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CHO Differential & & Selective Fungi Enriched Basic ordinary Yeast Anaerobic Media Characteristic All media must be sterile & the basic conditions for autoclaving Temperature = 121oC Pressure = 15 PSI Time = 20 minutes • • • 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 MOواحد يعيش والباقي يموت (تختارة) • Differential فيها مادة تخلي شكل الـ MOمختلف عن الـ MOالتاني غالبا ً يكون االختالف في اللون Characteristic • • • ميديا بستخدمها في التعرف على البكتريا عن طريق الميتابوليزم بتاعها ( )Sugar fermentation Nutrient Agar MAC Plate Media Chocolate Agar Blood Agar Nutrient Broth Sabouraud’s media Peptone H2O Liquid Media Thioglycolate Media Cooked Meat Nutrient Agar Slant Christensen Deep Agar Urea Solid & Simmon Gelatin Slant Citrate Media L-J Loffler’s Media Serum Enriched media Enrichment media Selective media Contain growth factors & other complex organic substances like blood, serum, etc Better for Fastidious Pathogenic bacteria Provides nutrient & environmental conditions that favor the growth of certain organisms but not suitable for others. Characteristic media Selenite broth that used for Salmonella 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) Differential media Blood Agar MacConkey’s media Simmon’s Citrate media Differentiate ( ) the bacteria by change in the color of the growing colonies Used to test the organism for Triple sugar iron (TSI) Metabolic activity Or Metabolic products Or Metabolic requirements Lysin iron agar (LIA) Useful in identification of the type of the bacteria. Sulfide indole motility (SIM) Nutrient Broth Cultivation of bacteria meat extract & Pepton & 0.5% NaCl, neutral PH light yellow transparent fluid Fluid Basic Ordinary Media Peptone Water Indole Production Base for sugar media 1% Pepton + 0.5% NaCl + water Agar Plate N.B + 1-5 – 2 % Agar Nutrient Agar Semisolid Basic Ordinary Media N.B + 10-15 % Gelatin Agar Slant Deep Agar Gelatin Media Gelatin Liquefaction Isolation of bacteria (Streaking for isolation) Short Storage (3-6 weeks) Prolonged Storage (6 months) Proteolytic activity Enriched Blood Chocolate Loffler’s Agar Agar serum Lowentsein Blood Agar Alpha Beta Gamma Incomplete (partial) & green zone Complete & Clear zone No Change Streptococcus Staph. aureus Pneumonia St. Pyogenes Differentiate M.O according 2 Hemolytic activity N. Agar 100 C 55 C 5-10 % Sheep or Ox Blood St. Faecalis Chocolate Loffler’s Serum Lowenstein Jensen H. Inflenza & Neisseria Corynbacterium Diphtheria Mycobacterium TB Heated sheep blood Horse serum Malachite green (Selective& Enriched) 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 MAC Simmon’s Citrate Selective Differential Inhibits the growth of Gm+Ve due to the presence of Crystal violet & Bile salts Differentiate ( ) bacteria on the basis of a color change reaction Gm –Ve bacteria grow well Lactose Neutral red MAC contains: 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 Lactose Fermenters Example of Non-Lactose Fermenters E. Coli & Klebsiella Salmonella & Shigella & Proteus species & Pseudomonas aeruginosa Pink Colony Colorless Broth media + Sugars (Glucose & Galactose & Lactose & Mannose & Maltose) + Phenol Red (Yellow in Acidic PH & Purple/Red in Basic PH) + Durham's tube (Gas indicator) Non-Fermenter Fermenter-Acid Producer Fermenter-Acid & Gas Producer No fermentation & PH indicator remains Purple Fermentation with the production of acid (Yellow color) but no gas Fermentation with the production of acid (Yellow color) and gas (Bubbles in Durham tube) Yeast & Fungi Incubation 25C for 10 days Fluid Sabouraud’s 4% Glucose & 5.6 Acidic PH Sterility Test (Saliva &Candida) Aerobic Anaerobic Incubation 30-35C for 7 days Facultative Sterility Test Thioglycollate Methylene green Na Thioglycollate Methylene blue Cystine Resazorine (Reducing agents) (O2 indicator) Small amount of Agar (Decrease oxygen diffusion) Cooked meat for Anaerobic ONLY Glutathione Lactose Sucrose Glucose FAS Na2S2O3 TSI Characteristic media Used in Identification of Enteric organisms Casein Starch Gelatin Hydrolysis 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 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. 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 Urease H2S Hemolysin Ammonia Oxidase Catalase Nitrate Production Acid Or Gas 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 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 Hemolysin Production Test the ability of the organism to produce: Exoenzyme Hemolysin which has destructive effect on the blood cells Blood Agar Beta Alpha Gamma Complete & Clear zone Incomplete (Partial) & green zone No Change St. Pyogenes St. Pneumonia St. Faecalis 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 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 Sugar (CHO) Fermentation Test the ability of the organism to produce: Acid or Acid & Gas upon sugar fermentation CHO Media No Fermentation Fermentation Fermentation No Acid No Gas Acid Production / No Gas Acid & Gas Production Phenol Red = Red Phenol Red = Yellow Phenol Red = Yellow Bubble in Durham’s tube 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 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 MR VP Indole Citrate IMViC IMViC tests used for Identification & Differentiation of Enterobacteriaceae (Klebsiella & Enterobacter & E. Coli) ( All are Lactose Fermenters) The presence of Enterobacter & Klebsiella The presence of E. coli Does not Indicate fecal contamination because they are widespread in soil & grass Indicate fecal contamination of food & water 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. MR Test VP Test 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 Klebsiella and Enterobacter E. Coli Produce neutral products from glucose (Ethyl alcohol & Acetyl methyl carbinol) PH rise above 6.2 Adding MR indicator Yellow color (Negative MR test) Produces acidic products from glucose PH drop below 4.4 Adding MR indicator Cherry red color (Positive MR test) Vogas ProskaurTest 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) Klebsiella and Enterobacter E. Coli Produce neutral products from glucose (Ethyl alcohol & Acetyl methyl carbinol) PH rise above 6.2 Adding Barritt's A & B Pink color (Positive VP test) Produces acidic products from glucose PH drop below 4.4 Adding Barritt's A & B Slight Yellow or (No Change) (Negative VP test) 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+) 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) Eosin-Methylene blue medium • Lactose / Esoin & MB • Permit differentiation between enteric lactose fermenters and non-fermenters • Alos in identification of E. coli • Lactose fermenter: purple black • Non- Lactose fermenter: colorless • E.coli: metallic green sheen 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)
