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Some of our staphylococcus epidermidis will take up

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Some of our Staphylococcus epidermidis will take up to 10 Minutes to fizz. So be very wary of Negative Catalase Results on any Unknown. Request a YGC Plate!
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Gram Negative Biochemical Tests The Game Plan: You’ll do an Oxidase Test to determine whether your Bacterium is Pseudomonas or an Enteric. You’ll confirm Pseudomonas by plating on F Agar. You’ll then determine whether your Enteric is E. coli or Enterobacter by performing an IMViC (which is actually four separate Biochemical Tests). Oxidase (Atlas Page 87) “Does this Bacterium have the Enzyme Cytochrome Oxidase?” Aerobes, Facultative Anaerobes, and Microaerophiles have the Enzyme Cytochrome Oxidase in their Electron Transport Chains to transfer Electrons to Water. We’ll be checking for the Presence of this Enzyme by adding a reduced Artificial Electron Donor (Tetramethyl-p-Phenylendiamine). If Cytochrome Oxidase is present it will oxidize this Artificial Electron Donor and change it to a dark Blue. The Oxidase Test is fairly easy. Get a Piece of Filter Paper and use a Sterile Toothpick and transfer a large Goober of Bacteria. You want a large, visible Smear. Then add a Drop of the Oxidase Reagent (Light Blue). If your Bacterium is Oxidase Positive, the Goober of Bacteria will turn dark Blue (and it will do so within about 10 Seconds) . This means you have Pseudomonas . If your Bacterium is Oxidase Negative, the Goober will remain icky Latex Colored. This means you have an Enteric Bacterium, either E. coli or Enterobacter . However, keep in Mind that given sufficient Time -- say a few Minutes -- these Bacteria will also turn dark Blue. The Oxidase Test is a wonky Biochemical Test. Keep this in Mind if you get goofy F Agar or IMViC Results. F Agar (Fluorescence Agar) “Does this Bacterium produce fluorescent Siderophores?” Some species of Pseudomonas secrete the fluorescent, Water-soluble Pigments Pyocyanin (G= blue-green pus) and Pyoverdin (G= green pus), which fluoresce Blue- Green and Green, respectively under Ultraviolet Light (or even under the Fluorescent Lights in the Lab). These Pigments are termed Siderophores (G= star [iron] holders) and they are Chelating Agents (G= claw) that latch onto Iron in the Environment and make it more readily available to the Bacterium. Iron is a Key Component of Cytochromes and Proteins involved in the Electron Transport Chain. Pseudomonas can be induced to produce these Pigments by Growth on an Iron-deficient Medium, such as F Agar (“F” for Fluorescence). Once you’ve demonstrated that your Pseudomonas is Oxidase Positive, go ahead and streak it onto an F Agar Plate (Brown Stripe) and incubate it at 30°C until the next Lab. A Positive -- and we’ve rarely encountered Duds in MIC 101 -- will reach out and slap you in the Face with Fluorescence next Lab. You won’t really need the UV Light to see this Fluorescence but we’ve got it in the Prep Area, just in case. F Agar is a very Reliable Medium.
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