Media! - Summer 2020 - HCC.pdf

Media! Media Terms: 1) Media – Material used for the growth of microorganisms Types of Media: - Broth = Liquid - Solid = Broth + Agar - Types of Solid Media: Plate, Slant, Deep Types of Media Components: - Nutrient – general purpose media - Selective and Differential Media Components 2) Inoc...

Media! Media Terms: 1) Media – Material used for the growth of microorganisms Types of Media: - Broth = Liquid - Solid = Broth + Agar - Types of Solid Media: Plate, Slant, Deep Types of Media Components: - Nutrient – general purpose media - Selective and Differential Media Components 2) Inoculate – The process by which microorganisms are added to media 3) Colony – a group of genetically identical bacteria Methods to Inoculate Media Agar Plate – - Streak to isolate/Quadrant Method/Streak plate method - Purpose is to obtain isolated, individual colonies Broth – - Dip – n – Swish Method Agar Slant - - Fish Tail method Agar Deep - - Straight Line Stab Streak To Isolate Method Streak To Isolate Method - Use an inoculating loop Streak To Isolate Method Streak To Isolate Method Streak To Isolate Method Figure 1.16 Bacterial colonies on agar Bacterium 6 Bacterium 5 Bacterium 4 Bacterium 3 Bacterium 2 Bacterium 1 Bacterium 7 Bacterium 8 Bacterium 9 Bacterium 10 Bacterium 11 Bacterium 12 Streak To Isolate Method Figure 6.8 Characteristics of bacterial colonies-overview Dip – n – Swish Method - Used to inoculate broth - Use an inoculating loop Inoculating Loop Broth Fish Tail Method - Used to Inoculate an Agar Slant - Use an inoculating loop Slant Straight Line Stab - Used to Inoculate an Agar Deep - Use an Inoculating Needle Deep Bench Top Culturing Microorganisms • Culture Media – Six types of general culture media • • • • • • Defined media Complex media Selective media Differential media Anaerobic media Transport media © 2012 Pearson Education Inc. Figure 6.11 Slant tube containing solid media Slant Butt Figure 6.13 The use of blood agar as a differential medium Beta-hemolysis Alpha-hemolysis No hemolysis (gamme-hemolysis) Figure 6.15 Use of MacConkey agar as a selective and differential mediumoverview Figure 6.14 The use of carbohydrate utilization tubes as differential media Durham tube (inverted tube to trap gas) No fermentation Acid fermentation with gas 3 Reasons why we inoculate differential and selective media 1. To culture all bacteria present and see which if any predominates. 2. To differentiate species by certain characteristic responses to media ingredients. 3. To selectively encourage growth of species of interest while suppressing normal flora. Differential media - media that helps us separate bacteria based on a metabolic process (enzyme tests, carbohydrates) Selective media – media that contains an ingredient that encourages the growth of some bacteria, but discourages the growth of other bacteria. MSA – Mannitol Salt Agar, For isolation and differentiation of Staphylococcus species Differential – mannitol sugar Indicator – phenol red Selective – 7.5% NaCl Yellow = acidic = (+) for mannitol fermentation Orange/red = neutral, no reaction Hot pink = alkaline = (-) for mannitol fermentation MacConkey Agar – used to isolate and differentiate gram negative organisms Differential – lactose fermentation Selective – bile salts and crytal violet - pH Indicator: neutral red Hot pink colonies = (+) for lactose fermentation Colorless colonies = (-) for lactose fermentation Blood agar – Differential based on hemolysis, isolation and cultivation of fastidious bacteria Beta – complete breakdown of hemoglobin; clearing of medium; Gamma or nonhemolytic; no hemolysins present; no change in medium Enterococcus faecalis Alpha – incomplete breakdown of hemoglobin; Starch Agar – to check for the presence of amylase via starch hydrolysis. Reagent – Iodine; Bacillus sp. Halo = (+) for Amylase No Halo = (-) for Amylase EMB – Eosin Methylene Blue Agar, isolate Gram neg. bacteria Selective – eosin; methylene blue; inhibitory to Gram + organisms Differential – lactose fermentation Metallic green/blue black colonies- (+) vigorous lactose fermentors, acidic Dark Purple – (+) slower fermentors acidic Light pink/colorless – (-) not fermenting Phenol Red Broths: used to determine carbohydrate fermentation with or without gas production (Durham tube); tubes are read as: A for acid (yellow); (+) for carbohydrate fermentation NR for no reaction; and K for alkaline (pink) (-) for carbohydrate fermentation G for gas (trapped bubbles in the Durham tube); Reversion – best to read tubes 18-24 hours. SIM tubes – sulfide, indole, motility Hydrogen sulfide (H2S) production leads to blackening of medium Black = (+) H2S No black = (-) H2S Indole is a by-product of the breakdown of tryptophan reagent: Kovac’s Red = (+) for Indole Yellow = (-) for indole Motility – determined by cloudiness away from the stab line Example of how to write the reaction: S+ I+ M-