Media!

Figure 1.16 Bacterial colonies on agar Bacterium 6 Bacterium 7 Bacterium 5 Bacterium 8 Bacterium 4 Bacterium 9 Bacterium 3 Bacterium 10 Bacterium 2 Bacterium 11 Bacterium 1 Bacterium 12

Figure 6.8 Characteristics of bacterial colonies-overview

Figure 6.9 Streak plate method of isolation-overview

Culturing Microorganisms Culture Media Majority of prokaryotes have not been grown in culture medium 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 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 medium-overview

Acid fermentation with gas 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 To culture all bacteria present and see which if any predominates. To differentiate species by certain characteristic responses to media ingredients. 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 Selective – 7.5% NaCl Indicator – phenol red 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 Alpha – incomplete breakdown of hemoglobin; Beta – complete breakdown of hemoglobin; clearing of medium; Gamma or non-hemolytic; no hemolysins present; no change in medium Enterococcus faecalis

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); NR for no reaction; and K for alkaline (pink) G for gas (trapped bubbles in the Durham tube); (+) for carbohydrate fermentation (-) for carbohydrate fermentation 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-