end-products </li></ul>
  42. 42. Methyl Red <ul><li>Methyl Red indicator used to detect pH changes (reacts at a pH~4) </li></ul><ul><li>Red color in the broth indicates acid end-products and is a positive test </li></ul><ul><li>EC MR+ </li></ul><ul><li>EA MR- </li></ul>
  43. 43. Voges-Proskauer <ul><li>Glucose fermentation </li></ul><ul><li>Acid end-products </li></ul><ul><li>Conversion of these acid end-products to non-acid end-products </li></ul>
  44. 44. Voges-Proskauer <ul><li>Barrits Reagents A & B </li></ul><ul><li>Detects acetoin, a non-acidic end-product </li></ul><ul><li>Rose-colored band at the top of the broth </li></ul><ul><li>EC VP- </li></ul><ul><li>EA VP+ </li></ul>
  45. 45. MRVP Test
  46. 46. Citrate Utilization <ul><li>Citrate as a sole carbon source </li></ul><ul><li>Simmons citrate agar slant </li></ul><ul><li>Bromthymol blue – pH indicator </li></ul>
  47. 47. Citrate Utilization <ul><li>Blue slant – positive test </li></ul><ul><li>Green slant – negative test </li></ul>
  48. 48. Citrate Utilization
  49. 49. Litmus Milk <ul><li>Skim milk </li></ul><ul><li>Lactose </li></ul><ul><li>Milk proteins – casein, lacto-albumin </li></ul><ul><li>pH indicator is none other than litmus </li></ul>
  50. 50. Litmus Milk <ul><li>Lactose fermentation - pink </li></ul><ul><li>Litmus reduction - white </li></ul><ul><li>Gas formation – cracks/fissures </li></ul><ul><li>Curd formation – acid vs. rennet </li></ul><ul><li>Proteolysis – purple/brown, whey-like </li></ul><ul><li>Alkaline reaction – unchanged or deep purple </li></ul>
  51. 51. Litmus Milk
  52. 52. Litmus Milk
  53. 53. Nitrate Reduction Test <ul><li>Nitrates to Nitrites (NO 3 to NO 2 ) </li></ul><ul><li>Anaerobic Process </li></ul><ul><li>Nitrate Broth </li></ul><ul><li>Sulfanilic Acid (Solution A) </li></ul><ul><li>Alpha-napthylamine (Solution B) </li></ul>
  54. 54. Nitrate Reduction Test <ul><li>Mix solutions A & B </li></ul><ul><li>Red color develops? </li></ul><ul><li>No color develops? Significance? </li></ul><ul><li>Add Zinc Powder? </li></ul><ul><li>Color? </li></ul><ul><li>No color? </li></ul>
  55. 55. Nitrate Test
  56. 56. Catalase <ul><li>Degrade H2O2 using catalase </li></ul><ul><li>Phenomenon of aerobic bacteria </li></ul><ul><li>TSA </li></ul><ul><li>H2O2 </li></ul>
  57. 57. Catalase <ul><li>Bubbles? </li></ul><ul><li>No bubbles? </li></ul>
  58. 58. Catalase
  59. 59. Oxidase <ul><li>Cytochrome oxidase C activity </li></ul><ul><li>Phenomenon of aerobic respiration </li></ul><ul><li>TSA plate </li></ul><ul><li>p-aminodimethylaniline </li></ul><ul><li>Artificial electron donor </li></ul>
  60. 60. Oxidase <ul><li>Black color develops? </li></ul><ul><li>No color or pink coloration? </li></ul>
  61. 61. Oxidase, at the bottom
  1. A particular slide catching your eye?

    Clipping is a handy way to collect important slides you want to go back to later.

×