Microbiology Lab 5

2. Both crystal violet and safranin are basic stains and may be used to do simple stains on Gram-positive and Gram-negative cells. This being the case, explain how they can be used in a differential staining technique. (Hint: What extra steps are in the Gram staining procedure that are not used in a simple staining procedure? Which step accounts for the differences?)

It is the ability of Gram-positive cells to retain the crystal violet with the use of the mordant iodine when subjected to alcohol decolorization.

What is the counterstain in a Gram stain?

Safranin

Mordant

A compound that fixes a primary stain to cells. It literally means "to bite," so it causes a stain to "bite" into a surface more tightly. In the case of a Gram stain, KI-I2, is the mordant, and large complexes of crystal violet-iodide are formed inside the cell after its addition to the slide.

Crystal violet-iodine complex

A large, insoluble form of crystal violet and iodide anion that forms after addition of Gram's iodine. The complex is too large to escape through the small pores of a thick peptidoglycan layer that has been compressed by ethanol treatment. It is small enough, however, to escape through the relatively large pores of a Gram-negative bacterium whose out membrane has been solubilized by ethanol.

Peptidoglycan

A mesh-like layer of six-carbon sugars NAG and NAM that are crosslinked by 3-5 amino acids. This meshwork forms a thick outer cell wall in Gram positive bacteria that is interlaced with teichoic acid for rigidity. Gram negative bacteria, by contrast, have a thin peptidoglycan layer that is covered by a second lipid membrane known as the outer membrane.

Counterstain

A second stain that is used to detect cells that became clear during the previous decolorization step. All cells are stained by the counterstain including cells that were not previously decolorized, but the effect on the color of these (positive) cell types is negligible. The real value of a counterstain is to visualize the previously decolorize (negative) cell types.

What kind of stains are used then during a capsule stain?

An acidic or negative stain (Ex. Congo red) and a basic or positive stain (Ex. Maneval's stain)

3. If you saw large, eukaryotic cells in the preparation made from your gum line, they were most likely your own epithelial cells. Are you Gram-positive or Gram-negative? (You can make a good guess about this even if you didn't see your cells. Hint: Why do you see the differences when you stain bacterial cells; what structure is different? Do our cells have this structure?)

Animal cells tend to stain Gram-negative. It is the thick peptidoglycan layer of Gram-positive walls that makes them able to retain the crystal violet during decolorization. In fact, most cells, prokaryotic and eukaryotic, are Gram-negative.

Over-decolorize

Application of solvent for too long of a period of time such that cells that would normally stain positive appear negative. It is not really an all-or-nothing phenomenon but is rather a rate-dependent phenomenon. In other words, in the case of Gram staining, even Gram positive bacteria can lose their C.V.-Iodine complexes if ethanol treatment lasts too long.

Under-decolorize

Application of solvent for too short a period of time such that cells that would normally stain negative appear positive. Since decolorization occurs at a faster or slower rate depending of cell surface, ethanol treatment must last long enough for negative cells to actually decolorize. Too short treatment with ethanol means decolorization will not be complete.

Where would you stain then during a capsule stain?

Around the cell

What is the primary stain of the Gram stain?

Crystal violet

1. Predict the effect of the following "mistakes" made when performing a Gram stain. Consider each mistake independently. a. You do all the other steps in the correct order, but you fail to add the iodine. What color will Gram negative cells be (purple or pink or colorless)? Explain WHY!

Crystal violet stain is purple. If the KI-I2 is accidentally omitted, then no crystal violet-iodine complex is formed. When you decolorize the cells with ethanol, the crystal violet alone is small enough to get washed out, so all of the cells become colorless. Then, when you stain the cells with safranin, the cells will stain pink, which is the correct result for a Gram-negative cell.

1. Predict the effect of the following "mistakes" made when performing a Gram stain. Consider each mistake independently. a. You do all the other steps in the correct order, but you fail to add the iodine. What color will Gram positive cells be (purple or pink or colorless)? Explain WHY!

Crystal violet stain is purple. If the KI-I2 is accidentally omitted, then no crystal violet-iodine complexes are formed. When you decolorize the cells with ethanol, the crystal violet alone is small enough to get washed out, so all of the cells become colorless. Then, when you stain the cells with safranin, the cells will stain pink, which is not the correct result for a Gram-positive cell.

1. Predict the effect of the following "mistakes" made when performing a Gram stain. Consider each mistake independently. b. You do all the other steps in the correct order, but you fail to apply the decolorizer. What color will Gram positive cells be (purple or pink or colorless)? Explain WHY!

Crystal violet stain is purple. The iodine leads to the formation of large, insoluble crystal violet-iodine complexes inside the cells, which would stain all of the cells purple. You forget to decolorize with ethanol, so the cells remain purple. Then, you stain the cells with safranin, which stains them pink. Though, the cells are already purple, so they would appear dark purple, which is the correct result for a Gram-positive cell.

1. Predict the effect of the following "mistakes" made when performing a Gram stain. Consider each mistake independently. b. You do all the other steps in the correct order, but you fail to apply the decolorizer. What color will Gram negative cells be (purple or pink or colorless)? Explain WHY!

Crystal violet stains purple. The iodine leads to the formation of large, insoluble crystal violet-iodine complexes inside the cells, which would stain all the cells purple. You forget to decolorize with ethanol, so all the cells remain purple. Then, you stain the cells with safranin, which stains them pink. Though, the cells are already purple, so they would appear dark purple, which is the incorrect result for a Gram-negative cell.

1. What is the purpose of emulsifying the bacteria in serum in this staining procedure? (We will skip this step in lab, but why do they have it in the procedure? It has a purpose, what is it?)

Encapsulated cells may be slippery. Serum helps them stick to the slide, so they don't wash off as easily.

2. Some oral bacteria produce an extracellular "capsule." Of what benefit is a capsule to these cells? (What are the functions of the capsule?)

In the oral environment, a capsule helps them adhere to the tooth/gum surfaces. In internal environments, capsules protect the cells against phagocytosis and antibodies.

Why are the cells not heat-fixed during a capsule stain?

It would cause the cells to shrink, leaving an artifactual white halo around them that might be interpreted as a capsule

1. Why is the acidic stain Congo Red used? (Think about charges) What does it stain?

Negatively charged dye stains the background since the negative cell membrane repels the negative dye.

3. Is the capsule stained by the dyes? (Yes or No) Describe what the capsule should look like under the microscope when the capsule staining procedure is used.

No, the capsule is uncharged and, therefore, is not stained by either dye. The capsule appears as a clear halo around the outside of the cell.

2. Why is the basic Maneval's stain used? (Think about charges) What does it stain?

Positively charged dye stains the cell since the negatively charged cell membrane attracts the positively charged dye. The background turns from red-to grayish-blue due to a pH change.

Decolorization

Removal of the primary stain using a solvent such as 95% ethanol. This is the critical step in most differential staining procedures that distinguishes one class of cells from another.

1. Predict the effect of the following "mistakes" made when performing a Gram stain. Consider each mistake independently. d. Reversal of crystal violet and safranin stains. (All other steps are performed in the correct order) (Hint: what would your clothes look like if you dyed your clothes pink then dyed them purple?) What color will Gram positive cells be (purple or pink or colorless)? Explain WHY!

Safranin stains the cells red. Iodine-safranin-iodine complexes form, and the cells remain red. Then you would decolorize the cells with ethanol. The peptidoglycan layer shrinks and traps the safranin-iodine complexes, so the Gram-positive cells appear red. Crystal violet is then added, which is a purple stain. The purple stain on top of the red-stained cell makes it appear dark purple, which is the correct result for a Gram-positive cell.

1. Predict the effect of the following "mistakes" made when performing a Gram stain. Consider each mistake independently. d. Reversal of crystal violet and safranin stains. (All other steps are performed in the correct order) What color will Gram negative cells be (purple or pink or colorless)? Explain WHY!

Safranin stains the cells red. Iodine-safranin-iodine complexes form, and the cells remain red. Then you would decolorize the cells with ethanol. This would make large holes in the membranes of the Gram-negative cells, so the safranin-iodine complexes are washed away. This would make the Gram-negative cells colorless. The crystal violet stains the cells purple, and the Gram-negative cells appear a lighter purple, which is not the correct result.

Structural stains

Stains that identify particular cell attributes such as a capsule, an endospore (if the conditions are right for its production), and a flagellum.

5. If you did a negative stain would you be able to see a capsule? (Yes or No) Explain WHY?

The background is stained, but because neither the cell nor the capsule are stained, they are indistinguishable. In this case, the unstained capsule will make the unstained cell appear larger than if the slide were treated with a simple stain.

What would the appearance of the cells be once the acidic and basic stains are used during a capsule stain?

The capsule would look like a white halo around the cell

1. Predict the effect of the following "mistakes" made when performing a Gram stain. Consider each mistake independently. c. You do all the other steps in the correct order, but you fail to apply the safranin. What color will Gram negative cells be (purple or pink or colorless)? Explain WHY!

The crystal violet is purple. Adding iodine leads to the formation of the crystal violet-iodine complexes which is the reason these cells stain purple. Decoloring with ethanol makes large holes in the cell membranes, so the crystal violet-iodine complexes are easily washed away, making the Gram-negative cells colorless. Since safranin was omitted, the Gram-negative cells remain colorless, which is not the correct result for a Gram-negative cell.

1. Predict the effect of the following "mistakes" made when performing a Gram stain. Consider each mistake independently. c. You do all the other steps in the correct order, but you fail to apply the safranin. What color will Gram positive cells be (purple or pink or colorless)? Explain WHY!

The crystal violet is purple. Adding iodine leads to the formation of the crystal violet-iodine complexes which is the reason these cells stain purple. Then, you would decolorize with ethanol. The peptidoglycan layer shrinks and traps the crystal violet-iodine complexes, so the Gram-positive cells stain purple. Since we forgot the safranin, the cells stay purple. Maybe they are a slightly lighter shade of purple, but it is too hard to tell. This is the correct result for a Gram-positive cell.

Primary stain

The first stain in a multi-step staining procedure.

4. If you did a simple stain would you be able to see a capsule? (Yes or No) Explain WHY!

You will just see the cell, not the capsule, because the capsule doesn't stain, and the background is not stained. In this case, the capsule is indistinguishable from the background.

Gram stain

a differential stain in which a decolorization step occurs between the application of two basic stains

differential stains

allow a microbiologist to detect differences between organisms or differences between parts of the same organism. Not only allow determination of cell size, morphology, and arrangement (as with a simple stain) but information about other features as well.

Capsules

composed of mucoid polysaccharides or polypeptides that repel most stains since they are nonpolar