Endospore Staining Lab Report
Endospore and Capsule Cell Staining
Allison Lui
Partner: Mary Chagin
BIOL-235 W07
Dr. Runco
2/10/15
Introduction
The purpose of this lab is to learn what endospore and capsules are, and how to identify them under a microscope using capsule and endospore staining methods.
Capsules are found only in select bacteria, and serve a protective purpose. Made out of sugars and proteins, they are antiphagocytic, which prevents other cells to engulf the bacteria through phagocytosis. It helps the bacterial cell to adhere to host cells, and can help in the formation of biofilm. For the capsule staining, the bacteria Klebsiella Pneumoniae was utilized. It is a facultative anaerobe, meaning it can survive with or without oxygen present. …show more content…
Its known for its capsule, made out of polysaccharides and proteins. It surrounds the entire bacteria, making treatment an infected patient difficult. Capsule staining is a combination of negative staining and simple staining. In the negative staining, the nigrosin is used to darken the background. This is a result of the negatively charged dye and negatively charged bacteria. The opposite charges force the glass of the slide to be stained, but the bacteria and its capsule to be colorless. In the simple staining, crystal violet is used. The highly resistant capsule will resist the stain of the dye, resulting in a clear halo around the cells that have capsules.
The only role of an endospore is to help the bacteria survive, and its production is stimulated when conditions are unfavorable.
It has two basic cycles: vegetative and sporulation. In the vegetative cycle, a vegetative cell called a sporangium exists. An endospore forms within the cell. When it is fully formed, the endospore lyses out of the sporangium. In the sporulation cycle, the endospore exists. It can withstand extreme temperatures, chemicals, and radiation for thousands of years. Once conditions are favorable again, the endospore germinates and the cycle repeats. In the endospore staining, the bacteria Staphylococcus aureus was used. Due to its hardy nature, heat is used to impart the malachite green stain into both the endospore and vegetative cells. In this lab, the Schaeffer-Fulton method is used, which uses a steam bath to heat the stain. Water is then used to decolorize the vegetative cell, but not the endopore. Safranin is used as a counterstain to give the vegetative cells a pink color, so both are visible under the …show more content…
microscope.
The hypothesis for the capsule staining is that the bacterial cells that are surrounded by capsules will have a clear halo around them, while the endospore staining will cause the endospores to have a green color. This will be possible only if done properly, and viewed under a microscope. Materials
Capsule Staining
Nigrosin
Agar culture of Klebsiella Pneumoniae
Microscope slides (2)
Bunsen burner
Striker
Crystal violet
Water
Inoculating loop & needle
Bibulous paper
Microscope
Endospore Staining
Nutrient agar culture of Staphylococcus aureus
Inoculating loop & needle
Microscope slide
Bunsen burner
Striker
Endospore staining kit
Malachite green
Safranin
Water
Glass beaker
Iron ring
Wire mesh
Bibulous paper
Microscope
Methods
Capsule Staining A clean microscope slide was obtained.
A drop of nigrosin was added to the near end of the slide. A small drop of K. pneumoniae was added next to, but not touching, the nigrosin. The inoculating loop was used to spread the K. pneumoniae into the nigrosin. Any clumps that were in the mixture were broken up using the loop. A second microscope slide was obtained, and held at an angle against the first horizontal slide. The second slide was pushed backwards towards the mixture of the nigrosin and K. pneumoniae until it made contact. The liquid was then dragged towards the opposite end of the slide, spreading the mixture along the bottom slide. The slide was allowed to air dry. Once dried, it was heat fixed by quickly passing it over the bunsen burner flame three times. The slide was then flooded with crystal violet, and let stain for one minute. It was then washed with water and blotted dry with bibulous paper. Finally, it was observed under the microscope. The stained specimen was located under the lowest objective by using the coarse adjustment knob. While keeping the specimen centered and in focus using the fine adjustment knob, the objectives were increased in power until the oil immersion objective was reached. A drop of oil was added to the slide before viewing under the oil immersion
objective.
Endospore Staining A clean microscope slide was obtained. Two loopfuls of S. aureus were placed onto the slide. It was allowed to air dry, then it was heat fixed by quickly passing it over the bunsen burner flame three times. Malachite green was saturated onto the bacteria. Meanwhile, a steam bath was prepared. A beakerful of water was placed on top of a wire mesh, iron ring, and a bunsen burner. Once the water began to boil, the microscope slide rack with the slide on top was placed onto the beaker. If the slide began to dry out, additional malachite green was added to keep the smear wet. The slide was steamed for five minutes. The slide was then allowed to cool slightly so it would not crack. It was then counterstained with safranin for thirty seconds, rinsed off with water, and blotted dry with bibulous paper. Finally, it was observed under the microscope. The stained specimen was located under the lowest objective by using the coarse adjustment knob. While keeping the specimen centered and in focus using the fine adjustment knob, the objectives were increased in power until the oil immersion objective was reached. A drop of oil was added to the slide before viewing under the oil immersion objective.
Results
Capsule staining of K. pneumoniae
Endospore staining of S. aureus
Discussion & Conclusion
This lab used capsule and endospore staining to identify each under the microscope. Capsules, which surround the bacterial cell, are used to protect against phagocytosis, to help stick to host cells, and in the formation of biofilm. In capsule staining, a mixture of negative and simple staining is utilized to produce a clear halo around a capsule when seen under the microscope. An endospore is a protective covering that is used to help bacteria withstand extreme temperature, chemicals, and radiation. It alternates between the vegetative and sporulation life cycle. In endospore staining, the Schaeffer-Fulton method is used to impart a green color to endospores, and safranin is used to impart a pink color to vegetative cells, which can be seen when viewed under the microscope.
Allison Lui
Partner: Mary Chagin
BIOL-235 W07
Dr. Runco
2/10/15
Introduction
The purpose of this lab is to learn what endospore and capsules are, and how to identify them under a microscope using capsule and endospore staining methods.
Capsules are found only in select bacteria, and serve a protective purpose. Made out of sugars and proteins, they are antiphagocytic, which prevents other cells to engulf the bacteria through phagocytosis. It helps the bacterial cell to adhere to host cells, and can help in the formation of biofilm. For the capsule staining, the bacteria Klebsiella Pneumoniae was utilized. It is a facultative anaerobe, meaning it can survive with or without oxygen present. …show more content…
Its known for its capsule, made out of polysaccharides and proteins. It surrounds the entire bacteria, making treatment an infected patient difficult. Capsule staining is a combination of negative staining and simple staining. In the negative staining, the nigrosin is used to darken the background. This is a result of the negatively charged dye and negatively charged bacteria. The opposite charges force the glass of the slide to be stained, but the bacteria and its capsule to be colorless. In the simple staining, crystal violet is used. The highly resistant capsule will resist the stain of the dye, resulting in a clear halo around the cells that have capsules.
The only role of an endospore is to help the bacteria survive, and its production is stimulated when conditions are unfavorable.
It has two basic cycles: vegetative and sporulation. In the vegetative cycle, a vegetative cell called a sporangium exists. An endospore forms within the cell. When it is fully formed, the endospore lyses out of the sporangium. In the sporulation cycle, the endospore exists. It can withstand extreme temperatures, chemicals, and radiation for thousands of years. Once conditions are favorable again, the endospore germinates and the cycle repeats. In the endospore staining, the bacteria Staphylococcus aureus was used. Due to its hardy nature, heat is used to impart the malachite green stain into both the endospore and vegetative cells. In this lab, the Schaeffer-Fulton method is used, which uses a steam bath to heat the stain. Water is then used to decolorize the vegetative cell, but not the endopore. Safranin is used as a counterstain to give the vegetative cells a pink color, so both are visible under the …show more content…
microscope.
The hypothesis for the capsule staining is that the bacterial cells that are surrounded by capsules will have a clear halo around them, while the endospore staining will cause the endospores to have a green color. This will be possible only if done properly, and viewed under a microscope. Materials
Capsule Staining
Nigrosin
Agar culture of Klebsiella Pneumoniae
Microscope slides (2)
Bunsen burner
Striker
Crystal violet
Water
Inoculating loop & needle
Bibulous paper
Microscope
Endospore Staining
Nutrient agar culture of Staphylococcus aureus
Inoculating loop & needle
Microscope slide
Bunsen burner
Striker
Endospore staining kit
Malachite green
Safranin
Water
Glass beaker
Iron ring
Wire mesh
Bibulous paper
Microscope
Methods
Capsule Staining A clean microscope slide was obtained.
A drop of nigrosin was added to the near end of the slide. A small drop of K. pneumoniae was added next to, but not touching, the nigrosin. The inoculating loop was used to spread the K. pneumoniae into the nigrosin. Any clumps that were in the mixture were broken up using the loop. A second microscope slide was obtained, and held at an angle against the first horizontal slide. The second slide was pushed backwards towards the mixture of the nigrosin and K. pneumoniae until it made contact. The liquid was then dragged towards the opposite end of the slide, spreading the mixture along the bottom slide. The slide was allowed to air dry. Once dried, it was heat fixed by quickly passing it over the bunsen burner flame three times. The slide was then flooded with crystal violet, and let stain for one minute. It was then washed with water and blotted dry with bibulous paper. Finally, it was observed under the microscope. The stained specimen was located under the lowest objective by using the coarse adjustment knob. While keeping the specimen centered and in focus using the fine adjustment knob, the objectives were increased in power until the oil immersion objective was reached. A drop of oil was added to the slide before viewing under the oil immersion
objective.
Endospore Staining A clean microscope slide was obtained. Two loopfuls of S. aureus were placed onto the slide. It was allowed to air dry, then it was heat fixed by quickly passing it over the bunsen burner flame three times. Malachite green was saturated onto the bacteria. Meanwhile, a steam bath was prepared. A beakerful of water was placed on top of a wire mesh, iron ring, and a bunsen burner. Once the water began to boil, the microscope slide rack with the slide on top was placed onto the beaker. If the slide began to dry out, additional malachite green was added to keep the smear wet. The slide was steamed for five minutes. The slide was then allowed to cool slightly so it would not crack. It was then counterstained with safranin for thirty seconds, rinsed off with water, and blotted dry with bibulous paper. Finally, it was observed under the microscope. The stained specimen was located under the lowest objective by using the coarse adjustment knob. While keeping the specimen centered and in focus using the fine adjustment knob, the objectives were increased in power until the oil immersion objective was reached. A drop of oil was added to the slide before viewing under the oil immersion objective.
Results
Capsule staining of K. pneumoniae
Endospore staining of S. aureus
Discussion & Conclusion
This lab used capsule and endospore staining to identify each under the microscope. Capsules, which surround the bacterial cell, are used to protect against phagocytosis, to help stick to host cells, and in the formation of biofilm. In capsule staining, a mixture of negative and simple staining is utilized to produce a clear halo around a capsule when seen under the microscope. An endospore is a protective covering that is used to help bacteria withstand extreme temperature, chemicals, and radiation. It alternates between the vegetative and sporulation life cycle. In endospore staining, the Schaeffer-Fulton method is used to impart a green color to endospores, and safranin is used to impart a pink color to vegetative cells, which can be seen when viewed under the microscope.