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Biological test

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Bacteriological Examination of WaterObjectives• Carry out a presumptive test for the presence of coliform bacteria in a water sample• Determine the most probable number (MPN) of bacteria in a positive presumptive sample• Carry out a confirmed test to begin isolation of bacterial colonies• Carry out a completed test using a Gram stain and morphologyIntroductionWe consider fresh-water streams, lakes and ground water to be polluted when some conditionmakes the water unsafe for human recreation or consumption.We usually think of two forms ofpollution: toxic chemicals or pathogenic microorganisms. Probably the largest single source ofpotentially pathogenic microbes is animal feces (including human), which contains billions ofbacteria per gram. Although most intestinal microbes are non-pathogenic, some cause entericdisease. The organisms which cause typhoid fever (Salmonella typhi), cholera (Vibrio cholera),and bacterial dysentery (Shigella flexneri) are examples of enteric diseases caused by bacteria.In addition, some viral and protozoan pathogens are spread through water contaminated byfeces.How can we know if a water sample contains any of these pathogens?To test for eachorganism separately would be extremely costly and time-consuming. Therefore, a simple rule isfollowed: if a water sample contains any microorganism common to animal intestines, it shouldnot be consumed, because it may contain enteric pathogens.Water testing for microbiological safety rests on the ability of microbiologists to detect coliformbacteria.The word “coliform” refers to any bacterium that is likeEscherichia coliin the followingcharacteristics: 1) it is a small, gram-negative rod; 2) it does not contain spores; 3) it fermentslactose with the production of acid and gas; 4) it produces a green metallic sheen on EMB agar.E. coli, which is found in large numbers in the feces of all animals, lives longer in water thanmost intestinal pathogens do. Therefore, if noE. coliare present, there should be no intestinalpathogens present in the water sample. For this reason, testing for coliform organisms isperformed daily by municipal water departments and waste-water (sewage) treatment plants.The bacterial examination of water to ascertain its potability (suitability for drinking) has beenstandardized into three tests. The first, or presumptive test, is a screening test to sample waterfor the presence of coliform organisms. A series of lactose fermentation tubes are inoculatedwith the water sample. If the presumptive test is negative, no further testing is performed, andthe water source is considered microbiologically safe for drinking. If, however, any tube in theseries shows acid and gas, the water is considered unsafe and the confirmed test is performedon the tube displaying a positive reaction. The presumptive test is also designed to estimate theconcentration of coliform organisms, called the most probably number (MPN)in the watersample.The confirmed testis a second screening procedure in which a gram-negativeselective medium is used (like EMB). This also allows for the differentiation of coliform(producing a green metallic sheen) from non-coliform colonies. The completed test is performedon a typical, well-isolated colony to reaffirm gas production in lactose, and to determine themorphology and gram reaction of the isolate from a nutrient agar slant.Bacteriological Examination of WaterPage 1 of 10

Procedures:(1) Presumptive Test and MPNMaterials3 triple-strength lactose tubes with brom-thymol blue6 regular-strength lactose tubes with brom-thymol blueSterile water collection bottles for community sampling (or “doped” water samples)10 ml and 1 ml sterile pipets1. Collect approximately 50 ml of water to be tested (from pools, streams, ocean, etc.) oryou may use the “doped” samples provided in class.Record the source and date ofcommunity samples or the sample number of the “doped” sample to be tested.2. Vigorously shake the water sample to be tested by moving it 25 times through a 12-inch arch.3. Transfer 10 ml of the sample into each of the three triple-strength lactose tubes.Always use aseptic technique(Appendix 1) in the water inoculations and label thetubes with the amount of water sample tested.4. Transfer 1 ml of the sample into each of three regular strength lactose tubes. Usingthe same pipet, transfer 0.1 ml to each of the three remaining regular-strength lactosetubes.5. Incubate all tubes at 37oC until the next laboratory session.(2) Confirmed TestMaterials1 EMB agar plate1. Examine the tubes from the presumptive test and determine if any hasproduced an acid/gas reaction. If so, this is a positive presumptive test. If no gas ispresent in any of the Durham tubes, this is a negative presumptive test.2. Determine the number of tubes positive for acid/gas in each of the three volumecategories. Determine the MPN of your water sample by comparing these numbers tothe MPN Determination chart accompanying this exercise (Appendix 2). Complete thetable for the presumptive test as shown below and enter it into your laboratoryworksheet.3. From any tube showing 10% gas production or more, streak one loopful of the brothonto an EMB plate using the isolation streaking technique. Incubate the plate at 37oCuntil the next laboratory session.Bacteriological Examination of WaterPage 2 of 10

(3) Completed TestMaterials1 lactose tube with brom-thymol blue1 NA slantGram stain reagents1. Draw or describe your EMB plate in your laboratory worksheet. Carefully examine theplate, looking for well-isolated coliform colonies. Typically,E. colicolonies appear witha metallic green sheen on EMB. From one of these colonies, set up your completedtest by inoculating a lactose fermentation tube and a NA slant. Incubate them at 37oC.until the next laboratory session.2.After this incubation, check the lactose tube for acid and gas production. If no gas ispresent, this is a negative completed test.Prepare a Gram stain from the NA slant(Appendix 3).If the organism is a non-spore producing Gram negative rod and thelactose broth shows an acid/gas reaction, this is a positive completed test.Recordyour results in the table below.Include a drawing of your Gram stain.Results:Presumptive TestRecord the source of the water sample tested and the number of tubes in each category thatproduced acid/gas reactions.Determine MPN and record in the following format in yourlaboratory notebook.SampleSource10 ml tubes1.0 ml tubes0.1 ml tubesMPNOcean Water3 out of 31 out of 31 out of 375Confirmed TestDescribe or draw the EMB plate here:Bacteriological Examination of WaterPage 3 of 10

Completed TestRecord the results of your lactose broth and Gram stain below.Additionally, draw your Gramstain results below.Sample SourceGas Produced?Gram Stain ReactionSpores Present?Ocean WaterNoNoneNoDiscussion questions:1. Was your water sample potable (safe to drink)?2. Why do we look forE. coliin water?Why notSalmonella typhi?3. Another Gram negative rod that is frequently found in contaminated water samples isEnterobacter aerogenes. SinceE. aerogenesif found in the soil and on plants in addition tobeing present in fecal material, its presence in water does not always indicate fecalcontamination. What series of tests would differentiateE. colifromE. aerogenes?Bacteriological Examination of WaterPage 4 of 10

Water Bacteriology AbstractWrite a report regarding the safeness of your water sample.In it, provide your recommendationfor whether you would allow people to utilize the water in your collection area.Include the following in your abstract on your water sample collected:A brief introduction (including hypothesis begin tested),Description of methodsoDate and location where you collected your sample.Why this location wasselected.(Thursday Sep, 7, 2017) (San Clemente Beach, California, USA) thereason the location was selected because we wanted to test if ocean watercontains bacteriaoTypes of tests run on your samplePertinent resultsoResults of tests (MPN, Gram + or -, etc.).Discussion/conclusion - what do your results mean? (i.e. are there coliform bacteriapresent?)oIs the water safe for consumption, play, etc.oDiscuss the conditions that may have caused the results that you got.oIf you need to consume the water, what would your recommendation be?oIf you deem it unsafe, what recommendations would you offer before you allowpeople to use the water again?The Next page was left it contains the procedure.I left it to Help you to do the abstractBacteriological Examination of WaterPage 5 of 10

APPENDIX 1: Aseptic TransferThe word “aseptic” means without contamination. When cultured microorganisms are transferred fromone container to another, aseptic techniques are used to ensure that no contaminating organisms from theair or nearby surfaces are introduced. These techniques are designed to keep the cultures in a “pure” state,i.e., one that contains only one microbial species or strain. Aseptic practices also protect the culturehandler and others who may come in contact with the live cultures or equipment used in the process.Procedure:1.Clean the work area with tabletop disinfectant before starting.2.Practice transferring phantom “bacteria” between various empty culture tubes as follows:Hold two empty tubes in your left (or non-dominant) handHold the inoculating loop in your right (or dominant) handFlame the inoculating loop with a Bunsen burner and approximately 1 inch of the adjacentnichrome wire until it becomesbright orange.Quickly remove the caps from both empty tubes with the smallest fingers of your right hand whilestill holding the loop with your thumb and forefinger. Take care not to touch the opening of thecaps against the skin of your palm. Try to keep the open sides of the caps facing downward tominimize airborne contamination. Do not set the caps on the tabletop because airborne microbescan easily fall into the upturned caps.Pass the open necks of the uncovered tubes back and forth through the flame twice to incinerateany contaminating microbes that may be adhering to the opening.Carefully insert the sterilized inoculating loop into the “pure” culture tube and pantomimeremoving a very small amount of bacteria from the surface of the agar. Take care not to touch thesides of the tubes with the non-sterilized parts of the wire loop or handle. Work quickly and try tohold the tubes at a horizontal orientation to minimize airborne contamination.Pantomime introducing the bacteria into the new tubes and inoculating the media containedwithin.Flame the open necks of the glass tubes by passing them back and forth through the flame twice.Recap the tubes, taking care not to touch the opening of the tube or caps with your hands.Flame the inoculating loop to kill any remaining bacteria before setting the loop down on yourtabletop.1.Use caution and care when operating the Bunsen burners and hot plates. Make sure that paper towels,textbooks, alcohol, the gas hose, electric cords, and other lab apparatus are not close to the Bunsenburner flames.2.Tie back long hair to prevent it from contacting the Bunsen burner flame, hot plates, or fromcontaminating cultures.Bacteriological Examination of WaterPage 6 of 10

3.If you smell gas in the laboratory, check for a leak in the burner tubing or an open gas stopcock.Inform your instructor immediately!APPENDIX 2: MPN Determination from Multiple Tube TestMost Probable Numbers (MPN) Index for Various Combinations of Positive andNegative ResultsWhen Three 10-ml Portions, Three 1-ml Portions, and Three 0.1-ml Portions AreUsedNumber of tubes giving positivereaction out ofMPN Indexper 100 ml95% Confidence Limits3 of 10 mleach3 of 1 mleach3 of 0.1 mleachLowerUpper000<3<0.50013<0.590103<0.5131004<0.5201017121110712311111336120113362009136201143372101534421120789220214472212810150300234120301397130302641538031043721031175142303121203038032093153803211503044032221035470330240361,300331460712,4003321,1001504,800333>2,400Sources:Standard Methods for the Examination of Water and Wastewater, 13th Edition New York.American Public Health Association, 1971.Standard Methods for the Examination of Water and Wastewater,12th Edition New York.American Public Health Association, 1967, p. 608.Bacteriological Examination of WaterPage 7 of 10

Bacteriological Examination of WaterPage 8 of 10

APPENDIX 3: Gram StainingFrom:Laboratory Manual for Bio 15 – General Microbiology. Revised June 2016. J. Clark, M.Friedrich, E. Ininns, A. Sirulnik, A. Bandekar, and R. Wrightsman. Saddleback College,Department of Biological Sciences. Professor Monica Friedrich’s version.The Gram stain is named after the Danish physician who developed it, Hans Christian Gram. Gram wasworking with other doctors in Berlin to identify the cause of pneumonia. He was experimenting withdifferent methods to stain lung tissue to make the bacteria easier to see under the microscope. One methodhe developed did not stain all bacteria equally; some bacteria retained the first dye added and some thesecond dye. This staining method showed that there were two different types of bacteria causingpneumonia. This stain developed is now called the Gram stain and it identifies two large groups ofbacteria:Gram-positive and Gram-negative. The difference in staining outcome is due to differences inthe structure of the cell wall of these two types of bacteria. The Gram stain has been used as a key test inthe initial identification of bacteria ever since the late 1880’s.The Gram stain is a compound stain, in that more than one dye is used in the procedure. It is alsoconsidered a differential stain, because it allows the distinction between different types of bacteria basedon their cell wall structure. There are 4 steps to the Gram stain:1.Primary stain – Gram’s crystal violet (purple)2.Mordant – iodine3.Decolorizer – acetone-alcohol4.Counterstain or secondary stain – safranin (pink)The Gram stain differentiates between the two different cell wall structures that bacteria have. (Seechapter 4 in “Microbiology” by Tortora,et al.)Gram-positive cell walls contain multiple layers ofpeptidoglycan. Gram-negative cell walls contain a single layer of peptidoglycan and an additional, outerlipid membrane external to the peptidoglycan. Both stains used in the procedure are basic dyes that carry apositive charge in aqueous solutions.These dyes will bind to the outer layer of the cell, whether it isGram-positive or Gram-negative. Gram-negative bacteria will be decolorized more quickly than Gram-positive bacteria, because the acetone-alcohol decolorizer disrupts the lipid outer membrane in the Gram-negative cell wall, washing away the crystal violet. In contrast, in Gram-positive cells the thickerpeptidoglycan layer retains the crystal violet stain longer during the decolorizing step. Thus, after thedecolorizing step, Gram-positive cells remain purple, while Gram-negative cells are colorless. The finalstep is a secondary counterstain which is used to give color to decolorized bacteria, making them visibleunder the microscope. As a basic dye, safranin will give a pink color to all bacteria, however only Gram-negative bacteria will appear pink because the purple stain retained by the Gram-positive bacteriaoverpowers the pink color of the safranin stain. (See chapter 4 in “Microbiology” by Tortora,et al.)Theoutcome of Gram staining results in Gram-positive cells stained purple and Gram-negative cells stainedpinkish red.The Gram stain is the most commonly used stain in the microbiology laboratory so it will beimportant to learn how to perform this stain in an accurate, repeatable manner. There are several factorsthat can affect the outcome of the Gram stain. Errors in staining can result in false-positive staining (aGram-negative cell that is purple) or false-negative staining (a Gram-positive cell that is pink). Thefollowing table outlines factors that can affect Gram stain results.Bacteriological Examination of WaterPage 9 of 10

Table I.Troubleshooting the Gram-stain procedure.ProblemWhat HappensAppearanceToo much decolorizerG+ cells decolorizeFalse negative (G+ are pink)Too little decolorizerG– cells fail to decolorizeFalse positive (G– are purple)Water remains on slide afterthe first wash stepG+ cells stain weaklyFalse negative or mixedreaction (some G+ & G–)High cell density on the slideDifficult to decolorize evenly;G– cells fail to decolorizeFalse positive (G– are purple,or mixed reaction)Culture is old(cell wall is weak)G+ cell decolorizes easilyFalse negative (G+ are pink)Over heat-fixation(damages cell wall)G+ cell decolorizes easilyFalse negative (G+ are pink)Type of organism(ex.Mycobacterium)Does not retain the stain wellMixed reaction, some cellsappear pink other purple oreven spotted!Materials:Gram’s crystal violetBunsen burnerGram’s iodineInoculating loopAcetone-alcoholdH2O, squirt bottleSafranin4 clean microscope slidesBacterial culturesGram Stain Procedure:1.Make a smear with an appropriate amount of bacteria, air dry fully, and heat fix with flame.2.Flood with Gram’s crystal violetand stain for 1 minute. (primary stain)Rinse with dH2O, then tap the slide to remove excess waterTake care not to touch glass slide with dropper bottle3.Flood with Gram’s iodineand stain for 1 minute. (mordant stain)Rinse with dH2O, then tap the slide to remove excess water4.Carefully decolorize with acetone-alcoholfor 5 secondsusing a washing motion until the purplecolor stops coming off the slide.Rinse immediately with dH2O, then tap the slide to remove excess water5.Flood with safraninand stain for 1 minute. (counter stain)Rinse with dH2O, then tap the slide to remove excess water6.Gently pat dry between 2 paper towels, do not rub!7.Dispose of the stain and rinse water in the dish into the designated liquid waste container. Rinsethe dish and rack with tap water and put all stains away, beforegetting out your microscope.8.Observe under oil immersion, 1000x. Make sure to clean the objective lenses when you are done.Bacteriological Examination of WaterPage 10 of 10

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