All media must be sterile & the basic conditions for autoclaving 

1. All media must be sterile & the basic conditions for autoclaving   Temperature = 121oC  Pressure = 15 PSI  Time = 20 minutes

2. Ordinary • أكتر ميديا بتستخدم في نمو البكتريا • Enriched • غنية فيها اضافات البكتريا محتاجاها علشان تنمو وبالذات البكتريا الممرضة • Enrichment • فيها حاجات تزود العدد بتاع البكتريا اللي موجودة بكمية قليلة علشان أزود عددها عن الـ N. Flora • Selenite broth  allow growth of Salmonella & prevent E. Coli • Selective • فيها مادة تخلي MO واحد يعيش والباقي يموت (تختارة) • Differential • فيها مادة تخلي شكل الـ MO مختلف عن الـ MO التاني غالباً يكون الاختلاف في اللون • Characteristic • ميديا بستخدمها في التعرف على البكتريا عن طريق الميتابوليزم بتاعها (Sugar fermentation )

7. Cultivation of bacteria • meat extract & Pepton & • 0.5% NaCl, neutral PH • light yellow transparent fluid • Indole Production • Base for sugar media • 1% Pepton + 0.5% NaCl + water

8. N.B + 1-5 – 2 % Agar N.B + 10-15 % Gelatin

10. Differentiate M.O according 2 Hemolytic activity N. Agar  100 C 55 C  5-10 % Sheep or Ox Blood

14. Simmon Citrate Agar • Upon citrate utilization the PH of the media will be increased causing change in color of the media into  blue • Due to Bromothymol Blue • Ability of MO to use citrate as carbon source for energy. • Degrade citrate producing CO2 which react with Na & water forming Na carbonate (alkaline product) which change color or BTB from green into deep prussian blue

20. Broth media + Sugars (Glucose & Galactose & Lactose & Mannose & Maltose) + Phenol Red (Yellow in Acidic PH & Purple/Red in Basic PH) + Durham's tube (Gas indicator)

24. Cooked meat for Anaerobic ONLY Glutathione

25. Characteristic media Used in Identification of Enteric organisms

27. Starch Hydrolysis • Test the ability of the organism to produce: • ExoenzymeAmylase which breaks down the Starch (Complex CHO of large molecule  Cannot pass through the cytoplasmic membrane) into Monosaccharide (MS = Simple can be used by the organism)  Inoculate the Organism in Starch agar + add I2  Amylase producing organism is surrounded by a clear zone(MS) while the remaining of the media will stain with the violet color

28. Casein Hydrolysis • Test the ability of the organisms to produce: • Proteolyticexoenzymes (Proteinase which hydrolyze casein) • Casein  Main protein of milk Responsible for the white color of milk. • Hydrolysis of casein  Form more soluble & transparentcompounds (peptides &aa) • Upon growing the organism on casein media the area surrounding the proteinase producing organism will appear transparent. • Casein hydrolysis is called Peptonization or Proteolysis.

29. Gelatin Hydrolysis (Liquefaction) • Test the ability of the organism to produce: • ExoenzymeGelatinsae which liquefy gelatin. • Gelatin hydrolysis (Liquefaction) is indicated by: • loss in ability to solidify even after refrigeration

31. Catalase Production • Test the ability of the organism to produce: • Catalase enzyme that degradatesH2O2 O2 + H2O + Air bubbles. • H2O2 is added to the bacterial media • Presence of gas bubblesmeans that the organism produces catalase

32. Oxidase Production • Test the presence of Cytochrome Cin the respiratory chain. • Aerobic organisms with Cytochrome Ccan oxidize amines to form coloredproducts. • This Test is specific for Pseudomonas Aeruginosa. • Wet F. Paper with • 1% N,N,N',N' Tetra methyl - P-Phenylene-Diamine (TMPD) (KovacOxidase reagent) • allow to dry & Pick bacterial colony with sterile toothpick  add to F. Paper • A purple color is produced

33. Hemolysin Production • Test the ability of the organism to produce: • ExoenzymeHemolysin which has destructive effect on the blood cells

34. Urease Production • Test the ability of the organism to produce: • Urease enzyme which splits urea in urea media to form Ammonia + CO2 • Accumulation of Ammonia will produce alkaline PH  Turns the color of indicator (phenol red) into Pink

35. H2S Production • H2S from Organic S or Inorganic S • Hydrogen Sulfide is detected by iron salt. • The presence of black precipitate is indication of H2S production. • Inoculate media peptone iron agar or TSI (Na2S2O3) • Black color will indicate H2S production

36. Sugar (CHO) Fermentation • Test the ability of the organism to produce: • Acid or Acid & Gas upon sugar fermentation

37. Nitrate Reduction • Test the ability of the organism to produce: • Nitrate reductaseenzyme which can reduce nitrate into nitrite  Inoculate organism into nitrate broth  Incubate at 37 C for 48 hrs  Add 1 ml of coupling reagent (sulfanilic acid & 1 ml of dimethyl alpha naphthyl amine reagent) If the organism produce nitrate reductase the nitrate in the media will be reduced into nitrite & Color become red precipitate

38. Ammonia Production • Test the ability of the organism to: • Degradate the organic nitrogen in the protein into ammonia.  Inoculate organism in 4% peptone water  Incubate at 37 c for 2, 4, 7 days  Add Nessler’s reagent. Appearance of Yellow-Orange or brown color indicates +Ve test

39. IMViC tests used for  Identification & Differentiation of Enterobacteriaceae (Klebsiella & Enterobacter & E. Coli) ( All are Lactose Fermenters)

40. Indole Test • Test the ability of organism to break down tryptophan into indole. • Incubate tryptophan (Peptone) broth media with the tested organism. • The Presence of indole can be detected by Kovac’s reagent • (Para DimethylAminobenzaldehyde in amyl alcohol) Kovac's reagent (yellow color) reacts with indole & produce (red color) on the surface of the test tube.

41. MR Test

42. VP Test

43. Methyl Red Test • Test the ability of organism to ferment the glucose & produce acids which will change the color of M.R (PH indicator) into red color

44. VogasProskaurTest • Test the ability of organism to ferment the glucose & produce neutral products which will change the color of indicator into Pink color • The reagents used for the VP test are • Barritt's A (Alpha-Napthol) & Barritt's B (Potassium-Hydroxide)

45. MR & VP tests is done on MR-VP broth media • (contains glucose & peptone) • MR & VP tests: • E. Coli is (MR+/VP-) • Klebsiella & Enterobacteraerogenes is (MR-/VP+)

46. Citrate Test • Test the ability of organism to utilize citrate as its only source of carbon. • Simmon’s Citrate media used in this test • Bacteria can break citrate into organic acids & CO2 CO2 form a basic compound (Na2CO3) Adding Bromothymol Blue Detects the presence of Na2CO3 by turning into blue (+Ve test)

47. Eosin-Methylene blue medium • Lactose / Esoin & MB • Permit differentiation between enteric lactose fermenters and non-fermenters • Alos in identification of E. coli

48. Lactose fermenter: purple black • Non- Lactose fermenter: colorless • E.coli: metallic green sheen

49. Motility Test • The medium contain triphenyltetrazolium which is reduced into red color by the stabbed bacterial growth. • Motile bacteria appear as diffused growth (with red color) • Non-Motile bacteria appear as single line of growth (the original stabbed line with pin color)