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Various Methods of Staining

Ben Charles

21002693

Abtin Roostan and Emma Sabo

BIOL 240 Section 18

September 27, 2022

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Introduction

Staining is a procedure that is used with the goal of observing cells, whether it be through

highlighting a cell, highlighting parts of a cell, or highlighting the area surrounding a cell. The

procedure uses carbol fuchsin to solubilize the cell wall, while using heat to increase the

penetration of the stain. The experiments conducted seek to demonstrate the effectiveness of the

three selected staining methods, those being the gram stain, the spore stain, and the negative

stain.

The gram stain is considered to be the most widely used stain when observing bacterial

life. The gram stain is used to differentiate between gram-positive and gram-negative bacteria.

Gram positive bacteria possess a thick cell wall made up of many strands of peptidoglycan that

overlap. Gram negative bacteria possess a thin layer of peptidoglycan (Wessner et al., 2021).

Gram staining uses this key difference to distinguish between the two types of bacteria. First, a

primary stain is used on the organisms. Next, the mordant is applied so that the primary stain is

fixed within the cells. A decolorizing agent is then used to wash the stain. Finally, a counterstain

is applied to the sample. The gram-positive bacteria will not have the primary stain washed away

by the decolorizing agent, and so will retain their original color. The gram-negative bacteria

bacteria will be decolorized, and will then be stained by the counterstain. As a result, the two will

be distinguishable when observed.

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In harsh environmental conditions, certain bacteria condense their cellular components

into an endospore, a structure that is highly resistant to environmental influences. The spore stain

is a method of staining that is specialized for spores. Ordinary dyes are resisted by spores. The

stain is driven into the cell, typically using heat. Once the spore stain has been applied, a

counterstain can be applied to such that the remnants of the vegetative cells can be observed.

The negative stain is used in instances where it is preferable to not use heat-fixation, due

to the risk of distorting the cells. In addition, certain bacteria can be difficult to stain, in which

case the negative stain is more effective. The negative stain uses an acid dye nigrosin, in which

the chromophores are negatively charged. The dye is repelled by the outer surface of bacteria,

forming a deposit around the cell, resulting in clear cells with a colored background.

Materials and Methods

Please refer to Department of Biology, 2022, Biology 240L Fundamentals of Microbiology

Laboratory Manual, Experiment 4: The Gram Stain, pp. 42-44, Experiment 6:

The Spore Stain (Bartholomew and Mittwer Cold Stain Method), pp. 49-50, Experiment 7: The

Negative Stain, pp. 52

Results
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Figure 1: Microscope slide observation from a sample of Escherichia Coli at a magnification of

1000X.

Figure 2: Microscope slide observation from a sample of Staphylococcus epidermidis at a

magnification of 1000X.
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Figure 3: Microscope slide observation from a sample of Clostridium Sporogenes at a

magnification of 1000X.
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Figure 4: Microscope slide observation from a sample of Bacillus Brevis at a magnification of

1000X.
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Figure 5: Microscope slide observation from a sample of Bacillus Brevis at a magnification of

1000X

Discussion:

In the gram stain experiment, the results were as expected, in that the S. epidermidis cells

were observed to be purple, while the E. coli cells were observed to be pink. Due to the greater

number of peptidoglycan chains present in the cell wall of the gram-positive S. epidermidis cells

the primary crystal violet stain is not washed away by the 95% ethyl alcohol solution. The

thinner layer of peptidoglycan in the gram-negative E. coli cells results in the ethyl alcohol

solution washing the primary stain out of the cells. As a result, the E. coli cells are colorized by
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the Safranin counterstain, while the S. epidermidis cells maintain the color from the crystal violet

primary stain. The S. epidermidis appears purple, while the E. coli cells appear pink.

In the spore stain experiment, the primary stain, malachite green, was forced into the cell

using heat. The sample was then counterstained with safranin. When the slide was then observed,

the spores appeared green, while the vegetative cells appeared red. This suggests that the spores

absorbed the malachite green dye as a result of the heat fixing, whereas the safranin was only

absorbed by the vegetative cells as the proteinaceous outer layer of the endospore provides too

much resistance against the dye (Bellido et al., 2022).

In the negative stain experiment, acid dye nigrosin, a dye with negative chromophore

charges, was used for the stain. For the process, heat fixing was not used, as it was not necessary

with a negative stain, in addition to heat often distorting the membranes, DNA, RNA, and

proteins of the cells (Russel, 2003). This results in clear cells against a coloured background.

The gram stain is effective for its ability to differentiate between gram-positive and

gram-negative cells. The spore stain is effective for its ability to penetrate the outer layer of the

endospore. The negative stain is effective, as it does not use heat to potentially distort the cells

being observed.

Questions:

1. A simple stain would color all the cells on a slide with the same dye, whereas a

differential stain colors all the gram positive cells on a slide with one dye, and all the

gram negative cells on a slide with a different dye.

2. Young cultures must be used for gram staining, as cell walls in aged cultures may be

distorted, which would inhibit the gram-positive cells’ ability to ignore the ethyl alcohol

solution.
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3. Gram’s iodine fixes the primary stain in the bacterial cells, and so without this mordant,

the decolorizing agent would be effective on both the gram-positive and gram-negative

cells. As a result, all the cells on the slide would appear pink.

4. A simple stain uses a dye with positive chromophores, while a negative stain uses a dye

with negative chromophores. The dyes used in a simple stain are attracted to the negative

charges in bacterial cell envelopes, and so the dyes stain the cells. The dyes used in a

negative stain are repelled from the negative charges in the cell envelopes, resulting in the

cells remaining clear, while the surrounding area is colored by the dye.

5. Heat can often distort the size and shape of cells. The negative stain does not require heat

fixing, and so it is optimal to use this stain when determining the accurate size of a

microorganism is critical.

6. Heat fixing removes any unwanted organisms from the slide, in addition to fixing the

bacterial cells to the slide. Heat fixing too much can result in distorting the cell, or in

some cases, killing the cell. We overheat-fixed the spore stain because the outer layer of

the spore is much more resistant than a cell. As a result, the heat is necessary to allow the

dye to penetrate the cell.

7. The vital components of a vegetative cell are condensed into the endospore. By studying

the endospore of a cell, the characteristics of the cell can be understood, and so the

endospore of a cell can be compared to other endospores to find similarities and

differences between them

8. The endospore serves to carry the vital components of the cell in a secure manner,

protecting them from unfavorable environmental conditions. The samples used in the

experiment are 48 hours old to allow the endospore time to form.

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References:

Wessner et al., (2021). Microbiology. Wiley

Russel A. D., (2003). Lethal effects of heat on bacterial physiology and structure. Sage Journals

Calvo et al., (2022). General Characteristics of Bacteria. Encyclopedia of Infection and

Immunity