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The Staining Method(S) Tricia Halim

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The purpose of these experiments is to view bacterial cells under a microscope by staining the cells and accommodating their characteristics with the stains used as different bacteria cells have different characteristics, depending on the environment in which they are exposed in. Experiment 4 covers the process of Gram stain with the use of two organisms which are Staphylococcus Epidermis (Se) and Escherichia coli (Ec). Gram stain is a differential staining technique as it is used to differentiate types of bacteria, depending on their ability to maintain a specific stain. The bacteria are divided into two groups, which are the Gram-positive bacteria and the Gram-negative bacteria. We can differentiate the two bacteria through the different stain color in the final product.

The bacteria are first stained with crystal violet and followed with iodine which helps bacteria to retain the crystal violet, at this stage all bacterial cells are purple. When alcohol rinsing takes place, all gram-negative cells turn clear in color due to having thin peptidoglycan walls, where gram positive cells will remain purple. Safranin is then added to counterstain the bacteria, turning the gram-negative bacteria pink and Gram-positive purple, this distinction of color can be seen when the bacterial cells are observed under a microscope. Experiment 5 uses Carbolfuschin stain, which like gram stain, is a differential stain. In this experiment however, the two organisms under investigation is the organism Bacillus megaterium (Bm) and Mycobacterium Tuberculosis (Mt).

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Although Carbolfuschin is a differential stain like grams’, it is used for bacteria that does not readily stain with simple stains due to their waxy cell walls that contain huge amounts of lipoidal material (mycolic acid) that prevent simple stains to penetrate. Carbolfuchsin is used as it has a high affinity for mycolic acids found in these organism’s cell membranes. The bacterial smear is also heated during the experiment to help in the infiltration of the stain. Like experiment 4, alcohol is also used for decolouration, and cells that are not acid-fast (those that does not have wax-like cell walls) will turn clear where the acids that are acid-fast will appear red under a microscope. The smear is further counterstained with methylene blue to color in the non-acid fast bacteria blue. Lastly, Experiment 6 is the Spore stain or is also known as the (Schaeffer-fulton method). In this method, the organisms under scrutiny are Bacillus Cereus (Bc) and Clostridium Sporogenes (Cs), they. The purpose of this experiment is to visualize bacterial endospores under the microscope. Endospores are formed from vegetative bacteria that are exposed to prolong hours of hostile conditions which sporulation occurs, creating thick and impermeable walls for these cells, allowing them to withstand heat, radiation chemicals and desiccation. Since endospores resist traditional stains (methylene blue, crystal violet and carbolfuschin), malachite green is used with the help of heat to stain. Once malachite green successfully penetrates the cell, it is then counterstained with safranin to give the color difference to cells that are in vegetative phase and to endospores. Under the microscope, endospores will be green in color where vegetative cells will be red in color.

This is under 1000x magnification. Figure 2 (experiment 4)Figure 2 above, is Escherichia coli (Ec), it has a rod cell shape and it is stained pink which indicates it’s a Gram-negative cell. This is under 1000x magnification. Figure 3 (experiment 5)Figure 3 above, is Mycobacterium Tuberculosis, it has a rod cell shape and it has a red color showing that its an acid-fast cell. This is under 1000x magnification. Figure 4 (experiment 5)Figure 3 above, is Bascillus Megaterium, it has a rod cell shape and it has a blue color showing that it’s a non-acid-fast cell. This is under 1000x magnification. Figure 5 (experiment 6)Figure 3 above, is Bascillus Cereus (Bc), it has a rod cell shape and it is mostly in reddish pink color with spots of greens showing that it id mostly a vegetative cell with some endospores. This is under 1000x magnification.


Experiment 4 focuses on a staining method called the Gram stain which was discovered by Hans Christian Joachim Gram in 1884 who is a Danish Physician and scientist (“Gram Stain Technique”, n. d. ). The gram stain is a differential stain in which it divides bacteria into two groups which is the gram positives and the gram negatives. Gram stain is very important in the field of microbiology in ways that is it the preliminary step in the first classification and characterization of bacteria through light microscope (“Gram Stain Technique”, n. d. ).

Furthermore, today, Gram stain is known to be used as a crucial step during the screening of infectious agents in specimens, for example, by getting direct smears from patients suffering a disease (“Gram Stain Technique”, n. d. ). The stains on the bacteria after staining with the gram stain is based on the cell’s peptidoglycan layer thickness in their cell membrane (Bruckner, n. d. ). The peptidoglycan is composed of mostly the polysaccharide N-acetyl glucosamine and N-acetyl muramic acid, the peptidoglycan layers forms a cross link of peptides with the help of transpeptidase enzyme which results to the thickness and strength of their cell wall (“Gram Stain Technique”, n. d. ). Cells that has a thicker peptidoglycan layer will be keep the crystal violet process even after the decolorization with alcohol. Where on the other hand, cells that has a thinner peptidoglycan will lose its purplish color from the crystal violet when decolorized by alcohol but will turn pink once it is counterstained by safranin to allow for an ease of viewing under the microscope. A key factor to consider when doing gram stains is the age of the culture being used. The most ideal condition is when a fresh culture is used to do the staining, as the older the culture is, the culture’s ability to retain the primary stain deteriorates and as a result Gram positive organism will appear to be like a Gram-negative organism and a Gram-negative organism will appear to Gram-variable (“The Gram-Stain Tutorial”, n. d. ). At the end of the experiment the results of the stain under the view of a light microscope shows that, Staphylococcus epidermis (Se) is a gram-positive organism (purple in color) and the Escherichia coli is a gram-negative organism (pink in color) due to (Se) having a thicker peptidoglycan layer.

Experiment 5 covers the method of Acid-fast stain or also known as the Kinyoun method. This method was discovered by Ziehl and then was modified by Neelsen and hence it is also called the Ziehl-Neelsen staining method. The acid-fast stain, like the gram stain is a differential stain, however, it is used to distinguish between bacteria that are non-acid fast and those that are acid-fast—acid-fast bacteria do not readily stain when the gram or simple stain method was applied. This is because these organisms have waxy cell walls, especially organisms being part of the genus Mycobacterium (“Acid Fast Stain”, n. d). The waxy cell walls are close to being impermeable as they contain huge amounts of fatty acids, mycolic acid and lipids (“Acid Fast Stain”, n. d). Due to this layer in the cell wall, the Acid-fast stain method uses Carbol Fuschin instead of Carbon fuschin and iodine. Carbon fuschin has phenol that solubilizes the lipids in the cell wall, heat is also applied to help for further penetration into the cell wall (Aryal, 2018). An acid alcohol is also used to decolorize the cells, the cells that are not acid-fast will turn clear and will have to be counterstained with Methylene blue and will be seen as blue under light microscope. On the contrary, the cells that are acid-fast will stay red in color due to the Carbol fuschin. The cells that are acid-fast will not be affected by the decolorization using alcohol, this is because of their lipoidal material inside their cell wall that prevents alcohol from penetrating into the cell (Aryal, 2018). The acid-fast method plays the main role in the discovery of the disease tuberculosis that comes from the mycobacterial Mycobacterium Leprae and until today, acid-fast stain is still used as a tool by pathologists to identify disease causing organisms (Parry, n. d. ). During the lab, the results of the experiment was exactly like what was expected. Mycobacterium tuberculosis can be seen as rod and appear to be red in color, showing that it is an acid-fast bacterium, where on the other hand, Bascillus megaterium can also be seen as rods but is blue in color as it is not an acid-fast bacteria.

Lastly, experiment 6 discusses the Spore stain (Schaeffer-Fulton method) and like the two experiments above, it is also a differential stain that stains endospores. Bacillus are the genera of bacteria that forms endospores and by forming endospores bacteria can survive even in extreme conditions. When bacteria like Bascillus and Clostridium are exposed in environments of high stress, specifically when there is a lack of nutrient for them to grow, they tend to become dormant as a survival mechanism. Since Gram stain or the simple stain wouldn’t work on these bacteria due to their protein coats made from keratin, a different staining technique using malachite green is used with the help of heating (“Endospore Stain”, n. d. ). No alcohol is used in this method, but instead water was used to decolorize the dye malachite green from the cell wall of vegetative cells (cells that are still actively growing) but the dye will stay in dormant cells/endospores (Tankeswar, 2015). A counterstain of gram’s safranin is then used to color in the vegetative cell to be red and the spores remain green. This result is expected in the experiment, where both Bascillus cereus and Clostridius Sporogenes will have spores in them and hence both red and green will be present when seen under the light microscope. Today, spore-stain plays an important role in clinical biology where it is used to analyze body tissues or fluids of patients for spores, especially Bascillus and Clostridium which causes diseases such as tetanus and anthrax (Reynolds et al, 2009).

During the experiments, errors could occur quite easily in which would result to not being able to see anything under the light microscope. Errors from heat fixing can cause the cells morphology to change and causes the cell to decolorize more readily (Sutton, 2009). Another possible error would be the excessive washing which could wash of the dyes off the cells. The last possible cause of error in this lab would be excessive counterstaining, it could possibly replace the crystal violet during the gram stain and hence causes the gram-positive cells to appear gram-negative, and hence the counterstain can not be left on for longer than 30 seconds (Sutton, 2009).

Study Questions to be Included with Report 1: (each is worth 2 marks)

  1. Discuss two differences between a simple and differential stain such as the Gram stain? Gram stain uses two different stains, one to stain and another to counterstain, it allows for the distinguishing between 2 different types bacteria based on the elements of their cell wall. Simple stain on the other hand only uses 1 type of stain, it colors the organism in one color and won’t show the different parts of the cell nor the types of the cells present.
  2. You will notice for Experiment 4, a 12-18 h culture was used. Explain why a young culture must be used for the Gram staining technique and possible outcome if the culture is too old? Young cultures must be used as its cell walls is still very strong, and so that the crystal violet can stick to its cell walls, since in the old cultures, the cell walls break down causing the staining process to be inaccurate. When older cultures are used, the gram-positive organism can appear to be gram-negative or gram-variable where the gram-negative may appear as gram-variable, this causes inaccuracies.
  3. A student completed the Gram stain technique but neglected to use the Gram’s iodine. Discussing the function of Gram’s iodine describe the final appearance or color of the cells. Gram’s iodine acts as an agent/mordant that fixates the crystal violet unto the cell wall of the bacteria. Without Gram’s iodine, when the cells are decolorized with alcohol, all the crystal violet will run off both the gram-positive and negative cells, so all the cells will turn clear. And when the counterstain is added (safranin) all the cells will be pink in color, making it impossible for us to distinguish the different cells.
  4. What chemical (or property) is responsible for the acid-fast property of Mycobacteria? Discuss how this property relates to the acid-fast procedure with specific reference to heating, and staining. How do you think the acid-fast nature contributes to it virulence? The property responsible for the acid-fast property of Mycobacteria are the waxy cell walls that contain huge amounts of lipoidal material (mycolic acids). The mycolic acids make the mycobacteria more resistant to dehydration and chemical damage. Carbolfushcsin is used as a stain as it contains phenol that solubilizes the cell wall and heat helps in penetration of the cell wall. Carbonfuschsin is also more soluble in the cell wall lipids than in the acid alcohol, so that when decolorization occurs using the acid alcohol, the carbolfuschin that penetrates through the cell wall will stay and stain the cells.
  5. Name two pathogenic acid-fast bacteria and the diseases they cause. Mycobacterium leprae which causes leprosy which affects the skin, periphery and mucosal surfaces in organisms another acid-fast bacteria is Mycobacterium tuberculosis which causes tuberculosis which is a disease that affects the lungs and is highly infectious.
  6. All four techniques for staining required that you heat-fixed the slide prior to staining. Give two reasons for heat-fixation. What would be the outcome if you heat-fixed too much? It allows the bacteria to be “fixed” to the slide as it coagulates the bacterial proteins. It is also a way to kill the bacteria without destroying it. And it also denatures the bacterial enzyme in prevention for them to digest the cell parts. Too much exposure to heat fixing will lead to the distortion of the cells and hence the cell won’t be of use for viewing under the microscope.
  7. Explain how endospores within the vegetative cells can be used to help differentiate a species of microorganism? Not all bacteria, in fact, most types of bacteria cannot change into the form of an endospore. Endospores also contain dipicolinic acid and it is a spore-specific chemical that helps in maintaining the dormancy of endospores. When seen under light microscopy it is also hard to detect the endospores due to its impermeability of its walls, leaving endospores colorless unless a specific stain is used and the other cells that are not endospores to be colored.
  8. What is the function of an endospore and how does this relate to the age of the culture you used in the lab? The endospore is tough, dormant and non-reproductive with its main function to ensure the survival of the bacterium in hostile conditions. The older the culture is, the more endospores there is and the younger the culture the more vegetative cells present, the reason being that in older cultures, the nutrients becomes limited and the cells can’t grow anymore as it lacks nutrients, hence it turns into an endospore for survival.
  9. What is a typical component of a capsule and what role do capsules play in the disease forming process? The capsule can be considered as a virulence factor as it contributes to the enhancement of a bacteria to cause disease. This is because the capsule prevents phagocytosis from happening. It also protects the bacteria against desiccation as it contains water.


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