Environmental Mutagenesis Lab Manual PDF

+-----------------+-----------------+-----------------+-----------------+ | Index | | | | +=================+=================+=================+=================+ | S.no | Date | Experiments | Remarks | +---...

+-----------------+-----------------+-----------------+-----------------+ | Index | | | | +=================+=================+=================+=================+ | S.no | Date | Experiments | Remarks | +-----------------+-----------------+-----------------+-----------------+ | 01 | 15-10-2024 | LAB NO: 1 | | | | | Isolation of | | | | | soil-degrading | | | | | bacteria | | +-----------------+-----------------+-----------------+-----------------+ | 02 | 22-10-2024 | LAB NO: 2 | | | | | | | | | | Degradation of | | | | | benzene from | | | | | soil | | | | | microorganisms | | | | | by cotton soak | | | | | method. | | +-----------------+-----------------+-----------------+-----------------+ | 03 | 22-10-2024 | LAB NO: 3 | | | | | | | | | | Degradation of | | | | | hydrocarbons | | | | | (octane) by | | | | | soil | | | | | microorganisms | | +-----------------+-----------------+-----------------+-----------------+ | 04 | 4-12-2024 | LAB NO: 4 | | | | | | | | | | Degradation of | | | | | hydrocarbon | | | | | (diesel) by | | | | | soil bacteria | | | | | using spray | | | | | plate | | | | | technique. | | +-----------------+-----------------+-----------------+-----------------+ | 05 | 11-12-24 | LABNO:5 | | | | | | | | | | UV radiation | | | | | induces | | | | | mutagenesis in | | | | | bacteria | | +-----------------+-----------------+-----------------+-----------------+ **LAB NO: 1** Date: 15-10-2024 **OBJECT**: Isolation of soil-degrading bacteria **INTRODUCTION**: Soil-degrading bacteria are microorganisms that break down organic matter in soil. This process is essential for nutrient cycling and maintaining soil health. Soil bacteria can transform certain compounds into more or less mutagenic forms. For instance, some bacteria can degrade complex pollutants into simpler compounds. Greasy soil, often contaminated with hydrocarbons from oil spills or industrial activities, poses a significant environmental challenge. However, nature has its solution: soil-degrading bacteria. These microscopic organisms can break down complex hydrocarbon molecules into simpler, less harmful substances. Several types of bacteria are particularly effective in degrading the hydrocarbons found in greasy soil. ***Pseudomonas*:** This genus of bacteria is well-known for deleting a wide range of pollutants, including hydrocarbons. They can often be found in contaminated environments.   ***Bacillus*:** Bacteria in this genus are highly versatile and can degrade various organic compounds, including hydrocarbons. They are often used in bioremediation processes. **MATERIAL AND METHOD** **Sample Collection** 1. **Soil Sample:** A 1gram sample of greasy soil was collected from a petrol pump. 2. **Media:** Nutrient agar plates were prepared and sterilized. **Serial Dilution and Plating** 1. **Serial Dilution:** A serial dilution of the soil sample was performed in sterile saline solution, ranging from 10^-1^ to 10^-6^. 2. **Plating:** 0.1 mL of the 10^-4^ ,10^-5^ , and 10^-6^ dilutions were aseptically spread onto nutrient agar plates. 3. **Incubation:** The plates were incubated at 37°C for 24 hours.  **Isolation and Identification** 1. **Colony Selection:** Colonies with distinct morphological characteristics, such as color, shape, and texture, were selected for further analysis. 2. **Oxidase Test:** The oxidase test was performed to differentiate between *Pseudomonas* and *Bacillus* species. A small amount of bacterial growth was transferred to a filter paper impregnated with an oxidase reagent. A color change to purple within 10 seconds indicated a positive result for *Pseudomonas*. 3. **Pure Culture Isolation:** Selected colonies were streaked onto fresh nutrient agar plates and slants to obtain pure cultures. 4. **Storage:** Pure cultures were stored on nutrient agar slants at 4°C for future use **RESULT AND DISCUSSION:** The experiment aimed to isolate soil-degrading bacteria, specifically *Bacillus* and *Pseudomonas* species, from greasy soil. Serial dilution was employed to dilute the soil sample and reduce the bacterial load, allowing for the isolation of individual colonies on nutrient agar plates. After incubation, various bacterial colonies were observed on the plates. Based on colony morphology and biochemical (oxidase) tests, *Pseudomonas* and *Bacillus* species were identified. The serial dilution technique effectively reduced the bacterial load, facilitating the isolation of individual colonies. The oxidase test is a rapid and reliable method for differentiating between *Pseudomonas* and *Bacillus* species. *Pseudomonas* species are oxidase-positive, while *Bacillus* species are oxidase-negative **LAB NO: 2** Date: 22-10-2024 **OBJECT**: Degradation of benzene from soil microorganisms by cotton soak method. **INTRODUCTION**: Hydrocarbons, particularly those derived from petroleum, are ubiquitous in the environment. They can enter the environment through various sources, including Accidental Spills, Industrial Activities, Urban Runoff, and Natural Seeps. Hydrocarbons, particularly polycyclic aromatic hydrocarbons (PAHs), are known to be mutagenic and carcinogenic. They can cause DNA damage, leading to mutations that can contribute to cancer and other health problems. The degradation of benzene by microorganisms is a vital ecological process contributing to environmental health, human well-being, and sustainable development. By harnessing the power of these tiny organisms, we can protect our planet and safeguard future generations The cotton-soaked method offers a simple and effective approach to studying benzene\'s biodegradation by soil bacteria. By understanding the factors influencing degradation rates, we can optimize bioremediation strategies to clean up contaminated sites. **MATERIAL AND METHOD** Day 1 Using the isolated culture of *bacillus* and *pseudomonas* stab on nutrient agar to make a master plate and incubate for 24 hours at 37^o^C Day 2 With the help of a replicator stamp the culture from the master plate to the below plates - Positive control (a minimal media plate +glucose) - Negative control (a minimal media plate) - Test plate (a minimal media plate) Now, make 4 wells in each plate by using the borrower Put the soaked benzene cotton balls using sterile forceps in each well of the test plate. Cover the plates with scotch tape to provide anaerobic conditions Incubate the plates for 4 days at ambient temperature. **OBSERVATION** +-----------------+-----------------+-----------------+-----------------+ | **Culture | **MM+ Glucose** | **MM** | **MM** | | code** | | | | | | **Positive | **Negative | **Test plate** | | | control** | control** | | +=================+=================+=================+=================+ | *bacillus* | **-ve** | **-ve** | **-ve** | +-----------------+-----------------+-----------------+-----------------+ | *pseudomonas* | **-ve** | **-ve** | **-ve** | +-----------------+-----------------+-----------------+-----------------+ | | |  | | +-----------------+-----------------+-----------------+-----------------+ **Keys :** \+ degradation \- no degradations **RESULT AND DISCUSSION:** As observed, the experimental setup did not yield positive results for benzene degradation by the tested bacterial strains, *Bacillus* and *Pseudomonas*. All wells, including the test wells with benzene-soaked cotton, remained negative, indicating no visible bacterial growth or benzene degradation. **LAB NO: 3** Date: 22-10-2024 **OBJECT**: Degradation of hydrocarbons (octane) by soil microorganisms. **INTRODUCTION:** Hydrocarbons, such as octane, are significant environmental pollutants primarily resulting from industrial activities, oil spills, and automobile emissions. Their persistence in the environment poses a substantial risk to ecosystems due to their toxicity and potential to bioaccumulate. Bioremediation, using microorganisms to degrade these pollutants, is a promising eco-friendly approach for mitigating such contamination.\ Soil microorganisms, particularly species such as *Bacillus* and *Pseudomonas*, are well-known for their hydrocarbon-degrading abilities. These microorganisms utilize hydrocarbons as a carbon source, breaking them down into less harmful substances. This study aims to evaluate the ability of *Bacillus* and *Pseudomonas* to degrade octane by growing them on minimal media, simulating hydrocarbon exposure, and observing their growth patterns **METHODOLOGY** DAY 1[:] 1. Using the isolated culture of *bacillus* and *pseudomonas* stabbing was done on nutrient agar to make a master plate. 2. Then the plate was incubated for 24 hours at 37^o^C. Day2: By using a sterile replicator , do replication on two plates of minimal media. Then place the filter paper on the lid and spray them then covered that plates with lid having sprayed filter paper inside Then incubate the plates for 3-4 days at 37^0^C **OBSERVATIONS** **Minimal media** **observation** ------------------- -------------------------------------- ------------------------ Plate 1 Fungal contamination on filter paper  Plate 2 Fungal contamination on filter paper **RESULT AND DISCUSSION** During the experiment, fungal contamination was observed on the filter paper in both minimal media plates (Plate 1 and Plate 2). This contamination could have interfered with the assessment of hydrocarbon degradation by the bacterial isolates.\ The fungal contamination on the filter paper suggests that the experimental conditions favored fungal growth, potentially outcompeting the bacterial isolates. This outcome highlights the importance of maintaining aseptic conditions during the experimental setup.\ Despite the contamination, the use of minimal media demonstrates its potential to differentiate microbial growth in response to hydrocarbon exposure. The presence of *Bacillus* and *Pseudomonas* on nutrient agar indicates their viability, but replication on minimal media with hydrocarbon vapors could not be effectively evaluated due to fungal interference.\ Future experiments should focus on improving aseptic techniques and potentially incorporating antifungal agents to prevent contamination. Additionally, using vapor-phase hydrocarbons in a closed system or alternative methods for hydrocarbon exposure could enhance the reliability of the results. This study underscores the challenges and critical considerations in designing experiments for microbial hydrocarbon degradation. **LAB NO: 4** Date: 4-12-2024 **OBJECT**: Degradation of hydrocarbon (diesel) by soil bacteria using spray plate technique. **INTRODUCTION:** Hydrocarbons, such as diesel, are significant environmental pollutants due to their widespread use in transportation and industrial applications. Their persistence in the environment leads to contamination of soil and water, posing serious ecological and health risks. Bioremediation, the use of microorganisms to break down hydrocarbons, is an environmentally friendly alternative to conventional remediation methods. Certain bacteria, such as *Bacillus* and *Pseudomonas* species, are known for their ability to degrade hydrocarbons by utilizing them as a carbon source. These bacteria secrete enzymes capable of breaking down complex hydrocarbon chains into simpler, less harmful compounds. This experiment aims to investigate the potential of soil bacteria, specifically *Bacillus* and *Pseudomonas*, to degrade diesel through the spray plate technique. By observing bacterial growth on minimal media supplemented with diesel, the study seeks to evaluate their ability to metabolize this hydrocarbon under controlled conditions. **METHODOLOGY** DAY 1[:] 3. Using the isolated culture of *bacillus* and *pseudomonas* stabbing was done on nutrient agar to make a master plate. 4. Then the plate was incubated for 24 hours at 37 degrees. DAY2: 5. Using a replicator, a replica of growth was prepared by taking an impression from the master plate on three plates of minimal media under strictly sterile conditions. 6. Mark the plates as test, positive control, and negative control. 7. On test and negative control plates, spray the diesel. 8. On positive control plate, the spray was not done, 9. Cover the lids and incubate for 3 days at 37 degrees. **OBSERVATION:** **Agar plates** **Growth** ------------------ ------------ ------------------------ Positive Control **+++**  Negative Control **++** Test ++++  **[KEY:]** ++ \-\-\-\-\-\-\-- less growth +++ \-\-\-\-\--growth ++++ \-\-\-\-\--more growth **RESULT AND DISCUSSION:** These strains of organisms are not capable of degrading diesel. The results show differing levels of bacterial growth across the experimental groups. The positive control confirmed bacterial viability on the minimal media. The key observation is the increased growth on the test plate (bacteria + diesel) compared to the negative control (bacteria + diesel). *However, this increased growth does not definitively prove diesel degradation* **LAB NO: 5** Date:11-dec-24 **OBJECT**: UV radiation induces mutagenesis in bacteria **INTRODUCTION:** **METHODOLOGY** **EXPECTED OBSERVATION** **cultural observations** **Condition** **Colony Morphology** **Growth Density** ------------------------------- ----------------------------------------------------------------------- --------------------------------- **Negative Control** Well-defined, smooth-edged colonies of uniform size and pigmentation. Consistent and abundant growth. **Positive Control (10 min)** Smaller, irregular colonies with possible pigmentation changes. Moderate reduction in growth. **Test Plate (20 min)** Significantly smaller or abnormal colonies; some areas lack growth. Sparse or minimal growth. **Microscopic observations (gram staining)** **Condition** **Appearance** **Other Indicators** ------------------------------- ----------------------------------------------------------------------- ------------------------------------------- **Negative Control** Uniform pink, rod-shaped cells with normal morphology. None **Positive Control (10 min)** Mostly intact pink rods; occasional deformed or fragmented cells. Some clumping or irregular arrangements. **Test Plate (20 min)** Few cells with pronounced abnormalities (e.g., filamentation, lysis). Ghost cells or debris from dead bacteria. This study highlights the vulnerability of bacterial strains to UV radiation and its potential to induce both sub-lethal and lethal mutations, which could have implications in microbial evolution, adaptation, and sterilization techniques.