Intracellular space the gram stain exploits this

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Basic Blueprint Reading and Sketching
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Chapter 2 / Exercise 6
Basic Blueprint Reading and Sketching
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intracellular space. The Gram stain exploits this characteristic by using the dye combinations of Crystal violet and Iodine. Crystal violet is retained by the thick peptidoglycan cell wall and forms a stable complex with iodine (upon its addition) effectively trapping the dyes in the cell. The resulting mixture is a purple coloration of the cell. Thus Gram­positive cells appear purple.
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Chapter 2 / Exercise 6
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Gram­negative bacteria have a relatively thin peptidoglycan layer followed by an outer membrane composed of lipopolysaccharides (LPS). This distinguishing characteristic sets Gram­negative apart from Gram­positive bacteria. During the Gram staining procedure, Gram­negative bacteria will initially retain the crystal violet dye. However, by next washing the cells with alcohol, a step referred to as the decolorization wash, the LPS and thin peptidoglycan layers of Gram­negative bacteria are unable to retain the dye and the outer membrane is depleted of its color. Importantly, Gram­positive bacteria remain unaffected by this decolorization step. In order to visualize the now unstained bacteria, a secondary (counterstain) dye called safranin is added. By counterstaining with the positively charged Safarnin dye, Gram­negative bacteria now retain a pink color. 05:56 12:12
Figure 3.8 Gram Staining . (A) Gram­positive bacillus (B) Gram­negative bacillus and (C) a mixed culture of Gram­positive coccus and Gram­negative bacillus are shown. As the Gram stain distinguishes between bacteria with a thick peptidoglycan wall and those without (or very thin), this is referred to as a differential stain . Differential staining is a generalized term used for any staining technique that separates specimens into further subgroups. This process most often utilizes at least two dyes. One of the disadvantages of the Gram stain is the requirement of the cells to be fixed (attached) to a glass slide. Tightly adherent cells are required to prevent sample loss during the staining and wash steps. The most common method to fix a sample is via heat fixation . By this process, samples are added to a glass slide and then passed through a flame until all liquid in the sample has been removed. Alternatively, chemical fixation strategies are also available and include the use of paraformaldehyde, ethanol or methanol. Although these processes fix the sample to the slide it also kills the microorganism. As such, characteristics related to motility (movement) are not possible. Wet mount is a basic form of sample preparation for viewing live samples. A small liquid culture (usually just a drop) containing a microorganism of interest is prepared, added to a slide and then covered with a glass coverslip. The coverslip is present to both protect the objective and the specimen while also holding the microorganism in place. Note: heat fixing is not performed. Wet mounts are used to observe the motility and behavior of an organism.

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