BIO 310, Lab Section
Professor Gonzalez-Pedrosa
March 1, 2018
Title of Report: An Introductory Exposure to Differential Staining: Acid-Fast Staining
Introduction/Content
The purpose of acid-fast staining is to properly distinguish and classify species of bacteria into acid-fast
or non-acid fast categories. Acid-fast organisms contain mycolic acid within its cell walls, which is a lipid-
based waxy material that retains carbolfuchsin (the primary stain) and is resistant to the acid-alcohol
solution that is used during the decolonization phase. These retention and resistance abilities allow the
cells to be stained red (or a similar shade), allowing viewers to determine that the cells are, in fact,
belonging to an acid-fast organism and contain mycolic acid. Non-acid fast organisms, on the other hand,
will stain blue, as the cells do not contain mycolic acid and the cells only retain the color of the
counterstain (methylene blue).1
Acid-fast organisms can also be classified as a weak Gram-positive. 1 In this case, the color of the stained
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cells are not the indicator, as acid-fast organisms will be red. This is because of the processes in which
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the cellular makeup retain or do not retain stains. In Gram-positive cells, the primary stain (crystal violet)
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will adhere to the exposed layer of peptidoglycan and stain the cells red, and in acid-fast cells, the
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primary stain (carbolfushsin) will be retained by the mycolic acid and stained blue.
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There are a number of staining methods within acid-fast staining; however, the two discussed in this
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experiment are the Ziehl-Neelson (ZN), or steaming method, and Kinyoun (K) methods, or cold method.
The ZN method differs slightly from the K method in that the smears, after both being heat-fixed, are put
through a steaming process. The slide is placed on a metal mesh over a beaker of boiling water, as a
means to potentially break down the lipids of the mycolic acid. The K method, which is used during this
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experiment, will not include this steaming process.
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Materials
The following materials were utilized during this experiment:
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Compound light microscope
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Bacteria samples in agar: Mycobacterium phlei and Staphylococcus aureus
New and clean glass slides
Inoculating loop
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Marking pencil
Burner light, Bunsen burner, and tubing
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Test tube clamp
Distilled water
Stains: carbolfuchsin and methylene blue
Acid alcohol (95% ethanol + 3% HCI)
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Immersion oil and cleaning spray
Staining tray and metal slide holder
Bibulous paper book
Kimwipes
Cell phone stopwatch
Methods/Procedure
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The following procedures outline the steps taken during this experiment:
1. Obtain all additional materials required for the experiment that are not found at the lab station.
This will include: a microscope, clean slides, bibulous paper, and Kimwipes.
2. Prepare a smear of each bacteria by: labeling the slide appropriately using the marking pencil;
applying a drop of water in two places (where the intended smears will be); obtaining the
bacterial samples using antiseptic techniques; and allowing each smear to air dry before heat
fixing the slide.
3. For this experiment, we will be following the Kinyoun (cold; non-steaming) method. Once the
smears are prepared, place the slide on the slide holder of the staining tray, and cover the
smears with the primary stain, carbolfuschsin. Set a timer and allow the stain to sit for 15
minutes.
4. After 15 minutes, hold the slide with a test tube holder at a 45 degree angle and rinse the slide
with distilled water for roughly 30 seconds.
5. Continue to hold the slide at this angle, and rinse the slide with the acid-alcohol solution. Rinse
untill the runoff is completely clear. This will take about 30-45 seconds.
6. Continue holding the slide, and rinse again with distilled water to remove any leftover acid-
alcohol solution.
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7. Rest the slide in the staining tray on the slide holder, and apply the counterstain, methylene
blue, to each smear. Keep this stain on for 2 minutes.
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8. After 2 minutes, hold the slide at a 45 degree angle and rinse the slide for a final time with
distilled water.
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9. Using the bibulous paper book, place the slide inside the pages and blot completely dry.
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10. Once the slide is dry, place the slide on the stage and focus on the smears using the 100x
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objective lens. Apply 1-2 drops of the immersion oil to each smears to allow for optimal focus
and light capture.
11. Once the slide is in focus, attempt to photograph the samples, sketch the shape and
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arrangement, and determine what smear is acid-fast or non-acid fast. Dispose of all slides in a
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sharps container at the end of the experiment.
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Results
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For this experiment, slides were prepared with a smear from each bacteria samples on the same slide.
Photographs and the respective drawing are below. Mycobacterium phlei are rod-like in shape, whereas
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the Staphylococcus aureus sample are cluster (essentially coccus) in shape.
It is also important to note that the Mycobacterium sample that was used in lab was Mycobacterium
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phlei and not Mycobacterium smegmatis, which is listed on the supplemental lab questions handout.
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Mycobacterium phlei Photograph and Drawing
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Staphylococcus aureus Photograph and Drawing
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Conclusions/Discussion
When preparing the smears, it was tangibly noticed that the Mycobacterium phlei sample was stickier in
texture, compared to the S. aureus sample. The stickiness caused the smear process to be slightly more
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difficult when trying to create a homogenous mixture.
Referring to the pictures and drawings above, it can be determined that the Staphylococcus aureus
smear was properly stained. These cells are blue and considered non-acid fast due to inability to retain
the primary stain after the decolorization phase. Considering the Mycobacterium phlei, these cells are
the acid-fast organisms. Once under 100x magnification, it was difficult to find a sample that had
appropriately retained the primary stain. As the smear was being created, there was some difficulty
distributing the bacteria evenly with the distilled water, which left the smear with a textured surface.
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Due to this, the majority of the smear was rinsed away during the decolorization process and the
staining process was not uniformly successful.
Questions
1. The major chemical (structural) differences among bacteria that may explain the distinction
between acid-fast and non-acid fast is the quantity of mycolic acids found in the cell walls of
acid-fast bacteria.1 Mycolic acid is essentially a waxy (or lipid) substance that offers acid-fast
organisms under layer of protection. Due to this, acid-fast organisms will be more likely to retain
the primary stain (carbolfuchsin) and be resistant to the decolorization phase. The end result will
be red stained cells.
2. Acid-fast staining is likely not used as widely compared to Gram-staining due to the fact that only
a limited amount of bacteria contain mycolic acid. It would be particularly useful to utilize the
acid-fast techniques over the Gram staining method when acid-fast organisms are assumed, or if
an organism from the Mycobacterium genus is specifically provided.
3. Heating the bacterial smear during the ZN stain promotes entry of carbolfuchsin into the acid-
fast cell wall by creating a chemical reaction (heat + stain) that breaks down the lipid compound
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in the mycolic acid and allows the stain to enter the cell walls of the organism. 1
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4. The K acid fast stain procedure is referred to as the cold method because it does not involve a
steaming process. Unlike the ZN methods, the K methods involves adding the primary stain
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directly to the bacterial smears on the slide, rather than applying a piece of bibulous paper, the
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primary stain, and steam.
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References/Literature Cited
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1. Leboffe, Michael J, Pierce, Burton. Microbiology : Laboratory Theory and Application, Brief. 3 rd Edition.
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Morton Publishing Company. 2016.
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