V] · The presence of total coliform bacteria, including Klebsiella, Escherichia, Serratia,...

DOCUMENT RESUME

ED 204 572 CE 029.495

TITLE Performing Analyses for Waterborne Bacteria. Module13. vocational Education Training in EnvironmentalHealth Sciences.

INSTITUTION Consumer Dynamics Inc., Rockville, Rd.SPONS AGENCY Office of Vocational and Adult Education (ED),

waShington, D.C.PUB' DATE (813CONTRACT 300-80-0088NOTE 36p.: For related documents see CE 029 482-507.AVAILABLE FROM National Technical Information Service, U.S. Dept.

Commerce 5285 Port Royal Rd., Springfield. VA22161.

EDR9 PRICE MF01/PCO2 Plus Postage.DESCRIPTORS Competency Based Edudation: Educational Equipment:

*.Environmental Education: *Environmental Technicians;Health Conditions:Learning ACtivitiesI *MeaSurementTechniques: programed Instructional Materials:Health: *Sanitation: Tetts: Vocational Education'

IDENTIFIERS *Bacteria: Ehvironmental'Health:, *Water Quality,

ABSTRACTThis module, one of 25,o7Orocational educatiOn

training for careers in environmental lealth-occupationsc containsself - instructional materials on performing analyses for waterbornebacteria. Following guidelines for students instructors end an-ihtroductio*.that explains What'the studentilllearnare three1.esiOns: (Wnaming, sterllizing.andasSembiing thecOmponent partsof a membrane filter testilni.: (2) suctioningawater-sample'through:1C-teibrane filter, :using aseptic'techniques4 and preparing themembrane filter,for-incubatiot: and, :(3) determiDinuthe number oftotal'coliform Organitis in a water-Sample, Each ledton- contains .

objectives`,. recommended Methods and locations for ,practice,perforMance criteria,. equipmentand4upOlieS toiperforva task,detailedstep-bp-step-instrUttions for learnitO'i task, andPerfPrmance exercises.TWO performance tests cover preparing watersamples forybacteriOlogicalantly4s:and deteriining the number;. ofm1Cro-organisms in a-water sample (CT)

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Vocational Education Training in Environmental Health Sciences

PerformingAnalyses forWaterborne Bacteria

Module 13U S. DEPARTMENT OF HEALTH,

EDUCATION & WELFARENATIONAL INSTITUTE OF

EDUCATION

THIS DOCUMENT HAS BEEN REPRO-DUCED EXACTLY AS RECEIVED FROMTHE PERSON or ORGANIZATION ORIGIN-ATING IT POI, '13 OF VIEW OR OPINIONSSTATED DO NUT NECESSARILY REPRESENT OFFICIAL NATIONAL INSTITUTE OFEDUCATION POSITION OR POLICY

U.S. DEPARTMENT OF EDUCATION, Office of Vocational and Adult Education

2

FOREWORD

The Curriculum and Instruction Branch of the Office of Vocationaland Adult Education, U.S. Department of Education, identified aneed to improve the training opportunities for vocational educationstudents interested in pursuing careers in environmental health.To fulfill that need, Consumer Dynamics, Inc., a Rockville,Maryland, based company, was awarded the contract to developperformance-oriented, competency-based modules in the environmentalhealth sciences.

PERFORMING ANALYSES FOR WATERBORNE BACTERIA is one of the modulesin the series, "Vocational Education Training in EnvironmentalHealth Sciences." The module content is based on selectedmaterials in the environmental health field. The module isintended to supplement existing course materials.

CONTENTS

FOREWORD

USING THESE SELF-INSTRUCTION MATERIALS 1

Guidelines For Students 1

Guidelines For Instructors 2

INTRODUCTION 4

Background 4

What You Will Learn 5

LESSON ONE 6

Objective 6

Where And How To Practice 6

How Well You Must Do 6

Things You Need 6

Getting There--Steps 8Exercises 12

LESSON TWO 14

Objective 14



Things You Need 14

Getting There--Steps ,15Exercises 22

LESSON THREE 23

Objective 23



Things You Need 22

Getting There--Steps 24

Exercises 27

PERFORMANCE TEST 29Preparing Water Samples for Bacteriological Analysis 29Determining The Number of Micro-organisms In A Water

Sample 30

REFERENCES 32

A iii

USING THESE SELF-INSTRUCTION MATERIALS

This ec '-instruction learning package or module is designed toallc students and instructors flexibility of use. Althoughpr ntended for use in existing training programs, themo( be used by anyone interested in learning new skills orpe old ones. Therefore, two sets of guidelines are

e set addressed to students and the other setaddrel instructors. First, find out how you, the student,should L materials in this book.

GUIDELINES FOR STUDENTS

Take the When you pick up this book and work through it,Performarr_ your goal will not be a letter grade or a highTest as F., score on an exam. Instead, you will work topretest. develop skills that you can measure. You will

not have to worry about how well someone else isdoing. Before you start work on this module, youshould, first, find out if you have sufficientskills to start training by reading through thesection called PERFORMANCE TEST. If you thinkyou can do all or most of the items in this test,ask your instructor to obtain the necessaryequipment and supplies. You should have had acourse in high school biology, or have gained theequivalent knowledge through on-the-job training;know how to use laboratory pipets, and havelearned how to use a stereo microscope.

Work on partsyou need topractice.

Work straightthrough eachlesson in theorder pre-sented.

If you do everything well, according to thecriteria in the Performance Test guidelines, youwill not need to spend time working on thismodule. If after taking the Performance Test youdiscover there are parts you need to practice,follow the key to each item in FOR FURTHER STUDY.

Should you decide to completely work through thismodule, begin with the INTRODUCTION and gostraight through each of the three lessons. Thelesson begins with the OBJECTIVE of the training.Follow the instruction for each part in the orderpresented. Practice each step in a lesson untilyou can do it according to the criteria statedfor the step. At the end of a lesson, do theEXERCISES. When there are audiovisuals listed atthe end of a lesson, ask your instructor for helpin obtaining them.

1


Take the Finally, after you have mastered all of thePerformance exercises in each lesson, ask your instructorTest as a to watch you do each item in the Performanceposttest. Test. The items in the Performance Test are

intended for use as a posttest to evaluate thequality of your performance. Turn now to thePerformance Test.

GUIDELINES FOR INSTRUCTORS

Approach The approach of these materials is to provide thestudent with the opportunity to learn skills forusing membrane filter filtration equipment, andfor determining the number of indicator organismsin a standard sample volume. The equipment youhave available may be somewhat different fromthat presented in the lessons. If such is thecase, you may need to write supplementaryinstructions to point out the equipment differ-ences. The skills tested on the Performance Testare designed for use with any make and model ofinstrumentation.

Independent Students can work independently and at their ownStudy pace. Depending on the time frame you set for

completing each lesson, you may want to start agroup off in each lesson with a demonstration andinformal presentation.

As a Labora-tory Workbook

GeneralITEiTTUEtions

2

Alternatively, you may choose to use this moduleas a laboratory workbook in a structured labora-tory session. With this option, you may allowstudents greater access to your assistance, espe-cially in watching them perform the pre- andposttest portions of the training.

Read through each lesson to anticipate what equip-ment and supplies you will need to make availablefor students to use. Also, order any audio-visuals or reading materials you think may pre-sent a complementary perspective to the trainingin this module. Use the items in the PerformanceTest as the minimum requirements for gaugingsuccessful completion of the training.


Specific Students are required to use sterile, buffered,Instructions distilled water. The following instructions from

NTOTC* are provided to aid in the preparation ofthat water:

*See References.

1. Prepare stock solution of potassium dihy-drogen phosphate (02PO4) by dissolving34.0 g of this chemical in 500 ml ofdistilled water and adjusting its pH to 7.2with 1 N Na0H. Dilute to 1 liter in avolumetric flask.

2. Prepare stock solution of magnesium sulfate(MgSO4.7H20) by dissolving 50 g of thischemical in 500-600 ml of distilled waterand, after complete dissolving, bring thefinal volume to 1 liter in a volumetric flask.

3. Prepare working solution of dilution water byadding 1.25 ml of the potassium dihydrogenphosphate stock solution and 5 ml of themagnesium sulfate stock solution to eachliter of distilled water to be used in thepreparation of dilution water.

4. Sterilize the buffered water in 1-liter quan-tities. Pour the water into a 1-liter Erlen-meyer flask and stopper with a cotton plug.Autoclave for 15..minutes at 121° C.

3

INTRODUCTION

BACKGROUND

Even before it was known that specific micro-organisms in watercause certain diseases, scientists believed water transferreddisease. Microbiologists who study disease causes found that fecalcoliform bacteria, normally present in human intestines, are amongthe micro-organisms that cause cholera, typhoid fever, anddysentery.

The presence of total coliform bacteria, including Klebsiella,Escherichia, Serratia, EnviHia, and Enterobacteria are indicatorsof water quality. But the tests for total coliform bacteria notonly indicate the presence of fecal coliform from the intestines ofwarm-blooded animals but also other coliforms that live in soil.Consequently, total coliform testing is very limited in tracinghuman disease sources. For this reason, only fecal coliform aretested where nonpotable (nondrinkable) water sources are used forrecreational swimming or bathing. Bacteriological testing fortotal coliform organisms is routinely performed in evaluating thequality of potable water supplies. Therefore, the focus of LessonThree of this module is on total coliform testing; fecal coliformtesting is also included as part of an exercise in this lesson.

There are several methods for performing a microbiologicalexamination for total coliform in drinking water. You will learnthe membrane filter technique, which involves suctioning a standardquantity of water through a flexible porous filter. The filter isplaced on an absorbent pad containing nutrients on which thebacteria feed during incubation at a constant temperature:. The

filter can be viewed with the naked eye or with a microscope ifthere are only a few colonies present; however, if there are many,use of a stereo microscope is recommended. The results of thistest indicate the presence of total coliforms (fecal plus nonfecalforms), and their numbers are expressed as bacterial colonies.

4

INTRODUCTION

WHAT YOU WILL LEARN

When you finish working through the steps and exercises in thismodule, you will be able to perform an examination of drinkingwater for the presence of indicator bacteria (coliform).

Using a portable water quality field test kit, you will learn howto perform this function in three lessons:

o Lesson One

You will be able to name, sterilize, and assemble the componentparts of a membrane filter test unit.

o Lesson Two

. You will be able to suction a water sample through a membranefilter, using aseptic techniques, and prepare the membranefilter for incubation.

o Lesson Three

You will be able to determine the number of total coliformorganisms in a water sample.

L'5

LESSON ONE

OBJECTIVE

You will be able to name, sterilize, and assemble the componentparts of the membrane filter test unit.

WHERE AND HOW TO PRACTICE

In the microbiological testing of water, it is important that it becarried out as aseptically (free from micro-organisms) as pos-sible. You should practice this lesson on a workbench that hasbeen thoroughly cleaned end has room enough to sprerd out thevarious components of the filtration unit. The specific testingprocedure and equipment you will be using are designed for fielduse. Under actual conditions you may be required to perform thesetests on the tailgate of a station wagon or van. It is importantthat you learn to minimize any potential sources of contamination.

HOW WELL YOU MUST DO

You must be able to accurately name all the parts of the filtrationunit and disassemble, sterilize, and assemble the filtration unitin 20 minutes.

THINGS YOU NEED

You will need the following equipment:

o a portable water laboratory, such as the Millipore ModelXX63-001-50 or equivalent,* containing at a minimum:

- a filtration unit composed of a funnel, membrane filterholding assembly, and a receiving flask

- a hand-operated or battery-powered pump

- an alcohol lamp

- plastic tubing with adaptors

- sterile, buffered, distilled water, 1,000-ml minimum (see"Guidelines for Instructors")

- stainless steel forceps

*Use of a particular brand of equipment is not intended tc be anendorsement of the product by the U.S. Department of Education.

6

10

INTRODUCTION

- sterile, packed petri dishes, absorbent pads, membranefilter

- Endo broth, nutrient media or equivalent, suitable forculturing total coliform

- sterilized stainless steel cup or glass graduated cylinder.

o methanol for sterilizing the filter holder and forceps

o matches.

Instruction 1: Before proceeding with Lesson One, plug in yourincubator and let it reach the appropriate incubation temperaturefor the organism you are culturing.

Instruction 2: Have your instructor demonstrate the propertechnique for flaming or sterilizing the forceps.

Instruction 3: Now turn to the next page and begin work on LessonOne, "Getting There--Steps."

11 7

LESSON ONE

GETTING THERE--STEPS

STEP 1

Open the portable water lab.Identify the major componentsof the lab: water filtrationunit (1); syringe or hand-operated pump (2) for drawingwater through the filtrationunit; portable incubator(3) for incubating membranefilters in the field; anda supply of membrane fil-ters (4) , petri dishes (5) ,

and nutrient media broth (6).This equipment is suitablefor both laboratory and fieldtesting.

STEP 2

Locate the filtration unitand remove it from the labkit. The filtration unitis composed of a funnel (1)and a membrane filter holdertwist-lock collar (2), en-closed by a stainless steelhydrosol (receiver flask)(3); suction is appliedthrough plastic tubing at theside of the membrane holdingassembly (4). Remove thehydrosol by inserting thebutt end of the forceps be-tween the hydrosol and thefilter holder. Place thehydrosol on the workbench.The funnel and filter holderare shown locked together in

(5).

8

KEY POINT 1

Identify the major components ofthe portable water lab.

KEY POINT 2

(3)

(5)

Use the butt end of the forcepsto pry open the stainless steelfilter holder.

LESSON ONE

STEP 3 KEY POINT 3

The funnel is secured tothe membrane filter holderby a twist-lock collar (1).When tightening or releasingthe twist lock, firmly holdthe sides of the funnel withone hand and twist the collarcounterclockwise to releasethe funnel from the filterholder. Unscrew the twistlock and remove the funnel.Examine the depression (2)in the holder where a mem-brane can be placed; thenreassemble the funnel tothe filter holder base bytwisting the collar clock-wise. Remove the funnel by unscrewing

the twist lock.

STEP 4 KEY POINT 4

Before a water sample canbe filtered, the funnel andfilter holder must be steri-lized. Carefully pour one-half of a capful of metha-nol on the asbestos ring (1)that surrounds the top ofthe filter holder base.Do not spill the alcoholdown the side or onto thetable; flaming may start afire in your work area.

13

(1)

Pour one-half of a capful ofmethanol on the entire asbestosring as you rotate the entireunit slowly.

LESSON ONE

STEP 5

Quickly but carefully lightthe methanol. Do not leanover the unit when lightingit. Wait 30 seconds and thencarefully replace the hydro-sol over the funnel and burn-ing asbestos ring. After 2or 3 minutes, carefully re-move the hydrosol and rinseout the funnel thoroughlywith sterile, buffered, di-stilled water to remove anytoxic residue. Place the hy-drosol under the filter hol-der to catch the filteredwater.

STEP 6

Sterilize the forceps by dip-ping the ends into alcoholand by flaming. Set them onclean dry toweling so thepoints do not contact thepaper. Separate the fun-nel from the filter holder.Set the funnel on clean drytoweling. Pick up the packetcontaining the membrane fil-ters and absorbent backuppads. Open the flap and, us-ing the cooled forceps, graspa flexible filter (white withlines) (1) by the outer 3/16 -inch of its circumference.Place the filter, grid sideup (2), into the depressionin the filter holder.

10

KEY POINT 5

After 30 seconds, replace thereceiver flask over the holderflask and wait 2-3 minutes.

KEY POINT 6

Unlock the sterilized funnel andload the holder with a membranefilter; retain the funnelaseptically.

to:4;

LESSON ONE

STEP 7 KEY POINT 7

Carefully replace the funneland tighten the twist lock.Pick up the hydrosol and at-tach it to the bottom of thefilter holder base. In this

position the receiver flaskwill capture the filtrate(water passing through themembrane filter).

Make sure the twist lock istightened.

11

LESSON ONE

EXERCISES

Instruction 1: Referring to the equipment and/or drawings in thelesson, label the following drawings. You must be able to name

each part of a label in the drawing and describe in your own wordshow a part works or what a label means.

Instruction 2: Practice each step in Lesson One until you can dothe following in less than 20 minutes:

o disassemble, sterilize, and assemble the filtration unit in

an aseptic manner

o flame sterilize a forceps and aseptically grasp a membranefilter by the outer 3/16-inch of its circumference

o place a membrane filter into the filter holder and assemble the

filtration unit.

12'6

LESSON ONE/EXERCISES

Instruction 3: When you can perform each of the above correctlywithout referring to this book or to the manufacturer's operatinginstructions manual, begin work on Lesson Two.

13

LESSON TWO

OBJECTIVE

You will be able to suction a water sample through a membranefilter, using aseptic techniques, and prepare the filter forincubation.


This lesson will be carried out in the same location and manner asdescribed in Lesson One. Carefully perform each step in the ordergiven. Any other sequence may prevent an even bacterial dispersionon the membrane filter.


You must be able to suction a water sample through a membranefilter, using aseptic techniques to prevent contamination of thesample by organisms from other samples, or from fecal-contaminatedequipment or supplies. You must also be able to disassemble thefilter holding assembly, aseptically remove the filter, distributethe media broth evenly on the absorbent pad, and place the filterin the petri dish so that air is not trapped by the filter.

THINGS YOU NEED

In addition to the equipment you used in Lesson One, you will needthe following:

o ampules, containing Endo broth media or Endo broth preparedfrom powdered media

o water, containing fecal organisms, 10-ml minimum

o sterile, graduated pipets and 100-ml capacity cylinder

o clean and dry 4-ounce bottles, 5.

Instructions: Now turn to the next page and begin work on LessonTwo, "Getting There--Steps."

14

LESSON TWO


STEP 1

Prepare a positive blank sam-ple, using water from sourcescontaining fecal organisms.Use a sample of raw or un-chlorinated sewage watercollected in a dry, clean,sterilized 125-ml sample bot-tle containing sodium thio-sulfate. For a label, placea piece of autoclave tape ormasking tape on the bottle.Add 1.0 ml of fecal organismcontaining water to a clean,sterilized bottle and 100 mlof distilled water. Labelthe bottle: "Positive Blank,1:100 Dilution."

STEP 2

Prepare a negative blanksample. Obtain a dry, clean,sterilized bottle that con-tains sodium thiosulfate.For a label, place .a pieceof autoclave tape or mask-ing tape on the bottle.Label the bottle: "NegativeBlank, Distilled Water."Fill the bottle up with dis-tilled water.

KEY POINT 1

Prepare a positive blank, sampleusing water containing fecalorganisms.

KEY POINT 2

Prepare a negative blank sample,using distilled water.

15

LESSON TWO

STEP 3 KEY POINT 3

Prepare a petri dish con-taining an absorbent padsaturated with media broth.Open a pouch containing back-up absorbent pads and fil-ters. Do not handle the pad.Remove a pad (1) with- ste-rilized flamed forceps andplace it into the openedpetri dish (2) as shown inthe Key Point. Break an am-pule containing media anddistribute it evenly over thesurface of the pad. If youprepared media broth in bulkamount, pipet 1.8 ml of ste-rilized (heated) media ontothe pad, using a sterile*pipet.

Never touch the absorbent pad.

STEP 4 KEY POINT 4

Attach a piece of suctiontubing (1) to the perpendi-cular side inlet valve (2) ofthe stainless steel syringe(3). Pump the syringe sever-al times to check for valveblockage. If there is noblockage, insert the plug endof the suction tubing intothe side vent hole (4)located in the center of thefilter holder base.

*Free from fecal organisms.

16

Test the syringe before attachingthe suction tube to the filterholder.

20

LESSON TWO

STEP 5

Pour approximately 10 ml ofsterile, buffered, distilledwater into the funnel. Donot apply suction to thefiltering unit. Observe thefunnel and holder assemblyfor leakage. If any leakageoccurs, unscrew the unit andinspect the base of the fun-nel for debris or damage. If

the 0-ring is damaged, youwill not be able to draw suc-tion. Reassemble as in Steps6 and 7 of Lesson One. If

the unit continues to leak,ask your instructor forassistance.

STEP 6

Using sterilized forceps,remove a membrane filterfrom its shipping package.Center the filter in therecessed area in the filterholder. Close the filter-pad package and lay it asidein a dry area.

KEY POINT 5

Pour approximately 10 ml ofsteri'e, buffered, cistilledwater into the funnel.

KEY POINT 6

Do not touch the membrane filterat any time.

2117

LESSON TWO

STEP 7

Locate a clean, sterilized100-m1 graduate cylinder orgraduated stainless steelcup provided in the waterlab. Uncap the negativeblank sample. If jou usethe graduated cylinder, liftit to eye level and 'slowlypour 100-m1 of the negativeblank sample into it untilthe bottom (1) of the menis-cus touches the 100-m1 line.Gently pour the 100-m1 sampleinto the funnel. Pour slowlyand close to the rim of thefunnel to avoid splashing.As a matter of routine prac-tice, rinse the cylinder se-veral times with sterile,buffered, distilled waterand pour each rinsing indi-vidually into the funnel.Allow a 5-second drainageperiod before shaking offthe last drop.

KEY POINT 7

Observe the meniscus when fill-ing a graduated cylinder.

STEP 8 KEY POINT 8

To generate suction, pumpthe syringe until all of thesample flows through themembrane and into the re-ceiving flask. The filterwill have a semidry look whensuction is complete.

18

Apply suction by pumping with asyringe plunger.

90titi

LESSON TWO

STEP 9

Rinse the funnel three timeswith sterile, buffered, di-tilled water. Use about 2 ml

for each rinse and flush thewalls of the funnel to removeall residual water sampledroplets. Then, with thesyringe, generate a suctionafter each rinse and allowall of the water to passthrough the membrane beforeapplying the next rinse.

STEP 10

After drawing off all thewater with the suction, re-lease any negative pressurethat may have built up inthe filtration unit bygently removing the suctiontube adaptor from the filterholder base.

STEP 11

Holding the funnel with onehand, turn the twist lock andgently separate the funnelfrom the filter holder.Without touching any of theinside surfaces of thefunnel, hold it in one handwhile you gently lift themembrane off the filterholder using the flame-sterilized forceps. Onceyou remove the membrane,temporarily replace thefunnel on the filter holderbase.

KEY POINT 9

Rinse the funnel with sterile,buffered, distilled water.

KEY POINT 10

Release negative pressure toprevent damage to the membranefilter.

KEY POINT 11

Hold the funnel aseptically.

19

LESSON TWO

STEP 12

Remove the cover of the pre-pared petri dish. With itsgrid or inked side up, slidethe membrane filter acrossthe top of the open petridish until its far edge justenters the dish and settleson the absorbent pad. Using

a slight rolling motion,center the filter on top ofthe media-saturated absorbentpad. If air pockets occur,pick up the membrane filterby its edge and reroli. If

you are still unable to elim-inate the air pockets trappedbetween the filter and thepad, ask your instructor forassistance.

STEP 13

Once the membrane filter isproperly positioned, tightlyclose the petri dish. Re-move the tape label from thesample bottle and place iton the petri dish cover.Invert the dish, record thetime in your notebook, andthen place the inverteddish into a portable in-cubator with the temperatureset at 35 +0.50 C. Thistemperature should be main-tained for 22-24 hours forculturing total coliformbacteria.

20

KEY POINT 12

With a rolling motion, slide themembrane filter into the petridish.

KEY POINT 13

Incubate the inverted petri dishat 35 +0.50C for 22-24 hours.

LESSON TWO

STEP 14 KEY POINT 14

As a routine habit, replace Rinse the funnel after each use,the funnel and rinse the and flame the forceps after eachfunnel with generous amounts use.of sterile, buffered, dis-tilled water after suctioninga sample through a membranefilter. Sterilize theforceps after using them.

STEP 15 KEY POINT 15

Filter the positive blankafter the negative blank.Repeat Steps 3 through 13.Water from the followingsources shall be filteredin this order:

(1) negative blank

(2) potable water

(3) thawd ice

(4) any sample that mayyield a high colonycount after in ubationon media broth

(5) positive blank.

Filter first those samples leastcontaminated.

21

LESSON TWO

EXERCISES

Instructions: Check your aseptic technique.

Part A: Prepare Positive Blanks

Obtain five dry, clean, sterilized bottles containing sodiumthiosulfate. Replace a piece of autoclave or masking tape oneach. Add the following amounts of unchlorinated sewage water:

o Bottle 1 -- 50 ml (1:1 dilution)

o Bottle 2 -- 10.00 ml (1:10 dilution)

o Bottle 3 -- 1.00 ml (1:100 dilution)

o Bottle 4 -- 0.10 ml (1:1,000 dilution)

o Bottle 5 -- 0.01 ml (1:10,000 dilution)

Add enough sterile, buffered distilled water to Bottles 1 through 5to make 100 ml in each bottle. Label each bottle with the dilutionratio.

Part B: Filter Samples

Repeat Steps 3 through 13. Filter bottles in the numberedsequences. In actual practice, do not run less contaminatedsamples after those that may contain more fecal organisms. SeeStep 15. Remember to use generous amounts of sterile, buffereddistilled water to rinse between samples.

Part C: Incubate Samples

Incubate these samples and use them in Lesson Three. When youcount the colonies, you should find that the number of coliformcolonies decreases from Bottle 1 through Bottle 5. Without'counting the colonies, however, you should see there is a decreasein the total number of organisms present from Bottle 1 through 5.If not, repeat the exercises and pay particular attention to howyou handle sterilized equipment and materials, and that you placethem in sterile areas when you set them down. You may need toincrease the dilution to 1:100,000 by adding 0.01 ml of raw sewageto 1,000 ml of sterile, buffered distilled water. Furtherdilutions can be made by first diluting the raw sewage water withsterile, buffered distilled water and then taking an aliquot ofthat and adding it to 100 ml or 1,000 ml of sterile, buffereddistilled water.

22

LESSON THREE

OBJECTIVE

You will be able to determine the number of total coliformorganisms in a water sample.


This lesson will be carried out in the same location and manner asdescribed in Lessons One and Two.


You must be able to identify total coliform by their goldenmetallicsheen with a greenish tint, and generally semisphericalshape, and to calculate the density of total coliform coloniespresent in a 100 ml sample of water.

THINGS YOU NEED

You will need the following equipment:

o wide-field stereo microscope

o fluorescent light source

o petri dishes incubated in Lesson Two.

Instructions: Now turn to the next page and begin work on LessonThree, "betting there--Steps."

23

LESSON THREE


STEP 1 KEY POINT 1

After incubating a membranefilter for 22-24 hours at35 +0.50 C, carefully re-move a petri dish from theincubator. Rough handlingor jarring of the plate cancause spattering of dropletsand cause difficulty incounting. Gently turn thedish over (right side up).If there are only a fewcolonies on the filter, youmay be able to count themwithout magnification.Dozens of colonies will re-quire magnification to beread accurately. Take thedish to the microscope areaof the workbench to countthe colonies.

Handle incubated petri dishescarefully.

STEP 2 KEY POINT 2

Remove the cover from thepetri dish and place thebottom plate on the stage(1) of a wide-field stereomicroscope. Adjust themagnification to 10x.Position and adjust a cool,white fluorescent lightsource so the light fallsas vertically as possibleon the membrane filter.(An an angle of 600 to800 is best.)

24

Position the plate bottom onthe stage of the stereo micro-scope under a fluorescent lightsource.

LESSON THREE

STEP 3

Look into the stereo micro-scope and examine the entiresurface of the membranefilter for the presenceof indicator organisms. The

indicator organism coloniesare pink to dark red andhave a golden metallic sheenwith a greenish tint. The

metallic sheen may coveran entire colony or onlythe center. The size ofthe colonies varies.

STEP 4

Microscopically scan themembrane filter with a backand forth movement over thegrids and count allcolonies having a sheen.

KEY POINT 3

Using a stereo microscope,examine the surface of themembrane filter for the presenceof indicator organisms (totalconform).

KEY POINT 4

Use the grid system on the sur-face of the membrane filter tolocate colonies along thecounting path.

d425

LESSON THREE

STEP 5

If you observe numerous col-onies on the membrane filter,even if they do not have asheen, you must count theseas well; if there is a totalcolony count (of all typecolonies) greater than 200,discard this filter becausea count of 200 colonies in-terferes with the validity ofthe test. If this occurs onyour plate, you will haveto dilute your sample thesame way you dilute the posi-tive blank to reduce thebackground colonies. Thecolonies counted from eachdilution series then can beadded together to get atotal coliform count.

STEP 6

Report the colony count per100 ml of the standardwater sample. Refer to theequation in Key Point 6 toget your total colony count.Refer to the water quality

standards that apply to yourarea when determining howmany colonies may be presentin a sample of potable orrecreation water.

26

KEY POINT 5

Dilute the sample if the colonycount is too high.

KEY POINT 6

Indicatororganisms x % dilution x 100counted

ml filtered sample

Indicator organisms/100 ml

Calculate the density of indi-cator bacteria present in thesampled water.

3o

LESSON THREE

EXERCISES

Instruction 1: Count the total coliform colonies present in theseven petri dishes you incubated in Lesson Two. In calculating theindicator organisms per 100 ml water sample, use the number ofmilliliters of the contaminated water sample actually used, i.e.,0.01 ml for the 1:10,000 dilution. Using actual counts, verifyyour findings in Lesson Two/Exercises. If the counts indicate adifferent result, repeat Lesson Two.

Instruction 2: If there are fewer than 200 colonies, count thetots number of bacteria colonies present on an incubated membranefilter and fill in the individual blank squares of the grid belowwith the number of colonies counted. Use the accompanying diagramto assist in identifying in which square a borderline colony may belocated.

Sca

pat

Pet

Pa

/ 2 0 00zO/

2.. 0/

> _

T < -

1_,.....> ._-< < -__<

<-- 4p

, k?

31

example

Fill-in

th

27

LESSON THREE/EXERCISES

Instruction 3: Substitute fecal coliform media broth in the petridish and incubate your cultures at 44.5 +0.200 C. This is thetest for fecal coliform. When examined under 10-20X magnification,all colonies exhibiting a blue color are fecal coliforms.Calculate the density of fecal coliform colonies present in thewater sample using the same formula presented in Key Point 6.

Instruction 4: Count the number of fecal coliform bacteria in yoursample (see Instruction 2). Calculate the density of fecalcoliform colonies in the water sample.

Instruction 5: Practice each step in Lessons One, Two, and Threeat some field location. Also practice using an automobile batteryto power your portable incubator and light for your microscope.

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PERFORMANCE TEST

Instructions: Check your skill level or progress by workingthrough each of the items in this test. If you can perform eachitem as required, place a check in the space provided. When all ofthe items are checked, you are ready to demonstrate your skills toyour instructor. You may use the following list if needed. You

will be considered trained in a skill after your instructorapproves your performance of each of the following items:

PREPARING WATER SAMPLES FOR BACTERIOLOGICAL ANALYSIS

No. 1 Sterlize the funnel and filter assembly, usingmethanol.

No. 2

No. 3

No. 4

Prepare a positive blank sample, using water con-taining fecal organisms.

Prepare a negative blank sample, using sterile,distilled water.

Add 1.8 ml Endo broth media (ampule or by pipetfrom a newly prepared batch) to an absorbent pad ina petri dish so that the media is evenly distributed.

No. 5 Aseptically place a new membrane filter into thefilter holder.

No. 6 Filter 100-ml sample and rinse the measuring con-tainer and funnel before aseptically removing themembrane filter.

No. 7 Place the semidry membrane filter onto a mediasaturated pad in a petri dish so that air is nottrapped under the filter.

No. 8 Identify the sample, invert it, and incubate it.

No. 9 Prepare the filtration equipment for the next sample.

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PERFORMANCE TEST

FOR FURTHER STUDY

If you could not perform one or more of the nine items above,review and practice the following lesson steps:

No. 1Lesson One, Step 4

No. 2Lesson Two, Step 1


No. 4ern Two, Step 3


No. 6Lesson Two, Steps 7 through 11




DETERMINING THE NUMBER OF MICRO-ORGANISMS IN A WATER SAMPLE

No. 1 Place an incubated sample on the stage of a stereomicroscope without damaging the culture.

No. 2 Using a diffuse light source and an appropriate.power of magnification, count all coliform colonies,using the scanning technique.

No. 3 Count all colonies up to 200.

No. 4 Calculate the number of indicator organisms per 100ml of sample.

30

PERFORMANCE TEST

FOR FURTHER STUDY

If you could not perform one or more of the four items above,review and practice the following lesson steps:

No. 1Lesson Three, Step 1

No. 2Lesson Three, Steps 3 and 4



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REFERENCES

Millipore Corporation, Biological Analysis of Water and Wastewater,Application Manual, AM302, Bedford, MA, March 1973.

Standard Methods for the Examination of Water and Wastewater, 14thed., American Public Health Association, 1975.

U.S. Army, Academy of Health Sciences, U.S. Army Health andEnvironmental Division. Bacteriological Tests on Water,Environmental Health Specialist Course, 322-91S10/20 (No date).

U.S. Army, Field Manual, FM 8-91S1/2, Soldier's Manual, MOS 915,Environmental Health Specialist, Washington, DC, August 1977.

U.S. EPA, National Training and Operational Technology Center,Office of Water Program Operations. A Prototype for Developmentof Routine Operational Procedures for the Collection and Handlingof Drinking Water Supplies, February 1978.

U.S. EPA, Quality Criteria for water, Washington, D.C., July 1976.

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V] · The presence of total coliform bacteria, including Klebsiella, Escherichia, Serratia,...

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