Miramar College

Biology 205 Microbiology

Lab Exercise 6: The Smear and Simple Staining

Background
Smears
The preparation of a good smear is the backbone of all of the staining techniques you will master throughout this
course. A good smear consists of three major things:
1. Heat fixing/adhering the cells to the slide so that they are not washed off during subsequent staining
procedures.
2. Heat fixing gently so as not to cause distortion and artifacts to the slide.
3. Preparing a thin smear, because the thickness will determine whether or not you can visualize individual cells,
their arrangement or details regarding Gram reaction or internal structure.
Simple staining
The use of a single stain or dye to color a bacterium is called a simple stain (Figure 1). These types of dyes, called basic
or positive dyes, are positively charged, containing cationic chromophores. Because all cells are negatively charged
(-70mV), there is an attraction between the positive dye and the negative cell. Some common basic dyes used in
staining are methylene blue, crystal violet and basic fuchsin. However, not all dyes are positively charged. Acidic or
negative dyes are negatively charged, containing anionic chromophores, and do not stain bacterial cells. They are
usually used for negative staining protocols and you will get experience with this in future labs. Common acidic dyes
are nigrosin, India ink and eosin.

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Figure 1: Simple stain of Staphylococcus aureus using the basic/positive dye Methylene Blue. Note the staphylococcal
morphology and arrangement of the cells.
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Staining microbial cells is important because without stain, most bacterial cells are extremely difficult to see. Staining
allows you to visualize them and make observations as to their morphology (i.e. individual cell shape) and arrangement
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(i.e. how the cells adhere to one another). Bacteria are morphologically categorized as either cocci (singular: coccus)
when they are round; bacilli (singular: bacillus) when they are rod-shaped; or spirilla (singular: spirillum) if they are
spiral-shaped. Although there are other bacterial morphologies, including the characteristic corkscrew-shaped
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spirochetes, the three mentioned above are by far the most common. As such, you should be able to identify, describe
and correctly name cocci, bacilli and spirilla. In addition, bacteria are characterized based on the arrangement of cells.
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Cells that are arranged in pairs are described with the prefix diplo-, cells that are arranged in chains are strepto- and
cells arranged in clusters are staphylo- in arrangement. It is important to note that morphology and arrangement are
determined microscopically and must be identified in areas of your field of view where the cells are not crowded
together because of a too thick smear. Overcrowding should not be confused with true staphylo- arrangements.
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You’ll learn in lecture that these arrangements are the result of bacterial reproduction or binary fission. Because fission
of the cells is sometimes incomplete, bacterial cells may be physically attached to one another- causing the
aforementioned bacterial arrangements. Tetrad and staphylo- arrangements of cells result from binary fission along
two planes, and are only possible when cocci reproduce. For this reason, you will never see bacilli or spirilla arranged in
tetrads or clusters.

Introduction
Today, you will stain an environmental sample from Lab Exercise 4, as well as an avirulent strain of Corynebacterium
diphtheriae, the etiologic agent of Diphtheria. You will want to notice the following features of C. diphtheriae:

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 • Pleomorphism: This refers to the irregular shapes of C. diphtheriae. It is usually a bacillus, but may be club-
shaped, needle-shaped or sperm-like in its appearance.
• Palisade arrangement: This refers to the parallel arrangement of bacilli. It is sometimes referred to as the
“picket fence” arrangement and is common in the Corynebacterium genus.
Make sure to also describe your bacterial samples in terms of their morphology and arrangement.

Objectives
1. Prepare bacterial smears for the microscopic visualization of bacteria.
2. Perform a simple staining procedure.
3. Compare the shapes and arrangements of bacterial cells.

Smear Protocol
Table supplies Individual supplies
Solid culture of Corynebacterium diphtheriae Body broth cultures from Lab 4
Glass microscope slides
Inoculating loop
1. Prepare your lab bench by removing extraneous items and cleaning the surface with table disinfectant.
2. Wipe a microscope slide clean with a paper towel or kimwipe.
3. Using a permanent marker, draw a circle about the size of a dime on the bottom of the slide.
4. Transfer culture to the TOP of the slide, within the circle drawn in step 3.

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5. Transfer culture to top of slide:
From the solid culture:

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1. Place one drop of water from your loop on the center of the circle

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2. Transfer a small amount of solid inoculum into the drop of water and disperse thoroughly.

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3. Spread both into a thin area about the size of a dime.
4. Allow smear to air dry.
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From the liquid culture:
1. Place one to two loopfuls of liquid suspension on the center of the circle.
2. Spread the suspension into a thin area about the size of a dime.
3. Allow smear to air dry.
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6. When the smear is completely dry, heat-fix by quickly passing the smear over the flame two to three times.
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7. You should make at least one smear from a solid inoculum and one smear from a liquid inoculum.
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Simple Stain Protocol

Individual supplies
Heat-fixed slides (see protocol above)
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Methylene blue dye

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Staining tray
Water bottle
Bibulous paper
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1. As diagrammed in Figure 2, place your slide on the staining tray and cover the smear with methylene blue dye for 1
minute.
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Figure 2: Diagram of placement and staining of microscope slide in staining tray.

2. Gently wash the smear by flooding it with water to remove excess stain.
3. Using bibulous paper, as diagrammed in Figure 3, blot dry but do not wipe the slide.

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 Figure 3: Blotting the stained microscope slide with bibulous paper.
4. Once the slides are dry, examine them microscopically.

Data Collection and Analysis

1. Draw observations of cells from your body broth/environmental sample and of C. diphtheriae, taking care to
indicate total magnification. Make sure to properly describe the morphology and arrangement of the cells you
observe.

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DISCUSSION
1. How does smear preparation of cells from a liquid medium differ from preparation of cells from a solid medium?

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2. Why is it important to limit the quantity of cells used to prepare a smear?

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3. For preparation of a smear on a slide, what is the purpose of heat fixation? What problems can arise when the slide

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is heated in a flame?

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4. What causes a stain to adhere to bacterial cells? Why are all colored dyes not necessarily useful for simple staining?
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