MICROPARA LAB REPORT IMMUNITYYYYYYYYYYYYYYYYYYYYYYY

MICROPARA LAB REPORT IMMUNITYYYYYYYYYYYYYYYYYYYYYYY

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  Differential Staining: GramStaining Canto GPA, Cava LAA, Dalida KA, Dofitas BGC, Duran RG, Espinosa AA, Eufemio JXY Group 2, Section C, College of Nursing, West Visayas State University, 5000 Iloilo City, Philippines Corresponding author e-mail: [email protected] ABSTRACT Gram staining is an essential technique in microbiology that helps classify bacteria into two groups: Gram-positive and Gram-negative, based on differences in their cell walls. This laboratory experiment aimed to distinguish between two bacterial samples using the Gram staining method and microscopic observation. The procedure involved applying a series of dyes—crystal violet, Gram’s iodine, ethyl alcohol, and safranin—to highlight differences in bacterial cell structures. The results showed that Specimen A appeared deep purple and had a clustered, round shape, identifying it as a Gram-positive bacterium, Staphylococcus aureus. In contrast, Specimen B appeared pinkish-red and had a rod shape, confirming it as a Gram-negative bacterium, Escherichia coli. These findings are consistent with the known characteristics of these bacteria, demonstrating the reliability of Gram staining in bacterial identification. This technique is widely used in clinical and microbiological studies to quickly identify bacteria and assist in selecting appropriate antibiotic treatments. Keywords: bacteria, Escherichia coli, microbiology, morphology, Staphylococcus aureus
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  INTRODUCTION ​ Gram stainingis a crucial component and a vital staining technique in microbiology. A gram stain is a staining technique that helps diagnose harmful bacteria. Under a Gram stain, different kinds of bacteria change one of two sets of colors (pink to red or purple to blue) under a special series of stains and are categorized as “gram-negative” or “gram-positive,” accordingly. (Professional, 2024) This works by differentiating between different bacteria by the chemical and physical properties of their cell walls. The technique got it’s name from Danish bacteriologist Hans Christian Gram, who first introduced it in 1882, mainly to identify organisms causing pneumonia (Tripathi and Separa, 2023). To prepare a slide smear, a drop of suspended culture is transferred onto a microscope slide using an inoculation loop, with minimal culture to avoid excess collection. The culture is spread into a thin film, air-dried, or gently heated to ensure adhesion. Gram staining follows, starting with crystal violet application, iodine fixation, and decolorization using ethanol-acetone solvents. If the bacteria is Gram positive, it will retain the primary stain (crystal violet) and not take the secondary stain (safranin), causing it to look violet/purple under a microscope. If the bacteria is Gram negative, it will lose the primary stain and take the secondary stain, causing it to appear red when viewed under a microscope. (Bruckner, 2007) Moreover, gram staining offers numerous benefits that make it a valuable tool in microbiology. This type of staining is known for its rapid identification of bacterial pathogens. It provides immediate results which helps identify harmful bacteria in samples like mucus from the lungs and fluid from the wounds (Fujisaki et al., 2024). Therefore, it is a crucial treatment in an environment where quick action is needed, for instance, emergency rooms and intensive care units, where timely interventions can significantly affect patient recovery (Yoshimura et al., 2023). Additionally, this type of staining helps prevent the overuse of broad-spectrum antibiotics by enabling targeted treatment based on the Gram reaction (Yoshimura & Ogura, 2023). Research shows that Gram staining is effective in tracking the success of antimicrobial treatments, with changes in staining patterns indicating the success of the treatment (Fujisaki et al., 2024).​ ​ In addition, gram staining has also contributed to the clinical field in its application in identifying and determining the appropriate antibiotics to treat certain infections after diagnosis, coupled with the relative ease in performing the technique it proved useful in decreasing delays in providing initial antibiotics (Thairu et al., 2014). Gram staining also proves to be a useful diagnostic tool, if utilized properly, in identifying causative microorganisms concerning making informed decisions in clinical settings for planning interventions, such as surgeries (Wouthuyzen-Bakker et al., 2019). The primary objective of this laboratory report is to classify two given bacterial samples, labeled specimen A and specimen B, according to their Gram reaction and to distinguish their morphological characteristics. By observing the staining results and cell shapes under the microscope, the classification of the bacteria as Gram-positive or Gram-negative, as well as their specific morphologies, can be determined. MATERIALS AND METHODS The following materials used in the experiment are the following: Two compound microscopes, two (2) glass slides and coverslips for each bacterial culture (E. coli and S. aureus), a wire loop, pasteur pipette, solutions of Gram’s iodine, Safranin O, Crystal Violet, Cedar wood oil, Ethyl Alcohol, and distilled water, slide rack, and an alcohol lamp.
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  Figure 1. SchematicDiagram of the Gram Staining Procedure. The procedure for this activity includes the following steps: To begin the experiment, the workspace was first cleaned to ensure a sterile environment. Two glass slides were prepared for each bacterial culture, and a drop of standard saline solution (NSS) was placed on each slide. An inoculating loop was then sterilized by heating it in a flame until it became red-hot, followed by a brief cooling period to prevent heat from killing the bacteria (The Australian Wine Research Institute, 2011). Once cooled, the sterilized loop was used to transfer E. coli and S. aureus separately onto their designated slides. Each bacterial specimen was carefully mixed with the NSS to create a thin smear, which was then left to air dry completely before proceeding to the next steps. After the samples had dried, a catch basin was prepared to serve as a foundation for staining.The slides were carefully placed in the basin, and crystal violet was applied to cover each sample fully. The stain was allowed to sit for one minute before being rinsed off with distilled water. Once the samples had dried, Gram's iodine was applied in the same manner, ensuring complete coverage of the samples. The iodine was left to react for one minute, after which the slides were rinsed again with distilled water. Following
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  this, ethyl alcoholwas gradually added until no violet color remained. The slides were then immediately rinsed with distilled water and gently tapped to remove excess liquid (Tiprana & Nishant, 2023). The staining process was repeated using Safranin O, ensuring that each sample was fully covered and left to soak for one minute. Afterward, the slides were rinsed with distilled water and carefully patted dry. Once completely dried using a paper towel and air exposure, the samples were examined under both the high power objective (HPO) and oil immersion objective (OIO) lenses to observe the bacterial structures. RESULTS AND DISCUSSION Gram staining is a differential laboratory test that helps identify bacteria into two groups: Gram-positive, and Gram-negative (MedlinePlus, 2021). This Gram-staining method was developed by Hans Christian Gram in 1884 (Aryal, 2022), it was a differential staining method based on the structural differences of the bacterial cell walls, specifically the thickness of the peptidoglycan layer and the presence or absence of an outer membrane (Rowden, 2022, Steward, 2019). Table 1. Specimen A and B under Different Magnifications Test Specimen High Power Objective (400x) Oil Immersion Objective (1000x) Notes Specimen A Specimen A exhibited a deep purple color and a clustered cocci or round-shaped, in clusters or clumps, indicating that it is a Gram-positive bacteria. Specimen B Specimen B exhibited a reddish to bright pink color, indicating that it is a Gram-negative bacteria. The results of the Gram-staining experiment successfully demonstrated the expected characteristics of Gram-negative and Gram-positive bacteria. When the team observed specimen A under the High Power and Oil Immersion objectives, it appeared as purple, round-shaped cells in clusters, therefore classifying it as a Gram-positive cocci bacteria. On the other hand, specimen B displayed pinkish-red in color, with rod-shaped cells, indicating that it is a Gram-negative bacilli bacterium. These findings were consistent with the known structural differences between the two bacteria species.
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  CLASSIFICATION OF SAMPLES Afterconducting the Gram staining activity, the team observed distinct classifications for the bacteria tested. Specimen A was identified as Staphylococcus aureus and classified as Gram-positive, displaying a purple coloration with round-shaped cells clustered together after staining. These findings align with the results reported by Mueller and Tainter (2023), which confirmed the same classifications using the same bacterial samples. In contrast, Specimen B was identified as Escherichia coli was classified as Gram-negative, as it exhibited a pinkish-red color with rod-shaped cells when viewed under the High-Power Objective (HPO) and Oil Immersion Objective (OIO) lenses. GRAM-POSITIVE BACTERIA The observed staining results can be explained by the differences of the structures of the bacteria, more specifically the differences in bacterial cell wall composition (Rowden, 2022, Steward, 2019). Gram-positive bacteria, in this case S. aureus, garners a deep violet or purple stain because of its thick peptidoglycan cell wall layer (Steward, 2019). During the staining procedure, after the sample was collected, the Gram stain was applied, turning the specimen sample purple. The process involves four reagents: crystal violet, Gram's iodine, ethanol, and safranin. After the Gram’s iodine is applied, the crystal violet molecules bind to the bacteria’s cell walls, forming a large complex that becomes trapped within the thick peptidoglycan layer (Aryal, 2022). The solvent, either ethanol or acetone, is the decolorizer and has a crucial part in determining what the type of bacteria is in a stained sample. In Gram-positive bacteria, the thick peptidoglycan layer becomes dehydrated and shrinks, causing it to retain the color of the crystal violet (Aryal, 2022). Bacteria that remain purple are classified as Gram-positive. Last is the safranin counterstaining, however, since Gram-positive bacteria have already retained the purple stain, they do not take up the pink counterstain (LibreTexts, 2021). Thus, they remain purple when viewed under the microscope. GRAM-NEGATIVE BACTERIA Gram-negative bacteria appears as a pale reddish color when it is observed under the microscope after the process of gram staining. It is due to having a far thinner layer of peptidoglycan and an outer membrane rich in lipopolysaccharides (Aryal, 2022). The structural differences explain their inability to retain the purple color from the crystal violet stain. Initially, the crystal violet stains all bacterial cells purple, and Gram’s iodine further forms interactions creating a complex dye. However, during decolorization, the ethanol introduced disrupts the outer membrane of the Gram-negative bacteria, allowing the crystal violet-iodine complex to be washed off completely (Steward, 2023). This leaves the Gram-negative bacteria to be clear and colorless during this stage. The final step of the staining, the safranin counterstaining, then gives the Gram-negative bacteria its notable red or pink color (Bruckner, 2007).
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  OTHER GRAM-POSITIVE BACTERIA Whenperforming a gram staining test, gram-positive bacteria will show blue or purple after. They have thick cell walls, which means that they retain the dye in gram testing, causing them to show a blue color when viewed under a microscope (Rowden, 2022). All bacteria can be classified based on their shape and behavior of growth (Sizar et al., 2023). In our experiment, we identified S. aureus as Gram-positive bacteria. Some other examples are Staphylococcus epidermidis and Staphylococcus saprophyticus. These bacteria belong to Staphylococcus strain classification of bacteria, in which they are notably round-shaped and usually grow in clumps or clusters which is visible after the Gram-staining process (Bush, 2019). In contrast, Streptococcus is another group of bacteria, though they are similar to the Staphylococcus where they are also round-shaped, they instead grow in chains rather than in clusters. Some examples of Streptococcus bacteria are: Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae (Sizar, Leslie & Unakal, 2023). Additionally, Bacilli are a group of bacteria that are notably rod-shaped, and it is further divided based on their ability to produce spores. Bacillus and Clostridia are spore-forming, while Listeria and Corynebacterium are not. Gram-positive bacteria that produce spores can survive in the environment for many years compared to nonspore-forming bacteria. Some examples of rod-shaped Gram-positive bacteria are: Bacillus antracis, Clostridia botulinum, Corynebacterium diphtheria, and Listeria monocytogenes (Sizar et al., 2023). OTHER GRAM-NEGATIVE BACTERIA Similarly, Gram-negative bacteria can also be classified based on their shape. Neisseria meningitidis and Neisseria gonorrhoeae are both Gram-negative bacteria that are cocci or round-shaped (Oliveira & Reygaert, 2023). In our experiment, Specimen B was classified as Gram-negative and observations displayed that it is rod-shaped or a bacilli bacteria. Some examples of rod-shaped bacteria are: Escherichia coli, Salmonella typhi, and Vibrio cholerae (Aryal, 2022). Additionally, these rod-shaped bacteria are also classified as enterics or Enterobacteriaceae because these bacteria mostly infect and cause fatal diseases related to the gastrointestinal tract (Oliveira & Reygaert, 2023) These bacteria share several characteristics that classify them as Gram-negative bacteria. All have a thin peptidoglycan layer and an outer membrane containing lipopolysaccharides (LPS), which contribute to their structural integrity and antibiotic resistance. Due to this composition, these bacteria appear pink or red after Gram staining. Additionally, all possess a periplasmic space, which contains enzymes involved in nutrient processing and resistance mechanisms (Richard, Sawers, et al., 2013). CONCLUSIONS ​ The experiment successfully differentiated Gram-positive and Gram-negative bacteria based on their structural composition. After analyzing the specimens under different magnifications (see Table 1.), it is evident that each specimen is of different structures. Specimen A, which is S. aureus, exhibited a deep purple color and a clustered cocci shape. Therefore, it indicates that it is a Gram-positive bacteria with a thick peptidoglycan layer that retained the crystal violet. On the other hand, Specimen B, which is E. coli, exhibited a reddish to bright pink color. Hence, it indicated that it is a Gram-negative bacteria with a thinner peptidoglycan layer and an outer membrane that allowed crystal violet to be washed away during decolorization, and taking up safranin counterstain instead. These findings align with the established bacterial characteristics. In addition, the results showcased the effectiveness of Gram staining in microbiological identification.
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  Moreover, Gram stainingis a crucial technique in microbiology that is used to categorize bacteria into Gram-positive and Gram-negative based on the composition of their cell walls. This is vital in clinical settings, as it helps diagnose bacterial infections like pneumonia and sepsis (Matsuura et al., 2024). Additionally, it aids in antibiotic selection, as Gram-negative bacteria often exhibit more excellent resistance due to their protective outer membrane (Paray et al., 2023). Beyond clinical applications, Gram staining is significant in microbial research and plays a key role in ensuring the safety of food and water, as well as maintaining quality control in the pharmaceutical and industrial sectors. Its speed makes it an integral method in both medical diagnostics and microbiological studies. Gram staining is a widely used microbiological technique, but it has significant limitations that can affect its diagnostic reliability. While the technique often demonstrates high specificity, with some studies reporting up to 100%, its accuracy decreases in the presence of concurrent conditions such as crystal arthritis, which complicates result interpretation (Stirling et al., 2017). Furthermore, Gram staining is less effective in tissue samples due to nonspecific staining of connective tissues, making bacterial visualization more challenging (García-Aguilar et al., 2024). These limitations can lead to misdiagnoses and inappropriate antimicrobial therapies, potentially compromising patient outcomes (Thairu et al., 2014). Therefore, Gram staining should be treated as a supplementary diagnostic tool rather than a standalone method. To address these challenges, it is recommended to combine Gram staining with advanced diagnostic techniques, such as molecular methods or culture-based analyses, to ensure a more accurate and comprehensive identification of infections. The Gram-staining experiment highlights the significance of differentiating bacteria based on their cell wall structure, which plays an important role in bacterial identification and treatment. Understanding the distinction between Gram-positive and Gram-negative bacteria provides valuable insights into their characteristics, antibiotic resistance, and pathogenicity. The successful identification of S. aureus and E. coli in this experiment further strengthens the reliability of Gram staining as a fundamental microbiological technique. These findings emphasize the importance of proper bacterial classification, hence, it can aid in proper disease diagnosis and treatment planning. REFERENCES Ansar Ahmad Paray, Manju Rawat Singh, & Mir M. (2023). Gram Staining: A Brief Review. International Journal of Research and Review, 10(9), 336–341. https://doi.org/10.52403/ijrr.20230934 Aryal, S. (2022). Gram Staining: Principle, Procedure, Interpretation, Examples and Animation. Microbiology Info.com. https://microbiologyinfo.com/gram-staining-principle-procedure-interpretation-examples-a nd-animation/ Australian Wine Research Institute. (2022). Aseptic technique. The Australian Wine Research Institute. https://www.awri.com.au/industry_support/winemaking_resources/laboratory_methods/m icrobiological/aseptic/
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  Bruckner, M. Z.(2007, May 16). Gram staining. Microbial Life Educational Resources. https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html Bush, L. (2019). Overview of Gram-positive bacteria. MSD Manual Consumer Version; MSD Manuals. https://www.msdmanuals.com/home/infections/bacterial-infections-gram-positive-bacteri a/overview-of-gram-positive-bacteria BYJUS. (2020). Gram Positive Bacteria - Characteristics And Structure. BYJUS. https://byjus.com/biology/gram-positive-bacteria/ Cleveland Clinic. (2022, March 16). Gram Stain: What It Is, Purpose, Procedure & Results. Cleveland Clinic. https://my.clevelandclinic.org/health/diagnostics/22612-gram-stain Fujisaki, R., Yamaoka, T., & Nishiya, H. (2024). Can Gram-Stained Sputum Be an Effective Therapeutic Marker of Effectiveness of Antimicrobial Agents in Bacterial Pneumonia and Bronchitis? Medical Research Archives. https://esmed.org/MRA/mra/article/view/5110 Garcia-Aguilar, K., Gopar-Cuevas, Y., Chavez-Briones, M.-L., Ortega-Martinez, M., & Jaramililio-Rangel, G. (2024). MHSCongress. Mhscongress.com. https://mhscongress.com/page/proceedings/30 Hartline, R. (2022). Gram Stain. Biology LibreTexts. https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_Laboratory_Manual_(H artline)/01%3A_Labs/1.10%3A_Gram_Stain Matsuura, H., Arimoto, K., Takahashi, Y., & Kishimoto, M. (2024). Combination of a multiplex pneumonia panel and Gram staining for antimicrobial selection to treat lower respiratory tract infection. Pneumonia, 16(1). https://doi.org/10.1186/s41479-024-00125-z MedlinePlus. (2021). Gram Stain: MedlinePlus Medical Test. Medlineplus.gov. https://medlineplus.gov/lab-tests/gram-stain/ Nunez, K. (2019, December 18). Gram-Positive Bacteria Overview, Interpreting Test Results. Healthline. https://www.healthline.com/health/gram-positive#types Oliveira, J., & Reygaert, W. C. (2023, August 8). Gram Negative Bacteria. Nih.gov; StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK538213/ OUYANG, Z., ZHAI, Z., QIN, A., LI, H., LIU, X., QU, X., & DAI, K. (2015). Limitations of Gram staining for the diagnosis of infections following total hip or knee arthroplasty. Experimental and Therapeutic Medicine, 9(5), 1857–1864. https://doi.org/10.3892/etm.2015.2315
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  Richardson, D. J.,Sawers, G., & Van Spanning, R. J. M. (2013). Periplasmic Electron-Transport Systems in Bacteria (pp. 400–406). Academic Press. https://doi.org/10.1016/B978-0-12-378630-2.00218-8 rowden, adam. (2022, September 29). Gram-positive and gram-negative: What is the difference? Www.medicalnewstoday.com. https://www.medicalnewstoday.com/articles/gram-positive-vs-gram-negative#gram-positi ve-types Sherrell, Z. (2022, September 30). Gram-positive bacteria: Characteristics, treatment, and examples. Www.medicalnewstoday.com. https://www.medicalnewstoday.com/articles/gram-positive-bacteria#vs-gram-negative-ba cteria Sizar, O., Unakal, C. G., & Leslie, S. (2023). Gram positive bacteria. Nih.gov; StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK470553/ Steward, K. (2019, August 21). Gram Positive vs Gram Negative. Technology Networks; Technology Networks. https://www.technologynetworks.com/immunology/articles/gram-positive-vs-gram-negativ e-323007 Stirling, P., Tahir, M., & Atkinson, H. D. (2018). The Limitations of Gram-stain Microscopy of Synovial Fluid in Concomitant Septic and Crystal Arthritis. Current Rheumatology Reviews, 14(3), 255–257. https://doi.org/10.2174/1573397113666170329123308 Thairu, Y., Nasir, I. A., & Usman, Y. (2014). Laboratory Perspective of Gram Staining and its Significance in Investigations of Infectious Diseases. Sub-Saharan African Journal of Medicine. https://journals.lww.com/ssjm/fulltext/2014/01040/laboratory_perspective_of_gram_staini ng_and_its.2.aspx Tripathi, N., & Sapra, A. (2022). Gram Staining. PubMed; StatPearls Publishing. http://www.ncbi.nlm.nih.gov/books/nbk562156/ Wouthuyzen-Bakker, M., Shohat, N., Sebillotte, M., Arvieux, C., Parvizi, J., & Soriano, A. (2019). Is Gram staining still useful in prosthetic joint infections? Journal of Bone and Joint Infection, 4(2), 56–59. https://doi.org/10.7150/jbji.31312 Yasuo Kunugiza, Tamaki, M., Miyamoto, T., Tsuji, S., Koichiro Takahi, Nishikawa, M., Yoshida, A., Nomura, K., Iwamoto, K., Toshitaka Fujito, Kentaro Toge, Ishibashi, T., Okada, S., & Tomita, T. (2023). Gram staining of the preoperative joint aspiration for the diagnosis of
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  infection after totalknee arthroplasty. Journal of Joint Surgery and Research, 1(1), 175–178. https://doi.org/10.1016/j.jjoisr.2023.07.005 Yoshimura, J., Ogura, H., & Oda, J. (2023). Can Gram staining be a guiding tool for optimizing initial antimicrobial agents in bacterial infections? Acute Medicine & Surgery, 10(1), e862. https://doi.org/10.1002/ams2.862 List of Member Contribution 1.​ Canto, GPA - Helped in performing the gram staining technique, wrote the observations, helped in answering the questions in the results and discussion, synthesized all the answers in the results and discussion, proofread and formatted the paper. 2.​ Cava, LA - helped in performing the gram staining technique, wrote the abstract, discussed the benefits of gram staining and identified the objective of the experiment in the introduction, wrote the summary of the experiment findings, limitations and recommendations, as well as the final remarks in the conclusion. 3.​ Dalida, KA - Helped perform gram staining. Discussed why gram-negative bacteria stains red and gave more examples of gram-positive bacteria. 4.​ Dofitas, BGC - helped in performing the gram staining technique, wrote the introduction, and the key terms. 5.​ Duran, RG - assisted in performing the Gram staining technique, wrote the materials and methods, and provided a schematic diagram, then discussed examples of Gram-negative bacteria, identified Gram-positive and Gram-negative samples from the activity in the discussion section, and explained the significance of the Gram staining technique in the conclusion. 6.​ Espinosa, AA - helped in performing the gram staining technique, discussed why gram-positive bacteria stains deep violet, discussed the interpretation of results in the conclusion, contributed to the editing of Table 1. 7.​ Eufemio, JXY - helped in performing the gram staining, used the microscope to observe the specimen, discussed the history, short definition, and summarized procedure in lab report introduction.