PROJECT REPORT ON

QUALITY CONTROL OF MICROBIAL QUALITY OF WATER

TRAINING PLACE: INSTITUTE OF TECHNOLOGY-BHU, VARANASIDURATION:

17th JUNE TO 10th JULYSUBMITTED TO:

Dr. RASHMI SHARMA (H.O.D)

(Dept. of Biotechnology)SUBMITTED BY:

HIMANSHU KUMARB.E.(BIOTECHNOLOGY)

VII SemesterRoll. No. 07BT40(8428)

INSTITUTE OF ENGINEERING & TECHNOLOGY, KHANDARI CAMPUS, AGRA. ACKNOWLEDGEMENTI am very thankful to DR. P. N. TIWARI. He has always been the sources of inspiration and has enabled me to complete my project efficiently by his valuable guidance, constructive criticism, innovation ideas and moral support.

I also want to thank Mr. Manish Mishra for his valuable advice and guidance throughout the training period. On the same note I also want to thank Mr. Alok Sharma, Mr. Sandeep Sharma, Ms. Pooja for helping me in better understanding of Microbial Quality of Water handing. They gave me appropriate knowledge of understanding and identification of microorganism in Q.C. and encouraging me to complete the training successfully.

I would also like to thank Dr. Sanjeev Sharma Director of the Institute of Engineering and technology, Agra and Mrs. Rashmi Sharma H.O.D. of Biotechnology and Training and placement coordinate Mr. Diwakar Tiwari and all faculty members and friends for helping me.

At last special thanks to my family who directly and indirectly took a keen interest in assembling my internship, without my family support its impossible for me to complete this project.

(HIMANSHU KUMAR)

CONTENTS MICROBIAL QUALITY OF WATER

GENERAL

TEST FOR TOTAL AEROBIC MICROBIAL COUNT

TEST FOR TOTAL COMBINED YEAST/MOLD COUNTS

ENRICHMENT FOR PATHOGENS

TEST FOR PATHOGENS

TEST FOR E.Coli.

TEST FOR SALMONELLA

TEST FOR PSEDOMONAS AERUGINOSA

TEST FOR STAPHYLOCOCCUS AUREUS

TEST FOR TOTAL VIABLE COUNTS IN WATER FOR INJECTION

MICROBIAL LIMITS OF DM/PURIFIED WATER

REFERENCES

ABBREVIATIONS

DECLARATIONTITLE:- MICROBIAL QUALITY OF WATER.

Objective:- To lay down a procedure for monitoring of microbiological quality of water.

E H S Precautions:- Use all safety protective equipment like hand gloves & safety goggles during analysis.

Procedure:-

1. Sampling Procedure:-

1.1 Collect the sample daily as per the sampling schedule.

S.O.P ( Standard operating procedure)

1.2 For sampling purpose sterilize sample bottle or flask capacity to hold 250-ml water. Properly classed with stopper or plug followed by derminum foil/butter paper.

1.3 For UGT sample 5 drop of sodium Thio-sulphate is added in sampling bottle and sterilized it for deactivation of unlorine.

1.4 Before collecting the sample drain out the water from tap line for about 10 to 15 liter.

1.5 Wear pre sterilized hand gloves & sanitize hand gloves with 70% IPA (Isopropyl Alcohol) prior to sample collection.

1.6 Clean the outer surface of sampling bottle or flask with 70% IPA to avoid external contamination.

1.7 Open the sterile bottle or flask by removing the lap/stopper or plug of flask in such a way that bare hand should not touch to the inside of the plug of bottle or flask.

1.8 Collect 250ml sample from the user point taking precaution to avoid contamination.

1.9 User point is the point from where water is taken for actual use, for example has pipe, which is either connected, to the reactor for charging or to the centrifuge.

1.10 Collect the sample and immediately clause the lap/stopper or plug. Cover the bottle. Flask with aluminum foil/butter paper.

1.11 Label the sample bottle/ flask its identification and date of collection of sample.

1.12 Bring the sample to the microbiology laboratory for analysis.

1.13 Analyze the sample with in two hour of collection, if the sample is not analyzed within two hours then keep it at 2-8C and analyze them within four hours.

1.14 Allow the sample to attain the room temperature before analysis if it is kept in refrigerator.

2. General:-

2.1 As per specification, water is analyzed for total aerobic microbial count and total combined yeast/ molds count.

2.2 As additional in house requirements, water is also tested for pathogens (Eschericnia coli, Salmonella SP. Psedomonas aeruginase and stephylococus aureus)

2.3 Filtration method is used for analysis of D.M water for total bacterial and fungal count and for pathogens in water samples.

2.4 Pour plate method is used for analysis of Raw water / Portable for total bacterial and fungal count and pathogens are tested through membrane filtration method.

2.5 Filtration method is used for analysis of water for injection for total bacterial and fungal count and for pathogens in water samples.

3. Test for total Aerobic microbial count:-

3.1 Take 1ml of the sample into a sterilized filtration assembly containing 0.45 membrane filter and filter using vacuum.

3.2 After filtration transfer the membrane filter with the sterile forceps on to soybean casein digest agar for total aerobic microbial count.

3.3 Keep the Petri dish of soybean casein digest agar in incubator.

4. Test for total combined yeast/mold counts:-

4.1 Take 1ml of the sample into a sterilized filtration assembly containing 0.45 membrane filter and filter using vacuum.

4.2 After filtration transfer the membrane filter with the Sterile forceps on to Sabouraud Dextrose agar for total combined yeast/mold count.

4.3 Keep the Petri dish of sabouraud dextrose agar in incubator at 20 to 25C for 5-7 days in inverted position.

4.4 At the competition of incubation period , count the no. of colonies developed.

4.5 Calculate the no. of CFU 1ml of the sample and report the results.

5. Enrichment for Pathogens:-

5.1 Filter 100ml of water sample by 0.22 or 0.45 micron membrane.

5.2 Transfer the membrane aseptically to 100 ml of or soybean casein digest media or nutrient broth.

5.3 Incubate the tube at 30 -35C for 18-24 hours (Solution A)

5.4 Observe after incubation, if growth is present carry out the primary test.

6. Test for Pathogens:-

6.1 Primary Test of Escherichia Coli:-

6.1.1 Add 1ml from enrichment culture (Solution A) to 100ml Macconkey broth.

6.1.2 Incubate at 42 -44C for 24 48 hours.

6.1.3 If the content of the tube shows acid and gas formation, carry out the confirmatory test. Acid production is indicated by change in colour of the broth from purpose to yellow.

6.2 Secondary test:-

6.2.1 Subculture on a plate of macconkey agar and incubate at 30-35C for 18 to 72 hours. Growth of brick red, generally non-mucoid colonies of gram-negative rods, sometimes surrounded by a precipitation Bile zone indicates the possible presence of E. Coli.

6.2.2 Confirm the presence of E-Coli by inoculating the suspected colonies into 5ml of peptone water and incubating at 30-35C for 24 hours to test innate, add 0.5ml of kavacs reagent, shake well, and allow standing for one minute. If a red color is produced in the reagent layer enable is present. Simultaneously if require steak the colonies on EMB agar at 30-35C for 18-48 hours. Growth of blue black colonies with metallic sheen confirms the presence of E. coli. The product passes the test if such colonies are not seen or if the confirmatory biochemical tests are negative.

Note:- Carry out a control test by sub culturing a 24 hours old broth culture of E .Coli ATCC 8739/ MTCC 1687 on macconkey agar, EMB Agar and macconkey broth. The test is invalid if the results dont indicate that the control contains E.Coli.

Kovacs reagent :

P-dimethylaminobenzaldehyde

5ml

Amyl Alcohol

75ml/litres

HCL concentrated

25ml/litres

Or

Use kavacs reagent manufacture by Himedia

7.2 Primary test for salmonella:-

7.2.1 Shake the container and transfer 0.1ml of enriched culture (Solution A) to 10ml of Rappaport vassiliadis Enrichment broth and incubate 18 to 24 hours.

7.2.2 Secondary test:- Subculture on xylose lysine deoxycholate Agar or Bismuth Sulphite agar or Brilliant green agar incubate at 30-35C for 18 to 48 hors. If any colonies conforming to the description in Table 111 are produced carry out the confirmatory test.

Table 111

Medium

Description of Colony

(1) Xylose lysine Deoxycholate

Well developed Red colonies,

with or without Black Centers.

(2)Bismuth sulphite agar

Black or green colonies

(3) Brilliant green agar

Small transparent colorless or

pink to white. Opaque (Surrounded by pink to red zone)

7.2.3 Subculture any colonies showing characteristic given in Table 111 on triple sugar iron agar using surface and deep inoculation. Incubate at 30-35C for 18 24 hours. The presence of salmonellae is provisionally confirmed if, in the deep culture but not in the surface culture. There is a change of color from red to yellow accompanied by gas formation with or without blackening. Precise confirmation may be carried out by appropriate biochemical; & serological tests.

7.2.4 The preparation being examined passes the test, if in the primary test, cultures of the type described do not appear or if, in the secondary test. The confirmatory biochemical and servo tests are negative.

Note:- Carry out a control test by sub culturing a 24 hours old broth culture of salmonella enteric SSP. Enteric Serotype abony NCTC 6017/MTCC 3858 on triple sugar iron agar. The tests is invalid if the results do not indicate that the control contains S. abony.

Interpretation for TSI Slants :-

Tube Slant ButtGasH2SInferenceRemarks

1Orange

RedOrange

Red--Un-inoculated Control-

2PinkYellow-SligntDextrase fermentation and H2S production (Slignt)

3YellowYellow+-Dextrase and lactase/sucrase fermentationOther Enterobacteria

4PinkBlack++Dextrase fermentation and abundant H2S productionCharacteristics of Salmonellae.

8. Primary Test for Psedomonal aeruginasa:-8.1 Subculture on a plate of cetrimide agar by streaking agar by streaking from solution A and incubate the plates at 30-35C for 18 to 72 hours. If no growth of microorganism is detected, the preparation being examined passes the test.

8.2 Secondary test:- If growth of colonies of gram-negative rods occurs, proceed further suspected colonies on Pseudomonas agar for flouresein and Pseudomonas aga for Pyocyanim.

Mor phological characteristics of P. Aerugfinose on selective agar media

Selective media

1. Pseudomonas agar medium for detection of flouresein.

2. Pseudomonas agar medium for detection of Pyocyanim.

3. Cetrimide Agar

Characteristic Colonial Morphology:

1. Generally colorless to yellowish

2. Generally greenish

3. Generally greenish

Florescence in U. V. light:-

1. Yellowish

2. Blue

3. Greenish

8.3 oxidase test:- Smear the suspected colony on the oxidase test dise (N N dimethyl p phenylene diamine oxalate). The test is positive if purple color is produced within 5 to 10 seconds. Positive oxidase test and Gram negative bacilli indicates the presence of P. aeruginase. If purple color is not produced within 5 top 10 seconds or produced later on indicates the absence of P. aeruginase.

9. Primary Test for Staphylococcus aureus:-

9.1 Streak the enriched culture on Vogel Johnson Agar or mannital salt agar or baired Parker agar.

9.2 Cover and invert the dishes and incubate at 30-35C for 18 -72 hours.

9.3 If any colonies confirming to the description as given below, indicates the presence of staphylococcus aureus.

MediumColony CharacteristicsGram Stain

Vogel Johnson AgarBlack coloies surrounded by yellow zonePositive cocci in clusters

Manital salt AgarYellow colonies with yellow zonePositive cocci in clusters

Baired Parker AgarBlack Shiny, surrounded by clear zonePositive cocci in clusters

9.4 If typical colonies observed, perform coagulase test.

9.5 Coagulase Test:-

9.5.1 Transfer suspected colonies to the tube containing 5ml mammalion Plasma/ rabbit/ horse with or without additives.

9.5.2 Incubate on water bath at 35C.

9.5.3 Examine the tubes at 3 hours and subsequently at suitable interval up to 24 hours. If no. coagulation in any degrees of sample meets the requirement of absence of S. aureus.

10. Test for Total viable counts in water for injection:-

10.1 Filter about 100-100ml water for injection through sterile 0.22 or 0.45 micron membrane filter separately for TAMC and TYMC.

10.2 Put the filter paper on freshly prepared and solidified soybean casein digest agar plate for total Aerobic microbial count and sabouraud dextrose agar for total combined yeast/ molds count.

10.3 Take precaution while transferring the filter paper that no air bubble that no air bubble should be entrapped in between agar surface and filter paper.

10.4 incubate the plate in inverting position at 20 to 25C for 5-7 days for total combined yeast / molds count and 30-35C for 3-5 days total aerobic microbial count.

10.5 count no. of colonies which develop on surface preferatty on colony counter under magnification.

10.6 calculate the No. of total colony farming unit/100ml and report the results.

10.7 Analysis for pathogens is done as per point no. 5 and 6.

11. Microbial limits of DM/ Purified water ( Release criteria):-

11.1 For total aerobic microbial count not more than 100 cfu/ml.

11.2 for total combined yeast/ molds count: NMT 10cfu/ml.

11.3 Pathogens : Absent

11.4 Refer the guide lines given below for avert & action limit.

For TBCLoop systemNon Recirculation system

Alert limitLess than 40 cfu % mlLess than 60 cfu/ml

Action limitLess than 60 cfu / mlLess than 80cfu/ml

12. Records and Trends analysis of Data:-

12.1 Record the samples for microbial quality of water in format no. R/MB/62/03/03

12.2 Record the observations in format no. R/MB/157/00/10.

12.3 When observation of primary test for specified organisms (pathogens) are secondary test and record the observations of format No. R/MB/158/00/10

WATER TREATMENT

Water is mostly used for industrial & municipal purpose. In order to ensure the quality and quantity of water for these purposes, it is extremely important to monitor water supply through out taking all the aspects into consideration. The quality and quantity of water are important in the location of chemical plant. For this purpose surface water as well as ground water may be used, but supply must be adequate and continuous through out the year. The treatment of water to which it is subjected depends on the purpose for which the treated water has to apply. Head water contains dissolved salts of magnesium present as bicarbonates sulphates and chlorides. We use the water treatment process to remove the impurities like (bicarbonates sulphates and chlorides) of hard water. Working and sanitation of equipments involved in water treatment are as follows:

Sources of water: Bore well is the source of the water the are four in number and water is pumped out with the help of bore well pumps. Then the pumped out water sent to plant by pipelines. The water pumped out is known as raw water, which is stored and chlorinated and passes through sand filter, ACF.

1. Sand filter: It removes suspended particles present in water. It is cleaned daily by reverse water flow & yearly sand charged.

2. ACF (Activated Carbon Filter): It removes chlorine added as a disinfectant, from water. It is sanitized weekly & yearly it is replaced. These filters have various alternative layers of media and carbon, which are responsible for the removal of chlorine from water. There are 3 ACF filters.

3. Softener Filter: It removes hardness based on sodium base cationic exchangers. It is charged by 33% brine solution. It is sanitized by formaldehyde soln.(0.5%) and by reverse flow. Softener filters are three in no. that works alternatively.

WATER TREATMENTWater (Bore well)

Chlorine (6ppm)

Raw water storage tank

Sand filter

AFC (Activated Carbon Filter)

(Chlorine removes)

SF (Softener)

(.2% Nacl are used for charging)

R.O. System

R.O. water Storage tank

(addition of cl2 8ppm)

Micron Filter

UV Light

Product line

Flow diagram of water treatment

Difference between Treated Water and Raw Water

RAWTREATED WATER

COLORNo colorNo color

ODORNo odorNo odor

TASTENo tasteNo taste

TOTAL SOLIDS>2000