Pius Lab 5 Negative contrast stain 1

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Feb 20, 2024

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Lab 6 Differential Endospore Stain that separate the spores from vegetative cells and viewing of endospores. Purpose: The purpose of this lab is to perform endospore staining technique to determine whether endospores are present in a bacterial sample, to see and investigate endospores before and after the counterstaining. The purpose of completing endospore stain is to distinguish between bacterial spores and other vegetative cells as well as spore producers and non-spore producers (Microbugz, 2019). Hypothesis: In this lab I expect to see positive endospores, which would be green in color once it has been stained with the main stain, malachite green. I expect the Cellular Morphology and Arrangement to be Bacilli shaped and arranged in chains. Introduction A differential stain called the endospore stain is used to see bacterial endospores. Some bacterial genera, including Bacillus, produce endospores. Endospore is a tough, inactive and non- reproductive structure produced by certain firmicute phylum bacteria (4.5A: Endospores, 2017). Bacteria are able to live in harsh environments by producing spores. Heat, desiccation, chemicals, and radiation don't harm spores. After being subjected to unfavorable conditions for 6 to 8 hours, bacteria can produce endospores. A vegetative cell is a cell that ordinarily divides and develops an endospore. Spores are dehydrated and biologically inactive. They can continue to function for a very long time. Spores can develop into a vegetative cell within 90 minutes of exposure to favorable conditions (Microbugz, 2019). Since there aren't many spore-forming species, it's important for the clinical microbiologist to identify endospores while examining a patient's bodily fluid or tissue. The possibility of a certain bacterial species causing the infection can be reduced by knowing whether or not it produces spores. There are actually two main genera of pathogenic (disease-causing) spores: Bacillus and Clostridium. Botulism, gangrene, tetanus, c-diff, and anthrax are only a few of the deadly and fatal diseases caused by Bacillus and Clostridium species (Hartline, 2022). Endospores can develop in a variety of locations within the vegetative cell. They could be terminal (end of the cell), central (middle of the cell) or subterminal (near the end of the terminal). The diameter of endospores can vary from that of the vegetative cell. The vegetative cell will experience a "swelling" in the area where they are greater in diameter. These endospore traits can be utilized to identify the organism because they are shared by all spore-forming species (Microbugz, 2019). Spore creation begins with the bacterial DNA being copied, then the fore spore is formed by squeezing the cellular plasma membrane between the replicated chromosomes. The second cellular membrane is then extended to encompass the fore spore with calcium and dipicolinic acid, resulting in the formation of a cortex between the inner and outer membrane. The endospore is finally enclosed by the exterior spore coat before being released (Basta & Annamaraju, 2022).

Spores have thick keratin protein coatings that make them extremely resistant to common staining techniques. Malachite green, the main stain used in the endospore stain process, is heated to penetrate the cells. Given that the vegetative cells have been disrupted by heat. Malachite green is water-soluble and does not attach well to the cell, the vegetative cells are better able to absorb the counterstain since the malachite green is easily rinsed from them (Microbugz, 2019). The goal of sporulation is to create a cell type known as a "endospore" (referred to hereafter as a "spore") that is essentially metabolically inert and may endure harsh environmental circumstances until favorable growth conditions are again (Tan & Ramamurthi, 2013). Malachite green, which serves as the major stain, adheres to the spore walls of endospores and mature spores as well as the cell walls of vegetative cells. In fact, the stain actually comes right out of the cell walls of vegetative cells when thoroughly washed with water. Malachite green must be heated in order to pass through the spore walls' limited permeability. The spore wall is made up of several different substances, including calcium and keratin protein, although peptidoglycan is found deeper in the wall. The endospore wall's outer layer of keratin is stain- resistant. The spore wall will become more permeable to the malachite green when the cells are heated, allowing it to adhere to the peptidoglycan. The overlaying spore wall becomes less permeable as the smear cools, making it impossible for the malachite green to escape once it has been introduced to the slide over steam. When the slide cools down, the malachite green is cleansed of the vegetative cells with water as steam is used as the heat and they turn transparent (1.12: Endospore Stain, 2022). Safranin solution is then applied on the slide, its used for counterstaining which stains the vegetative cells or cells that did not make endospores. Safranin solution will render everything red except the endospores (Endospore Lab Video, n.d.). Methods: Materials Required: 1. Malachite green solution (Endospore Lab Video, n.d.). 2. Safranin solution (Endospore Lab Video, n.d.) 3. Microscope slides (Endospore Lab Video, n.d.). 4. Staining tray (Endospore Lab Video, n.d.). 5. Paper towel. 6. Water. 7. Soap. 8. Spraying bleach solution. 9. Hibiclens. 10. Latex gloves. 11. Mask. 12. Oil immersion. 13. Bibulous paper 14. Lens paper Procedure :

1. When entering the lab ensure you have all your hair tied up, closed shoes to cover entire foot and have a protective clothing on top of your home cloths. Sterilize the working table by spraying bleach solution and wipe with a paper towel. 2. Remove jewelry that are on your hands to the wrist, roll up your sleeves to perform hand washing. Turn on the faucet and adjust the water temperature to your comfortable temperature, wet your hand with water and apply anti bacteria hand wash soap and scrub your hands to include 4 inch above the wrist. Scrubbing to remove the dirt for at least 1 minute then rinse the soap with running water. If you touch the sink or anything repeat the whole hand washing process and dry your hands with a paper towel and discard it to the correct bin and pick other clean paper towels and shut the faucet off and dispose the paper towels in the disposal bin. Do not touch your face, eye and mouth while working in microbiology lab. 3. Carefully put on your face mask and wear latex gloves. 4. Gather all the materials and instruments needed for the lab work i.e. Hot plate, beaker from the storage. malachite green solution, safranin solution, staining tray, microscope slides from the shelf and Microscope from the shelf 20. 5. To have the plate hot for use, plug in the power cable to the electric switch and turn it on. Put a beaker of water on the hot plate and boil until steam is coming up from the water. 6. Turn the hot plate heat down so that the water is barely boiling but producing steam. 7. Place a wire stain rack over the beaker. Steam should be coming up through the wire rack. 8. Pick up your already heat fixed slide on top of the wire strain carefully with the side containing the specimen facing up. 9. Open your malachite green solution bottle and squeeze up the solution and put on to the slide covering the entire slide with precaution not to over flood it and have the malachite green solution spill over to the boiling water below. Ensure the slide does not dry off the malachite green solution by putting more on it for 3 full minutes. Steam is used to help the malachite green solution to easily penetrate endospores since they are rigid structures and we need to have the inside stained of the endospore. 10. After 3 minutes are over, remove the wire stain containing the slide from the beaker and place it on the staining tray to cool down for a couple of minutes. 11. Pick up your slide and pour water which acts as the decolorizer on top and blow the slide until the water runs clear. The malachite green solution will only remain in the endospores. 12. Then open the safranin solution bottle and put the solution on the slide, it will stain the vegetative cells or cells that did not make endospores. safranin solution will render everything red except the endospores. Leave the safranin solution for 30 seconds.

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13. After the 30 seconds rinse off the safranin solution with water on both sides until the slide rinse clean, you will see all cells red in color except the endospores that will be green in color. 14. Blot your slide dry with a bibulous paper with the side that has the specimen facing up. The slide is ready to be viewed under a microscope. 15. Put up the microscope the distance from the edge should be greater than 6 inch plug the power cable one end on the microscope and other end to the power source lower the stage make sure the objective is red. If the microscope objective is not red use nose piece to select red objective turn on micro scope. 16. Using the coarse focus lower the stage. Insert the slide on the stage and fasten it with clips. Centre the stain into the light Adjust the stage to the highest level. 17. Begin to view your sample #7 the focus should be on scanning objective red use a fine focusing knob to view and use the iris to regulate the light. Start with a low-magnification objective to find the area of interest on your slide. Use the nose piece to turn to the next objective yellow use the fine adjustment to focus then move to the next objective blue once in focus turn in between blue and white objective. Using the phone take a picture of the viewed specimen under the microscope. Add a drop of immersion oil right where the light hit the slide move the white objective in place and use fine knob to focus. Always use the correct lens when doing oil immersion as it will be damaged. 18. If you do not successfully view your slide under the oil immersion. Use the coarse knob lower the stage move the objective between white and blue dab the oil out of white objective and check all other objectives and make sure they are free from oil. move the slide to a different spot using the nose piece turn from blue to yellow then to red objective start focusing from red objective then yellow, blue move the nose piece between blue and white add immersion oil then move to white and focus Once you have put oil in all spots the slide you cannot use it again you need to prepare another slide let the tutor know to provide another slide, go back to the beginning of the process and make another slide to view. 19. When done with microscope lower the stage remove the slide clean the microscope with lens paper use the nose piece to move objective from white to blue then to yellow then red. Dab the oil out from the oil immersion lens objective check all objectives to make sure they are free from oil use a new piece of lens paper each time you clean the objective. Immersion oil will penetrate and damage microscope components and objectives not suited for immersion. Remove excess oil using a lens cleaning tissue with a single sweep across the lens. Keep wiping the objective front lens with a clean piece of tissue for each wipe until no trace of oil. 20. When done, switch off the micro incinerator and remove power cable from the socket putting it away to storage table. Discard appropriately any disposable waste materials from the lab work and put away other materials and equipment to appropriate storage at the end of the lab work. Then disinfect the working table by spraying bleach solution and wipe with a paper towel. 21. Remove the gloves and face mask and discard in the appropriate disposal bin and perform hand washing. Turn on the faucet wet your hand with water and apply anti bacteria hand wash soap and scrub up to 4 inch above the wrist, scrubbing thoroughly for at least 1 minute then rinse the soap with running water and apply a dime size amount of hibiclens,

rub hands for 30 seconds then rinse it off. If you touch the sink or anything repeat the whole hand washing process and dry your hands with a paper towel and discard it in to the correct disposal bin. Use another paper towel to shut the faucet and dispose it. Roll down your sleeves and can leave the lab when done. Results: Conclusion: Restate: Explain: In the lab we will begin by preparing the working area and self. Sterilize the working area with bleach, roll up sleeves and remove jewelry and perform hand hygiene. When hands are dry wear on gloves and mask and gather all the materials and equipment’s to be used. To have the plate hot for use, plug in the power cable to the electric switch and turn it on. Put a beaker of water on the hot plate and boil until steam is coming up from the water. Turn the hot plate heat down so that the water is barely boiling but producing steam. Place a wire stain rack over the beaker. Steam should be coming up through the wire rack. Pick up your already heat fixed slide on top of the wire strain carefully with the side containing the specimen facing up. Open your malachite green solution bottle and squeeze up the solution and put on to the slide covering the entire slide with precaution not to over flood it and have the malachite green solution spill over to the boiling water below. Ensure the slide does not dry off the malachite green solution by putting more on it for 3 full minutes. Steam is used to help the malachite green solution to easily penetrate endospores since they are rigid structures and we need to have the inside stained of the endospore. After 3 minutes are over, remove the wire stain containing the slide from the beaker and place it on the staining tray to cool down for a couple of minutes. Pick up your slide and pour water which acts as the decolorizer on top and blow the slide until the water runs clear. The malachite green solution will only remain in the endospores. Then open the safranin solution bottle and put the solution on the slide, it will stain the vegetative cells or cells that did not make endospores. safranin solution will render everything red except the endospores. Leave the safranin solution for 30 seconds. After the 30 seconds rinse off the safranin solution with water on both sides until the slide rinse clean, you will see all cells red in color except the endospores that will be green in color. Blot your slide dry with a bibulous paper with the side that has the specimen facing up. View the specimen under microscope Put away the instruments and materials in the correct storage and finish by disinfecting the work surface with bleach and remove the gloves, and dispose them off at the correct bin. Perform hand washing after lab work. Turn on the faucet wet your hand with water and apply anti bacteria hand wash soap and scrub 4 inch above the wrist, scrubbing for at least 60 seconds then rinse the soap with running water. If you touch the sink or anything repeat the whole hand washing process, apply hibiclens, rub hands for 30 seconds then rinse it off and dry your hands with a

paper towel. Use the paper towel to shut the faucet and dispose it. Do not touch your face, eye and mouth while working in microbiology lab. After you are done leave the lab. Results Uncertainty Q1. Why are the cells colorless? What reaction is occurring to “repel” the stain? Due to the negative charge on bacterial cell surfaces, the stain will be rejected, making the bacteria stand out against a dark backdrop ( Welcome to Microbugz - Negative Stain , 2019). Q2. Explain the difference between the attractions of a negative stain and a positive stain (also including which stain is basic/acidic). Then, give the reagents that can be used in both a negative stain and a positive stain. Positive stain is usually basic, carries a positive charge, and adheres to the negatively charged surface. Negative stain is generally acidic, carries a negative charge, and is attached to the positive surface. Salts with a positive ion and a negative ion make up the stains. Depending on the kind of dye, the chromophore (the colored ion) may be a positive or negative ion the other, uncolored ion is known as the counterion. A basic dye is one in which the chromophore is a positively charged ion an acidic dye is one in which the chromophore is a negatively charged ion (OpenStax, n.d.). Crystal Violet, Iodine, Ethyl Alcohol and Safranin are the two substances most frequently utilized as staining reagents (Tripathi & Sapra, 2022). References Microbugz. (2019). Welcome to Microbugz - Endospore Stain . Austincc.edu. https://www.austincc.edu/microbugz/endospore_stain.php

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Hartline, R. (2022, June 12). 1.12: Endospore Stain . Biology LibreTexts. https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_Laboratory _Manual_(Hartline)/01%3A_Labs/1.12%3A_Endospore_Stain Endospore Lab Video . (n.d.). Www.youtube.com. https://www.youtube.com/watch? v=IKcciY0iqdE 4.5A: Endospores . (2017, May 7). Biology LibreTexts. https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_(Boundless )/04%3A_Cell_Structure_of_Bacteria_Archaea_and_Eukaryotes/ 4.05%3A_Specialized_External_Structures_of_Prokaryotes/4.5A %3A_Endospores Basta, M., & Annamaraju, P. (2022, February 2). Bacterial Spores . PubMed; StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK556071/ Tan, I. S., & Ramamurthi, K. S. (2013). Spore formation in Bacillus subtilis. Environmental Microbiology Reports , 6 (3), 212–225. https://doi.org/10.1111/1758-2229.12130

Pius Lab 5 Negative contrast stain 1

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