Differentiating Organisms Using a Gram Stain
Amanda
Lab Report 1
Microbiology Laboratory
Title – Differentiating Organisms using the Gram Stain
Introduction
The experiment conducted was based upon the known attributes of two different groups of bacteria, those that are gram positive, and those that are gram negative. Using a specific staining procedure, it is possible to differentiate the two types under a microscope
The gram stain method of differentiation is possible because of differences in the cell membrane between the two categories of bacteria. Gram positive cells have an extra thick layer that is made of highly-crosslinked peptidoglycans, which impart different properties. This layer makes the cells less susceptible to decolorization by Ethyl Alcohol. Using this property, cells with a thinner peptidoglycan layer can be stained a different color than gram positive cells within a culture.
The primary color is applied to the culture and imparts a purple color to all bacteria in your sample. Adding Gram’s iodine, a mordant used by the method’s namesake, makes the violet color more intense, and creates a bond with the primary stain that makes it less soluble. The third step is to add a decolorizing agent, Ethyl Alcohol. In gram negative cells the alcohol makes the outer cells porous, which allows the primary stain to be washed away. The gram positive cells have pores that are actually dried out by the alcohol, which makes the primary stain fix into the membrane, and not get washed away. The final stain is the counter stain that lends a different, pinkish color to the gram negative cells that have been washed of the primary stain.
Methods
The method used in this experiment was derived from Cappucino and Sherman (2011) as detailed in experiment 10 with substitutions for the unknown bacteria. Aseptic lab technique was followed. One slide of the #7 unknown bacteria and one slide which included both unknown #7 and unknown #2 were prepared using the following stain technique.
The initial
Lab Report 1
Microbiology Laboratory
Title – Differentiating Organisms using the Gram Stain
Introduction
The experiment conducted was based upon the known attributes of two different groups of bacteria, those that are gram positive, and those that are gram negative. Using a specific staining procedure, it is possible to differentiate the two types under a microscope
The gram stain method of differentiation is possible because of differences in the cell membrane between the two categories of bacteria. Gram positive cells have an extra thick layer that is made of highly-crosslinked peptidoglycans, which impart different properties. This layer makes the cells less susceptible to decolorization by Ethyl Alcohol. Using this property, cells with a thinner peptidoglycan layer can be stained a different color than gram positive cells within a culture.
The primary color is applied to the culture and imparts a purple color to all bacteria in your sample. Adding Gram’s iodine, a mordant used by the method’s namesake, makes the violet color more intense, and creates a bond with the primary stain that makes it less soluble. The third step is to add a decolorizing agent, Ethyl Alcohol. In gram negative cells the alcohol makes the outer cells porous, which allows the primary stain to be washed away. The gram positive cells have pores that are actually dried out by the alcohol, which makes the primary stain fix into the membrane, and not get washed away. The final stain is the counter stain that lends a different, pinkish color to the gram negative cells that have been washed of the primary stain.
Methods
The method used in this experiment was derived from Cappucino and Sherman (2011) as detailed in experiment 10 with substitutions for the unknown bacteria. Aseptic lab technique was followed. One slide of the #7 unknown bacteria and one slide which included both unknown #7 and unknown #2 were prepared using the following stain technique.
The initial
Cited: Cappuccino, J.G., and N. Sherman (2011) Microbiology: A Laboratory Manual. Pearson Benjamin Cummings (San Francisco).