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Activity 2. Bacterial Smear Preparation

This document discusses how to properly prepare a bacterial smear for staining and visualization under a microscope. It outlines the necessary precautions and steps, including using clean slides, making a thin smear, allowing it to air dry, and then fixing it through heat or methanol to adhere the cells. A well-prepared smear is thin and uniform yet contains enough cells for examination.

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Roan Eam Tan
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2K views5 pages

Activity 2. Bacterial Smear Preparation

This document discusses how to properly prepare a bacterial smear for staining and visualization under a microscope. It outlines the necessary precautions and steps, including using clean slides, making a thin smear, allowing it to air dry, and then fixing it through heat or methanol to adhere the cells. A well-prepared smear is thin and uniform yet contains enough cells for examination.

Uploaded by

Roan Eam Tan
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Activity 2: Bacterial Smear Preparation

ACTIVITY 2
BACTERIAL SMEAR PREPARATION

Before any staining or visualization of a bacterial sample can take place, a


good smear must first be made. Although not difficult, the preparation requires
adequate care. A properly prepared bacterial smear is one that withstands one or
more washings during staining without loss of organisms, is not too thick, and does
not result in excessive distortion due to cell shrinkage. The process of making a
smear for staining is an important skill in the laboratory, whether it is a public health,
clinical, or research laboratory.

To prepare a smear for staining, apply a small amount of sample to the center
of the microscope slide. Spread the sample evenly and thinly around the center of
the slide. A good quality stain is directly related to the quality of the smear. A well-
prepared smear will have a thin layer of cells to allow individual cells to respond to
the staining procedure.

By the end of this activity, you will be able to:

1. Discuss the precautions when making a smear; and


2. outline the steps for preparing a smear.

Precautions When Making a Smear

During the smear preparation process, there are some precautions you must take:

• Use clean, unscratched microscope slides.


• You will want to use a clean, unscratched
microscope slide because:
• Dirt, dust, and other debris can be
mistaken for microorganisms after the
staining process.
• Scratches on a slide can be confused
with microorganisms after the staining
process.
• Microorganisms can be washed off a
greasy slide during the staining
process.
• Grease or oil from the fingers on slides must be removed by washing
the slides with soap and water or scouring powders such as Bon Ami®,
followed by a water rinse and a rinse of 95% alcohol. After cleaning,
dry the slides and place them on laboratory towels until ready for use.
Note: Remember to hold the clean slides by their edges.

• Label the slides


• Proper labeling of the slide is essential. Write the organism's initials on
either end of the slide with glassware marking pencil on the surface on
which the smear is to be made. Ensure that the label does not come
into contact with staining reagents.

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Activity 2: Bacterial Smear Preparation

• Avoid making smears that are too thick.


• A smear that is too thick will result in
uneven staining and/or decolorization.
Too many cells on the slide will prevent
light from penetrating through the
smear, making interpretation very
difficult.

• Avoid making smears that are too thin.


• A smear that is too thin will not provide
enough cells for a thorough examination
of the specimen. With only a few cells
on the slide, searching for them can be
time-consuming.

Note: Smears require only a small amount of bacterial culture. A good smear is one
that, when dried, appears as a thin whitish layer or film. It allows newsprint to be
read through the smear. Different techniques are used depending on whether the
smear is made from a broth or solid-medium culture.

a. Broth cultures: Resuspend the culture by tapping the tube with your finger.
Depending on the size of the loop and the amount of culture growth, apply
one or two loopfuls to the center of the slide with a sterile inoculating loop and
spread evenly over an area about a dime's size. Set the smears on the
laboratory table and allow to air-dry.

b. Cultures from solid medium: Organisms cultured in a solid medium produce


thick, dense surface growth and are not amenable to direct transfer to the
glass slide. These cultures must be diluted by placing one or two loopfuls of
water on the center of the slide, in which the cells will be emulsified. Transfer
the cells using a sterile inoculating loop or a needle. Only the tip of the loop or
needle should touch the culture to prevent the transfer of too many cells. The
suspension is accomplished by spreading the cells in a circular motion in the
drop of water with the loop or needle. This helps to avoid cell clumping. The
finished smear should occupy an area about the size of a nickel and should
appear as a translucent or semitransparent, confluent whitish film. Allow the
smear to dry completely. Note: Do not blow on the slide or wave it in the air.

Steps to Making a Well-Prepared Smear

There are seven essential steps to making a well-prepared smear.

1. Wear appropriate personal protective equipment (PPE) to include gloves,


laboratory coat, and face and eye protection, as indicated in your laboratory's
SOP and safety manual.
2. Use a clean microscope slide, with a frosted edge.
3. Label the frosted edge of the slide, as indicated in your laboratory's SOP,
using a pencil or wax pencil.
Note: To prevent the sample identification from being washed away during the
staining process, do not use ink or marker.

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Activity 2: Bacterial Smear Preparation

4. Transfer specimen or culture to the center of the slide.


a. From a Broth Medium
Resuspend the sedimented cells in the broth culture by tapping the
culture tube with your finger. With a sterile loop, place one loopful of
culture on a slide. With a circular movement of the loop, spread the cell
suspension into an area approximately the dime size.
b. From a Solid Medium
Add one drop (10 µL) of sterile saline or sterile water to the center of
the microscope slide. Using a sterile loop or applicator stick, aseptically
pick a small amount of an isolated colony and gently mix it into the drop
of saline or water on the microscope slide, using circular motions. Mix
evenly to make a thin smear. Note: Mix carefully to minimize the
generation of aerosols.
5. Allow the smear to air dry completely.
Note: The thickness of the smear may affect the required drying time.
6. Fix the smear to the microscope slide. Note: Pass the air-dried slide in front of
the micro incinerator entrance or pass the slide through the outer portion of
the Bunsen flame to prevent overheating, which can distort the morphology
through the plasmolysis of the cell wall.

Methods for Fixing a Smear

There are two methods for fixing a smear, heat fixation and methanol fixation.

• Heat fixation should only be used for biosafety level 1 (BSL–1) organisms
because aerosols can be generated. Unless fixed on the glass slide, the
bacterial smear will wash away during the staining procedure. This is avoided
by heat fixation, during which the bacterial proteins are coagulated and fixed
to the glass surface. Heat fixation is performed by the rapid passage of the
air-dried smear two or three times over the flame of the Bunsen burner or in
front of a micro incinerator. While many texts discuss using a Bunsen burner
for sterilization and heat fixation, the American Society for Microbiology
(ASM)—one of the governing bodies that determines safe laboratory
procedures—changed the proscribed methods for heat fixation and benchtop
sterilization to utilize a micro incinerator instead of a Bunsen burner to reduce
the possibility of aerosolization of bacteria on the slide or loop.

Figure 2.1. Bacterial smear following fixation

• Methanol fixation is the preferred method, as it does not create aerosols and
has fewer cellular morphology changes.

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Activity 2: Bacterial Smear Preparation

Figure 2.2. Bacterial smear preparation

Important Reminders

• Allow the smear to air dry completely before fixing it with heat or methanol.
• Heat fixing before the smear has completely air-dried can cause
aerosolization and/or cell breakage, causing the cells to appear distorted after
staining.
• Methanol fixing the smear before it is completely dry can cause the specimen
to wash off the slide.
• Excessive heating can crack the microscope slide and/or rupture the cells of
the specimen.
• Fixing the smear kills the organisms and causes the cells to adhere to the
microscope slide, so they do not wash off during the staining process.

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Activity 2: Bacterial Smear Preparation

Take Note!

1. The bacterial smear should be heavy enough to leave a slight film but
not so heavy that you can plainly see the bacteria without a microscope.
Students sometimes err on the side of adding too much bacteria to a slide to
make sure there will be “enough” bacteria there for later visualization. This
has the potential to interfere with later staining procedures and produce false
results.

2. Heat fixing should warm the slide until it is hot to the touch but not to
the point of burning.
Overheating the slide during this step increases the potential for damaging the
cells. Damaged cells do not retain stains and produce inconclusive staining
results. Underheating of the slide does not allow the cells to affix to the glass.
Resulting washes or stains will rinse the bacteria off the glass, leaving few if
any bacteria present for later viewing.

References:

Cappucino, J. & Welsh, C. 2020. Microbiology: a laboratory manual, 12th ed.,


Pearson Education, Inc., New York

The Centers for Disease Control and Prevention, Division of Laboratory Systems.
(n.d.-b). CDC Train: Routine Microscopy Procedures: Microbiology
Curriculum. Www.Train.Org. Retrieved August 28, 2020, from
https://www.train.org/cdctrain/course/1046095/

Luis, LM.G. Page 5 of 5

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