HES 032-Microbiology and Parasitology

College of Nursing
RAD Learning (AY: 2023-2024 )

Group Leader: BADANA. DANICA KAYE

Group Members: A N D R A D A , S E A N F E R N A N
AQUE, MARY LEAN M.
ARREZA, NADINE CLAIRE V.
ASPERIN, CASANDRA ROSE

ACTIVITY NO. 4: Bacterial Smear Preparation

LEARNING OBJECTIVE: At the end of the laboratory period, the student should be able to:

1. Discuss the proper procedure of preparing a bacterial smear from a liquid and solid
bacterial culture media.
2. Explain the advantages and disadvantages of bacterial smear preparation.
3. Differentiate the two most common methods of fixation.
4. Identify the purpose of fixation.

DISCUSSION:

Before the microorganisms can be stained, they must be fixed (attached) to the microscope
slide. Fixing simultaneously kills the microorganisms and fixes them to the slide. It also
preserves various parts of microbes in their natural state with only minimal distortion.

When a specimen is to be fixed, a thin film of material containing the microorganisms is spread
over the surface of the slide. This film, called a smear, is allowed to air dry.

The two most common methods of fixation are heat fixation and methanol fixation. Heat
fixation is usually accomplished by passing the smear through a Bunsen burner flame. If not
performed properly, excess heat can distort the morphology of the cells. Methanol fixation,
which is accomplished by flooding the smear with absolute methanol for 30 seconds, is a more
satisfactory fixation technique. In general, fixation serves three purposes:

1. It kills the organisms.

2. It preserves their morphology (shape).
3. It anchors the smear to the slide

Compiled by: Claire Mae Caval, RMT

Microbiology and Parasitology – Instructor
Southwestern University PHINMA
Source:
https://bio.libretexts.org/Courses/College_of_the_Canyons/Bio_221Lab%3A_Introduction_to_Microbiology_%28Burke%29/17%3A_Smear_Pre
p_and_Simple_Stains/17.04%3A_Materials_and_Procedures

Compiled by: Claire Mae Caval, RMT

Microbiology and Parasitology – Instructor
Southwestern University PHINMA
 References:

Engelkirk, P., & Engelkirk, J. (2015). Burton's Microbiology for the Health and Sciences. 10th ed., Philadelphia:
Lippincott Williams & Wilkins
Tortora G., F. B. (2016). Microbiology an Introduction, 12th Edition. United States of America: Pearson Education

OBSERVATIONS:

As a nursing student in the lab, illustrate the outcome of the bacterial smear prepared:

STUDY GUIDE QUESTIONS:

1. What is the significance of smear preparation?

For various laboratory processes, including the Gram-stain, a smear must be prepared.
Making a smear is done to adhere the bacteria to the slide and stop the sample from slipping
away during the staining process. It causes bacteria to adhere to a slide so that they can be
stained and observed. It also kills them, rendering pathogenic bacteria safe to handle. In order
to stain and observe germs, it makes them stick to a slide. Additionally, it kills them, making
pathogenic bacteria manageable.

2. What will happen to the smear if too much heat is applied during heat fixation?
If too much heat is applied during the heat fixation process for a smear, several
undesirable effects can occur, including: Cellular Damage Excessive heat can cause significant
damage to the cells in the smear, Over-fixation can lead to the cells being overly fixed to the slide,
making it challenging to stain and observe them properly, Overheating can create artifacts in the
smear, which are misleading or unnecessary structures that ca obscure the actual features of
interest, and Prolonged exposure to high heat can damage the glass slide itself, causing it to crack
or warp, which can affect the quality of the smear and microscopy.
Compiled by: Claire Mae Caval, RMT
Microbiology and Parasitology – Instructor
Southwestern University PHINMA
 3. What will happen to the smear if heat fixation is neglected before staining the glass
slide?
If heat fixation is neglected before staining a glass slide in microbiology, the smear will
likely not adhere properly to the slide. Heat fixation is a crucial step that helps to kill the
microorganisms on the slide, attach them firmly, and preserve their cellular structures. Neglecting
heat fixation can result in the cells being easily washed away during the staining process, leading
to an inaccurate or unsuccessful staining procedure.

Compiled by: Claire Mae Caval, RMT

Microbiology and Parasitology – Instructor
Southwestern University PHINMA