Determining Unknown in Microbiology Lab

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Determining an Unknown Through
Deferential Stains and Biochemical Tests

There are many reasons for knowing the identity of microorganisms. The reasons range from knowing the causative agent of a disease in a patient, so as to know how it can be treated, to knowing the correct microorganism to be used for making certain foods or antibiotics. This study was done by applying all of the methods that have been learned so far in the microbiology laboratory class for the identification of unknown bacteria. The identification process can be completed with a series of deferential stains and biochemical tests. Creating a dichotomous key helps to limit the amount of biochemical tests done on an unknown organism and by observation and recording of data the unknown organism can be found. Materials and Methods

The procedure for all tests performed was taken from Leboffe and Pierce’s Microbiology: Laboratory Theory and Application. The first and easiest data to record is the color of the bacterial colonies on a nutrient agar plate. Growing the cultures on the agar can be done by streaking a plate so as to isolate the bacterial colonies. Then separate the two colonies that are different in growth on a TP plate by using the same streaking techniques with a sterile loop. Both bacteria will then undergo a simple stain and a gram stain. The simple stain uses the charged portion of a chromogen and allows it to act as a dye where it becomes positively charged as a result of picking up a hydrogen ion or losing a hydroxide ion. Thus, the cell becomes colored. The simple stain is useful for identifying the shape of the unknown bacteria. A gram stain is also performed on the unknowns. The gram stain first uses crystal violet as the primary stain. Iodine is then used as a mordant fixodent to lock the stain in. Ethanol is used to decolorize the cell. Safarin is then used as the counter stain in order to show a contrast between the two colors. If the bacteria cell is...
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