CC BY
11STANDART OPERATING PROCEDURE TO ANALYZE THE WATER FOR
CHEMICAL AND MICROBIOLOGICAL TESTS IN A MODERN WAY
1 2 Akhmatova Durdona , Akhmatov Alisher
1Faculty of Pediatrics, Tashkent Pediatric Medical Institute,Uzbekistan Faculty of Pharmacy,Tashkent Pharmaceutical Institute,Uzbekistan Email: septolede@mail.ru https://doi.org/10.5281/zenodo.8368087 Abstract. Water is one of the most vital resources on Earth. It is the foundation of life and plays a crucial role in sustaining both human and environmental health. However, despite its importance, water can become contaminated by a variety of pollutants, including chemicals, bacteria, viruses, and other harmful substances.Water testing is the process of analyzing a sample of water to determine its quality and identify any potential contaminants. This analysis can reveal a range of information, including the presence ofpathogens, the levels of nutrients or pollutants, and the water's overall health.There are many reasons why water testing is important. Perhaps the most significant is that it helps to protect human health. Waterborne diseases can cause serious illness or even death, and contaminated drinking water is a major source of these diseases. By testing water supplies for harmful pathogens such as E. coli and coliform bacteria, water treatment facilities can ensure that drinking water is safe for consumption.
In addition to protecting human health, water testing is also critical for protecting the environment. When water is contaminated, it can have devastating effects on aquatic ecosystems, including fish and other wildlife. Water testing can help identify the sources of pollution and inform management strategies that can reduce or eliminate these impacts.
Keywords: Bacterial Endotoxin Test,pathogens,Salmonella species,Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus,aciditu,alkalinity,ammonium
Аннотация. Вода - один из самых жизненно важных ресурсов на Земле. Это основа жизни и играет решающую роль в поддержании здоровья как человека, так и окружающей среды. Однако, несмотря на свою важность, вода может быть загрязнена различными загрязняющими веществами, включая химические вещества, бактерии, вирусы и другие вредные вещества. Вот почему тестирование воды так важно.Тестирование воды - это процесс анализа пробы воды с целью определения ее качества и выявления любых потенциальных загрязнителей. Этот анализ может выявить целый ряд сведений, включая наличие патогенных микроорганизмов, уровни питательных веществ или загрязняющих веществ, а также общее состояние воды.Существует много причин, по которым важно проводить тестирование воды. Пожалуй, самым важным является то, что это помогает защитить здоровье человека. Болезни, передаваемые через воду, могут привести к серьезным заболеваниям или даже смерти, и загрязненная питьевая вода является основным источником этих заболеваний. Проверяя источники водоснабжения на наличие вредных патогенов, таких как кишечная палочка и бактерии группы кишечной палочки, водоочистные сооружения могут гарантировать, что питьевая вода безопасна для потребления.
Помимо защиты здоровья человека, тестирование воды также имеет решающее значение для защиты окружающей среды. Загрязнение воды может иметь разрушительные последствия для водных экосистем, включая рыбу и другую дикую
природу. Тестирование воды может помочь выявить источники загрязнения и разработать стратегии управления, которые могут уменьшить или устранить это воздействие.
Annotatsiya. Suv yerdagi eng muhim resurslardan biridir. Bu hayotning asosidir va inson va atrof-muhit salomatligini saqlashda hal qiluvchi rol o'ynaydi. Biroq, uning ahamiyatiga qaramay, suv turli xil ifloslantiruvchi moddalar, jumladan kimyoviy moddalar, bakteriyalar, viruslar va boshqa zararli moddalar bilan ifloslanishi mumkin. Shuning uchun suvni sinovdan o'tkazish juda muhimdir.Suvni sinash-bu uning sifatini aniqlash va potentsial ifloslantiruvchi moddalarni aniqlash uchun suv namunasini tahlil qilish jarayoni. Ushbu tahlil patogenlar mavjudligi, ozuqa moddalari yoki ifloslantiruvchi moddalar darajasi va suvning umumiy salomatligi kabi bir qator ma'lumotlarni aniqlashi mumkin.Suvni sinovdan o'tkazish muhimligining ko'p sabablari bor. Ehtimol, eng muhimi, bu inson salomatligini himoya qilishga yordam beradi. Suv orqali yuqadigan kasalliklar jiddiy kasalliklarga yoki hatto o'limga olib kelishi mumkin va ifloslangan ichimlik suvi bu kasalliklarning asosiy manbai hisoblanadi. E. coli va koliform bakteriyalar kabi zararli patogenlar uchun suv ta'minotini sinab ko'rish orqali suv tozalash inshootlari ichimlik suvi iste'mol qilish uchun xavfsiz bo'lishini ta'minlashi mumkin.Suvni sinovdan o'tkazish inson salomatligini muhofaza qilishdan tashqari, atrof-muhitni muhofaza qilish uchun ham juda muhimdir. Suv ifloslanganda, u suv ekotizimlariga, shu jumladan baliq va boshqa yovvoyi tabiatga halokatli ta'sir ko'rsatishi mumkin. Suvni sinovdan o'tkazish ifloslanish manbalarini aniqlashgayordam beradi va ushbu ta'sirlarni kamaytiradigan yokiyo'q qiladigan boshqaruv strategiyalarini xabardor qiladi.
1.0 PURPOSE
To lay down the procedure for the analysis of water.
2.0 SCOPE
Applicable to all sampling points of water system.
3.0 RESPONSIBILITY Microbiologist
4.0 ACCOUNTABILITY Head of Department
5.0 PROCEDURE
Collect the sample as per Standard Operating Procedure for water sampling and analysis for chemical and microbiological parameters as per their specifications.
5.1 Chemical Analysis
Prepare the solutions/ reagents for chemical analysis.
5.1.1 Description
Examine the water physically such as color, odor.
5.1.2 Hardness
Take 100 ml sample add 2 ml of ammonia buffer pH 10.0, 50 mg of mordant black 11 mixture and add of 0.01 M disodium edetate until, a pure blue colour is produced. Measures the volume of disodium edetate used and calculate the hardness by the following formula.
Hardness as mg/L = ml of EDTA used x 1000 mg/L Sample volume
5.1.3 Total Suspended Solids (TSS)
Take the gouch crucible clean and dry in oven for one hour at 105°C, Cool the gouch crucible in desiccator and take the empty weight of gouch crucible and then filter the 30 ml water sample from the gouch crucible with the help of vacuum pump and calculate the TSS with the help of the formula.
TSS = W2-W1 x 1000 (mg/L) ml of solution taken
W1 : Weight of Gouch crucible before filtration
W2 : Weight of Gouch crucible After filtration
5.1.4 Total dissolved solids (TDS)
Measure the conductivity at 25 °C with a calibrated conductivity meter and convert the value in TDS by the following formula.
TDS in mg/L= conductivity in mS X 0.667 (Geographical factor of area)
5.1.5 Acidity
Take 10 ml sample freshly boiled and cooled sample, add 0.05 ml of methyl red solution and mix, the resulting solution is not red.
Interpretation of result: If the solution is in red colour the sample is Acidic
5.1.6 Alkalinity
Take 10 ml sample freshly boiled and cooled sample, add 0.1 ml of bromothymol blue solution and mix.
Interpretation of result: If the solution is in blue colour the sample is Alkaline.
5.1.7 Ammonium
Take 20 ml sample add 1 ml of alkaline potassium mercuri-iodide solution and allow standing for 5 minutes. When vertically viewed the solution is not more intensely colored than a solution prepared at the same time by adding 1 ml of alkaline potassium mercuri-iodide solution to a solution containing 2.5 ml of dilute ammonium chloride solution and 7.5 ml of the liquid being examined.
5.1.8 Calcium & Magnesium
Take 100 ml sample add 2 ml of ammonia buffer pH 10.0, 50 mg of mordant black 11 mixture and 0.5 ml of 0.01 M disodium edetate, a pure blue color is produced.
5.1.9 Heavy Metals
In a glass-evaporating dish evaporate 150 ml of sample to 15 ml on a water bath.
Standard solution
Into a small Nessler Cylinder, pipette 10.0 ml of lead standard solution (1ppm Pb).
Test Solution
Pipette 12 ml into a small nessler cylinder.
Procedure
To the cylinder containing the standard solution add 2.0 ml of the test solution and mix. To each cylinder add 2 ml of acetate buffer pH 3.5, mix, add 1.2 ml of thioacetamide reagent, allow to stand for 2 minutes and view downwards over a white surface, the colour produced with the test solution is not more intense than that produced with the standard solution.
5.1.10 Chloride
Take 10 ml sample add 1 ml of 2 M nitric acid and 0.2 ml of 0.1 M silver nitrate, the appearance of the solution does not change for at least 15 minutes.
5.1.11 Nitrate
Take 5 ml sample in a test tube immersed in ice add 0.4 ml of a 10% w/v solution of Potassium chloride, 0.1 ml of diphenylamine solution and drop wise with shaking 5 ml of sulphuric acid. Transfer the tube to a water bath at 50°C to allow standing for 15 minutes. Any blue colour in the solution is not more intense than that in a solution prepared at the same time and in the same manner using a mixture of 5.5 ml of nitrate free water and 0.5 ml of nitrate standard solution (2 ppm NO3).
5.1.12 Sulphate
Take 10 ml sample add 0.1 ml of 2 M Hydrochloric acid and 0.1 ml of barium chloride solution. The appearance of the solution does not change for at least 1 hour.
5.1.13 Oxidisable substances
Take 100 ml sample add 10 ml of 1 M sulphuric acid and 0.1 ml of 0.02 M potassium permanganate and boil for 5 minutes, the solution should remain faintly pink.
5.1.14 Residue on evaporation
Evaporate 100 ml sample to dryness into hot plate and dry to a constant weight at 105°C. The residue weighs not more than 1 mg (0.001%).
Residue on evaporation: W?-Wj_x 100 (mg/L)
ml of solution taken
W1 : Weight of Evaporating dish
W2 : Weight of Evaporating dish + Residue
5.1.15 Total Organic Carbon
Analyse the sample for TOC in a calibrated TOC Analyser as per SOP.
5.1.15.1 Alert and Action limit for Total Organic Carbon of water system
S.No. Type of Water Alert Limit (ppb) Action Limit (ppb)
1 Purified water 300.0 500
2 Water for injection 250.0 500
3 Pure Steam 250.0 500
5.1.15.2 If the TOC results are above alert and action limit, follow the SOP. 5.1.16 Conductivity
Take the 100 ml sample in a suitable container, and stir the test sample by maintaining the temperature 25°C ± 1°C, measure the conductivity with the help of calibrated conductivity meter.
Temperature and the respective Conductivity. Temperature (°C) Conductivity |iS cm" 1
0 0.6
5 0.8
10 0.9
15 1.0
20 1.1
25 1.3
30 1.4
35 1.5
40 1.7
45 1.8
50 1.9
55 2.1
60 2.2
65 70 75 80 85 90 95 100
2.4
2.5 2.7 2.7 2.7 2.7 2.9 3.1
5.1.17 pH
Take 100 ml of sample and add 0.3 ml of saturated KCL solution. Mix the solution well and then measure the pH with the help of Calibrated pH meter.
NOTE : If results are observed out of limit in chemical analysis of water, follow the
5.2 Microbiological Analysis
Analyse the water samples for Microbiological analysis as per specifications.
5.2.1 Pour Plate Method
Dispense one ml of sample into two petridishes. Approximately add 15-20 ml of R2A / Plate count Agar into each petridishes. Cool the media approximately 45°C (feel on the dorsal side of the hand, it should be bearable). Cover the petridish, mix the sample with the agar by tilting or rotating the dishes and allow the contents to solidify at room temperature. Invert the petridishes and incubate at 30-35°C for 5 days. After incubation, examine the plates for growth, count the number of colonies and express the average for the two plates in terms of the number of colony forming units per ml.
Related: Incubation Conditions for Common Media used for Fungus and Bacteria
5.2.2 Membrane Filtration Technique
The procedure gives the use of a single disposable/ autoclaveable filtration funnels and filter holder, using MILLIFLEX system.
Preparation of the Filtration apparatus
Operate the Milliflex as per its SOP. Use sample size as specified in the specification for filtration through the 0.45 m filter.
After completion of filtration of sample, rinse the filter with 100 ml sterile water remove the filter using sterilised forceps and transfer it immediately to the previously prepared petri-dish with appropriate medium (R2A agar/Plate count agar).
Place the membrane filter carefully so that the air should not be trapped inside the filter, as this will prevent nutrient medium from reaching the entire membrane surface. Replace the lid. Incubate the plates in upright position (in case of filter) at 30-35°C for 5 days. Count the number of colonies on the membrane and express the results as per specification.
5.2.3 Bacterial Endotoxin Test
Refer the SOP for bacterial endotoxin test.
5.2.4 Pathogens
The sample shall be tested for the following four specific pathogens.
(A) Salmonella species
(B) Escherichia coli
(C) Pseudomonas aeruginosa
(D) Staphylococcus aureus
SOP.
Filter 100 ml of water sample through the 0.45 membrane filter fixed on Milliflex system. After filtration remove the filter aseptically and put it in 100 ml Soyabean Casein Digest Medium and incubate at 30-35°C for 24-48 hours.
From Soybean Casein Digest Medium, inoculate sterile 10 ml volumes of the following enrichment broths using 1 ml of inoculated broth
1. Selenite Cystine Broth for Salmonella species.
2. Tetrathinate Broth for Salmonella species.
3. MacConkey's Broth for Escherichia coli
4. Cetrimide Broth for Pseudomonas aeruginosa
5. Giolitti Cantoni Broth for Staphylococcus aureus (use sterile liquid paraffin for anaerobic conditions).
Incubate the tubes for 24-48 hours at 30-35°C.
(A) Test for Salmonella species:
If growth is present in Selenite Cystine Broth and Tetrathionate Broth, inoculate the following selective media plates and incubate at 30-35° C for 24-48 hours for presumptive
identification of the pathogen.
Medium Description of Colony
Xylose-Lysine Deoxycholate agar medium Red with or without Black Centre
Bismuth Sulphite agar medium Black or Green colonies
Brilliant Green agar Small, transparent, colourless or pink to white Opaque (frequently surrounded by pink to red zone)
Confirmatory Test
From the selective media plates pick the suspected colonies and go for confirmatory tests with the following biochemical/media and by gram reaction.
Individually transfer the suspected colony by first streaking the slope of slant, of Triple Sugar-Iron Agar with inoculating loop and then stabbing with inoculating straight wire well in the butt.
Incubate at 30-35° C for 24-48 hours
After incubation, examine the tube of Triple Sugar Iron Agar Medium for the presence of microbial growth and for the following Physical characteristics.
(a) Slant Surface : Alkaline reaction (red color)
(b) Butt : Acid reaction (yellow color) and/or gas bubble (with or without concomitant blackening).
If the butt, slant of Triple Sugar Iron Agar shows growth and physical characteristics confirming to the above descriptions the presence of Salmonella species is indicated.
(B) Test for Escherichia coli
If the inoculated MacConkey's broth tube shows acid and gas formation, inoculate the following selective media plates and incubate at 30-35°C for 24-48 hours for presumptive identification of the pathogen.
Medium Description of Colony
MacConkey's Agar Brick red may have surrounding zone of precipitated bile.
ISSN: 2181-3337 | SCIENTISTS.UZ
INTERNATIONAL SCIENTIFIC JOURNAL SCIENCE AND INNOVATION
ISSUE DEDICATED TO THE 80TH ANNIVERSARY OF THE ACADEMY OF SCIENCES OF THE REPUBLIC OF UZBEKISTAN
Eosin Methylene Blue Agar |Metallic sheen with dark grey colonies
Confirmatory Test
From the selective media plates pick the suspected colonies and go for confirmatory tests into the following bio-chemicals/media and by gram reaction.
Add 0.1 ml of the contents of the tube showing acid and gas to tubes containing 10 ml of peptone water
From peptone water tube perform Indole test as follow
Add 0.5 ml of Kovac's reagent to peptone water tube, allow to stand for one minute, if a red colour is produced in the reagent layer indole is present
The presence of acid and gas in MacConkey's broth, in peptone water and indole, indicates the presence of Escherichia coli.
Presence of Escherichia coli shall be confirmed by Gram staining (Gram-ve rods) and by streaking a loopful of the MacConkey's broth, with acid and gas production on plates of MacConkey Agar, and Levine Eosin Methylene Blue Agar.
Incubate the plates at 30-35°C for 24-48 hours.
If after incubation, plates shows colonies of following characteristics presence of Escherichia coli is confirmed.
MacConkey's Agar: Brick red colonies with or without surrounding zone of precipitates.
Levine Eosin Methylene blue Agar: Colonies with characteristic of metallic sheen under reflected light and blue-black appearance under transmitted light.
(C) Test for Pseudomonas aeruginosa
If the inoculated Cetrimide broth tube shows growth with greenish/bluish pigmentation, inoculate the following selective media plates and incubate at 30-35°C for 24-28 hours for presumptive identification of the pathogens.
Medium Description of Colony
Cetrimide Agar Greenish colonies, which exhibit a greenish fluorescence under ultra violet light.
Pseudomonas Agar (For Pyocyanin) Colourless to yellowish, yellowish under ultra violet light.
Pseudomonas Agar (For Fluorescein) Colourless to yellowish, yellowish under ultra violet light.
Confirmatory Test
From the selective media plates pick the suspected colonies and go for confirmatory tests Streak suspected colony on Pseudomonas Agar for Fluorescenin (PAF) Detection and Psedomonas Agar for Pyocyanin (PAP) Detection using inoculating loop. Incubate the plates in inverted condition at 30-35°C for 24-28 hours. Simultaneously inoculate the suspected colony in 100 ml of Soyabean casein digest medium and incubate at 41° to 43°C for 18 to 24 hours.
After incubation, examine the plates and tube of Soybean casein digest medium for the presence of microbial colonies of Gram-Negative rods exhibiting following characteristics. Pseudomonas Agar for fluorescenin detection: Colorless to yellowish fluorescence under ultra violet light. Pseudomonas Agar for Pyocyanin Detection: Greenish colonies, which exhibit a blue fluorescence under ultra violet light. Soybean casein digest medium: Growth occurs.
If colonies are found confirming to above descriptions, Oxidase test shall be performed to confirm identification as follow:
With the aid of an inoculating loop, transfer suspected colonies to strip or discs of filter paper impregnated with N, N-dimethyl-p-phenylenediamine dihydrochloride.
If a Pink-Purple colour is produced within five to ten seconds, the presence of Pseudomonas aeruginosa is confirmed.
(D) Test for Staphylococcus aureus
If growth is present in Giolitti Cantoni (G.C) broth, usually characterized by black settled growth at the bottom of the broth under anaerobic conditions, inoculate the following selective media plates and incubate at 30-35°C for 24-28 hours for presumptive identification of pathogen.
Medium Description of Colony
Mannitol Salt Agar Medium Yellow colonies with yellow zones
Vogel Johnson Agar Medium Black surrounded by yellow zone
Baird Parker Agar Medium Black, shiny, surrounded by clear zones of 2-5 mm
Confirmatory Test
From the selective media plates pick the suspected colonies and go for confirmatory tests
If colonies are found confirming to the above descriptions identification shall be performed by a coagulase test as follow.
With the aid of an inoculating loop, individually transfer suspected colonies to separate tubes containing 0.5 ml of mammalian plasma (preferably rabbit or horse).
Incubate in a water-bath / incubator at 370C for 3 to 24 hours, in parallel with positive control using known strain of Staphylococcus aureus and negative control using Plasma alone.
Examine after 3 hours and at suitable intervals thereafter for the presence of coagulation.
If coagulation in any degree is observed, the presence of Staphylococcus aureus is indicated. And perform the gram staining for the presence of gram Positive cocci.
5.2.5 Coli forms
Filter 100 ml of test sample and transfer the filter to M-Endo agar and incubate at 35°C for 22-24 hrs count colonies that are pink to dark red with a green metallic surface sheen, the sheen may vary from pinpoint to complete coverage of colony. Report the as number of Coliforms colonies per 100 ml.
5.2.6 After completion of testing prepare a test report.
5.2.7 If the counts obtained are above the limits specified below investigate the
results and take necessary actions as per SOP.
5.3 Alert and Action limit for TAMC of water system
S.No Type of water Alert limit Action limit
1 Raw water 300 500
2 Soft water 200 500
3 Potable water 150 500
4 Drinking water 100 500
5 Purified water 50 100
6 Water for injection (100 ml) 3 10
7 Pure steam (100 ml) 3 10
REFERENCES
1. Singh, V.P. Challenges in meeting water security and resilience. Water Int. 2017, 42, 349359. [Google Scholar] [CrossRef][Green Version]
2. Annin, P. The Great Lakes Water Wars; Island Press: Washington, DC, USA, 2006. [Google Scholar]
3. Elshafei, Y.; Sivapalan, M.; Tonts, M.; Hipsey, M.R. A prototype framework for models of socio-hydrology: Identification of key feedback loops and parameterisation approach. Hydrol. Earth Syst. Sci. 2014, 18, 2141-2166. [Google Scholar] [CrossRef][Green Version]
4. Carpenter, S.R.; Stanley, E.H.; Vander Zanden, M.J. State of the world's freshwater ecosystems: Physical, chemical, and biological changes. Annu. Rev. Env. Resour. 2011, 36, 75-99. [Google Scholar] [CrossRef][Green Version]
5. Montanari, A.; Young, G.; Savenije, H.; Hughes, D.; Wagener, T.; Ren, L.; Koutsoyiannis, D.; Cudennec, C.; Toth, E.; Grimaldi, S.; et al. Panta Rhei-Everything Flows: Change in hydrology and society-The IAHS Scientific Decade 2013-2022. Hydrol. Sci. J. 2013, 58, 1256-1275. [Google Scholar] [CrossRef]
6. Falkenmark, M. The greatest water problem: The inability to link environmental security, water security and food security. Int. J. Water Resour. Dev. 2001, 17, 539-554. [Google Scholar] [CrossRef]
7. Falkenmark, M. Freshwater as shared between society and ecosystems: From divided approaches to integrated challenges. Philos. Trans. R. Soc. B Biol. Sci. 2003, 358, 20372049. [Google Scholar] [CrossRef][Green Version]
8. Bangladesh Planning Commission; General Economics Division. Bangladesh Delta Plan 2100, Government of the People's Republic of Bangladesh; Main Report BDP2100; Base Line Report, Water Resources; Government of Bangladesh: Dhaka, Bangladesh, 2018; Volume 1. [Google Scholar]
9. Kausher, A.H.M. Progression of policy developments towards sustainable water management in Bangladesh. In Proceedings of the 2nd International Conference on Water and Environmental Engineering (iCWEE2019), Dhaka, Bangladesh, 19-22 January 2019. [Google Scholar]
10. BWDB. Bangladesher Nad-Nadi, 2nd ed.; Bangladesh Water Development Board: Dhaka, Bangladesh, 2011.
11. JRCB. Joint Rivers Commission Bangladesh. 2018. Available online: http://www.jrcb.gov.bd/ (accessed on 27 December 2020).
12. Hossain, M.A.R. An overview of fisheries sector of Bangladesh. Res. Agric. Livest. Fish 2014, 1, 109-126. [Google Scholar] [CrossRef]
13. WHO. Safely Managed Drinking Water—Thematic Report on Drinking Water; World Health Organization: Geneva, Switzerland, 2017; Available online: https://apps.who.int/iris/bitstream/handle/10665/325897/9789241565424-eng.pdf (accessed on 24 October 2021).
14. Parvez, A.K. Bacteriological quality of drinking water samples across Bangladesh. Arch. Clin. Microbiol. 2016, 7, 9. [Google Scholar]
15. Acharjee, M.; Rahman, F.; Rahman, F.; Jahan, F.; Noor, R. Bacterial Proliferation in Municipal Water Supplied in Mirpur Locality of Dhaka City, Bangladesh. J. Clean-Soil Air Water 2014, 42, 434-441. [Google Scholar] [CrossRef]
16. Flanagan, S.V.; Johnston, R.B.; Zheng, Y. Arsenic in tube well water in Bangladesh: Health and economic impacts and implications for arsenic mitigation. Bull. World Health Organ. 2012, 90, 839-846. [Google Scholar] [CrossRef]
ISSN: 2181-3337 | SCIENTISTS.UZ
INTERNATIONAL SCIENTIFIC JOURNAL SCIENCE AND INNOVATION
ISSUE DEDICATED TO THE 80TH ANNIVERSARY OF THE ACADEMY OF SCIENCES OF THE REPUBLIC OF UZBEKISTAN
17. UN-Water. UN-Water Country Briefs Bangladesh 2013. Available online: http://www.unwater.org/publications/un-water-country-briefs-zambia/# (accessed on 24 October 2021).
