Lab 10 Puzzle with Two Sides

Lab 10 Puzzle with Two Sides Purpose: The purpose of this catalase lab is to determine if a urinary tract infection (UTI) is being caused by bacteria or yeast. The purpose of lactose lab is to perform lactose test to separate lactose- fermenting from lactose-nonfermenting gram-negative bacteria and the isolation of gram- negative bacteria. The purpose of endospore 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 using dichotomous keys essential is to distinguish between various species based on their observable characteristics with sequence of statements with two options in each stage that guide users to the proper identification make up dichotomous keys (National Park Service, 2018). My purpose for using the dichotomous key enables to identify objects and living things in the natural environment Hypothesis: In this lab I expect to see the catalase-positive Achromobacter spp a nonfermenting gram- negative bacilli when hydrogen peroxide is applied the Phenylethyl Alcohol Agar will show catalase positive for Achromobacter spp , that Couse Pneumonia and bacteremia which will bubble. I don’t expect to need to do endospore lab Achromobacter spp are non-spore producing 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. They don't create spores, but they along with some vibrios appears to be remarkably resilient, persevering through hardships and actively pursuing fresh food supplies Achromobacter - an Overview | ScienceDirect Topics, n.d.). Gram-negative bacteria can only be grown on EMB agar. Gram-positive bacteria are prevented from growing by the medium's methylene blue When lactose or sucrose is fermented, eosin dye and methylene-blue indicator react to alter color. The amide bonding of the dyes in an acidic environment results in blue-black colonies with a green metallic sheen in lactose-fermenting grow with a metallic sheen and a dark center, and non-lactose-fermenting like Achromobacter spp are colorless, gram-negative bacteria A selective medium called phenylethyl alcohol agar (PEA) is used to grow Gram-positive organisms. By interfering with DNA synthesis, the active component, phenylethyl alcohol, inhibits or significantly lowers the growth of Gram-negative organisms PEA also stops Proteus species from congregating on the agar's surface. When separating catalase-positive Achromobacter spp from (catalase-negative), the catalase test is crucial. Several drops of 3% hydrogen peroxide are flooded in order to complete the test. Taxonomy is the classification of organisms into globally accepted classification systems using a hierarchical approach that places each organism into ever more inclusive groupings. As one branches out to become a separate species, the groupings becoming more and more distinct from the place of origin. Categorize all living things into three broad groups, or domains: Eukarya, Archaea, and Bacteria. There is a second category known as a kingdom within every domain. Kingdoms are followed by phylum, class, order, family, genus, and species in sequence of increasing specificity (The Taxonomic Classification System | Biology for Majors I, n.d.). Numerous uses for taxonomic classification exist, including learning more about the

evolutionary past. Humanity lacks a complete understanding of the precise classes to which most species belong, especially when considering the estimated number of organisms found in nature (Khawaldeh et al., 2017). Eight primary levels make up the current taxonomic classification scheme, arranged from most inclusive to most exclusive: Domain, Kingdom, Phylum, Class, Order, Family, Genus, and Species (Taxonomy | Biology for Non-Majors I, n.d.). There are two terms, the system used to name organisms is known as binomial nomenclature. The genus name appears in the first word, while the species name appears in the second. Both words are italicized. When written by hand, the two words are underlined independently. The genus name is capitalized up to the first letter; all other letters are in small letter It is possible to shorten the genus name to simply the first letter. Occasionally, "sp."—a term that encompasses all species within the genus (Binomial Nomenclature (Article), n.d.). Materials Required: 1. Petri Dish with Eosin Methylene Blue (EMB). 2. Petri Dish with Phenylethyl alcohol (PEA). 3. 6 sterile swabs. 4. Urine sterile culture specimen bottle with lid 5. Urine 6. Malachite green solution. 7. Safranin solution. 8. Microscope slides. 9. Staining tray. 10. wire stain rack. 11. Beaker. 12. Hot plate. 13. Oil immersion. 14. Microscope #20. 15. Bibulous paper. 16. Lens paper. 17. Distilled water. 18. Marker. 19. Paper towel. 20. Water. 21. Soap. 22. Spraying bleach solution. 23. Hibiclens. 24. Latex gloves. 25. Mask. 26. Camera. 27. Hydrogen peroxide (H2O2) 28. 2X Petri Dish with MacConkey agar. 29. 2 Bacteria unknown A &B. 30. Micro incinerator. 31. Inoculation loop.

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.

Procedure: Lactose Lab A. 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. B. 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. C. Carefully put on your face mask and wear latex gloves. D. Gather all the materials and instruments needed for the lab work i.e. petri dish from the refrigerator, Micro incinerator from the storage, inoculation loop. E. Turn on the Micro incinerator by plugging it on to the power using the power cable and switch it on at the on/off button which will turn the light on at the switch to show it is on and let it warm for 10 mins. You know it is ready to use when the inside has an orange glow. F. Using a marker label the petri dish from the outside to read 1-2, 2-3,3-4 and 4- 41/2 in a square like shape at the far corners of the Petri dish with the numbers following each other at the corners (i.e. label 1 on the upper left corner, 2 at the left lower corner, 3 on the right lower corner and 4 on the upper right corner). G. Sterilize your inoculating loop by inserting it at the Micro incinerator without touching any sides for 10 second until the wire turn orange then remove the inoculation loop from the micro incinerator and let it cool down for 30 seconds. H. Open petri dish that has the bacteria unknown A insert your sterile inoculating loop in the petri dish and scoop a small sample, close it return to the incubator. Transfer the scooped bacteria to the labeled petri dish using the zig zag technique. Apply the scooped bacteria at the labeled petri dish number 1 along the line to number 2 using thin zigzag movements. I. When done drawing the zigzags from 1 to 2, sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. J. Using the sterile inoculating loop, dilute the bacteria by drawing a zigzag movement from number 2 to 3 by touching where the zigzag ended at the number 2 corner and draw along the line to corner number 3. Sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. K. Using the sterile inoculating loop, dilute the bacteria further by drawing a zigzag movement from number 3 to 4 by touching where the zigzag ended at the number 3 corner and draw along the line to corner number 4. Sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. L. Using the sterile inoculating loop, dilute the bacteria by drawing a zigzag

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. Procedure : Catalase test 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. petri dish from the refrigerator. 5. Open the petri dish with the specimen to be tested 6. Open the hydrogen peroxide bottle and collect some drops of the hydrogen peroxide using a Pasteur Pipette. Recap the hydrogen peroxide (1.18: Catalase Test, 2022). 7. Put some 2-3 drops of the hydrogen peroxide and observe for any bubbling of fizzing as soon as the hydrogen peroxide drops are poured (1.18: Catalase Test, 2022). 8. If it does bubble, it means it catalase positive and if it does not bubble, it means it is catalase negative (1.18: Catalase Test, 2022). 9. When done discard appropriately any disposable waste materials from the lab work and put away other materials Hydrogen peroxide and petri dish 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. 10. 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: All the possible results of an endospore stain will be for the color of spores will seem bluish- green and maintain the malachite green stain. Red staining by the Safranin counterstain is seen in vegetative cells. Spores will be present in some vegetative cells; the endospores will stain green, and the cells will stain red (Aryal, 2015). Fig A Endospore (Aryal, 2015). All possible results for lactose lab Species that ferment lactose will produce pink colonies. The pH indicator becomes pink as a result of the acidic byproducts that lactose fermentation produces, which lower PH. Conversely, non-lactose fermenters will produce opaque, off-white colonies. There will be variations in the growth rate of species even among lactose fermenters. Another method for further differentiating organisms in the MAC medium is growth rate. Finally, the appearance of certain species that create capsules varies. In total. On MacConkey medium, gram-positive bacteria are not able to form colonies. Colonies will form more slowly in weak lactose fermenters than in the others. Examples of slow lac fermenters are Citrobacter and Serratia . Lactose is used by encapsulated bacteria to create capsules. This results in colonies that look sticky and moist. Examples of organisms that generate mucoid colonies are Enterobacter and Klebsiella (PMC, n.d.).

The possible results for catalase test. The enzyme catalase is responsible for converting hydrogen peroxide into oxygen gas and water. Combine bacteria and H2O2. When bubbles form as a result of oxygen gas generation, the bacteria are catalase positive. If there are no bubbles, the bacteria are catalase negative (Rapid Tests | Lab10 | Virtual Edge | Molb 2021 | College of Agriculture and Natural Sciences, n.d.). Fig 4 catalase positive and negative (Aryal, 2022)

References Aryal, S. (2022, August 10).  Catalase Test- Principle, Uses, Procedure, Result Interpretation with Precautions . Microbiology Info.com. https://microbiologyinfo.com/catalase-test- principle-uses-procedure-result-interpretation-with-precautions/ Aryal, S. (2015, September 26). Endospore Staining- Principle, Reagents, Procedure and Result. Microbiology Info.com. https://microbiologyinfo.com/endospore-staining-principle- reagents-procedure-and-result/#:~:text=Result%20of%20Endospore%20Staining Aryal, S. (2015, September 26).  Endospore Staining- Principle, Reagents, Procedure and Result . Microbiology Info.com. https://microbiologyinfo.com/endospore-staining-principle- reagents-procedure-and-result/#:~:text=Result%20of%20Endospore%20Staining Aryal, S. (2022, August 10).  MacConkey Agar- Composition, Principle, Uses, Preparation and Colony Morphology . Microbiology Info.com. https://microbiologyinfo.com/macconkey- agar-composition-principle-uses-preparation-and-colony-morphology/ Binomial Nomenclature (article). (n.d.). Khan Academy. https://www.khanacademy.org/science/in-in-class-11-biology-india/ x9d1157914247c627:the-living-world/x9d1157914247c627:nomenclature-and- taxonomy/a/binomial-nomenclature#:~:text=There%20are%20two%20words%2C %20hence Hauser, A. R., Jain, M., Bar-Meir, M., & McColley, S. A. (2011). Clinical Significance of Microbial Infection and Adaptation in Cystic Fibrosis.  Clinical Microbiology Reviews ,  24 (1), 29–70. https://doi.org/10.1128/cmr.00036-10 Khawaldeh, S., Pervaiz, U., Elsharnoby, M., Alchalabi, A., & Al-Zubi, N. (2017). Taxonomic