Water Microbiology PDF

# Water Microbiology ## Introduction A branch of science which deals with studying water and microorganisms present in it. * **Freshwater:** 3% * **Marine:** 97% ## Aim To know potable and non-potable water. * **Potable Water:** * Colorless * Tasteless * Odourless * **Non-Potabl...

# Water Microbiology ## Introduction A branch of science which deals with studying water and microorganisms present in it. * **Freshwater:** 3% * **Marine:** 97% ## Aim To know potable and non-potable water. * **Potable Water:** * Colorless * Tasteless * Odourless * **Non-Potable Water:** * Polluted ## Water Pollution/Water Contamination - Water pollution or contamination can be categorized into physical, chemical, and biological pollution. ### Physical Pollution * Turbidity by sand or clay ### Chemical Pollution * By chemicals ### Biological Pollution * By MO * **Pathogenic:** * "Sewage H₂O" * Like viruses and bacteria ## Dissolved Oxygen [D-0]: The amount of O₂ which present to dissolve in an aquatic ecosystem. ### Measurement Methods 1. **Direct Method:** Oxygen meter 2. **Indirect Method:** Titration (ppm=mg/l) ### Procedures 1. Take a known volume of water (50-100 ml). Add 2 ml of fixing solution. * Fixing solution: [MnSO₄ (8g/l), KI + KOH (50g/l + 100g/l)] → 1 ml 2. Put a few drops of concentrated H₂SO₄ as an indicator until the colour of the sample becomes pale yellow color. 3. Titrate against 0.025 N of sodium thio-sulfate. 4. Titrate until the end point of the reaction. The pale yellow changes to colorless. 5. Calculate the amount of DO using the following formula: $D.O = \frac{V \times N \times 8 \times 1000}{Volume\ of\ water\ sample \times 2}$ * **Average (V):** (V₁ + V₂ + V₃) / 3 * **Notes:** * Repeat the titration (3-5 times) * Use an oxygen bottle to collect the sample or a well-sealed bottle. ## Analysis Of Drinking Water * **Chemical Analysis** * Dissolved Oxygen [D-0] * **Biological or Microbial Analysis** * Biological Oxygen Demand [B-O-D] ## Biological Oxygen Demand [B-O-D]: The amount of dissolved oxygen required for aerobic microorganisms to decompose organic matter in the aquatic ecosystem under certain time and incubation temperature (28-30 °C). ### Sources of Organic Matter: 1. Algae - Plank-ton 2. Dead organisms 3. Metabolism 4. Agricultural or sewage water ### Sources of D.O: 1. Plant 2. Algae * **Aquatic Organisms:** Waves → Diffusion of Oxygen ### Procedures for Determination of B-O-D: 1. Prepare two water samples from the same source. 2. Take one sample and measure its dissolved oxygen immediately by titration method. Record this as initial DO. 3. For the second water sample, put it in dark conditions for 5 days in an incubator at 28-30 °C. * **Why dark?** To prevent the growth of algae or phototrophic organisms to avoid increasing the amount of O₂. * **Why 28-30 °C?** Because it is the optimal temperature for microbial growth ("bacteria") and increases enzymatic activities. 4. Measure the final dissolved oxygen by titration method and record it as final DO. 5. Calculate B-O-D: $B-O-D = Initial\ D-O\ -Final\ D-O$ (ppm or mg/l). ### B-O-D Values: * 1-2: Very good water (potable) * 3-5: Moderate water (potable) * 6-8: Polluted water (non-potable) * 7-8: Very bad water (non-potable) ## Biological or Bacteriological Examination of Water Sample (1): ### Aim: To know potable and non-potable water by detecting bacterial growth. ### Factors Affecting Microorganisms in water 1. **Toxins:** Dye, heavy metals, pesticides, insecticides 2. **Environmental factors:** Physical, chemical (PH, T, P, nutrients) 3. Dissolved Oxygen **Sewage Wastes** * **Bacteria:** * **Pathogenic:** * Salmonella sp. * Shigella sp. * *Vibro cholera* sp. * **Non-Pathogenic:** * *E. coli* * *Streptococcus faecalis* * **Non-pathogenic bacteria:** Perfect sewage indicators. ### Why Don't We Use Pathogenic Bacteria? 1. Pathogenic causes serious diseases 2. It is very expensive to isolate it (media) 3. The survival time is very short. ### Non-Pathogenic Bacteria Turns To Pathogenic In Sewage Water * ***E.coli*:** * Coliform bacteria * G-ve * Facultative anaerobic * Non-spore formation * Can't use spore stain for examination. * Rod, bacilli * Fermentation lactose (acid + gas) * ***Streptococcus*, *Enterococcus*:** * G + ve * Cocci * Fermentation glucos (acid + gas) ### Detection of *E.coli* In Water Sample: 1. **Presumptive test:** Qualitative, +ve/-ve 2. **Confirmed test** 3. **Completed test** ### Procedures: 1. **Prepare lactose broth medium [LB]:** About 300 ml. * Peptone: 5 g/l * Lactose: 5 g/l * Beef: 3 g/l 2. Divide the volume of medium into two flasks, one containing 100 ml and the other 200 ml. 3. For the first flask (100 ml) duplicate the percentage of lactose. * **Prepare double strength lactose broth medium [DSLB]:** Then distribute it into 3 test tubes, each one containing 10 ml. 4. For the other flask (200 ml) it's called single strength lactose broth medium [SSLB]. Then distribute it into 2 groups, each group containing 3 test tubes, each tube containing 10 ml of media. 5. Put a Durham tube in an inverted position in each test tube to collect the gas if formed. 6. Sterilize the tubes in an autoclave. 7. Inoculate the water sample in the test tubes. * DSLB: 10 ml * SSLB: 3 tubes → 1 ml, 3 tubes → 0.1 ml 8. Incubate the tubes in an incubator at 35-37℃ for 24 hours. 9. Incubate the tubes again for 24 hours and observe the results. ### Observations: * **+ve result:** Gas formation after 24 hours * **-ve result:** No gas formation * If there is gas formation after 48 hours, it is not *E.coli*. It is another bacteria from the coliform group (doubtful result). * **The test is called MultiTubes Formation (MTF) due to using a lot of tubes.** * Group 1: UUU * Group 2: UUU * Group 3: UUU * **Physical Properties → Turbidity** ## Confirmed Test: * **Aim:** To confirm the presence of *E.coli*. * **Using selective medium:** * **Selective media:** Chemically modified media to activate the growth of G-ve and inhibit the growth of G+ve. 1. **Eosin methylene blue (E-MB)**: M-B inhibitor 2. **MacConkey medium:** Red/pink color, basic fuchsin 3. **EC medium:** Bile salts 1% ### Procedures: 1. Prepare media 2. Sterilize media in an autoclave at 121℃ + 1.5 PSI. 3. Pour media in St. Petridish and let it solidify. 4. Take a loopful of any tube with a +ve result, using a cotton swab. 5. Do streaking on the surface of the agar. 6. Incubate the Petridish in an incubator at 35-37 ℃ for 24 hours. 7. Examine the results. ### Examine The Result: * **E-MB:** * *E.coli*: Colored, small colonies with a green metallic sheen. * *Enterobacter*: Bacteria which appear with brown/black color. * **MacConkey Media:** * **Colorless:** Non-coliform * **Colored:** Coliform * ***E.coli*:** * Small colonies. * Nucleated. * Red/reddish color. ## Completed Test: * **To examine and check for *E.coli*:** 1. **By Gram stain:** Know morphological shape and type of bacteria (+ve) 2. **By spore staining:** (-ve). 3. **By lactose fermentation:** Forming gas or not. ### Preservation * **Short time:** By slant * **For long time:** -80℃, glycerol ### Procedures for Preservation 1. To increase the surface area so *E.coli* can grow on it for 2 months. 2. To decrease the presence of contamination 3. To increase the amount of media * **Procedures:** 1. Prepare the media 2. Pour it in St. tubes under aseptic conditions. 3. Lock the tubes with a plug. 4. Sterilize it in an autoclave. 5. Let it solidify in a tilted position. 6. Take a loopful of *E.coli* and do streaking. 7. Incubate it at 35-37 ℃ for 24 hours. ## Hydro Microbiology (5) ###+ve Result → Gas Formation * Group 1: 3 * Group 2: 3 * Group 3: 2 ## Quantitative Analysis of Water Sample: ### Counting of M-O Present In Water Sample ### Aim: 1. Count of bacterial cells/M-O present in the water sample. 2. Potable or non-potable water 3. Determine the efficiency of the pretreatment method. * "Radiation, Cl₂, biological method" * **Most Propable number:** (MPN) * **Standard Plate Count:** (SPC) * **Membrane Filter Technique:** (MFT) ### Most Propable Number: - Count/number of microorganisms present in 100 ml of the water sample. - Depends on the result of the presumptive test. * 3:1:3:1:2 → 1100 bacterial cells. * 3 groups of tubes → Clear sample * 411 → 3:1:3:1:3:1:2 → Turbid sample * **MPN:** Result of table x volume of inoculum of the excluded group. * 3:1:3:1:2: (Index) → 1100 bacterial cells/100 ml * **MPN = Result of table x volume of inoculum of the excluded group** * 3:1:3:1:2 → 1100 x 10 = 11000 bacterial cells. * **MPN₂ = result of table x volume** * 3:1:2:1:1 → 150 x 10 = 1500 b.c/100 ml * **Total** * (1500 + 11) / 2 = 755.5 cells. ### MPN: * **3:1:3:1:1** → Direct from table. MPN = 460 bacterial cells/100 ml. * **3:1:3:1:3:1:** MPN = table result x volume of inoculum of 1 ml. * 460 x 10 = 4600 cells/100 ml. * **3:1:2:1:2:** MPN = 210 cells/100 ml. * **3:1:3:1:0:** MPN = result of table x volume of inoculum of 1 ml. * 3:1:3:1:0 → 240 x 10 = 2400 cells/100 ml ## Standard Plate Count (SPC): ### Procedure: 1. Prepare nutrient agar medium. 2. Sterilize it in an autoclave at 121 °C + 1.5 PSI for 20 minutes. 3. Pour media into sterilized Petridishes and let it solidify. 4. Make a serial dilution of the water sample. * H₂O → 9 ml of sterile H₂O. * 10¹, 10², 10³, 10⁴ 5. Take 0.1 ml or 0.5 ml of water sample from each dilution and spread it on the media surface. 6. Incubate the plates in an incubator at 35°C for 24 hours 7. Count the number of formed bacterial colonies in each plate. 8. Determine the colony forming unit (CFU/mL) * CFU = No. of bacterial colonies / dilution x volume of inoculum. * **No. of colonies (30-300):** | Dilution | No. of Colonies | CFU of Countable Plates | |---|---|---| | 10¹ | 311 | | | 10² | 150 | | | 10³ | | | | 10⁴ | | | ## Membrane Filter Technique (MFT) (6): ### Aim: To count the number of bacteria cells in selective media (G-ve, *E.coli*) * **Count of only *E.coli* cells, not all viable cells:** * G-ve, *E.coli* ### Procedures: 1. Prepare a water sample into sterilized bottles. 2. Prepare selective media [MacConkey] and sterilize it in an autoclave at 121 °C + 1.5 PSI for 20 minutes. 3. Bring the filtration device parts (glass funnel, filter holder base, armed flask, and membrane filter). Sterilize all parts with 70% alcohol (ethanol). 4. Make filtration of water sample under aseptic conditions. * The volume of water used depends on the turbidity of the water sample. 5. After finishing the filtration process, wash the funnel using sterilized distilled H₂O. 6. Take the membrane filter by sterilized forceps and put it on the surface of solid selective media after pouring it and let it solidify. 7. Incubate the plate at 35-37 °C for 18-24 hours. 8. Count the bacterial colonies. * **Pore size:** 0.45 mm. * **Thickness:** 150 mm. * 80% of the surface is perforated: * Glass funnel * Water sample: * Clean: 500 ml * Turbid: 200 ml. * Membrane filter * Vacuum pump * Clips * Filter holder base * Armed flask ### Advantages: 1. Reducing time and material equipment. 2. Permanent method for the analysis of large volumes of water samples. 3. Gives information about the presence or absence of *coliform* bacteria in the water sample after 24 hours. 4. Isolation and counting for *coliform* bacteria. 5. A more effective and acceptable method. 6. Allows to remove bacteria static or bacteria sediments/agents from the water sample ## IMViC Tests (7) * **It is a series or group of biochemical experiments which differentiate between genera of coliform, specifically *E.coli* and *Klebsiella* sp.** * **I:** Indole Production Test * **M:** Methyl Red Test * **Vi:** Voges-Proskauer Test * **C:** Citrate Test * **Depends on a PH indicator:** Acidic/basic (microbial growth): ### Citrate Test (1) * **Bromothymol Blue** * **Basic:** Blue, bacterial growth → *Klebsiella* sp. * **Acidic:** Green, no bacterial growth → *E.coli* ### Procedures: 1. Prepare citrate agar medium. 2. Adjust the PH of the medium to a neutral PH (7). 3. Sterilize it in an autoclave at 121°C, 1.5 ATM for 15 minutes. 4. Pour the medium into sterilized Petridishes and let it solidify. 5. Prepare a control plate by preparing nutrient agar medium. Sterilize it. Then, poure it into sterilized Petridishes and let it solidify. 6. Inoculate both plates using the steps 4 and 5 with the bacterial culture. * Slant * Bacterial suspension * Citrate agar * N/A. 7. Incubate both plates at 35 - 37 °C for 24 hours. 8. Examine the bacterial growth. ### Observations: * **+ve (Positive) Result:** There is bacterial growth. The color of the medium is blue. *Klebsiella* sp. * **-ve (Negative) Result:** There is no bacterial growth. The color of the medium is green. *E.coli* ### Control Plates: * **N/A (-ve):** Invalid result. * **Citrate (-ve):** The bacterial inoculum is not viable enough. * **N/A (+ve):** It's *E.coli*. * **Citrate (-ve):** ### Comment * **The principle is to test the ability of bacterial cells to use citrate as a source of carbon in its metabolic pathways.** * **In citrate media, citrate presents as sodium citrate.** * ***Klebsiella* sp. have the ability to convert sodium citrate to NaHCO₃.** * **Note:** * 2- Ammonium Phosphate * NH₄OH * “NaHCO₃ and NH₄OH,” changes the PH to basic. This makes the media give a blue color. ## Additional Visual Information: * **Page 28:** Two petri dishes, one green and one blue. * **Page 29:** A round piece of filter paper with a grid pattern. * **Page 30:** A hand holding 3 test tubes filled with a reddish-brown liquid. * **Page 31:** An oxygen meter on a tiled floor. * **Page 32 & 33:** A hand holding a petri dish with a light orange-colored agar, marked “non-coliform bacteria.” * **Page 34:** A petri dish with red bacterial streaks on a white background. * **Page 35:** A photo of a petri dish partially covered by a yellow sticky note with the word “EMB” on it. * **Page 36:** A colony counter, which is a device used for counting colonies on an agar plate. * **Page 37:** A petri dish on a tile floor with a blue colored agar, divided into quadrants by black marker lines. The bottom of the image is marked “+ result” “*Klebsiella* sp”. * **Page 38:** A petri dish with a yellow-green colored agar and some bacterial culture marked “Control”. The bottom of the image is marked “Citrate agar media”.