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

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Presentation on theme: "All media must be sterile & the basic conditions for autoclaving "— Presentation transcript:

1 All media must be sterile & the basic conditions for autoclaving 
Basic ordinary Enriched Differential & Selective CHO Fungi & Yeast Anaerobic Characteristic All media must be sterile & the basic conditions for autoclaving   Temperature = 121oC  Pressure = 15 PSI  Time = 20 minutes

2 أكتر ميديا بتستخدم في نمو البكتريا Enriched
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 )

3 Plate Media Nutrient Agar Blood Agar Chocolate Agar MAC

4 Nutrient Broth Peptone H2O Cooked Meat Thioglycolate Media
Liquid Media Nutrient Broth Peptone H2O Cooked Meat Thioglycolate Media Sabouraud’s media

5 Solid & Slant Nutrient Agar Deep Agar Gelatin Media Loffler’s Serum
L-J Simmon Citrate Christensen Urea

6 Enriched media Enrichment media Selective media Differential media
Blood Agar Contain growth factors & other complex organic substances like blood, serum, etc Better for Fastidious Pathogenic bacteria Enriched media Selenite broth that used for Salmonella Provides nutrient & environmental conditions that favor the growth of certain organisms but not suitable for others. Enrichment media MacConkey’s media Simmon’s Citrate media Contains chemicals & dyes that inhibits the growth of certain bacteria While not interfere with the growth of other bacteria. Addition of bile salt to the media make this media selective to  Pathogenic Enteriococci (Salmonella & Shigella) Selective media Differentiate ( ) the bacteria by change in the color of the growing colonies Differential media Triple sugar iron (TSI) Lysin iron agar (LIA) Sulfide indole motility (SIM) Used to test the organism for  Metabolic activity Or Metabolic products Or Metabolic requirements Useful in identification of the type of the bacteria. Characteristic media

7 Cultivation of bacteria light yellow transparent fluid
meat extract & Pepton & 0.5% NaCl, neutral PH light yellow transparent fluid Fluid Basic Ordinary Media Nutrient Broth Peptone Water Indole Production Base for sugar media 1% Pepton + 0.5% NaCl + water

8 Semisolid Basic Ordinary Media (Streaking for isolation)
Nutrient Agar Agar Plate Isolation of bacteria (Streaking for isolation) Agar Slant Short Storage (3-6 weeks) Deep Agar Prolonged Storage (6 months) Gelatin Media Gelatin Liquefaction Proteolytic activity N.B – 2 % Agar N.B % Gelatin

9 Enriched Blood Agar Chocolate Loffler’s serum Lowentsein

10 Blood Agar Alpha Beta Gamma Incomplete (partial) & green zone
Streptococcus Pneumonia Beta Complete & Clear zone Staph. aureus St. Pyogenes Gamma No Change St. Faecalis Differentiate M.O according 2 Hemolytic activity N. Agar  100 C 55 C  5-10 % Sheep or Ox Blood

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12 Lowenstein Jensen Loffler’s Serum Chocolate
Mycobacterium TB Corynbacterium Diphtheria H. Inflenza & Neisseria Malachite green (Selective& Enriched) Horse serum Heated sheep blood

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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

15 Example of Non-Lactose Fermenters Example of Lactose Fermenters
Differential & Selective MAC Selective Inhibits the growth of Gm+Ve due to the presence of Crystal violet & Bile salts Gm –Ve bacteria grow well Differential Differentiate ( ) bacteria on the basis of a color change reaction MAC contains:  Lactose  Neutral red Simmon’s Citrate Upon citrate utilization the PH of the media will be increased causing change in color of the media into  blue Due to Bromothymol Blue Example of Non-Lactose Fermenters Example of Lactose Fermenters Salmonella & Shigella & Proteus species & Pseudomonas aeruginosa E. Coli & Klebsiella Colorless Pink Colony

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20 No fermentation & PH indicator remains Purple Non-Fermenter
Broth media + Sugars (Glucose & Galactose & Lactose & Mannose & Maltose) + Phenol Red (Yellow in Acidic PH & Purple/Red in Basic PH) + Durham's tube (Gas indicator) No fermentation & PH indicator remains Purple Non-Fermenter Fermentation with the production of acid (Yellow color) but no gas Fermenter-Acid Producer (Yellow color) and gas (Bubbles in Durham tube) Fermenter-Acid & Gas Producer

21 Fluid Sabouraud’s 4% Glucose & 5.6 Acidic PH Sterility Test
Yeast & Fungi Sterility Test (Saliva &Candida) 4% Glucose & 5.6 Acidic PH Incubation 25C for 10 days

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24 (Decrease oxygen diffusion)
Thioglycollate Aerobic Anaerobic Facultative Sterility Test Na Thioglycollate Cystine (Reducing agents) Small amount of Agar (Decrease oxygen diffusion) Methylene green Methylene blue Resazorine (O2 indicator) Incubation 30-35C for 7 days Cooked meat for Anaerobic ONLY Glutathione

25 Identification of Enteric organisms
Lactose Sucrose Glucose FAS Na2S2O3 TSI Characteristic media Used in Identification of Enteric organisms

26 Hydrolysis Starch Casein Gelatin

27 Starch Hydrolysis Test the ability of the organism to produce:
Exoenzyme Amylase 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:
Proteolytic exoenzymes (Proteinase which hydrolyze casein) Casein  Main protein of milk  Responsible for the white color of milk. Hydrolysis of casein  Form more soluble & transparent compounds (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 loss in ability to solidify even after refrigeration
Gelatin Hydrolysis (Liquefaction) Test the ability of the organism to produce: Exoenzyme Gelatinsae which liquefy gelatin. Gelatin hydrolysis (Liquefaction) is indicated by: loss in ability to solidify even after refrigeration

30 Production Catalase Oxidase Hemolysin Urease H2S Ammonia Nitrate
Acid Or Gas

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

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

33 Hemolysin Production Blood Agar Beta Alpha Gamma
Test the ability of the organism to produce: Exoenzyme Hemolysin which has destructive effect on the blood cells Blood Agar Beta Complete & Clear zone St. Pyogenes Alpha Incomplete (Partial) & green zone St. Pneumonia Gamma No Change St. Faecalis

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 Acid or Acid & Gas upon sugar fermentation
Sugar (CHO) Fermentation Test the ability of the organism to produce: Acid or Acid & Gas upon sugar fermentation CHO Media No Fermentation No Acid No Gas Phenol Red = Red Fermentation Acid Production / No Gas Phenol Red = Yellow Acid & Gas Production Bubble in Durham’s tube

37 Nitrate Reduction Test the ability of the organism to produce:
Nitrate reductase enzyme 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 The presence of Enterobacter & Klebsiella
IMViC Indole MR VP Citrate IMViC tests used for  Identification & Differentiation of Enterobacteriaceae (Klebsiella & Enterobacter & E. Coli) ( All are Lactose Fermenters) The presence of E. coli The presence of Enterobacter & Klebsiella Indicate fecal contamination of food & water Does not Indicate fecal contamination because  they are widespread in soil & grass

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 Dimethyl Aminobenzaldehyde 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 Klebsiella and Enterobacter (Ethyl alcohol & Acetyl methyl carbinol)
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 E. Coli Klebsiella and Enterobacter Produces acidic products from glucose  PH drop below 4.4 Adding MR indicator  Cherry red color (Positive MR test) Produce neutral products from glucose (Ethyl alcohol & Acetyl methyl carbinol)  PH rise above 6.2 Adding MR indicator  Yellow color (Negative MR test)

44 Vogas ProskaurTest E. Coli Klebsiella and Enterobacter
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) E. Coli Klebsiella and Enterobacter Produces acidic products from glucose  PH drop below 4.4 Adding Barritt's A & B  Slight Yellow or (No Change) (Negative VP test) Produce neutral products from glucose (Ethyl alcohol & Acetyl methyl carbinol)  PH rise above 6.2 Pink color (Positive VP test) (Color may take mins to develop)

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

46 Simmon’s Citrate media used in this test
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)

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