LAB REPORT 4 - Smear Preparation and Simple Staining

Using a staining reagent on bacteria is necessary when using a microscope because most bacteria have
no color, so they are difficult to see in the microscope field. Usually, the surface and cytoplasm of
bacteria are negatively charged so if a positive charge stain is smeared, a staining takes place. Once
stained, the bacteria may be observed and studied with respect to their shape, size and arrangement.

A stained bacterial smear and simple staining preparations have several steps. A glass slide is marked by
a pencil in the middle to know where to put the bacteria. A metal inoculation loop is sterilized then a
sterile water is dropped to form a film across the loop. Then the water is transferred into the circle
drawn by the pencil. A metal inoculating needle is flamed this time then let cooled for a while before
using it to transfer the bacteria on the glass slide. When the liquid completely evaporated on the glass
slide, heat fixed the bacteria on the glass slide. Lastly, two or three drops of the selected stain is put on
the bacteria. The technique is called the simple stain technique because only a single stain is used.

Guide Questions:

1. What are the two purposes of heat fixation?

Answer: Heat fixation is done so that the bacteria is ensured to be attached to the glass slide and heat
fixation is a way to kill bacteria without destroying it.

2. What is the purpose of simple staining.

Answer: The purpose of simple staining is to create a contrast between the bacteria and the
background. Since the surface of most bacterial cells is negatively charged, these positively charged
stains adhere readily to the cell surface.

3. Why are basic dyes more successful in staining bacteria than acidic dyes?

Answer: Basic dyes have a positively charge chromogen that forms an ionic bond with negatively
charged bacterial cell and thus colorize the bacterium. The advantage of using basic dyes is that basic
dyes allow you to directly see the cell. Acidic dyes have a negatively charged chromogen that is repelled
by the negatively charged bacterial cell and thus the background is colorized while the bacteria remain
transparent. Although the advantage of using acidic dye is that you can indirectly see the cell.

4. Name three basic stains

Answer: Three basic stains are crystal violet, carbolfuchsin and methylene blue.

5. Why is time an important factor in simple staining?

Answer: Time is an important factor because improper timing can produce false reactions. You can over
stain the bacteria resulting in making the bacteria to darken. You can under stain the bacteria resulting
in making the bacteria to lighten.

6. How would you define a properly prepared bacterial smear

Answer: It can be called a properly prepared bacterial smear is the bacteria are evenly spread out on the
glass slide in such a concentration that they are adequately separated from one another, the bacteria
are not washed off the slide during staining and lastly, the bacterial form is not distorted.
7. Why should you use an inoculating needle when making smears from solid media? An inoculating
loop from liquid media?

Answer: You should use an inoculating needle when making smears from solid media because only a
light scrape with a straight object is necessary. You should use an inoculating loop when making smears
from liquid media because there should not be too much to be transferred.

Reflection/Conclusion/Realization/Application