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Samplers, Dilution Kits, andSwab Test Kits User Guide

Notice

The information in this document is subject to changewithout notice and should not be construed as a commitmentby Millipore Corporation. Millipore Corporation assumes noresponsibility for any errors that may appear in this docu-ment. This manual is believed to be complete and accurate atthe time of publication. In no event shall Millipore Corpora-tion be liable for incidental or consequential damages inconnection with or arising from the use of this manual.

©1995, 1998, 1999 MILLIPORE CORPORATION. PRINTED INTHE UNITED STATES OF AMERICA. ALL RIGHTS RESERVED.THIS BOOK OR PARTS THEREOF MAY NOT BE REPRO-DUCED IN ANY FORM WITHOUT THE WRITTEN PERMIS-SION OF THE PUBLISHERS.

Millipore is a registered trademark of Millipore Corporation oran affiliated company.Coli-Count and Sterivex are trademarks of Millipore Corpora-tion or an affiliated company.Whirl-Pak is a trademark of Nosco International Inc.

Samplers, Dilution Kits, andSwab Test Kits User Guide

P15325, Rev. D, 8/99

SAMPLER DESIGN

Vent (on back of unit)

Outer LayerMilliporefilter trapsmicroorganisms

Inner LayerAbsorbent pad withnutrient mediumsupports growth ofmicroorganisms

IntroductionMillipore Samplers provide a means for simple, fastmicrobiological analyses of environmental waters,cooling tower waters, process waters, laboratorygrade and electronics waters, dialysis water, and foodand beverage products. Each sampler is constructedto combine both an intimate contact of a 0.45 µmMillipore membrane filter to a nutrient-pad, and theincorporation of an air-vent on the upper backportion of the paddle. This configuration allows forthe draw-through of 1 mL of sample to affix microor-ganisms to the filter surface for subsequent culturingwithin its transparent plastic case. The filter is grid-marked to aid in counting the microbial coloniesgrown on its surface. Each sampler assembly issterilized and packaged in a sealed plastic envelope.

Sampler TypesDescription Colour Code Catalogue

No. 25/pkColi-Count Sampler Blue MC0010025Yeast and Mold Sampler Yellow MY0010025Total Count Sampler White MT0010025Heterotrophic PlateCount Sampler Red MHPC10025(HPC)

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AccessoriesDescription Catalogue No.

Laboratory Incubator, 115V XX6350115

Laboratory Incubator, 230V XX6350230Sterivex-GS Filter Unit,0.22 µm, Sterile, 15/pk SVGS01015

Whirl-Pak Bag, 6oz., Sterile, 500/pk XX0500004Syringes, (polypropylene), 50mL, 5/pk XX1105005

Notes for Sample Use(a) Samples containing residual chlorine must be

neutralized with sodium thiosulfate (0.1 mL of10% solution/120 mL of sample) prior to testingwith the Millipore Samplers.

(b) Where exact counts are not required, samplescontaining an estimated microorganism level ofup to 400/mL may be tested with samplerswithout dilution. For exact counts, any samplecontaining more than 200/mL should be diluted.If a dilution level of 1:10 is required, simply fillSampler-case with the liquid up to the lowergraduated line (1.8 mL) add sterile water (orbuffer) to the upper line (18 mL), insert Samplerpaddle and proceed with test. For dilutions of1:100, pipette 0.18 mL into case and add sterilediluent to 18 mL mark. For higher dilutionsplease refer to the "Dilution Kits" section.

(c) For most water samples, the Sampler paddleshould be immersed in the sample for 30 seconds.If, however, the sample is viscous, the paddlemay require immersion for up to 2 minutes toallow the 1 mL aliquot to wet thoroughly, andpass through the membrane. If the entiremembrane is properly wetted, the filter willappear dark gray, (a very light gray for Coli-Count).

3Samplers, Dilution Kits, and Swab Test Kits

Notes for Sample Use, Continued(d) Do not immerse paddle longer than is required,

otherwise a loss of medium through the mem-brane and into the sample may occur.

(e) Sample dilutions should be made with a sterilephosphate buffer (pH 7.2). If this is unavailable,sterile, chlorine-free tap water may be used. Non-sterile dilution waters may be sterilized by meansof the Millipore Sterivex-GS unit fitted with a50mL syringe.

Testing Liquid Samples1. Remove the Sampler from its plastic bag and write

on the case with indelible marker the date, typeand location of sampling.

2. Pour sample liquid (or dilution) into the Samplercase, filling to the upper (18mL) graduation.

3. Insert the Sampler firmly into case containingsample, and carefully lay the unit with membranefacing down onto a flat surface. Make certain themembrane is uniformly wetted, and while in thisposition, the unit should not be agitated. Allow 30seconds for sample to be drawn through filter. Ifthe sample is viscous, the paddle should remain inthe case for additional time (up to 2 minutes).

4. Remove the paddle and, with a firm snap of thewrist, shake off the excess liquid. Empty the caseand reinsert the paddle. To prevent the paddlefrom drying out during incubation, it should beseated firmly in the case to form an air-tight seal.

5. Incubate the Sampler, gridded side down, using thetime and temperature specified in the table shownin the "Culture-Incubation Guide".

6. Remove Sampler from incubator. For examinationand counting please refer to the "Filter Examina-tion" section.

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Testing Solid SamplesSolid samples can be tested by one of the followingmethods:

1. Weigh a given amount of the solid in a Whirl-Pakbag. Dilute with 100 mL of sterile water orphosphate buffer. Seal the bag and knead with thefingers for at least 2 minutes. Allow the solids tosettle, aseptically pour the upper liquid layer intothe Sampler case. Test with the Sampler in theusual manner. This technique is especially suitedfor pasty samples.

2. If the solid sample consists of hard particle-typematerials such as nuts, the bag, diluent, andcontents may be shaken vigorously for 2 minutes.

3. A very useful and convenient method for dispers-ing the microorganisms into the water or buffer isto place the seated bag into a sonic bath and runfor a few minutes. This is recommended forparticulate-type solids.

DILUTION KITSThe Millipore Dilution Kits are designed to provide afast, simple, and convenient means to dilute watersamples prior to testing with the Millipore Samplers.Each kit consists of 25 plastic vials (cases) containing18 mL each of sterile, phosphate buffer, and 2 rigid,compartmentalized pouches containing sterile,micropipettes.

The latter are appropriately graduated for accuracy,and fitted with fine stainless steel plungers for ease indispensing the water samples.


Dilution Kit TypesDescription Catalogue No.

Dilution Kit (1:1000) (25 tests) MM30 100 25includes: 25 vials(18 mL)

sterile phosphatebuffer25 (18 µl) sterilemicropipettes

Dilution Kit (1:10,000) (25 tests) MM40 100 25includes: 25 vials (18 mL)

sterile phosphatebuffer25 (18 µl) sterilemicropipettes

Instructions For Dilution Kit UseWhen employing the Millipore Samplers, it is advisedthat samples containing a microorganism level inexcess of 200/mL should be diluted to avoid over-crowding on the paddle membrane. An excess oforganisms on the filter creates a competition for theavailable nutrients present and can result in bothdifficulty in colony counting, and an erratic count.Dilutions of 1:10, and 1:100 are described in "Notesfor Sample Use", section 4(b). For dilutions of 1:1000and 1:10,000 please note the following instructions.

Making 1:1000 and 1:10,000 dilutions:

1. Remove a vial of sterile phosphate buffer from theDilution Kit box.

2. Loosen the cap and leave it in place.

3. Peel back the tape on the flat pack of micropi-pettes just enough to clear one. In removing themicropipette, handle it only from the dispensingend of the package.

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Instructions For Dilution Kit Use,Continued4. Fill the micropipette avove the black line by

inserting the tip into the sample and pulling themetal wire upwards. Then push down to blackline to expel excess. Remove any drop on the tipby touching the inside of the sample container withthe end of the pipette.

5. Dispense the sample from the micropipette into thevial of phosphate buffer by pushing the metal wireall the way down. Make sure that the tip is justunder the surface of the buffer while dispensing.

6. Place the cap back on the vial and shake vigor-ously. Remove the cap, insert the paddle andproceed with the test. See steps 3-6, in the "TestingLiquid Samples" section.

Culture-Incubation GuideWhen performing coliform analyses with the Coli-Count sampler the time and temperature shownbelow must be followed. The other Samplers may beincubated at any temperature shown below as long asthe temperature is consistently followed. Ideally, themid-points of these ranges are preferable. Time ofincubation for the Samplers (except Coli-Count)should be at least 48 hrs. Incubation periodsexceeding this will be dictated by practical timelimitations. At all times, the incubation period shouldbe consistent.


Sample Incubation IncubationType Temperature Time

Coli-Count 35°C 22-24 hoursYeast and Mold 28°C-32°C 48-72 hoursTotal Count 25°C-35°C 48-72 hoursHPC Total Count 25°C-35°C 48-72 hours

Filter ExaminationAfter incubation is complete, remove paddle fromcase, and examine filter surface with either a stereo-scopic microscope with illuminator (at 10X-20X) oran illuminated magnifier (preferably ≥ 5X). Theappearance of the microbial colonies will vary,depending upon the Sampler used, and the organismsrecovered. Generally they will appear as follows:

■ Coli-Count Sampler Coliforms are blue incolor. Non-coliforms aregreen, gray, or creamcolor.

■ Yeast and Mold Yeast usually have asatiny, opaque, whitecolor. Molds will varydepending on length ofincubation time.Bacteriaare usually more glisteningand transparent.

■ Total Count/HPC May vary. Most coloniesare glistening, andtranslucient or transparent.Colors vary from colorlessto white, cream, yellow orred.

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Counting ColoniesColonies growing on the filter surface of Samplers arecounted as individual organisms. In recording yourcount when using the Coli-Count Sampler forcoliform (or fecal coliform) analyses, count only theblue colonies. Coliform and fecal coliforms arealways reported as the number per 100 mL sample.Therefore in indiluted samples, count the number ofblue colonies obtained and multiply this result by100. If the sample is diluted, multiply the 100 m Lcount by the apropriate dilution factor.

For all other samples, the count per mL is thegenerally accepted system for recording your results.Therefore, for non-diluted samples, the number ofcolonies observed on the fliter will be the numberrecorded (as sample count/mL). For diluted samples,the count obtained must be multiplied by the dilutionfactor.

For example: number of colonies on filter = 60

Sample dilution = 1:1000 (∴ dilutionfactor is 1000)

Sample count/mL = 60 X 1000 =60,000/mL

A rapid, approximate count can be made by compar-ing the filter to the examples shown in the "ColonyCount Comparison Chart" section.

SWAB TEST KITSThe Millipore Swab Test Kit is a self-containedsystem, incorporating Millipore Samplers, and unitscontaining 18mL of sterile, phosphate buffer intowhich are fitted swab assemblies. The system isdesigned for use is swab tests for measuring bacteriallevels on flat or irregular surfaces. They can be usedin a variety of applications in food production, food


SWAB TEST KITS, Continuedhandling establishments, and other facilities to assessthe efficiency of sanitation measures used to eradicatemicrobial contamination. The buffer units containneutralizing agents to counteract the adverse affect ofany residual chlorine or quaternary ammoniumcompounds that may be present on surfaces aftersanitization.

Swab Kit TypesDescription Catalogue No.

25 Tests

Coli-Count Swab Test Kit MCSK10025Yeast and Mold Swab Test Kit MYSK10025Total Count Swab Test Kit MTSK10025HPC Total Count Swab Test Kit MSSK10025

NOTE: Each of the above kits consists of one box of(25) of the appropriate Samplers, and onebox of (25) of the Swab Test Buffer Set(MMSB10025).

Instructions For Swab Test Kit UseIn testing the surfaces of equipment, cutting boards,etc., the Swab Test Kit provides an overall picture ofthe test site (employing one Swab Test only).Sampling an isolated spot may not be representativeof the whole test area. The use of a swab provideshigh efficiency in penetrating crevices, rough andcurved surfaces, and other hard-to-reach areas. Forsanitization monitoring, the application of the swab/Sampler combination therefore provides the methodof choice for sampling these critical areas.

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Instructions For Swab Test Kit Use,Continued1. Grasp the swab handle and, using an end-to-end

rocking motion, remove swab from buffer case.

2. Roll tip of swab on inside of case to wring outexcess buffer.

3. Randomly select 5 areas of the surface to bemonitored, then holding the swab firmly, draw aletter "M" on the surface, rotating the swab tipduring procedure. Each straight section of theletter "M" drawn should be 2" in length (thedistance between swab tip end and base ofhandle).

4. Repeat this test for the four remaining areasselected. Each letter "M" swabbed represents 8linear inches (2" X 4), thus 5 areas sampled isequal to 40 linear inches for each piece ofequipment or surface area sampled. This methodof sampling should be used each time the test isrun in order to provide an equivalent comparisonof results. Where the letter "M" cannot be applied(saw blades, slicers, etc.) swab 5 different areas of8 linear inches each.

5. After swabbing the five selected points, insertswab firmly into case, and shake 30 times todislodge organisms into buffer. Remove swaband discard.

6. Open test Sampler package, lift out Sampler, andcarefully remove "paddle" from case. Avoidtouching gridded filter surface.

7. Insert the Sampler paddle firmly into case contain-ing the buffer, and carefully lay the unit on a flatsurface with membrane facing down. Do notagitate unit after placing down, and make certainthe membrane is uniformly wetted.


Instructions For Swab Test Kit Use,Continued8. After 30 seconds has elapsed, remove paddle from

case and shake once or twice vigorously toremove excess buffer.

9. Reinsert Sampler firmly into dry Sampler case,marking date, type, and place of sampling onoutside of case with grease pencil or indeliblemarker.

10. Incubate unit, gridded side down, at appropriatetemperature and for appropriate time periodindicated in the "Culture Incubation Guide"section.

11. Count visible colonies and record results. Timemay be saved by comparing the Samplers withthose illustrated on comparison chart.

NOTE: Since only 1 mL from the 18 mL of buffercontaining the organisms was drawn throughthe Sampler filter, the realistic count for thearea swabbed would be the count obtainedX 18. However, this monitoring technique isto provide an index only of the efficiency ofsurface sanitization, therefore, the countobtained as is will suffice if this method isalways followed.


Colony Count Comparison ChartSmall Colonies

To obtain appoximate count, align Sampler withdrawing showing same density of colonies.

10

30

50


Small Colonies

100

300

TNTC*

*Toonumerousto count


Colony Count Comparison ChartLarge Colonies

To obtain approximate count, align Sampler withphoto showing same density of colonies.

10

30

50


Large Colonies

100

300

TNTC*

*Toonumerousto count


Technical AssistanceFor more information, contact the Millipore officenearest you. In the U.S., call 1-800-MILLIPORE

(1-800-645-5476). Outside the U.S., see your Milliporelaboratory catalogue for the phone number of theMillipore office nearest you. Or, look us up on theInternet at our World Wide Web site:

http//www.millipore.com


Standard WarrantyMillipore Corporation (“Millipore”) warrants its prod-ucts will meet their applicable published specifica-tions when used in accordance with their applicableinstructions for a period of one year from the date ofshipment of the products. MILLIPORE MAKES NOOTHER WARRANTY, EXPRESSED OR IMPLIED.THERE IS NO WARRANTY OF MERCHANTABILITYOR FITNESS FOR A PARTICULAR PURPOSE. Thewarranty provided herein and the data, specificationsand descriptions of Millipore products appearing inMillipore’s published catalogues and product litera-ture may not be altered except by express writtenagreement signed by an officer of Millipore. Repre-sentations, oral or written, which are inconsistentwith this warranty or such publications are not autho-rized and if given, should not be relied upon.

In the event of a breach of the foregoing warranty,Millipore’s sole obligation shall be to repair or re-place, at its option, the applicable product or partthereof, provided the customer notifies Milliporepromptly of any such breach. If after exercising rea-sonable efforts, Millipore is unable to repair or re-place the product or part, then Millipore shall refundto the customer all monies paid for such applicableproduct or part. Millipore shall not be liable forconsequential, incidental, special or any otherindirect damages resulting from economic lossor property damages sustained by any customerfrom the use of its products.

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