Microbiology Midterm- Lab 5: Preparation of Smears and Simple Staining

Why do we heat fix, advantages and disadvantages of this?

-To kill the bacteria (coagulated proteins from the cells will cause cells to stick to the slide). -To denature bacterial enzymes, preventing them from digesting cell parts, which causes the cell to break, a process called autolysis. - To preserve microbes with minimal shrinkage or distortion when stained. -May not kill all bacteria.

What did Bacillus megaterium look like in the simple stain?

-color: blue -morphology (shape): bacilli (rod-shaped) -arrangement of cells relative to one another: chains

What did Staphylococcus epidermis look like in the simple stain?

-color: blue -morphology (shape): cocci (spherical) -arrangement of cells relative to one another: clusters

How do you prepare a smear and heat fix a slide?

A.) Mark the smear areas with a marking pencil on the underside of a clean slide. B.) Solid Media: Place 1-2 loopfuls of water on the slide. C.) Solid Media: Transfer a very small amount of the culture with a STERILE inoculating loop. Mix with the water on the slide. B.) Liquid Media: Place 2 or 3 loopfuls of the liquid culture on the slide with a sterile loop. C.) Liquid Media: Spread the bacteria with the circle of the loop. D.) Allow the smears to AIR DRY at ROOM TEMP. E.) Heat fix the bacteria to your slide by passing the slide through the flame of Bunsen burner (or bac-incinerator) two or three times (not the side with the bacteria!!)

Which bacterium is a rod? Staphylococcus epidermis or Bacillus megaterium

Bacillus megaterium

Which bacterium is larger? Staphylococcus epidermis or Bacillus megaterium

Bacillus megaterium

Methylene blue can be prepared as a basic stain or and acidic stain. How would the pH of the stain affect the staining of bacteria?

Bacteria is slightly negative so it will attract the methylene blue as a basic stain and it will repel it as an acidic stain. Therefore, the basic stain would result in direct staining and the acidic stain would result in negative staining.

Why is basic stain used when staining bacteria?

Because bacteria has a negative charge, and you have to have a positive charge so that bacteria will stain.

Of what value is a simple stain?

It determines cell morphology, size, and arrangement.

Bacteria can be seen without staining. Why, then, was Koch's recommendation of fixing and staining important for the discovery of the bacterial causes of diseases?

It was important because fixing the preserves the cells and can be used for a long time. Staining allows us to see the bacteria easier.

In heat fixing, what would happen if too much heat were applied?

It would damage the cell's structure and distort its shape. It could also cause you not to be able to find the bacteria when viewing it under the microscope.

Can dyes other that methylene blue be used for direct staining?

Yes, basic dyes which are positively charged can be used because the cation of these dyes will form weak ionic bonds with the negatively charged bacteria.

Quality control staff in a sterilization unit of a hospital used a simple stain to determine whether bacteria were present in sterilized materials. A simple stain of sterile saline used for respiratory therapy revealed the presence of bacteria. Is the saline contaminated?

Yes, because there is a presence of bacteria.

What is a smear?

a thin film of bacterial cells on a slide

How do you perform a simple stain?

a.Use a clothespin to hold the slide b.Cover the smear with methylene blue 30-60 seconds. c.Wash excess stain off with distilled water being careful to not spray the bacteria directly. Let the water run clear. d.Place slide in the bibulous paper to dry. DO NO RUB!

What is the difference between a basic and an acidic stain?

basic stains have positive ions acidic stains have negative ions

What is a chromophore?

ion that is colored and is used in dyes

Another method of fixing smears is to use methanol instead of heat. How does alcohol chemically fix the bacteria?

kills bacteria; denatures bacterial enzymes which prevents autolysis of cells and denatures proteins

What is the purpose of heat fixing a smear?

to kill the bacteria, preserve microbes with minimal shrinkage or distortion, and to make cells stick to slide