All methods were performed following the constructed flowchart (above).
To observe the colony morphology, the original unknown culture was streaked for isolation on nutrient agar using the quadrant streaking technique, inverted, and incubated at 37° C for 48 hours. This allowed for observation of the colony morphology.
Separation of Gram-positive and Gram-negative
The original unknown culture was streaked for isolation on Columbia CNA agar and MacConkey agar using a quadrant streak, inverted, and placed in a 37° C incubator for 48 hours. CNA agar contains a mix of colistin and nalidixic acid, as well as sheep blood. If there is poor or no growth on CNA agar, then the organism was inhibited by colistin and nalidixic acid and is Gram-negative. However if there is good growth, the organism was not inhibited by colistin and nalidixic acid, and is Gram-positive. MacConkey agar contains bile salts and crystal violet. If there is poor growth or no growth, the organism was inhibited by crystal violet and/or bile, and is Gram-positive. If there is good growth, the organism was not inhibited by crystal violet or bile, and is Gram-negative.
This protocol was performed once using the culture on the Columbia CNA agar, and once using the culture on the MacConkey agar.
The Gram stain uses crystal violet as a primary stain, ethanol-acetone as a negative stain, and safranin as a counterstain. A small amount of the culture was heat-fixed to a glass microscope slide. The culture was flooded with methylene blue, and allowed to sit for one minute. Then, the slide was rinsed with distilled water. The slide was flooded with Gram’s iodine, then allowed to sit for one minute. The slide was rinsed with water, ...
... middle of paper ...
...ic tubes are yellow, oxidation and fermentation occurred, or fermentation only. If there is a slight yellow area at the top of the aerobic and anaerobic tubes, oxidation and slow fermentation occurred, or slow fermentation only. If there is green or blue in the aerobic and anaerobic tubes, there was no sugar metabolism, and the organism is nonsaccharolytic.
Oxidase Test Confirmation Test
The oxidase test uses tetramethyl-p-phenylenediamine (TMPD) as an indicator which changes to a dark blue/purple color once oxidized. A visible amount of the Gram-negative unknown was placed on a Q-tip. A 3 drops of TMPD was dropped onto the organism. After 20 seconds, the color of the organism was recorded. If the organism turns dark blue within 20 seconds, cytochrome c oxidase is present. If the organism does not change to dark blue within 20 seconds, cytochrome c oxidase is absent.
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