Name: Cruz, Denmark G.

Date:___________________
Group: 4 Score:__________________

EXERCISE NO. 2
PREPARATION OF BACTERIAL SMEARS

Morphological studies of bacteria can be done by examining either live or killed (fixed)
cells. The preparation of live bacterial cells can only demonstrate the outline and structural
arrangements of the cells. A more detailed study of the internal cell structures can be done on
stained cells, however, they must be properly attached or fixed to the glass slide and killed.
This exercise will guide the preparation of bacterial smear for staining.

OBJECTIVE
1. To enable the student to prepare bacterial smears.

MATERIALS
1. 1 broth culture
2. 1 agar slant culture
3. Glass slides
4. Inoculating loops
5. Alcohol lamp
6. Marking pen

PROCEDURE from LIQUID MEDIA

1. Wash a glass slide thoroughly with soap and water to remove the dirt and grease from
the surfaces. After rinsing the slide thoroughly, be sure to handle the slide by the edges
only.
2. Dry the slide thoroughly with lens paper preferably or by careful blotting on folded tissue
paper.
3. Get the nutrient broth culture and shake sidewise with care and transfer two loopful of
the organism on the center of the glass slide.
4. Spread the organisms over an area that is about the size of a ten-centavo coin. To
remember where the smear is, you can encircle it with a marking pen on the reverse
side.
5. Allow the slide to air dry by normal evaporation of the liquid.
6. After the smear has completely dried, pass the slide over the flame a few times to heat-
kill and fix the organism to the slide.
PROCEDURE from SOLID MEDIA

1. Wash a glass slide thoroughly with soap and water to remove the dirt and grease from
the surfaces. After rinsing the slide thoroughly, be sure to handle the slide by the edges
only.
2. Dry the slide thoroughly with lens paper preferably or by careful blotting on folded tissue
paper.
3. Place a loopful of water on the slide (you can use a dropper or get a loopful of water
from the faucet with an inoculating loop).
4. With an inoculating loop pick up a very small amount of organism from the surface of the
agar slant and mix them with the water on the slide. Spread the organisms over an area
that is about the size of a ten-centavo coin. Prepare a good emulsion.
5. Allow the slide to air dry by normal evaporation of the water.
6. After the smear has completely dried, pass the slide over the flame a few times to heat-
kill and fix the organism to the slide.

Video demonstration guide:

Kindly access the given URL below:
https://www.youtube.com/watch?v=5TmyxFvo9x4

QUESTIONS:

1. What is the purpose of fixation?

- The fixation's main objective is to stop the bacteria from washing away during the
staining process. It is also important for accurately and effectively creating bacterial
smears for microscopic investigation. There are two types of fixation: heat fixation and
chemical fixation. Heat-fixation of an air-dried layer of bacteria may preserve the whole
morphology of the cell but not the internal structures, while chemical fixation is employed
to preserve the morphology of bigger, more fragile microorganisms as well as their
intricate cellular substructure.

2. Why aseptic technique is important in bacterial smear preparation?

- The purpose of aseptic technique is to stop microorganisms from being mistakenly

discharged into the environment and/or from infecting lab users. Microbiology and
clinical diagnostics both depend on adhering to aseptic standards.