MCB LAB: post-lab 1

Based on the results from your last experiment indicate the Gram status and cell shape of the following bacteria: E. coli and S. epidermidis.

E. coli: Gram negative bacillus S. Epidermidis: Gram positive cocci

Often, cultures that are very old will stain Gram-negative even if they're normally Gram-positive. Offer an explanation for this by considering how the states of the cell wall might affect Gram staining.

Old cultures will often stain Gram negative, because as they age they lose their ability to retain the primary stain. As they age, the peptidoglycan layer of the cell wall decreases.

Describe the difference between simple staining and differential staining? Give example of each.

Simple staining is the process of staining with a single reagent (like methylene blue, crystal violet, and carbol fuschin), so to visulaize the morphology and arrangement of cells. For example, in week 1 of lab we stained a slide of Staphylococcus aureus with methylene blue. Differential staining uses two contrasting stains to separate cells into groups and to visualize cell structures. For example, capsule staining and spore staining will come to illustrate cells with structures of interest.

Why is the use of oil recommended while using the 100x objective lens?

The use of oil is recommended while using the 100x objective lens to decrease light refraction. With less light refraction, more light rays enter the objective lens and produce an image with higher resolution (the ability to make out two close objects).

Supposing you added the iodine before the crystal violet during the Gram staining. Provide an explanation on what would happen? How would Gram positive cells appear? How would Gram negative cells appear? Explain why you think so.

If I added the iodine before the crystal violet during the Gram staining, no primary stain would be present for the iodine to increase the cell's affinity to the stain. My Gram positive cells and Gram negative cells would appear pink. They would appear pink since applying the iodine before the crystal violet would not allow them to interact, since after applying the iodine there would be a wash. The crystal violet stain would be applied after but since the iodine was washed off previously, the stain would not have a strong affinity to the cell. It is essential that the crystal violet is added first, so that the iodine interacts with the stain and forms a CV-I complex. This CV-I complex interacts with the Gram positive's thick layer of peptidoglycan, is insoluble, and resistant to the decolorizing agent.

If you forgot to do an alcohol wash, how do you think your Gram stain results would be affected? Explain why.

If i forgot to do an alcohol wash in Gram staining, I believe I would not be able to make a stain that distinguishes Gram positive and negative cells. My primary stain would not be removed from Gram negative cells if I forgot this step. My counterstain would then not be able to interact with the decolorized cells (Gram negative cells), so both Gram positive and negative cells would appear purple. Also, alcohol wash is responsible for a more stringent retention of the CV-I complex in Gram positive cells.

Why is it important for you to flame the neck and mouth of your culture tubes during aseptic transfer procedure?

It is important to flame the neck and mouth of the culture tubes during aseptic transferring, because it prevents contamination.

Why are basic dyes more commonly used in bacterial staining procedures?

Basic dyes are more commonly used in bacterial staining procedures, because its positive charge has a strong affinity for the negative charge on the bacterial surface.