Water microbiology

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Water microbiology

  1. 1. Water MicrobiologyPurposes What is water and water microbiology? Why water should be tested microbiologically? How water could be sampled ? How transported ?
  2. 2. Different names are given to waters various forms:1. according to state• Solid - ice• Liquid - water• Gaseous - water vapor2. levitating particles• clouds• Fog• mist3. according to occurrence:• Groundwater• Fresh water• Surface water• Mineral water - contains much minerals• Brackish water• Dead water - strange phenomenon which can occur when a layer of fresh or brackishwater rests on top of denser salt water, without the two layers mixing. It is dangerous forship traveling.• Seawater• Brine
  3. 3. 4. according to uses• tap water• bottled water• drinking water• purified water:distilled waterdouble distilled waterdeionized water5. according to other features• soft water – contains less minerals• hard water – from underground, contains more minerals• distilled water, double distilled water, deionized water - contains no minerals• Water of crystallization — water incorporated into crystalline structures• Hydrates — water bound into other chemical substances• heavy water – made from heavy atoms of hydrogen - deuterium. It is in nature innormal water in very low concentration. It was used in construction of first nuclearreactors.6. according to microbiology• drinking water• wastewater• stormwater or surface water
  4. 4. Water cycle:The water cycle (known scientifically as the hydrologic cycle) refers to the continuousexchange of water within the hydrosphere, between the atmosphere, soil water,surface water, groundwater, and plants.Water moves perpetually through each of these regions in the water cycle consisting offollowing transfer processes:1. evaporation from oceans and other water bodies into the air and transpiration fromland plants and animals into air.2. precipitation, from water vapor condensing from the air and falling to earth or ocean.3. runoff from the land usually reaching the sea.
  5. 5. Water:• Water is essential for the maintenance of all life on Earth.• It also acts as the vector for many diseases caused by bacteria,viruses, protozoa and worms.• For water to be regarded as potable, i.e. of a quality fit andsafe for drinking, it must be:1. free from pathogens.2. it must not contain any other noxious substances such aschemical hazards including pesticides, insecticides orherbicides, artificial fertilizers or heavy metal ions.3.should not have an unpleasant odor or taste.
  6. 6. What are water-borne diseases?1. Among the bacterial infections that are spread by water are:• cholera•enteric fevers•dysentery.2. Among the viruses are:•Hepatitis A•poliovirus cause infections after drinking contaminated water.3. Among the protozoa are:•Amoebic dysentery is caused by the protozoan Entamoeba histolytica and isspread either by drinking contaminated water or by eating food such as freshfruit, salad or raw vegetables that have been washed in contaminated water.• Other protozoal diseases such as those caused by Giardia intestinalis(Giardia lamblia)Balantidium coli and Cryptosporidium species are spread in a similar fashion.4. Among the helminthes are:Schistosomiasis, also known as bilharzia, is a water-borne infestation causedby worms of the genus Schistosoma.
  7. 7. Water microbiology assist in providing theanswers :• How do we know that the water used is safe?• Can we drink it?• Is a particular beach okay for swimming?• Can it be used for irrigation?The answers to these questions are of vital public health importance to all of us.The Water Microbiology field can assist in providing the answers.How can I confirm that the water is microbiologically safe to use for theintended purpose?
  8. 8. 1. Total Coliform (TC)2. Faecal Coliform (FC)3. Faecal Streptococci (FS)4. EnterococciThis is achieved by conducting standard microbiological tests to assess thesanitary quality of water. These tests are designed to determine thepresence/absence of the following indicator organisms:Indicator Organisms:The tests results are compared to standards or guideline values that have beendesigned to protect human health.
  9. 9. Why do we concentrate on testing for these indicatororganisms?• It is impossible to test for every known pathogenic organism on aroutine basis.• Indicator bacteria have been studied extensively. Their detectionand enumeration employ simpler and more economic test that canbe performed routinely.• The presence of such organisms in a water sample suggest thatthe water has been compromised by faecal contamination andthat pathogens may be present.•Indicator is generally isolated and identified easily in laboratory.•It is less cost effective in the lab.
  10. 10. Aquatic Facilities Microbiological Water Sampling Technique1. General Rules of SamplingTake extra care to avoid contaminating the sample container and water sample.Do Not:• Contaminate the bottle by touching the inside of the bottle.• Contaminate the bottle lid by touching the inside rim.• Put the bottle lid on the ground while sampling.• Rinse the bottle.• Transport aquatic facility water samples with other water samples, e.g. effluent ordrinking water.Always:• Collect microbiological samples before collecting other samples.• Label the bottle before sampling.• Discard damaged or contaminated bottles. If in doubt throw it out and take sample in anew bottle.• Wash your hands thoroughly before and collecting samples.
  11. 11. 2. Labeling :• Sender reference number• Site code• Point of Collection• (Aquatic Facility Name and pool (ie toddler’s pool)• Source (ie Pool outlet)• Date and time of collection• Transport temperature (4C or ambient)• Authority or Company Name
  12. 12. 1) A student creates a pendulum effect in order to castthe bucket out as far as possible.2) A student aims to retrieve the top surface of the watersample.3. Sampling Collection Procedure:
  13. 13. 4) A student checks to make sure that the water samplehas not been muddied during retrieval.3) The student uses his arm like a crane to retrieve thesample bucket without picking up any bottom sediment.
  14. 14. 4. Sample Transportation:• Temperature :Once water samples for bacteria are collected, they should be immediately stored withina chilled insulation container (esky) preferably at a temperature between 1°C and 4°C.To chill the samples/container, use freezer ice bricks if available, or loose ice.The chilled temperatures are used to prevent the multiplication of bacteria whichmay result in false bacterial counts. Cool and dark conditions should also bemaintained throughout transportation to the laboratory.• Time:1. the aim of delivering the samples to the laboratory as soon as possible, or within 6hours of commencing sampling, whilst keeping the sample bottle temperatures at 4°C±2°C.2. Under exceptional circumstances (regional locations), the sampling and transport timemay exceed 6 hours but should never exceed more than 24 hours.
  15. 15. 5. Submitting Samples :• Parameters:All water test reports list the water quality parameters that were tested. The list includesonly those you asked the laboratory to analyze or the lab recommended for your watersample.• The number of parameters can vary from just a few to dozens of tests.• Results:The most important information on your water test report are the actual results that thelaboratory found for your water sample.The result for each test should be compared to the drinking water standard:1. maximum contaminant level (MCL) for that parameter.2. Sometimes, the lab reports a water test result as ―ND‖ (Not Detected), which meansthe lab was unable to detect any of that contaminant with its equipment.3. Similarly, some results may have a less-than sign (<) in front of a number.• UnitsConcentrations of contaminants are usually measured in water by a unit of weight suchas milligrams per liter (mg/L), or by a number, for example, number of bacteria per100 milliliters of water (#/100 ml).• Standards:This allows for an easy comparison of your result with the safe or recommendedmaximum level for each test parameter.
  16. 16. 6. Comments:Some water testing laboratories will include a brief explanation of your water test results.• It is often list those contaminants that did not meet the drinking water standard.• Occasionally, these comments also describe the potential harmful effects ofcontaminants that exceed the standard and how to remove these contaminants from thewater.•Some laboratories, however, do not provide comments, so you need to review theresults yourself. Do not rely on the laboratory to point out important information.
  17. 17. Lab. Title:Plate counting (pour plate technique)The Hanging Drop Slide and Bacterial Motility
  18. 18. Measurements of bacteria1. Plate Count:a. Spread (Streak) Plateb. Pour Plate2. Direct Observation on Slides- Petroff-Hausser Chamber Slide3. Most Probable Number (MPN)4. Filtration (Membrane filter technique)
  19. 19. IndirectCountPour Plate
  20. 20. Indirect viablecounts (also calledplate counts)Pour plate methodAdvantages•Sensitive•Only count viable•Accurate
  21. 21. Viable cell counting: plate count or colony count (alreadycovered in the lab)
  22. 22. Counting colonies…•Diluting cell suspensions before plating: serial dilutions.
  23. 23. Petroff-Hausser Chamber Slide
  24. 24. Petroff-Hausser Counting ChamberMeasurement of Microbial Growth - Measurement of cellnumbers - Direct microscopic counts• Inexpensive• Relatively quick• Gives information about size and morphology
  25. 25. Direct microscopic countIn direct microscopic counting: 1) dead cells are not distinguished fromliving cells; 2) small cells are difficult to see under the microscope; 3)precision is difficult to achieve; 4) we need a phase contrast microscope; 5)not a good method for cell suspensions of low density.
  26. 26. There Are More Accurate Methods toDetermine Turbidity Levels
  27. 27. Materials per Student• microscope• culture of P. aerugenosa, Escherichia coli, bacillus• lens paper and lens cleaner• immersion oil• clean depression slides and coverslips• petroleum jelly (Vaseline)• inoculating loop• toothpicks• Bunsen burner
  28. 28. PrinciplesMany bacteria show no motion and are termed non-motile. However, in anaqueous environment, these same bacteria appear to be moving erratically.This erratic movement is due to Brownian movement.1. Brownian movement: results from the random motion of the watermolecules bombarding the bacteria and causing them to move (i.e.: vibrationof water molecules). Like Sarcina sp.2. Gliding motility: Helical-shaped spirochetes have axial fibrils (modifiedflagella that wrap around the bacterium) that form axial filaments. Thesespirochetes move in a corkscrew- and bending-type motion. (simply slideover moist surfaces in a form of gliding motion) Treponema pallidum3. Swimming motility: True motility (self-propulsion) has been recognized inother bacteria and involves several different mechanisms. Bacteria thatpossess flagella exhibit flagellar motion.4. Tumbling motility: Like in Listeria monocytogenesThe above types of motility or non motility can be observed over a long periodin a hanging drop slide. Hanging drop slides are also useful in observing thegeneral shape of living bacteria and the arrangement of bacterial cellswhen they associate together.
  29. 29. Flagellum arrangement
  30. 30. Procedure:1. With a toothpick, spread a small ring of Vaseline around the concavity of adepression slide. Do not use too much Vaseline.2. After thoroughly mixing one of the cultures, use the inoculating loop toaseptically place a small drop of one of the bacterial suspensions in the centerof a covers lip.3. Lower the depression slide, with the concavity facing down, onto the coverslip so that the drop protrudes into the center of the concavity of the slide. Pressgently to form a seal.4. Turn the hanging drop slide over and place on the stage of the microscopeso that the drop is over the light hole.5. Examine the drop by first locating its edge under low power and focusing onthe drop. Switch to the high-dry objective and then, using immersion oil, to the90 to 100× objective. In order to see the bacteria clearly, close the diaphragmas much as possible for increased contrast.Note bacterial shape, size, arrangement, and motility. Be careful to distinguishbetween motility and Brownian movement.6. Discard your covers lips and any contaminated slides in a container withdisinfectant solution.
  31. 31. Bacteria1. Aeromonas in Finished Water by Membrane Filtration2. Total Coliforms and Escherichia coli in Water by Membrane Filtration Using aSimultaneous Detection Technique3. Escherichia coli (E.coli) in Water by Membrane Filtration Using Modifiedmembrane-Thermotolerant Escherichia coli Agar4. Enterococci in Water by Membrane Filtration Using membrane-EnterococcusIndoxyl-ß-D-Glucoside Agar5. Enterococci in Water by Membrane Filtration Using membrane-Enterococcus-Esculin Iron Agar6. Escherichia coli (E. coli) in Water by Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar7. Improved Enumeration Methods for the Recreational Water QualityIndicators: Enterococci and Escherichia coli (March 2000)
  32. 32. •Direct measurements of microbial growth: total and viable counts.•Advantages and disadvantages.a) Direct microscopic countb) Viable cell countinga. Direct microscopic count:on samples dried on slides or on samples in liquid using growth chambers.
  33. 33. Standard Coliform Most ProbableNumber (MPN) Test&Presence-Absence Coliform Test
  34. 34. Bacteriological Analysis of WaterThe principal means through which pathogenic microorganisms reach water supplies is fecalcontamination. The method for bacteriologic examination of water is designed to provide anindex of fecal contamination. Pathogenic microorganisms do not necessarily multiply inwater, and therefore they may be present in small numbers that are difficult to demonstrate inculture. Escherichia coli, other coliform bacteria, and enterococci, however, are not onlyabundant in feces but also usually multiply in water, so that they are present in large, readilydetectable numbers if fecal contamination has occurred. Thus, culture demonstration of E. coliand enterococci in water indicates a fecal source of the organisms. In water from sourcessubjected to purification processes (such as reservoirs), the presence of E. coli or enterococcimay mean that chlorination is inadequate. By bacteriologic standards, water for drinking (i.e.,potable water) should be free of coliform and enterococci.The term “coliform,” which refers to lactose-fermenting gram negative enteric bacilli, is nowobsolete except in sanitary bacteriology.
  35. 35.  There are two principal groups of coliform bacteria: the fecal coliform(which includes the bacterium Escherichia coli has been most studied)and the total coliform group, which includes the fecal coliform andconsists mainly of species from the genera Citrobacter, Enterobacter,Escherichia, and Klebsiella. The former are exclusively fecal in origin,whereas the latter, although commonly found in feces, also occurnaturally in Soils and waters. Only the fecal coliform is definitive indicators offecal pollution.In water bacteriology the total coliform are regarded as "presumptive"indicators of pollution but in Wastewater bacteriology, this group isconsiderably less importance, because many of them are non-fecal in originand they can multiply in the environment under suitable conditions,especially in hot climates. Thus their presence or numbers may not necessarilyrelate to either the occurrence or degree of fecal pollution
  36. 36. 1. Total Coliform (TC)2. Faecal Coliform (FC)3. Faecal Streptococci (FS)4. EnterococciThese tests are designed to determine the presence/absence of the followingindicator organisms:Indicator Organisms:The tests results are compared to standards or guideline values that have beendesigned to protect human health.
  37. 37. Principles• The number of total coliform (Enterobacter, Klebsiella, Citrobacter, Escherichia) in awater sample can be determined by a statistical estimation called the most probablenumber (MPN) test.• This test involves a multiple series of Durham fermentation tubes and is divided intothree parts: the presumptive, confirmed, and completed tests.A presumptive test for coliform is performed by inoculating a sample of water intotubes of lactose broth containing Durham tubes. After 24 to 48 hours of incubation at35°C, the tubes are examined for the presence of acid and gas as an indication of lactosefermentation. Other than coliform, few organisms found in water can ferment lactoserapidly with production of gas.Gaseous fermentation of lactose within 24 to 48 hours provides presumptive evidenceof the presence of coliform. The test must be confirmed, however, to exclude thepossibility that another type of organism provided the positive lactose result.
  38. 38. Confirmed testIs done by plating a sample of the positive lactose broth culture into a lactosebroth again and same result which may provide confirmation of thepresumptive test.Inoculating in peptone water at 42 °C or brilliant green lactose bile broth.Completed testGrowing on differential agar medium. Eosin methylene blue (EMB) agar isfrequently used. Coliform colonies ferment the lactose of EMB and consequentlyhave a deep purple color with a coppery, metallic sheenRequires inoculation of another lactose broth and an agar slant with isolatedcolonies from EMB. Gas formation in the lactose broth and microscopicdemonstration of gram-negative, non-spore former rods on the agar slant areconsidered complete evidence of the presence of coliform organisms in theoriginal sample.
  39. 39. Learning ObjectivesEach student should be able to1. Determine the presence of coliform bacteria in a water sample2. Obtain some index as to the possible number of coliform bacteria present in the watersample being tested3. List and explain each step (presumptive, confirmed, completed) in the multiple tubetechnique for determining coliforms in the water sample4. Perform the presence-absence coliform test MPNColiform Guide• Citrobacter• Escherichia• Enterobacter• Klebsiella
  40. 40. Materials per Group of Students:• (10) 10-ml single-strength lactose broth (SSLB) in Durham fermentation tubes (lauryl tryptosebroth or lactose broth)• (5 )10-ml double-strength lactose broth (DSLB) in Durham fermentation tubes• 125-ml water sample (each group of students should bring in their own from a possiblecontaminated water system) at room temperature. (If the water samples are collected early, theyshould be refrigerated until analyzed.)• petri plate containing Levine’s EMB agar (or LES Endo agar)• tryptic agar slant• 3 tubes brilliant green lactose bile broth (brilliant green bile broth 2%) or 2 tubes lauryl tryptosebroth containing Durham tubes• 1 sterile 10-ml pipette with pipettor• 2 sterile 1-ml pipettes• wax pencil• test-tube rack• 35°C incubator• inoculating loop and needle• Bunsen burner
  41. 41. Procedure for the MPN TestFirst Period•Presumptive Test1. Mix the bottle of water to be tested 25 times. Inoculate five of the double-strengthlactose (or lauryl tryptose) broth tubes with 10 ml of the water sample; five single-strength tubes with 1 ml of the water sample; and five single-strength tubes with 0.1 mlof the water sample. Carefully mix the contents of each tube without spilling any of thebroth by rolling the tubes between the palms of your hands. Using the wax pencil, labelall tubes with your (name, date, and the amount of water added).2. Incubate the three sets of tubes for 24 to 48 hours at 35°C.3. Observe after 24 ±2 and 48 ±3 hours. The presence of gas in any tube after 24 hoursis a positive presumptive test. The formation of gas during the next 24 hours is adoubtful test. The absence of gas after 48 hours is a negative test.4. Determine the number of coliforms per 100 ml of water sample. For example, if gaswas present in all five of the 10-ml tubes, only in one of the 1-ml series, and none in the0.1-ml series, your test results would read 5–1–0. Table indicates that the MPN for thisreading would be 33 coliforms per 100 ml of water sample.
  42. 42. Most Probable Number (MPN)These are gas-filledtubes, an indicationof bacterial growth(fermentation).Looking forsufficient dilutionthat ~half of tubesshow growth.Reciprocal ofthat dilution bacterialdensity.
  43. 43. • Second PeriodConfirmed Test1. Record your results of the presumptive test in the report for exercise 46.2. Using an inoculating loop, from the tube that has the highest dilution of water sampleand shows gas production transfer one loopful of culture to the brilliant-green lactosebile broth tube. Incubate for 48 ± 3 hours at 35°C. The formation of gas at any timewithin 48 hours constitutes a positive confirmed test.
  44. 44. Third PeriodCompleted Test1. Record your results of the confirmed test in the report.2. From the positive brilliant green lactose bile broth tube, streak a LES Endo orLevine’s EMB plate.3. Incubate the plate inverted for 24 hours at 35°C.4. If coliforms are present, select a well-isolated colony and inoculate a single-strength,brilliant green lactose bile broth tube and streak a nutrient agar slant.5. Gram stain any bacteria found on the slant.6. The formation of gas in the lactose broth and the demonstration of gram-negative,nonsporing rods in the agar culture is a satisfactorily completed test revealing thepresence of coliforms and indicating that the water sample was polluted. This is apositive completed test.
  45. 45. Questions:•Write scientific interpretations for your results, and final comments?•Why we depended on gas production rather than acid formation?•What is the bacteriological standard for potable water?•Why the bacteriological analysis of water depends on recognition ofcoliform and enterococci instead of direct detection of pathogenicbacteria?•Why the positive presumptive tests of water must be confirmed?•List at least three waterborne infectious diseases (bacterial name).•Compare between (water samples), and (which method was better amongthem).•Compare between (Fecal coliform and Total coliform)
  46. 46. Principles for Membrane Filter Technique:• This technique involves filtering a known volume (100 ml for drinking watersamples) of water through a special sterile filter.• These filters are made of nitrocellulose acetate or polycarbonate, are 150 μthick, and have 0.45 μ diameter pores. A grid pattern is typically printed onthese filter disks in order to facilitate colony counting.• When the water sample is filtered, bacteria (larger than 0.45 μ) in the sampleare trapped on the surface of the filter. The filter is then carefully removed,placed in a sterile Petri plate on a pad saturated with a liquid or agar-basedmedium, and incubated for 20 to 22 hours at 35°C.• it is assumed that each bacterium trapped on the filter will then grow into aseparate colony. By counting the colonies one can directly determine thenumber of bacteria in the water sample that was filtered.• Fecal streptococci are the Lancefield Group D streptococci that occur in thefeces of humans and other warm-blooded mammals.
  47. 47. • Total coliform colonies will be pink to dark red in color and will appear tohave a golden green metallic sheen. Fecal coliform colonies will appear blue,and fecal streptococci colonies will appear light pink and flat, or dark red.• In determining total coliform, the amount of water filtered should be enough toresult in the growth of about 20 to 80 colonies and no more than a total of 200bacterial colonies of all types. About 50 to 200 ml of unpolluted water is oftenadequate for such bacterial counts. Polluted water may contain so manycoliform that it will be necessary to dilute 1 ml or less of sample with about 50ml of sterile water. This is done in order to provide enough volume for uniformbacterial dispersion across the filter surface, in addition to providing anappropriately low coliform count.• Coliform density is expressed in terms of the number of coliform per 100 ml ofwater and is calculated according to the following formula:
  48. 48. • The number of coliform should be given to show two significant figuresper 100 ml.•The standard set for potable (drinking) water is a limit of 1 coliform per 100 mland an action limit of 4 coliform per 100 ml. An action limit means that thewater company or other provider must take immediate action to remedy theproblem(s) that is/are responsible for the presence of coliform.• From positive fecal coliform and fecal streptococci test results, one can befairly certain that the water pollution is from which fecal source. However, inorder to determine whether the fecal source is from human or animal feces,Public Health authorities rely on a ratio expressed as:
  49. 49. 1. Those samples showing a higher fecal coliform count than fecal strep count arelikely to contain wastes from humans. In most cases, the FC/FS ratio will be greaterthan 2. When the ratio is equal to or greater than 4, one can be assured that thepollution is from human fecal material.2. When it shows a higher fecal strep count than fecal coliform count, it is most likelythat the pollution is from animal origin.3. If ratios fall in between two and four, estimates must be made as to how close theratio is to either the human or animal value. The following table shows some typicalFC/FS ratios:
  50. 50. :Total Coliform Test1. Using the sterile forceps, place a sterile absorbent pad into each of threepetri plates. With the wax pencil, label these plates with your name, date,and TCT (total coliform test).2. Add 2.0 ml of M-Endo broth MF (or m-ColiBlue 24 broth) to the surface ofeach pad.3. Filter 1, 5, and 15 ml of the water sample, and add the membranesrespectively to each plate.4. Incubate the plates at 35°C for 22 to 24 hours.5. Count only those colonies that are pink to dark red with a metallic sheen. Usea plate containing 20 to 80 colonies and no more than 200 of all types ofcolonies. If the m-ColiBlue 24 broth is used, blue to purple colonies indicateE. coli. The total coliform count is given by the sum of red and blue colonies.6. Record your results in the report.
  51. 51. Fecal Coliform Test:1. Using the sterile forceps, aseptically insert sterile pads into three snap-lidpetri plates. Using the wax pencil, label these plates with your name, date,and FCT (fecal coliform test).2. Add 2.0 ml of M-FC broth to the surface of each pad.3. Filter 1, 5, and 15 ml of the water sample, and add the membranesrespectively to each plate.4. Snap the lids of the Petri plates, seal them with waterproof tape, and placethem in a Whirl pack bag.5. Incubate the plates in a 44.5° ±0.2°C water bath for 22 to 26 hours. Makesure the bags are beneath the surface.6. Count only blue-colored colonies on a plate containing 20 to 60 fecalcoliform colonies.7. Record your results in the report.
  52. 52. Fecal Streptococcus Test:1. Aseptically insert a sterile absorbent pad into each of three Petri plates.Using the wax pencil, label these plates with your name, date, and FST(fecal streptococcus test).2. Add 2 ml of KF streptococcus agar; allow the agar to cool.3. Filter water sample volumes of 1, 5, and 15 ml as per previous test. Place themembranes in the labeled Petri plates.4. Incubate these plates for 48 hours at 35°C.5. Count only those colonies that are light pink and flat, or smooth dark redones with or without pink margins. Use the plate containing 20 to 100colonies.6. Record the number of colonies in the report.
  53. 53. Membrane Filter Technique.(a) Total Coliform test: Total coliform on a membrane filter (M-Endo MF broth medium).Notice the dark red to purple colonies with a metallic sheen.(b) Fecal Coliform Test: Fecal coliform on a membrane filter (M-FC broth medium).Notice the blue-colored colonies.
  54. 54. (c) Fecal Streptococcus Test: Fecal streptococci on KF streptococcus agar. Notice thelight pink colonies.(d) HACH m-ColiBlue 24 Broth: The sum of red and blue colonies indicates totalcoliform, while the blue specifies E. coli.
  55. 55. HINTS AND PRECAUTIONS:(1)The broth media should be freshly prepared on the day of the exercise.(2) Water should not be used that is high in turbidity or contains a lot of algae.Coliform density is always expressed in terms of a 100-ml water sample. Ifthe water sample is diluted, the number of colonies must still be calculatedfor a 100-ml sample. Similarly, if less than 100 ml of water is filtered, thecoliform density must still be expressed in terms of 100 ml.(3) If the water sample will not be tested immediately, store it in the refrigeratorto prevent extra microbial growth.(4) When using the membrane filter technique, the 19th edition of StandardMethods for the Examination of Water and Wastewater suggests that thefollowing sample volumes be used for total coliform tests:
  56. 56. General Procedure for the Membrane Filter Apparatus
  57. 57. The membrane filtration procedure
  58. 58. Lab. Title:Detection and enumerationofBacteriophages in Wastewater
  59. 59.  Are inactive molecules outside of the host celland active only inside host cells. Basic structure consists of protein shell (capsid)surrounding nucleic acid core. Nucleic acid can be either DNA or RNA but notboth. Nucleic acid can be double-stranded DNA,single-stranded DNA, single-stranded RNA, ordouble-stranded RNA.
  60. 60. Poxvirus, DNA virus Mumps virus RNA Herpesvirus DNARhabdovirus HIV (AIDS)RNA RNABacteriophage Papillomavirusa DNA virusAdenovirusa DNA virus
  61. 61. Phage Multiplication Lysogeny a form of bacteriophage replication in which the viralgenome is integrated into that of the host and is replicated alongwith it. Lytic cycle a process of viral replication involving the bursting ofthe host cell and release of new viral particles. Viruses which infect bacteria are known as bacteriophage, andthose which infect coliform bacteria are called coliphage. The phages of coliform bacteria are found anywhere coliformbacteria are found. Concentrations of human viruses in raw sewage range from (103–107 /L). Concentration of coliphage in raw sewage ranges from (10to 100 /ml).
  62. 62.  There are many potential applications of bacteriophages asenvironmental indicators. These include use as indicators ofsewage contamination, efficiency of water and wastewatertreatment, and survival of enteric viruses and bacteria inthe environment. The use of bacteriophages as indicators of thepresence and behavior of enteric bacteria and animal viruseshas always been attractive because of the ease of detectionand low cost associated with phage assays. In addition, they can be quantified in environmental sampleswithin 24 hours as compared to days or weeks for entericviruses. Coliphage have been the most commonly used in thiscontext although other bacteriophages and cyanophage (i.e.,viruses of blue-green algae) have also been studied. Much ofthe justification for the study of coliphage behavior in naturehas been to gain insight into the fate of human pathogenicenteric viruses. As a result, more is probably known about theecology of coliphage than any other bacteriophage group.
  63. 63.  Coliphage in water are assayed by addition of a sample to soft oroverlay agar along with a culture of E. coli in the log phase of growth.The phages attach to the bacterial cell and lyses the bacteria. Thebacteria produce a confluent lawn of growth except for areas wherethe phage has grown and lyses the bacteria. These resulting clearareas are known as plaques. A soft agar overlay is used to enhancethe physical spread of the viruses between bacterial cells. To obtain optimal plaque formation it is important that the hostbacteria are in the log phase of growth. This ensures that all thephage attach to live bacteria and produce progeny. This requires thata culture of host bacteria is prepared each day that an assay isperformed. Usually, a culture is incubated the day before the assay toobtain a culture in the stationary phase. This is used to inoculate abroth which is incubated to obtain enough host bacteria in the logphase for the assay (this usually requires 2–3 hours of incubation in ashaking water bath at 35 to 37°C).
  64. 64. MANY VIRUSES CAUSE DISEASE IN ANIMALS Viruses that infect animal cells cause diseases. RNA viruses have RNA as their genetic material andresponsible for flu, cold, measles, mumps, AIDS, polio. DNA viruses have DNA as their genetic material and causehepatitis, chicken pox, herpes.
  65. 65. VIRAL DNA MAY BECOME PART OF HOST CHROMOSOME Viruses are packaged genes-can only reproduce insidecells. Lytic cycle-viral replication cycle resulting in therelease of new viruses by Lysis of host cell. Lysogeny cycle-a bacteriophage replication cycle inwhich the viral genome is incorporated into thebacterial host chromosome and the host cell is not lysesunless the viral genome leaves the host chromosome.
  66. 66. VIRAL LIFECYCLES
  67. 67. The Lysogeny state in bacteria.- A bacterial DNA molecule canaccept and insert viral DNAmolecules at specific sites on itsgenome.-This additional viral DNA isduplicated along with theregular genome and canprovide adaptivecharacteristics forthe host bacterium
  68. 68. Precautions: When collecting sewage samples always wear latexgloves. Raw sewage is a potent source of bacterial,viral, and fungal pathogens. Raw sewage rich in bacteriophage is best collected atmunicipal sewage treatment plants. Usually,collection is made through manhole access. As emphasized in previous points of this manual,using pipette inhibited!
  69. 69. 1. DO: Bacteria must be in the log phase of growth for optimal phage plaque formation. This meansthat a new culture must be grown under a defined set of conditions (temperature, shaking ornon-shaking) each time. Be sure to shake the tube containing overlay agar to get as much out of the tube as possible.2. DO NOT: Do not allow the bacteria and phage to set in the water bath too long (no more than 1–2minutes) or they will be killed by the heat. Do not allow the molten agar to set in the 45°C water bath for more than 1–2 hours as thewater evaporates causing lumps of agar to form.Potential hazards: Remember if you are handling sewage, it may contain pathogens. Handle with care.
  70. 70. Water pollutionWaste Water Treatment&purification system
  71. 71. Pollution categories1. Point source pollutionrefers to contaminants that enter a waterway through adiscrete conveyance, such as a pipe or ditch. Examples ofsources in this category include discharges from a sewagetreatment plant, a factory, or a city storm drain.2. Non-point source pollutionrefers to diffuse contamination that does not originate from asingle discrete source. As rainfall runs over the surface ofroofs and the ground, it may pick up various contaminantsincluding soil particles and other sediment, heavy metals,organic compounds, animal waste, and oil and grease.Some jurisdictions require storm water to receive somelevel of treatment before being discharged directly intowaterways.
  72. 72. Causes of water pollutionThe specific contaminants leading to pollution in water include:1. Pathogens Coliform bacteria are a commonly-used bacterial indicatorof water pollution, although not an actual cause of disease.Other microorganisms sometimes found in surface waterswhich have caused human health problems include: Cryptosporidium parvum Giardia lamblia Salmonella Novovirus and other viruses Parasitic worms (helminthes).
  73. 73.  Water used for drinking and cooking should be free ofpathogenic (disease causing) microorganisms that cause suchillnesses as typhoid fever, dysentery, cholera, andgastroenteritis. Whether a person contacts these diseasesfrom water depends on:Type of pathogenNumber of organisms in the water (density)Strength of the organism (virulence)Volume of water ingestedSusceptibility of the individual.Purification of drinking water containing pathogenicmicroorganisms requires specific treatment called disinfection.
  74. 74. 2. Chemical and other contaminants:a. Organic water pollutants include:• Detergents• Disinfection by-products such as chloroform• Food processing waste.• Insecticides and herbicides.• Petroleum hydrocarbons: fuels (gasoline, fuel oil) and lubricants (motor oil).• Volatile organic compounds (VOCs), such as industrial solvents, from improperstorage.b. Inorganic water pollutants include: Acidity caused by industrial discharges (especially sulfur dioxide). Ammonia from food processing waste. Chemical waste as industrial by-products. Fertilizers containing nutrients--nitrates and phosphates--which are found in stormwater runoff from agriculture, as well as commercial and residential use. Heavy metals from motor vehicles.
  75. 75. 3. Physical contaminants:Thermal pollution: Thermal pollution is the rise or fall inthe temperature of a natural body of water caused byhuman influence. A common cause of thermal pollution isthe use of water as a coolant for industrial manufacturers.Macroscopic pollution--large visible items polluting thewater--may be termed "floatables":Trash (e.g. paper, plastic, or food waste)Shipwrecks, large derelict ships
  76. 76.  According to a 2007 World Health Organization report, 1.1billion people lack access to an improved drinking watersupply. 88% of the 4 billion annual cases of diarrheal disease areattributed to unsafe water and inadequate sanitation andhygiene, and 1.8 million people die from diarrheal diseaseseach year. The WHO estimates that 94% of these diarrheal cases arepreventable through modifications to the environment,including access to safe water. Simple techniques for treatingwater at home, such as chlorination, filters, and solardisinfection, and storing it in safe containers could save ahuge number of lives each year.
  77. 77. Sewage treatmentIs the process of removing contaminants from wastewater. It includes physical,chemical and biological processes to remove physical, chemical and biologicalcontaminants.Sampling: Sampling of water for physical or chemical testing can be done byseveral methods, depending on the accuracy needed and the characteristics ofthe contaminant.1. Physical testing:Temperature, solids concentration and turbidity.2. Chemical testing:Analytical chemistry. Many published test methods are available for both organicand inorganic compounds. Frequently-used methods include pH,biochemical oxygen demand (BOD), chemical oxygen demand (COD),nutrients (nitrate and phosphorus compounds), metals (including copper,zinc, cadmium, lead and mercury), oil and grease, total petroleumhydrocarbons (TPH), and pesticides.3- Biological testing:plant, animal, and/or microbial indicators
  78. 78. Treatment steps1. Primary treatment:Can physically remove 20-30% of the BOD that is present inparticulate form, in this treatment particulate material isremoved by screening, precipitation of small particulates byaddition of alum and other coagulation agents, and settling intanks. The resulting solid material is usually called (Sludge).2. Secondary treatment:is used after primary treatment for the biological removal ofdissolved organic matter, about 90-95% of the BOD and manybacterial pathogens are removed by this process. Under aerobiccondition, dissolved organic matter will be transformed intoadditional microbial biomass plus carbon dioxide. Minerals inwater also may be tied up in microbial biomass. When microbialgrowth is completed, under ideal conditions the microorganismswill be aggregate and form a stable floc structure.
  79. 79. 3. Tertiary treatmentThe purpose of tertiary treatment is to provide a final treatment stage to raise the effluentquality before it is discharged to the receiving environment (sea, river, lake, ground, etc.).More than one tertiary treatment process may be used at any treatment plant. Ifdisinfection is practiced, it is always the final process. It is also called "effluent polishing".FiltrationSand filtration removes much of the residual suspended matter. Filtration over activatedcarbon removes residual toxins.DisinfectionThe purpose of disinfection in the treatment of wastewater is to substantially reduce thenumber of microorganisms in the water to be discharged back into the environment.The effectiveness of disinfection depends on the quality of the water being treated (e.g.,cloudiness, pH, etc.), the type of disinfection being used, the disinfectant dosage(concentration and time), and other environmental variables. Cloudy water will betreated less successfully since solid matter can shield organisms, especially fromultraviolet light or if contact times are low. Generally, short contact times, low doses andhigh flows all militate against effective disinfection. Common methods of disinfectioninclude ozone, chlorine, or ultraviolet light. Chloramines, which is used for drinkingwater, is not used in wastewater treatment because of its persistence.
  80. 80. Water purification system:ScreeningCoagulationSedimentationFiltrationDisinfection
  81. 81. Sources of water1. Groundwater2. Upland lakes and reservoirs3. Rivers, canals and low land reservoirs4. Atmospheric water generation5. Rainwater harvesting or fog collection6. Desalination of seawater by distillation or reverseosmosis
  82. 82. Purification steps1. ScreeningRemoving of large particle or macroscopic contaminant suchas bottle and paper …etc.2. Coagulation: In rapid mixerFlocculation: is a process which clarifies the water. Clarifyingmeans removing any turbidity or color so that the water isclear and colorless.Coagulants / flocculating agents that may include:1. Aluminum sulphate.2.PolyDADMAC is an artificially produced polymer
  83. 83. 3. Sedimentation:Waters exiting the flocculation basin may enter the sedimentationbasin, also called a clarifier or settling basin.
  84. 84. It is a large tank with slow flow, allowing floc to settle to the bottom. The sedimentationbasin is best located close to the flocculation basin so the transit between does notpermit settlement or floc break up.
  85. 85. 4. Filtration:After separating most floc, the water is filtered as the final step to remove remaining suspendedparticles and unsettled floc.
  86. 86. 5. Disinfection (Chlorination) :Disinfection is accomplished both by filtering out harmful microbes and also by addingdisinfectant chemicals in the last step in purifying drinking water. Water is disinfected to killany pathogens which pass through the filters. Possible pathogens include viruses, bacteria,including Escherichia coli, Campylobacter and Shigella, and protozoa, including Giardialamblia and other cryptosporidia.
  87. 87. Aim of lecture What is disinfectant? Types of disinfectant Mechanism of each method What is the health effects of its bye-product How can MIC be determined? How differentiate between MIC and MBC? How determine exposure time of disinfectant ?
  88. 88.  Disinfection mechanismo Cell wall corrosion in the cells of microorganisms.o Changes in cell permeability.o Protoplasm or enzyme activity (because of a structuralchange in enzymes).o Change in essential structure of cell (DNA)These disturbances in cell activity cause microorganisms to:I. Unable to multiply. This will cause the microorganisms todie out.II. Oxidizing disinfectants also demolish organic matter inthe water, causing a lack of nutrients.
  89. 89.  Chemical disinfectant- Chlorine (Cl2), Chlorine dioxide (ClO2), Hypo chlorite (OCl-)- Ozone (O3)- Halogens: bromine (Br2), iodine (I)- Bromine chloride (BrCl)- Metals: copper (Cu2+), silver (Ag+)- Potassium permanganate (KMn4O)- Fenols, Alcohols- Soaps and detergents- Hydrogen peroxide- Several acids and bases Physical disinfectant- Ultraviolet light (UV)- Electronic radiation- Gamma rays- Sounds- Heat
  90. 90. I. ChlorinationIn chlorination, chlorine gas, sodium or calcium hypochlorite is added towater. A minimum reaction period of 20 minutes is also required foreffective water disinfection.Why is Chlorine Added to Tap Water? Chlorination is effective against many pathogenic bacteria, but atnormal dosage rates it does not kill all viruses, cysts, or worms.When combined with filtration, chlorination is an excellent way todisinfect drinking water supplies.Factors affecting chlorine efficiency: Interaction between chlorine andthe microorganisms results in an effective disinfection process: Contact time varies with chlorine concentration. The type of pathogens present and dose pH Temperature of the water.
  91. 91. The Benefits of Chlorine: Potent Germicide Taste and Odor ControlChlorine disinfectants reduce many disagreeable tastes and odors. Chlorineoxidizes is naturally occurring substances such as foul-smelling algaesecretions, sulfides and odors from decaying vegetation. Chemical ControlChlorine disinfectants destroy hydrogen sulfide (which has a rotten egg odor)and remove ammonia and other nitrogenous compounds that haveunpleasant tastes . They also help to remove iron and manganese from rawwater. Biological Growth ControlChlorine disinfectants eliminate slime bacteria, molds and algae thatcommonly grow in water supply
  92. 92. How Chlorine Kills Pathogen: Upon adding chlorine to water, two chemical species, knowntogether as “free chlorine,” are formed. These species,hypochlorous acid (HOCl, electrically neutral) andhypochlorite ion (OCl-, electrically negative), behave verydifferently. Hypochlorous acid is not only more reactive than thehypochlorite ion, but is also a stronger disinfectant and oxidant.The ratio of hypochlorous acid to hypochlorite ion in water isdetermined by the pH. At low pH (higher acidity), hypochlorous acid dominates. High pH hypochlorite ion dominates.
  93. 93. 2- Chlorine Dioxide:Chlorine Dioxide is an chemical which is used for waterdisinfection, which replaces chlorine in more andmore applications due to its multiple advantages: Its disinfection force is stronger and independentupon water’s pH value. Due to its specific chemistry,no by-products can develop. The much longer half-life affords better depot actionin treated water. In opposition to chlorine, Chlorine Dioxide is able toremove biofilm in pipe systems and tanks to abolishgrowth of Legionella.
  94. 94. What are the health effects of chlorine by-products?ChloroformBromodichloromethaneChlorodibromomethaneChloroacetic acidDichloroacetic acidTrichloroacetic acidDichloroacetonitrile
  95. 95. 3- Ozonisation Ozone is unstable gas comprising of three oxygen atoms, the gaswill readily degrade back to oxygen, and during this transition afree oxygen atom, or radical is form. Ozone has a negativecharge, and upon reaction, the particles are neutralized and willprecipitate. Ozone has greater disinfection effectiveness against bacteriaand viruses compared to chlorination.1. Oxidizing properties2. Reduce the concentration of iron, manganese, sulfur3. Reduce and eliminate taste and odor problems. Ozone is unstable, and it will degrade over a time frame rangingfrom a few seconds to 30 minutes. The rate of degradation is afunction of water chemistry, pH and water temperature.
  96. 96. How Ozone Kills Pathogen: The free oxygen radical is highly reactive and shortlived, under normal conditions it will only survive for milliseconds, and during this time frame it will oxidize virtuallyany chemical species. For example it will oxidize iron, manganese and mayother heavy metals, it will crack the carbon doublebond of organic molecules such as dissolved proteins. Itwill also oxidize the proteins in the cell wall ofbacteria, the cilia of protozoa, the shell of virus. Infact ozone has 7 times oxidizing capacity of freechlorine, also does not produce any toxic residuals.
  97. 97. 4. UV radiation disinfection mechanisms is based on a physical phenomenon whereby short wave UVradiation acts on the genetic material (DNA) of the microorganismsand the viruses, destroying them rapidly without producing any majorphysical or chemical changes in the treated water. UV inactivation is thought to occur as a result of the directabsorption by the microorganism of the UV radiation, bringing aboutan intracellular photochemical reaction that change the biochemicalstructure of the molecules (probably of the nucleic acids) that areessential to the microorganism’s survival. It has been shown thatirrespective of the duration and intensity of the dosage, theexpending of the same total energy will result in the same degree ofdisinfection. The ultraviolet radiation system often include activated carbonfilter to remove metals and particulates. Effectiveness of an ultravioletradiation system depends on the intensity of the lamp. Theminimum dose of UV light to inactivate bacteria is 38 mWs/cm2 set bythe NSF International.
  98. 98. Properties of ultraviolet radiationUV wavelengths are very similar to those of sunlight. The most importantparameters of UV radiation relating to water disinfection are: Wavelength: The germicidal portion is between 240 and 280 nm(nanometers) with maximum disinfecting efficiency existing at close to 260nm. Condition of the water Intensity of radiation Type of microorganisms Exposure time Municipal potable water supplies are usually chlorinated to provide a residualconcentration of 0.5 to 2.0 ppm. Here, in Kurdistan, the recommendedconcentrations for water disinfection are 0.7 ppm and 1.7 ppm (in the case ofepidemic water born disease).
  99. 99. 1) Estimation of MIC1. Place two sets of nine sterile tubes in a rack and label them set forChlorine (Cl2) and set for Chlorine dioxide (ClO2).2. With a 5-ml pipette add 2 ml of sterile broth to each tube.3. Add 2 ml of the Chlorine (Cl2) and Chlorine dioxide (ClO2) tothe first tube of each sets of. Discard the pipette. Theconcentration of chlorine and chlorine dioxide in the first tube is(2ppm and 1.5ppm) respectively.4. Take a fresh pipette, introduce it into the first tube (chlorine:2ppm and Chlorine dioxide: 1.5 ppm), mix the contentsthoroughly, and transfer 2 ml from this tube into the second tube(chlorine: 1ppm and Chlorine dioxide: 0.75ppm). Discard thepipette.5. With a fresh pipette, mix the contents of the second tube andtransfer 2 ml to the third tube (chlorine: 0.5ppm and Chlorinedioxide: 0.38ppm). Discard the pipette.6. Continue the dilution process through tube number 7. Theeighth and ninth tubes receive no chlorine and chlorine dioxide.
  100. 100. 7. After the contents of the seventh tube are mixed, discard 2 ml of broth so thatthe final volume in all tubes is 2 ml.8. From the plate culture of E. coli prepare a suspension of the organism in 5 ml of(chlorine free distilled water) equivalent to a McFarland 0.5 standard.9. With a fresh pipette, mix the contents of the tube well. Add 0.1 ml of this E. colisuspension to the chlorine and chlorine dioxide containing broth tubes 1through 7 and to the growth control tube.10. Shake the rack gently to mix the tube contents and place the tubes in theincubator for 18 to 24 hours.11. All sets were read visually and MIC values were recorded as the lowestconcentration of the chlorine and chlorine dioxide treatments that had novisible turbidity depending on positive control and negative control test tubes.12. MBC (Minimum bactericidal Concentration) was determined by transferring0.1ml of MIC test tubes and spread on Mueller-Hinton agar. After incubationtime MBC was recorded for each of samples (sewage and artificiallycontaminated water) as a lowest concentration of chlorine and chlorine dioxidethat had bactericidal activity if growth not obtained on the agar plate (MIC isMBC), however if bacterial growth noticed on the agar plate it means that theMIC is not MBC and the treatment was with bacteriostatic activity.
  101. 101. 2) The effect of recommended concentration of Chlorine andChlorine dioxide (XINIX) water disinfectants on the testedisolates with different contact time:To evaluate the antibacterial activity for recommendedconcentration of Chlorine dioxide (XINIX) and chlorinedisinfectants to the normal and contaminated water, weprepared two set of artificially contaminated sterilizedchlorine free water by two different bacterial cell density(CFU/ml) depending on (0.5 McFarland Standard ) foreach tested bacterium (E. coli, Salmonella sp.) then eachof chlorine and Chlorine dioxide (XINIX) product wereadded separately to different contaminated wateraccording to recommended concentration in the standardsheet for both normal and contaminated water. The testedwater (contains: Bacterium inoculums+ recommendedconcentration for each of chlorine and XINIX) wascultivated on Muller Hinton Agar at (5 min) intervals till 1hour and incubated for 24 hours.
  102. 102. Escherichia coli
  103. 103. Aim General characters of Enterobacteriaceae Type of fermentation Lactose and non- lactose fermenter Laboratory diagnosis of Escherichia coli Culture media Biochemical tests
  104. 104. General Characteristics: Gram negative bacilli, Facultative anaerobic over 40genera. Non spore former, some of them non-motile andother motile by peritrichous flagella. Habitat (Colon of human and other warm bloodedanimals). Antigenic structure: All have somatic antigen (O-Ag) Motile genera have flagellar antigen (H-Ag) Capsular former like Klebsiella & Salmonella are with (K-Ag and Vi-Ag) respectively. Pili antigen (P- and S-Ag).
  105. 105.  Enterobacteriaceae are characterized by: Catalase positive, Oxidase negative. All member of this family are able to reduceNitrate(NO3) to Nitrite (NO2) All are able to degrades sugar (glucose) bymeans Embedn Meyerhof pathway and cleavepyrovic acid to yield formic acid in formic acidfermentation, end product (Mixed acids endproduct and Butanediol or acetoin end product)from this fermentation are distinguish by(MR=methyl red , and VP=Voges proskauer).
  106. 106. Fermentation pathway1- The majority carryout (mixed acid fermentation) (Aceticacid, lactic acid, succinic acid, formic acid and ethanol)distinguish by (Methyl red test (+ve) result) :Escherichia coli2- (Butanediol fermntation) the major product are(Butanediol, ethanol and CO2) distinguish by (Vogesproskauer test +ve result):Enterobacter sp.Serratia sp.Erwinia sp.Klebsiella sp. Citrobacter sp
  107. 107.  Lactose fermentation useful for distinguish morepathogenic from less pathogenic or non pathogenicgenera:DistinctionofPotentialEntericpathogensbylactose fermentationLactosefermenter(+ve) Lactosenon-fermenter(-ve)E.coli Shigella Non-motileNoH2SproducerKlebsiella YersiniaEnterobacterCitrobacter Proteus MotileH2SproducerSerratia Salmonella
  108. 108. Culture media for Enterobacteriaceae:1- MacConkey agar & Deoxycholate citrate bile salt agar:Both are (Selective and Differential) How? Selective:inhibit all Gram-positive bacteria, and differential betweenlactose fomenter (appears with pink colonies color) andNon-lactose fomenter (appears with colorless colonies)2- Eosin Methylene blue (EMB):E. coli appears green metallic sheen while other pathogenic generaare colorless colonies.3- S.S. agar: (Salmonella-Shigella agar):Salmonella appears as colorless colonies with black center whileShigella with colorless colonies without black center. Otherslactose fermenter genera are with pink colonies color.
  109. 109. MacConkey agar EMB DCA
  110. 110. Some important medically important genera in the familyEnterobacteriaceae especially those which cause waterborne diseases are:a. Lactose fermenter Enterobacteriaceae:1. Escherichia coli:Five strains categories of pathogenic E. coli are recognized: Enterotoxigenic (ETEC). Enteroinvasive or "Shigella-like" (EIEC) Enteropathogenic (EPEC). Enterohemorrhagic (EHEC). Eteroaggregative (EAEC).Laboratory diagnosis: Colonial morphology: lactose fermenter, Small pink colonies onMacConkey agar, S.S. agar, Deoxycholate citrate bile salt agar whilecolorless on Hektoen enteric agar, EMB (eosin-methylene blue appearswith green metallic sheen. IMViC test and Kligler iron agar KIA
  111. 111. Lactose fermenter Non – Lactose fermenteron Hektoen Enteric agarYellow –orange color Green colony
  112. 112. Biochemical tests Tryptophan hydrolyses (Indole)
  113. 113. Methyl red Voges- Proskauer testsMR VPMixed acid Fermn. Butanediol fermn.
  114. 114. Citrate utilization Sodium citrate as carbon source
  115. 115. Kligler Iron agar KIALactose and glucose fermn.Deamination
  116. 116. b. Non- lactose fermenter Enterobacteriaceae:1.Salmonella sp.:2. Shigella sp.
  117. 117. Vibrio cholerae
  118. 118. Purpose of Lab. General info. Of V. cholerae Laboratory diagnosis Enrichment media Selective media Biochemical test Serological test
  119. 119. General characters Gram-negative, facultative anaerobe, straight or curvedrods bacilli, motile by means of a single polar flagellum(Monotrichous), with 2 chromosomes. Vibrio are typically marine organisms; most species require2-3% NaCl or a sea water base for optimal growth. Vibrio are one of the most common organisms in surfacewaters of the world. They occur in both marine andfreshwater habitats and in associations with aquaticanimals. Most species are Oxidase-positive Can ferments glucose, sucrose, and mannitol
  120. 120. General characters Modes of transmissions (fecal contamination of waters,food for human & animal like shellfish and shrimp). Infectious does: Water (infectious dose = 109), Food (infectious dose = 103) Person-to-person Most frequent causative cholera: El Tor biotype of O1 V.cholera serotype Ogawa. Divided into two types according to (O-Ag) in the cell wall. Non O1- group: cause sporadic disease or non pathogen. O1-group: cause epidemic disease, and with two biotypes(Based on biochemical reaction):
  121. 121. Pathogenic species disease1. V. cholera Cholera2. V. parahaemolyticus (saltfriend)Diarrhea associated with eating raw or improperlycocked seafood3. V. vulnivicus (Halophile) Cellulites especially in shellfish handlersPathogenic Vibrio
  122. 122. Classification scheme:
  123. 123. Holding or transport media.1Venkataraman - ramakrishnan (VR) medium:20g Sea Salt Powde5g Peptone dissolved in 1L of distilled water.2. Cary-Blair medium: This most widely-used carrying media. This is a buffered solutionof sodium chloride, sodium thioglycollate, disodium phosphate and calcium chlorideat pH 8.4.3. Autoclaved sea wateEnrichment media1. Alkaline peptone water at pH 8.62. Monsurs taurocholate tellurite peptone water at pH 9.2In epidemic area: Clinical judgment enough.In pandemic area: detection require the following media and methods :
  124. 124. 1- Using Culture media:A- Non-selective.1Alkaline bile salt agar (BSA): The colonies are very similar to those on nutrientagar..2Alkaline meat Extract Agar (MEA) smooth, opaque, and cream colored.B- Selective media1. MacConkey agar: Colorless colonies because non- lactose fermenter.2. Hektoen Entric agar : green color appearance.3. Monsurs gelatin Tauro cholate trypticase tellurite agar (GTTA) medium: Choleravibrios produce small translucent colonies with a greyish black centre with halo.citrate,thiosulphate,containsmediumThis.mediumusedwidelymostlyThis:TCBS.4bile salts and sucrose. Cholera vibrios produce flat 2-3 mm in diameter, yellownucleated colonies.
  125. 125. HEA MAcC. Agar
  126. 126. GTTA TCBS
  127. 127. 2. Direct microscopy. Microscopy is preferred only after enrichment, as this process reveals thecharacteristic motility of Vibrios and its inhibition by appropriate antiserum.3. Biochemical test:A- OxidaseB- T.S.I. (Triple Sugar Iron) gar: (slant A/ A Butt) without gas and H2S.C- Citrate, Ornithine, Mannitol fermentation positive.
  128. 128. 4. Serological methods:a. Agglutination test: Diagnosis can be confirmed as wellas serotype was done by agglutination with specific sera.Agglutination of bacterium by polyvalent O1 and non O1-antisera.b. Titer: By detecting a rise of antibody titer in acute andconvalescent phase sera.

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