Which of the 3 differential stains would likely be the first used when identifying an unknown bacterium? Explain. The Gram stain would likely be the first one used to identify an unknown bacterium since most bacteria have a slight negative net charge and would therefore bind the positively charged crystal violet primary dye of a Gram stain. A counterstain can then be used to identify gram negative bacteria so that the unknown bacterium can easily be categorized according to its Gram status. What is the function of a mordant? A mordant is a solution that is used to fix the stain inside the bacterium.
For differential staining, how does a counterstain differ from a primary stain? A counterstain is a second dye that is used to differentiate bacteria based on the staining properties of their cell walls. How do gram-positive and gram-negative bacteria differ in cellular structure? How does this contribute to their differential staining properties? The cell walls of gram positive bacteria contain more peptidoglycans which cause the bacteria to retain the violet primary dye more, preventing being counterstained. Gram negative bacteria on the other hand, possess thinner walls with an outer layer of lipopolysaccharides.
Washing with alcohol disrupts this layer so that the primary dye leaks out, allowing the second dye to stain these bacteria. Which is the most critical step in the Gram-stain procedure? Why? If this procedure is done incorrectly, how might that affect the final results? The most critical step in the Gram-stain procedure is the decolorizing step since washing with alcohol for too long may cause the violet stain to be washed away even if the bacterium is Gram positive, thus leading to the misclassification of the bacterium as Gram negative.
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On the other hand, a decolorization step that is too short might cause Gram negative bacteria to appear as Gram positive, since the stain has not been completely washed away. How does culture age affect the results of a Gram-stain? False results may also be obtained if the culture used is old. Cell walls may lose integrity with age so even Gram positive bacteria may appear negative when they fail to retain the violet dye. How does culture age affect the results of a spore stain?
Old cultures may not stain properly since the endospores will probably become more dehydrated and therefore more resistant to penetration of chemicals such as the dyes used for staining. Why must smear thickness be considered before performing a Gram stain? Thick smears will make it difficult to see individual cells as well as their arrangements. More importantly, thick smears may trap the primary stain so that they are not effectively removed during decolorization, thus leading to falsely classifying Gram negative bacteria as Gram positive. What color are bacterial endospores after a Gram stain is performed?
What does this tell you about the physical properties of endospores? Endospores appear colorless after Gram staining, showing how resistant endospores are to penetration of chemicals. Bacillus anthracis, the causative agent of anthrax, is an endospore former. Why does this trait enhance its capabilities as a biological weapon? The fact that it forms highly resistant endospores means that it can withstand conditions that would normally kill other bacteria. Resistance to heat, radiation or chemical disinfectants make it an ideal biological weapon because it is not easily killed.
The results from the Gram staining experiment revealed the growth of Staphyloccus aureus and Micrococcus luteus – both Gram-positive species as shown by the purple stains they acquired. Unfortunately, Serratia marcescens and Pseudomonas aeruginosa failed to grow so we were not able to make any observations on these species. Both are Gram-negative bacteria, so if they had been successfully grown, we would have seen them as pink-stained shapes. It could be argued that the bacteria did grow but counterstaining failed (hence, they could not be seen), but if they were present they should have shown up with the primary stain.
However, we only observed purple dots which retained their color even after decolorization, so we conclude that the said bacteria failed to grow in the first place. From the results obtained, what we can conclude about the stained bacteria is that their cell walls have a thicker layer of peptidoglycans, that’s why they were able to retain a violet color after Gram staining. Bacillus megaterium was successfully stained to show that it is an endospore-forming bacterium, as evidenced by the malachite green-stained dots (indicating the endospores) and safranin-stained vegetative cells which appeared pink.
References: Caprette , D. R. (2005). Laboratory Module in Microbiology — Advanced Laboratory Bios 318. Retrieved Feb. 25, 2008, from http://www. ruf. rice. edu/~bioslabs/bios318/staining. htm Department of Pathology and Laboratory Medicine. (2007). The Department of Pathology and Laboratory Medicine. Retrieved Feb. 25, 2008, from http://www. musc. edu/pathology/Micro/GramProcedure/Step5. htm Wikipedia. (2008). Wikipedia: Staining. Retrieved Feb. 25, 2008, from http://en. wikipedia. org/wiki/Staining_(biology)