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EXPERIMENT 1 Gram decolorizer shrinks and tightens bacteria’s peptidoglycan layer. This will INTRODUCTION degrade the bacteria that allows the counterstain...

EXPERIMENT 1 Gram decolorizer shrinks and tightens bacteria’s peptidoglycan layer. This will INTRODUCTION degrade the bacteria that allows the counterstain to penetrate out of the wall. STAINING TECHNIQUES FOR In which the Gram-positive bacteria resist, BACTERIA which enables it to be distinguished from Gram-negative bacteria. Wherein thick In the observation of bacteria, light and mesh-like layers that make up the cell microscopy can be used. However, the walls were discovered through the lack of pigmentation of most species microscope. Additionally, the Safranin resulted in the need for an improvement in solution is used to contrast stain the contrast. This can simply be solved via the microorganisms that do not preserve staining techniques using various dyes their primary color. As a result, it gives and reagents. The different staining Gram-positive bacteria a purple color that procedures can also give emphasis to is shown by heat-fixing the smear that certain cell parts, differentiate different helps to kill the bacteria through the flame bacterial types, preserve the specimen, that allows it to air dry. and to a certain extent, be used to detect a specific type of bacteria. GUIDE QUESTION METHOD 1.​ What will happen if bacterial smears are heat fixed without air drying? - Heat-fixing bacterial smears is a crucial step in preparing samples for microscopy that is to say, if smears are heat-fixed without first allowing them to air dry, the following issues can arise: Cell Lysis: Rapid heating can cause cells to burst, distorting their morphology and making it difficult to assess shape and arrangement. Poor Staining: Lysis can result in the loss of internal components, compromising the staining process and leading to unreliable RESULT results. Inconsistent Results: Distorted cells lead to variability in staining, The obtained result appeared in the smear complicating comparisons between is a purple color using a microscope under samples. oil-immersion objective, which means it distinguished as Gram-positive bacteria. 2.​ Explain the differences in the Dropping and washing the chemical Gram reaction exhibited by solutions repeatedly is an important step Gram positive and Gram in assessing if the microbiological culture negative cells. is Gram-positive examined under a microscope. Gram Crystal Violet is a dye - Gram-positive bacteria appear purple that easily colors Gram-positive S. aureus or blue due to their thick peptidoglycan due to their thick peptidoglycan wall. cell wall, which retains the primary stain, crystal violet. In contrast, Gram-negative ​ Principle: Removes the crystal bacteria stain pink or red because they violet-iodine complex. have a thinner peptidoglycan layer. ​ Effect: ○​ Gram-positive: Retain 3.​ What is meant by a Gram purple due to thick variable reaction? peptidoglycan. - A Gram variable response happens ○​ Gram-negative: Become when bacteria produce inconsistent results colorless due to thinner in the Gram staining procedure, which peptidoglycan and outer means they do not consistently appear as membrane. Gram-positive (purple) or Gram-negative (pink). There are some factors that can Counterstaining (Safranin): hinder the variability of gram staining Principle: Provides contrast to the reactions such as the structure of their cell primary stain. wall can influence how the dye is retained Effect: during staining. a.​ Gram-positive: Remain purple. 4.​ Why is it recommended to use b.​ Gram-negative: Take up 18–24-hour old bacterial safranin, appearing pink or cultures particularly in Gram red. staining technique? - For accurate Gram staining, it is 6.​ How do Gram-positive and recommended to use 18–24-hour-old Gram-negative bacteria differ bacterial cultures. Cultures that are too from each other? young may lack fully developed cell wall Gram-positive and Gram-negative characteristics, while older cultures may bacteria differ in several key ways: show altered morphology. 1.​ Cell Wall Structure: 5.​ Explain the principle of each ○​ Gram-positive: Thick step in the Gram staining peptidoglycan layer (up to technique and what happens to 90%), no outer membrane; a Gram positive and a Gram retain crystal violet stain, negative cell after every step. appearing purple. ○​ Gram-negative: Thinner Crystal Violet Staining: peptidoglycan (10-20%), ​ Principle: Primary stain that plus an outer membrane penetrates all cells. containing ​ Effect: Both types appear purple. lipopolysaccharides (LPS); do not retain crystal violet, Iodine Treatment: appearing pink after ​ Principle: Mordant that forms a counterstaining. complex with crystal violet. 2.​ Antibiotic Susceptibility: ​ Effect: Both remain purple, but the ○​ Gram-positive: More complex is more stable in susceptible to antibiotics Gram-positive cells. targeting cell wall synthesis ○​ Gram-negative: Generally Decolorization (Alcohol): more resistant due to their  outer membrane acting as METHOD a barrier. 7.​ In what ways does the Gram staining technique aid in the identification of bacteria? - Gram staining categorizes bacteria as Gram-positive or Gram-negative based on their cell wall structure. Gram-positive bacteria retain the crystal violet stain, appearing purple, while Gram-negative bacteria absorb a counterstain, appearing pink. This color differentiation allows for rapid identification under a microscope and aids in narrowing down potential bacterial species. Moreover, Gram staining reveals bacterial structure and arrangement, enhancing identification accuracy. Experiment 2 RESULT As the results demonstrated, E. coli INTRODUCTION revealed a pink color after the staining STAINING TECHNIQUES FOR FUNGI process– a distinguishing characteristic of Gram-negative bacteria. The decolorizer The Lactophenol Cotton Blue (LPCB) acts on the thin peptidoglycan layer and staining method is a widely used the outer membrane of the Gram-negative technique for the microscopic examination bacteria, the latter being degraded. When of fungi. This method combines the the cells are dehydrated by the alcohol, properties of phenol and lactic acid, which the pores in the peptidoglycan layer kills and preserves fungal structures shrink, trapping the crystal violet-iodine respectively, with the staining capabilities complex inside the cell and causing the of cotton blue. The LPCB staining method cell to lose its violet stain. Gram-negative is particularly useful because it provides a bacteria that have been decolorized clear contrast between the fungal maintained the Safranin counterstain, elements and the background, allowing for which gives the cells a pink to reddish detailed observation of the fungal appearance. stain. Gram-negative morphology. This method is commonly bacteria that have been decolorized. As used in mycology for the routine depicted in the photomicrographs, the examination of clinical specimens, bacteria mostly exhibited a scattered environmental samples, and for the distribution of bacillus bacteria. The identification and study of filamentous bacterial arrangement, on the other hand, fungi and yeasts. was inconsistent, multiple arrangements can be seen in the figure, primarily - LCPB enhances visualization of fungal displayed single-celled arrangement, structure by binding the cotton blue with however, clusters and clumps can also be the fungal spore cell wall consisting of observed. chitin. The blue color creates contrast with GUIDE QUESTION the background, and this intense blue 1.​ What is the role of each enhances visibility and highlights structural component in the LPCB stain details. (phenol, lactic acid, and cotton blue)? 4.​ What safety precautions must - Phenol serves as a disinfectant, be taken when handling LPCB effectively eliminating unwanted stains? microorganisms to facilitate the growth of - Given that LCPB stain is toxic, it is fungi. Lactic acid is then employed to essential to follow proper laboratory preserve the fungal structures, ensuring protocols, including wearing appropriate their integrity during observation. Finally, personal protective equipment (PPE), cotton blue is used to stain the chitin in the preparing slides correctly, and disposing of fungal cell walls, enhancing visibility and materials properly. allowing for clearer examination of the fungal morphology. 5.​ Can Lactophenol Cotton Blue (LCPB) stain be used effectively 2.​ Why is it important to aseptically for staining bacterial strains? If collect the fungal material from not, what are the recommended midway between the colony stains for bacterial center and edge? observation?" - Aseptic sampling from the fungal colony is crucial. This ensures the structures are - Lactophenol Cotton Blue (LCPB) is not typically effective for staining bacterial representative and mature, aiding strains, as it is specifically designed for accurate results (Kali, 2014). Careful slide fungi. For bacterial observation, preparation and appropriate sampling recommended stains include: locations enhance the visibility of morphological details. Since fungal growth 1.​ Gram Stain: Differentiates bacteria into Gram-positive often originates from the edges of the and Gram-negative based inoculated plant tissue, it is advisable to on cell wall properties. isolate by collecting hyphae from the 2.​ Methylene Blue: A simple periphery of the fungal colonies. stain that provides a basic contrast for bacterial cells. 3.​ Crystal Violet: Often used 3.​ How does LPCB staining in the Gram staining enhance the visualization of process; it also serves as a fungal structures? primary stain for some bacterial types.  4.​ Safranin: Commonly used 2. Swab the body of the squid, shrimp, or as a counterstain in Gram fish using a sterile cotton swab or staining. inoculating loop and inoculate in zigzag 5.​ India Ink: Useful for motion on one part of the agar plate. observing capsules around Inoculate the other half of the agar plate certain bacteria. for the other part of the samples Do not go over the surface which has been swabbed EXPERIMENT 3 already. ISOLATION OF BIOLUMINESCENT 3.Wrap the inoculated plates with paper BACTERIA and incubate the plates in an inverted position (upside down) at room INTRODUCTION temperature in the dark. Quorum sensing is a bacterial 4.Check for growth of bioluminescent communication system using chemical colonies in the dark room after 8 h of signals to coordinate responses to incubation. Incubate for 12 h or further if environmental changes. It requires bioluminescence was not observed. multiple bacteria to achieve a critical signal concentration, enabling them to 5. Take photos of the plates with work collectively for tasks like bioluminescent bacteria under bright light bioluminescence. For instance, Vibrio and in the dark. Do not use flash fischeri bacteria living in the Hawaiian photography; instead, try long exposure to bobtail squid emit light to help the squid highlight the bioluminescence. 6.Store the evade predators and attract prey. In plates in the refrigerator if positive (not in return, they receive nutrients in a the freezer). specialized compartment. This light production relies on quorum sensing RESULT through acyl-homoserine lactone (AHL) signals, activating bioluminescence genes 3.1. Bioluminescence Observation on like lux and the enzyme luciferase. In the Pterocaesio chrysozona in Bright Light open ocean, diluted AHL concentrations prevent light emission. Figure 5 illustrates the exposure of Pterocaesio chrysozona (Goldband METHOD fusilier, locally "Dalagang bukid") to bright light, focusing on bacterial isolation from 1. Label the agar plates with your group two sites: the gills (A) and eyes (B). number and the name of the sample on Samples were streaked on a petri dish the periphery of the bottom plate. Divide and incubated for 8, 12, 18, and 24 hours. the plate using a marking pen depending No changes occurred at 8 hours, but on the number of samples. Draw a line colonies began forming at the eyes (B) by down the center of the plate, dividing each 12 hours. By 18 hours, colonies were plate into halves. One side will be for visible at both sites, with more prominent growing bacteria from the outside surface growth in the eyes. At 24 hours, colonies of the sample, and the other half will be for expanded significantly, partially covering growing bacteria from the inside surface of the dividing line, indicating bacterial the sample. growth potential. However, bioluminescence could not be observed under bright light conditions. 3.2 Bioluminescence Observation on Pterocaesio chrysozona in Dark Light 3.4 Bioluminescence Observation on Figure 6 depicts Pterocaesio chrysozona Selaroides leptolepis in Dark Light under dark light conditions, with bacterial isolation from the gills (A) and eyes (B) Bacteria from S. leptolepis, previously streaked on a petri dish and incubated for cultured under light, were incubated in 8, 12, 18, and 24 hours. No visible darkness to observe bioluminescence at changes or bioluminescence were 8, 12, 18, and 24 hours. Bioluminescence, observed at 8 hours. By 12 hours, seen as blue-green light, was observed colonies began forming but showed no only in section A (gill samples). At 12 bioluminescence. At 18 and 24 hours, hours, moderate luminescence was colonies expanded significantly but still detected, concentrated at the streak's lacked bioluminescent activity. This edges. However, the glow diminished over absence is attributed to the failure of time, ceasing entirely by 24 hours. No bacteria to produce quorum sensing signal bioluminescence was observed in section molecules needed to activate the lux B (eye samples) throughout the incubation operon for bioluminescence. The period. This highlights the presence of Photobacterium Agar medium used bioluminescent bacteria in the gills but not facilitates quorum sensing, but the in the eyes. conditions did not induce bioluminescent activity. GUIDE QUESTION 1.​ What is bioluminescence? 3.3 Bioluminescence Observation on Bioluminescence is the light produced by Selaroides leptolepis in Bright Light living organisms through chemical reactions in their cells. Most Figure 7 shows the growth of bioluminescent organisms are found in the bioluminescent bacteria from Selaroides ocean, including marine fish, jellyfish, and leptolepis (Salay Ginto) cultured from the bacteria, while some, like fungi and gills and eyes under light conditions over fireflies, live on land. Freshwater 8, 12, 18, and 24 hours. Yellowish environments contain very few translucent colonies appeared denser bioluminescent species. over time, with gill samples showing partial growth by 8 hours and becoming more 2.​ How does the presence of compact by 24 hours. Eye samples bioluminescent bacteria showed no growth at 8 hours but exhibited influence the growth and minimal growth at 12 and 18 hours, with survival of nearby significant growth at 24 hours. non-bioluminescent bacteria? Bioluminescence was not observed due to Bioluminescent bacteria influence the bright environment, as it depends on non-bioluminescent bacteria through oxygen regulation in cells containing indirect effects and symbiosis. They can luciferin and luciferase in specialized light distract predators, signal toxicity, and organs. assist in mate selection. Their presence also supports the "bioluminescence shunt hypothesis," as luminous marine snow bioluminescent bacterial biosensor that and fecal pellets are more visible and detects Cd, As, Hg, and Cu in water. This linked to higher trophic levels. biosensor, utilizing freeze-dried bacteria in a disposable card, showed excellent 3.​ Describe the benefits that the stability, maintaining detection for 10 days squid and bioluminescent with a 3% reproducibility of the bacteria get from each other. bioluminescence signal. The bacteria act as bioreporters, emitting light in response - A symbiotic relationship exists between to specific pollutants, offering a rapid, bacteria and squids, exemplified by cost-effective, and real-time solution for counterillumination. The bioluminescent water quality assessment and pollution bacteria help squids camouflage control. themselves from prey by emitting light downward in response to moonlight and EXPERIMENT 4 starlight. In return, the bacteria colonize the squid’s light organ, gaining food, BACTERIA ISOLATION USING EMB shelter, and protection. During the day, the AGAR squid buries itself in sand to increase bacterial density for effective nighttime INTRODUCTION counterillumination. This cycle continues Enterobacteriaceae is a significant group throughout the squid's life. of bacteria found naturally in the intestinal tract or introduced through contaminated 4.​ How does bioluminescence food and water. Within this family, various occur? Show the simplified genera exhibit varying disease-causing chemical equation. potential, with Salmonella and Shigella being recognized as pathogenic. Others, like Escherichia and Enterobacter, along with Klebsiella and Proteus to a lesser extent, are considered part of the normal intestinal flora and are generally non-pathogenic. It's important to note that - Bioluminescence is a natural under certain conditions, all can potentially phenomenon where living organisms cause diseases. The isolation and produce and emit light through a chemical identification of enteric bacteria from reaction involving luciferin and the enzyme feces, urine, blood, and luciferase. When luciferin oxidizes, usually fecal-contaminated materials play a cri6cal in the presence of oxygen, it generates role in diagnosing enteric infections. While light and produces oxyluciferin as a Enterobacteriaceae share morphological byproduct. and metabolic similarities, laboratory procedures rely on distinguishing them 5.​ Describe a biotechnological based on differences in biochemical application of bioluminescence. activities. Cite an article or paper. METHOD - Bioluminescence has innovative applications in biotechnology, particularly in biosensors and environmental monitoring. A study introduced a 1. Label each EMB agar plate with your group number and sample name along the 3.1.2 Enumeration of Water Sample bottom periphery of each plate. Isolates in the EMB Agar (24-hour Observation) 2. Prepare a serial dilution using a saline solution by transferring 9 ml of saline Microorganisms from Pasig River water solution to each of the four tubes. were cultured on EMB media for 24 hours. After 18 hours, few colonies were 3. Inoculate the first tube with 1 ml of observed, but colony numbers increased wastewater from the Lagoon, mixing well. significantly with continued incubation, Repeat this process for the subsequent particularly at dilution factors 10⁻¹ to 10⁻⁴. tubes in the dilution series. At 10⁻⁵, minimal growth was observed, indicating substantial dilution of bacteria. 4. Open one of the labeled EMB agar Unexpectedly, the 10⁻³ plate had too many plates and streak the inoculated loop onto colonies to count, possibly due to the agar surface using a three-streak procedural errors like uneven cell patter. Secure the cover and base of the distribution. Variations in colony density plates with parafilm. and morphology suggest the presence of 5. Wrap the plates with paper, placing diverse bacteria with differing growth them in the inverted position. rates. 6. Incubate the plates for 24 hours at room 3.2. Morphological Assessment and temperature. Characterization of Bacterial Isolates on the EMB Agar Isolation Culture 7. After incubation, observe and document Plates bacterial growth on the EMB plates, no6ng the color of the colonies. Bacteria cultivated from the 10⁻¹ diluted Pasig River sample using the T-streak RESULT method were identified as coliforms from the Enterobacteriaceae family. Based on 3.1. Growth Observation and EMB agar color, phenotypic traits, and Quantification of Bacterial Isolates on colony morphology, the isolates were the EMB Agar Culture Plates putatively identified as Escherichia coli, Klebsiella pneumoniae, Enterobacter Figure 8 shows ten EMB agar plates (A to aerogenes, and Salmonella spp., J) prepared from Pasig River water corroborated by standard colony samples, representing serial dilutions from characterization protocols and references. 10⁻¹ to 10⁻⁵. The spread plate technique (A to E) was used for bacterial enumeration, while the quadrant streaking method (F to J) ensured proper isolation of colonies. After 18 hours, plates with 10⁻¹ to 10⁻³ dilutions showed minimal growth, while 10⁻⁴ and 10⁻⁵ had few or no colonies. Plates with more colonies indicate higher microbial loads. Observations were extended to 24 hours for detailed GUIDE QUESTION comparison and validation of results.  production. This suggests efficient 1. What is the mechanism by which enzymatic pathways for EMB agar discriminates between metabolizing lactose and lactose-fermenting and non-fermenting producing acidic byproducts. Such bacteria? traits, typical of enteric bacteria like Escherichia coli, reflect adaptation -​ Eosin Methylene Blue (EMB) agar to lactose-rich environments like is a selective, differential medium the gastrointestinal tract. that distinguishes lactose-fermenting from 4. Are there additional biochemical non-lactose-fermenting bacteria. tests available to offer a more thorough Lactose fermenters produce acid, characterization of E. coli isolates? lowering the pH and reacting with eosin and methylene blue dyes. -​ Yes, several biochemical tests can Strong fermenters, like Escherichia further characterize E. coli isolates. coli, form dark colonies with a The indole test detects indole metallic green sheen, weaker production from tryptophan, and fermenters appear pink, and the methyl red test confirms non-fermenters remain colorless. strong acid production from glucose fermentation. The 2. How do eosin and methylene blue in Voges-Proskauer test (acetoin EMB agar contribute to the assessment production) and citrate utilization of acid production? test are usually negative for E. coli. The urease test, often -​ Eosin and methylene blue in EMB negative, checks for urea agar act as pH indicators, breakdown. The triple sugar iron changing color in response to acid (TSI) agar test shows gas production during lactose production and typically no fermentation. Acid lowers the pH, hydrogen sulfide. These tests help causing strong fermenters like differentiate E. coli from similar Escherichia coli to form dark purple bacteria and confirm its metabolic or black colonies with a metallic traits. green sheen. Weaker fermenters produce pink colonies, while 5. How can these tests be applied to non-fermenters remain colorless or enhance our comprehension of the natural-colored. These color metabolic capabilities of various E. coli changes visually indicate lactose strains? fermentation and acid production. -​ Biochemical tests help identify 3. What insights into bacterial metabolic differences in E. coli physiology can be inferred from the strains. The indole test presence of a colony exhibiting a dark distinguishes indole producers, the center and a metallic green sheen? methyl red test measures acid production, and the -​ A colony with a dark center and Voges-Proskauer test detects metallic green sheen on EMB agar acetoin production. The citrate indicates strong lactose utilization test identifies strains fermentation and high acid that use citrate, while the urease  test and triple sugar iron test different body locations can be directly assess urease activity, sugar compared. To get a sterile sample, the fermentation, and gas production. bacteria are transferred using saline. Together, these tests provide insights into E. coli’s metabolic For incubation and observation, the plates diversity. are incubated upside down to prevent condensation from interfering with EXPERIMENT 4 bacterial growth. Gram’s iodine solution is then applied to visualize starch hydrolysis, Extracellular Enzymatic Activities of with clear zones around bacterial colonies Microorganisms - Starch Hydrolysis indicating where starch has been degraded by enzymes. The plates are INTRODUCTION incubated upside down to prevent condensation from causing interference Starch is a high molecular weight, with the growth of bacteria for incubation branching polymer composed of glucose and observation. To visualize starch molecules linked together by glycosidic hydrolysis, Gram's iodine solution is bonds. The degradation of this applied to view starch hydrolysis, with macromolecule first requires the presence clear zones around bacterial colonies of the extracellular enzyme amylase for its indicating that starch is degraded by the hydrolysis into shorter polysaccharides, enzyme. namely dextrins, and ultimately into maltose molecules. The final hydrolysis of RESULT this disaccharide, which is catalyzed by maltase, yields low-molecular-weight, 3.1. Growth Observation of B. soluble glucose molecules that can be megaterium and B. subtilis on Starch transported into the cell and used for Agar Plates energy production through the process of glycolysis. Table 1 summarizes the growth of Bacillus subtilis and Bacillus megaterium on starch METHOD agar plates. Yellowish translucent streaks were observed, and after applying Gram's In the inoculation phase, the B. iodine, clear halos formed around the megaterium and B. subtilis are divided on bacterial streaks. This indicates starch two sides of the starch agar plates. Half of hydrolysis by α-amylase enzymes, which each plate was divided to avoid cleave α-(1-4) glucosidic bonds in starch contaminating one bacterium with the to produce glucose. The violet tint resulted other and allowing the bacteria to grow on from iodine-starch interactions and iodide separate halves. Using the single-line concentration. streak method, consistent bacterial spread is assured throughout the plate to ensure 3.2. Growth Observation of Bacterial consistency in the experimental process. Sample Obtained from Human Mouth and Belly Button on Starch agar plates The flowchart also shows that the samples from the mouth and belly button are Table 2 compares the growth and inoculated onto separate plates. The morphology of oral (Region A) and experiment divides the plates in half so umbilical (Region B) microflora from three that the growth of the microbe from two individuals on starch agar plates, with and without Gram's iodine. Region A showed - Enzymes are crucial for cellular thinner, translucent streaks, while Region metabolism as they accelerate reactions B had darker, wider streaks. After iodine by lowering activation energy, ensuring application, clear halos indicating starch efficiency. They provide specificity, hydrolysis were prominent in Region A, directing metabolic pathways and especially for Subjects 1 and 2, due to preventing unwanted reactions. Enzyme amylase activity. Region B showed little to regulation, through cellular signals, helps no halos, suggesting its microorganisms cells adapt to changes, maintaining lacked amylase. homeostasis and ensuring efficient energy production and biomolecule synthesis for GUIDE QUESTION growth and survival. 1. What is starch, and what role does it 4. How might the findings from this play in microbial nutrition? experiment contribute to our understanding of microbial behavior - Starch, a carbohydrate composed of and function? glucose in linear (amylose) and branched (amylopectin) chains, serves as an energy - The findings from this experiment source for microorganisms. Microbes enhance our understanding of how produce enzymes like amylases to break microorganisms acquire nutrients, produce starch into simpler sugars, such as enzymes, and adapt to their environment. glucose, for energy and growth. This Studying enzyme production, like amylase process supports the carbon cycle and in starch degradation, reveals how has industrial applications, including microbes break down substances for bioethanol production and sweetener energy. This knowledge aids in microbial manufacturing. ecology, resource competition, and symbiotic relationships, while also 2. What is the purpose of flooding the informing biotechnological applications, starch agar plates with Grams Iodine improving industrial processes, and solution? managing microbial communities or infections - Flooding starch agar plates with Gram's iodine reveals starch degradation by EXPERIMENT 5 microorganisms. Iodine reacts with intact starch, producing a blue-black color. Areas CATALASE TEST where microorganisms have hydrolyzed starch using amylase remain clear, INTRODUCTION forming visible zones around colonies. This technique is used to identify amylase During aerobic respiration, production and study starch-utilizing microorganisms produce toxic byproducts microbes. like hydrogen peroxide (H₂O₂) and superoxides, which can be fatal if not enzymatically neutralized. Aerobes, 3. Why is the catalytic activity of facultative anaerobes, and enzymes essential to ensure and microaerophiles rely on enzymes such as regulate cellular metabolism? superoxide dismutase and catalase to degrade these substances, preventing cellular damage. Superoxide dismutase converts superoxides into the less harmful 3.2. Catalase Test: Observation of H₂O₂, which catalase further breaks down bubbles formation in S. aureus agar into water and oxygen. Strict anaerobes, and broth however, lack these enzymes, making oxygen lethal for them as they cannot The catalase experiment with S. aureus detoxify the harmful accumulation of H₂O₂ showed bubble formation, similar to E. when exposed to oxygen. coli, indicating the presence of catalase METHOD enzyme that decomposes hydrogen peroxide into water and oxygen. More 1. Label slides with the names of the bubbles were observed in the organisms. agar-cultured S. aureus compared to the broth-cultured sample. 2. Using a sterile loop, collect a small sample of the first organism from the 3.3. Catalase Test: Observation of culture tube and transfer it to the bubbles formation in S. marcescens appropriately labeled slide. agar and broth 3. Place the slide in the Petri dish. The catalase reaction of Serratia marcescens in both agar and broth media 4. Place one drop of 3% hydrogen showed bubble formation, indicating a peroxide on the sample. Do not mix. Place positive catalase result. The agar-cultured the cover on the Petri dish to contain any sample displayed pink-orange to red aerosols. pigmentation due to prodigiosin production, while the broth-cultured 5. Observe for the immediate presence of sample produced white bubbles. Both bubble formation. Record your results in reactions confirmed the presence of the the chart in the Lab Report. catalase enzyme in S. marcescens. 6. Repeat Steps 2 through 5 for the 3.4. Catalase Test: Observation of remaining test organisms. bubbles formation in M. luteus agar and broth RESULT The catalase reaction of Micrococcus 3.1. Catalase Test: Observation of luteus in both agar and broth cultures bubbles formation in E. coli agar and showed bubble formation, indicating a broth positive catalase result. As an obligate aerobe, M. luteus produces catalase to The catalase reaction experiment on E. metabolize oxygen, decomposing coli grown in different media showed hydrogen peroxide into water and oxygen, immediate bubble formation when which causes the observed bubbles. hydrogen peroxide was applied, indicating the presence of catalase enzyme. This GUIDE QUESTION reaction confirms that E. coli can metabolize hydrogen peroxide, producing 1. Explain the toxic effect of O2 on oxygen bubbles, and demonstrates a strict anaerobes. positive catalase result. - The toxic effect of O₂ on strict anaerobes arises because they lack the enzymes required to neutralize reactive oxygen antioxidant, they produce small molecules species (ROS), such as superoxide like glutathione and superoxide dismutase dismutase and catalase. When exposed to to scavenge ROS, further protecting O₂, strict anaerobes generate ROS (e.g., against oxidative stress. superoxide radicals, hydrogen peroxide) during metabolic processes. These ROS cause oxidative damage to cellular JOURNAL PRESENTATIONS components, including proteins, lipids, and DNA, ultimately leading to cell death. GROUP 1: Enhanced Isolation of Streptomyces from Different Soil 2. Illustrate the chemical reaction Habitats in Calamba City, Laguna, involved in the degradation of Philippines using a Modified hydrogen peroxide in the presence of Integrated Approach catalase. METHOD Collection of Soil Samples -​ Calamba City, Laguna, Philippines 3. Which bacterial strains can be -​ 25 total soil samples collected, 5 biochemically differentiated using the samples per site (400g, 0–20 cm catalase test? depth) -​ Riverside samples: collected 1 m - The table above laid out the away from the river and 15 m away observations recorded from the catalase from each other reaction experiment conducted in the four -​ Collection sites: dumpsite, (4) microorganisms grown in agar and riverside, garden, grassland, forest broth culture media. From this table alone, the group determined all microorganisms Pretreatment of Soil Samples showed positive results from the -​ Air-dried at room temperature (7 experiment as bubbles were observed to days) form from all the samples, which also -​ Manually crushed, then sieved indicated that these microbes contain the through a sterilized mesh enzyme catalase responsible for the -​ Soil-CaCO3 mixtures (10:1 w/w) decomposition of hydrogen peroxide. -​ Incubated at 30°C in a closed sterile Petri dish (2 days) 4. Account for the ability of -​ 20 g soil-CaCO3 mixtures streptococci to tolerate O2 in the suspended in a flask with 180 ml absence of catalase activity. distilled water -​ Incubated (28–30°C) on a rotary - The presence of peroxidase enzymes, shaker at 200 rpm for 30 mins. streptococci use enzymes like NADH peroxides to connect H2O2 into water, Standard serial dilution plate technique mitigating oxidative damage. Moreover, on starch casein agar (SCA) low oxygen metabolism, streptococci are -​ All pretreated soil samples were facultative anaerobes, relying primarily on serially diluted up to 10. fermentation, which generates fewer ROS -​ Serial dilution was vortexed for compared to aerobic respiration. With each tube.  -​ Plated an aliquot of 1 ml of every GROUP 2: ISOLATION OF dilution (in triplicate), overlaid with ACTINOMYCETES WITH approximately 20–25 ml of CELLULOLYTIC AND -​ SCA. ANTIMICROBIAL ACTIVITIES -​ Incubation (after gentle rotation): 28–30°C for 7 days. METHOD -​ The culture media: -​ 50 μg/ml of nystatin -The sampling sites at University of -​ 5 μg/ml of ampicillin Philippines - Diliman: lagoon, arboretum, -​ sunken garden -​ Quantified: -​ Total bacterial count (CFU/g of soil) - soil collection, air drying, sieving, wet -​ Total Streptomyces count (CFU/g heat exposure, phenol exposure, of soil) microorganism isolation -​ Purification of isolates: streaking colonies on new plates of SCA - suspension & dilution, plating, using sterile wire loops. incubation, colony observation, selection -​ Transferred to the SCA slants, and purification, maintenance stored at 4°C until further use. RESULT RESULT TYPICAL CHARACTERISTICS OF Optimal Conditions for Streptomyces ACTINOMYCETES COLONIES -​ Found in soils with higher altitudes, slightly acidic to alkaline pH - Dry Texture (6.7–7.1), and temperatures of With or without pigments 29–33°C. Mycelium Formation -​ Best growth observed in dumpsite - Aerial Mycelium and garden soils. - Substrate Mycelium Overall - A total of 385 distinct colonies were initially isolated from the soil samples -​ 367 bacterial colonies based on typical actinomycete colony observed. features. -​ 103 isolates identified as - Only 235 isolates were successfully distinct Streptomyces (28% purified. of total colonies). - The sampling sites were covered with Microscopic Observations diverse vegetation, influencing -​ All isolates are Gram-positive, abiotic andbiotic factors which affect filamentous, with branching cellulolytic microorganisms. mycelia. -​ 74% have straight or flexible - The study highlights urban green spaces (rectiflexibiles)spore chains. as a rich source of actinomycetes and Bacillus with the potential for cellulolytic and antimicrobial properties and potential industrial and medical applications.  Highest microbial contamination - Isolates showed moderately high -carrot antimicrobial and cellulolytic activities. Isolates that had cellulolytic activity Least microbial contamination on the primary screening may have been - tomato underestimated due to its ecological pressure in the sampled sites. Seven the primary focus of the GAPVF program isolated Streptomyces spp. had high in the Philippines? antimicrobial activities. - Reducing microbial contamination and ensuring consumer safety GROUP 3: Microbiological quality of fresh produce from open air markets Total produce sample and supermarkets in the Philippines -300 Type of agar used -​ MacKonkey agar Statistical test -anova Bell pepper, cabbage, carrot, lettuce, tomato Somatic phages NCR, laguna, pampangga Contamination from irrigation water and soil during cultivation GROUP 4: ANTIBIOTIC RESISTANT BACTERIA IN A RAW CHICKEN MEAT Main indicator for fecal contamination -​ E. coli