SHRI SHIVAJI SCIENCE COLLEGE, AMRAVATI

DBT STAR COLLEGE PROJECT ACTIVITY

ACTIVITY REPORT

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Microbiological Analysis of Drinking Water from Different Water Coolers in College Campus.


Activity Dates: 15/08/2024 to 15/09/2024

Type of Activity: Minor Research Project

Organizing Department: Department of Environmental Science

Program Coordinators: Dr. K. J. Gawai

Head of the Department: Dr. S. P. Ingole

External Collaborator (if any): NO

Objectives:

No of Beneficieries: 12

Classes Involved: B.Sc.II

Venue of the Activity: College Campus

Activity Report:

INTRODUCTION: Access to clean and safe drinking water is a fundamental requirement for maintaining public health, especially within institutions like college campuses where large populations are dependent on a centralized water supply. Drinking water can become a medium for the transmission of waterborne diseases if contaminated with harmful microorganisms such as bacteria, viruses, and protozoa. The presence of pathogens like Escherichia coli (E. Coli) coliform bacteria and Enterococci can indicate fecal contamination and pose significant health risks.
Microbial contamination in water can arise from various sources such as improper sanitation, leaking sewage systems, poorly maintained water distribution networks, or contamination during water storage. Therefore, regular microbial analysis of drinking water is essential to ensure its quality and safety for consumption. This analysis involves detecting and quantifying microorganisms that can be harmful to health, providing vital information to guide corrective measures if contamination is detected.
In the context of a college campus, where a high concentration of students, faculty, and staff rely on common water sources, the continuous monitoring of water quality becomes even more critical. Microbial analysis helps in identifying potential health risks, ensuring regulatory compliance, and maintaining overall campus well-being. This study aims to analyze the microbial quality of drinking water across different points within the college, assess compliance with health standards, and recommend necessary interventions if contamination is detected.
In the analysis of drinking water samples, identifying Gram-positive and Gram- negative bacteria is important for assessing contamination, ensuring safety, and complying with water quality standards. Here’s a breakdown of how each group of bacteria is involved in water safety Ensuring the absence of both Gram-positive and Gram-negative pathogenic bacteria is crucial for protecting public health and preventing outbreaks of waterborne diseases.
Routine water testing and analysis help ensure that drinking water is free from harmful bacterial contamination and safe for consumption.
Importance of microbial analysis in Drinking water :
Microbial analysis of drinking water is critical to ensuring public health and safety. The importance of this analysis lies in its ability to detect harmful microorganisms that can cause diseases if ingested through contaminated water. Here are the key reasons why microbial analysis of drinking water is important:
1. Preventing Waterborne Diseases:
−Microbial contamination in water can lead to diseases such as cholera , dysentery typhoid , gastroenteritis , and hepatitis A.
2. Protecting Vulnerable Populations:
−Children, the elderly, and people with weakened immune systems are especially susceptible to illnesses from contaminated water.
3. Ensuring Compliance with Safety Standards:
−Regulatory agencies such as the World Health Organization (WHO) and Environmental Protection Agency (EPA)set strict guidelines on acceptable microbial levels in drinking water
4. Maintaining Safe Drinking Water Supply:
−Microbial analysis is crucial for monitoring water treatment processes like chlorination, filtration, and UV treatment
5. Early Detection of Contamination:
−Routine testing allows for early detection of microbial contamination in water sources (rivers, lakes, groundwater).
6. Preventing Biofilm Formation in Water Systems:
−Some bacteria can form biofilms in water distribution systems, which can harbor pathogens and affect water quality.
7. Public Confidence in Water Quality:
−Microbial analysis promotes transparency and public confidence by demonstrating that water is routinely tested and meets safety requirements.
8. Avoiding Costly Outbreaks:
−Preventing waterborne disease outbreaks through microbial testing saves costs associated with healthcare, water treatment, and loss of workforce productivity.
9. Environmental Protection:
−By identifying microbial contaminants, water authorities can take measures to protect natural water sources from pollution and contamination, maintaining the ecological balance.

MATERIALS AND METHODS :
In order to evaluate the microbial quality of drinking water in the water coolers distributed in the college campus 5 water coolers were selected. The water samples were collected directly from the water coolers in a pre-sterilized 250 ml beakers and upon collection, drinking water samples were taken to the and analyzed same day or stored at refrigeration temperature in the beaker until tested.
Selected Water Coolers for microbial analysis of Drinking water in college campus :
1. Water Cooler in Panchayat Bhawan Building (P.B)
2. Water Cooler in Ground Floor of New Building
3. Water Cooler in Girls Hostel
4. Water Cooler Near Statistic Department
5. Water Cooler Near Sports Ground

Gram Staining technique is used to check the microbial quality of drinking water.
Following Materials used for the Gram Staining technique:
Pateridish, Conical Flask, Breaker, Glass Rod , agar-agar, Potato starch , Burner, wire gauge, Tripod stand, Distilled water , Cotton, Spatula, Measuring cylinder , Safranine , grams ayodine , crystal violet, 85% Alcohol , Microscope, Sliides , Coverslip.
Methodology used for the Gram Staining technique is as follows :
1. Washed all the instruments properly And allow it to dry
2. Sterilization and autoclave :
Sterilization refers to the process of eliminating or killing all forms of microbial life, including bacteria, viruses, fungi, and spores. It is a critical process in healthcare, laboratories, and industries to ensure that instruments, surfaces, and environments are free from any microorganisms that could cause infection or contamination.
Autoclave is a common device used for sterilization. It uses steam under pressure to kill microorganisms. The typical autoclaving process works at temperatures of 121°C (250°F) or 134°C (273°F) for varying time periods, depending on the item being sterilized. The high temperature and pressure allow the steam to penetrate materials and effectively sterilize them. Proper loading, cycle selection, and maintenance are important to ensure effective sterilization.
3. Preparation of nutrients agar plates :
The preparation of nutrient agar plates is a common procedure used to cultivate and grow bacteria in microbiological labs. Nutrient agar provides the essential nutrients and a solid surface for bacterial colonies to thrive. Here’s how we prepared nutrient agar plates:
Procedure:
1. Weigh the Nutrient Agar:
−Weigh out the required amount of nutrient agar powder according to the manufacturer’s instructions
2. Dissolve the Agar:
−Add the agar powder to the appropriate amount of distilled water in a large beaker or flask. Stir it to dissolve the powder thoroughly.
3. Heat the Mixture:
−Gently heat the mixture on a hot plate or burner to help dissolve the agar completely. Stir continuously to avoid burning at the bottom. Bring it to a gentle boil until fully dissolved.
4. Sterilize the Agar Solution:
−Transfer the dissolved nutrient agar solution to an autoclave or pressure cooker. Sterilize the solution at 121°C (250°F) for 15-20 minutes to kill any contaminants.
5. Cool the Agar
−After sterilization, allow the agar to cool to about 45-50°C. You can do this by letting the flask or container sit at room temperature for a while, but don’t let it solidify.
6. Pour the agar into Petri Dishes:
−Under aseptic conditions (near a burner along with swapping the nearby floor with alcohol), pour the cooled nutrient agar into sterile Petri dishes, covering the bottom to a depth of about 3-4 mm. Be careful to avoid bubbles.
7. Allow Agar to Solidify:
−Let the plates sit undisturbed at room temperature until the agar has completely solidified. This usually takes around 15-30 minutes.
8. Store the Plates:
−Once solidified, store the agar plates upside down (lid down) to prevent condensation from dripping onto the agar surface. You can store them in the refrigerator at 4°C for later use, but let them warm to room temperature before inoculation.
4. Pouring of water samples in agar plates : After solidifying the agar plates water samples from different water coolers were poured in separate agar plates and placed for incubation
5. Incubation :
Nutrient agar plates were placed at 37°c for 24 hr in incubator. Incubating agar plates involves growing microorganisms under controlled conditions. Following steps applied for the incubation purpose.
1. Label the plates: Always label the bottom of the agar plate (not the lid) with relevant information, such as the sample, date, and other identifiers.
2. Set temperature and time: Place the plates in an incubator at the appropriate temperature for the organisms you’re growing (e.g., 37°C for bacteria) and for the recommended time (commonly 24-48 hours for bacteria).
3. Monitor growth: Check the plates periodically for growth without opening them unless necessary.
6.Gram staining Technique :
Following procedure were followed for gram staining
1. Wash the slide with water and allow it to dry.
2. Take one drop of distilled water on the center of the slide with the help of needle .
3. Take a small growth from the slant culture mix it. Smear it across the slide so that the thin film is obtained.
4. Allow it to air dry this film of bacteria is known as smear. Fix the smear passing the slide over 1.w heating flame for 5 to 6 time cool the slide with tap water gently .
5. Cover the smear with crystal violet after 1 minute and wash the slide with water gently.
6. Then put the drop of gram iodine and allow it to react for 1 minute then wash the slide with tap water.
7. Decolorized the smears by applying alcohol until the violet colour comes out this procedure is carried out for only 30-40 second.
8. Wash the slide by tap water. Apply safranine and wash the slide after 1 minute. Allow it to air dry.
9. Then finally Observe the slide under microscope.

RESULTS AND DISCUSSION :
When slides were observed under microscope after gram staining the culture appers pink in colour in 80 % of the slides , 10% slides appears violet & 10% . slides appears pink as well as violet colour.
Study shows that if bacteria appear pink after Gram staining, they are Gram-negative. Gram-negative bacteria have a thinner peptidoglycan layer in their cell walls and an outer membrane, which causes them to lose the violet stain but retain the counterstain, usually safranin, making them appear pink.
Common examples of Gram-negative bacteria & their health risk include:
• Escherichia coli (E. coli)
• Salmonella : Causes salmonellosis, leading to gastroenteritis, fever, and, in severe cases, invasive infections that can spread beyond the intestines.
• Pseudomonas : Associated with hospital-acquired infections, especially in immunocompromised individuals, causing pneumonia, sepsis, and wound infections.
• Neisseria : Can cause bacterial meningitis and meningococcemia, which are rapid-onset, life-threatening conditions affecting the brain, spinal cord, and blood.

If a bacterial sample shows violet color under the microscope after Gram staining, it indicates that the bacteria are Gram-positive. This violet color is due to the thick peptidoglycan layer in the cell walls of Gram-positive bacteria, which retains the crystal violet stain used in the Gram-staining process.
Some common examples of Gram-positive bacteria & their health risk include:
• Staphylococcus : Can cause skin infections, pneumonia, endocarditis, and food poisoning.
• Streptococcus : Known for causing strep throat, scarlet fever, and severe conditions like necrotizing fasciitis (flesh-eating disease).
• Bacillus : Causes anthrax, which can manifest as a respiratory, gastrointestinal, or skin infection and is potentially fatal if untreated.
• Clostridium : Clostridium difficile can lead to severe gastrointestinal infections, especially following antibiotic treatment, causing colitis and life-threatening diarrhea.
Both Gram-positive and Gram-negative bacteria can be dangerous, but Gram-negative bacteria are often considered more dangerous.


Observation Table 1:

SLIDES NAMES TIME GRAM POSITIVE OR
NEGATIVE BACTERIA
P.B 24hr ✘
New Building Ground Floor 24hr ✘
Girls Hostel 24hr ✘
Statistics
Department 24hr ✔
Sports Ground 24hr ✔ - ✘


Observation Table 2:

SLIDES NAMES TIME GRAM POSITIVE OR
NEGATIVE BACTERIA
P.B 24hr Gram Negative
New Building Ground Floor 24hr Gram Negative
Girls Hostel 24hr Gram Negative
Statistics
Department 24hr Gram positive
Sports Ground 24hr Gram positive+ Gram Negative

CONCLUSION:
In conclusion, while most drinking water sources on the campus are safe, specific water coolers require attention to prevent potential health risks. Regular maintenance, cleanliness and microbial testing should be prioritized to maintain water quality across the entire campus. Further study is needed to identify the types of microbes present in water samples of water coolers.

Outcomes:

Photos:

Students Working for Microbial Ananlysis
Students Working for Microbial Ananlysis
Student observing Slide in Microscope 2
Student observing Slide in Microscope 2
Student observing Slide in Microscope 1
Student observing Slide in Microscope 1
Bacteria grown in Water sample from Sports Graund
Bacteria grown in Water sample from Sports Graund
Bacteria grown in Water sample Near Stat Dept.
Bacteria grown in Water sample Near Stat Dept.
Bacteria grown in Water sample from New Building Ground Floor
Bacteria grown in Water sample from New Building Ground Floor

Attendance Sheet: