Document L608QYzpqn4yLbyGeG4Mr24Q

IflU ssst nwl PLAINTIFF'S EXHIBIT DUP-667 EQUIPItEN and fpccedupes 7cp !:cs7.;:; m ^Tpnpp J AND COUNTING ASS7ST0S FT 5' s BY PHASE CONTRAST MICROSCOPY PS Stephen G. Beyer Ralph D. Zumvalde Thornes A. Brcvn Bureeu of Occupational Safety end Health 1014 Broadway Cincinnati, Ohio 45202 JULY 1969 U. S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service Consumer Protection end Environmental Health Service Environmental Control Administration DUP 1200011 DU 062539 table or cont-d.ts ..................................................................................................................................................................... , Equionent L'tt................................................. ^ Laboratory Set-uo............................................. \ Preliminary M-'croscoee Set-uo--*- -- .....--------------- .........-------------------------- j Microscope set up and the Procedure for obtaining Kohler Illumination------ 3 Detailed Instructions for Bausch and Lomb Binocular Microscopes---------- 5 Filter Mounting Set-up prior to mounting------------ ..............................---------------- 5 Mounting procedure--------------------- .......... -- ----------------.......------------------ 5 Microseone Precautions and Maintenance---------------- --------- ---------------------- ----------- -- 7 Counfng Procedure------------- --------------------------------------- ------------- .... ----------------7 Field Monitor Discussion---------------------------------------------------- J Calculation of Concentration------------------------*--*--------*-*-'------- 9 Addresses.......................... ...................... ........................................ ............. .............-......................... DUP 1200012 DU 062540 roRrv;E3 To determine concentrations o_f asbestos dust in air for eonoar-'sor wit-, established hytiene 'inits, a simple reorodv.eibl e method is requ-'red. A simo'e an'* inexpensive sa-->lir.q method uses d'soosab'e (reloadable ' desired) plasfc memb-ans filter holders, ore-loaded vich membrane filters. These membrane fi'ters say be rendered transoarent and the asbestos fibers counted with a phase-contrast microseooe at 430X (or &00X). This method has been used bv the Asbestosis Research Council in Creat Britain and the U.S. Public Health Service in the Urited States of America. It is specified in the proposed change for asbestos in the 1968 Threshold Limit Values (TLV) of the American Conference of Governmental Industrial Hygienists. Uniform oroeedures must be used for carrying out these counts is reproducible results are to be achieved. Detailed equipment ar.d procedures used by the U.S. Public Health Service are described here. \ DUP 1200013 DU 062541 - I- IVTPCCl'CT'CV For fever*1 ve*rs, set method for counting end siring airborne particulate matter, which has been deoosited upon membrane filters, has been used. The filters used in this technique are 37 mm diameter cellu lose ester membrane f1 Iters manufactured by Millipcre Corporation, (cat. mo. AAVP03700). The slide mounting technique refers only to these filters, end will not work for any others. The filters nay be purchased premounted in convenient field monitor eases, and are ready for sampling. The filters are nearly 1007. efficient for any asbestos dust, ever, for fibers v< th diameters much smaller than the 0.8 micron (u) pore sice. Samole nreoaration for electron end optical examination are both relatively sisole. Slide mounts of the filters ere semi-permanent. The following procedures and reecmer.dations will produce accurate results, if each step is performed exactly as indicated. FQOTFMTirr T-TST 1. Table 2. Adjustable chair 3. Phase contrast microscoo* 4.. Ribbon filament illuminator or (built-in illuminator) 5. Rye niece reticle (Portor. cyme) 6. Lens tissue 7. Small camels hair brush 8. Slides 9. Cover slips 10. Spatula 11. Tweezers 12. Scalpel 13. Wheaton Balsam bottlt LABORATORY SET-CP Tt i* imoortant to provide a suitable counting area, for the room in which the counting is to ba done has a great bear'ng upon the aieroscopist. !t should ba out of eh* way of normal traffic, and b* kept as frat from dust and smoke as oossible. The eables on which the microscopes art plsee should be as free from vibrations and shocks as notsibla. An adjustable chair will do much to add to the comfort of the counter. DUP 1200014 DU 062542 : MUMLiiLB MM - 2- ^5r,.TM'N'i:;v ''foncrnor $T~ "? Before any dust siring or counting en be done, orooer equioment Rust be <t hand. This secrler. describes the required equiomenc. The illuminator should be small and reasonably eomoact. Ribbon filament Illuminators are generally much easier to use. The illuminator must :r.eoroorate a condensing lens, so that the enlarged image of the lamo fila ment may be focused 'n the Diane of the substage condenser diaohragm. The Illuminator must also have an iris d'aohragm located as near as oossible to the condensing lens. The iris serves as a field diaohragm and is focused In the olane of the soeeimen. The microscooe used must have a substage condenser fitted vich an iris diaohragm. This iris serves rs an aoerture diaohragm. The microscooe should have a calibrated reticle inside the non-adjustable eyeotece. The reticle should bs small enough that all the counting field is in focus (if the flat field oblective and eyeoieces are used, essentially the en tire field will be in focus). The nicroscooes must be equioned with ohjse contrast accessories. Using ohase contrast, even objects with almost the same refractive index as the mounting media can be seen. Light waves which travel through such a soeeimen are retarded by a fraction of a wave length to oroduee a change in ohase. The ohase contrast ootieal system reveals these ohase changes as light or dark contrast against the back ground. This orocedure was written by a user of Bausch and Lomb ohase-equioned microstores, so that microseoot set-uo oroeedures eorresrend with these. However, any good mienoscooe with ohase contrast accessories will be satisfactory. For continual routine examination of filters, the binocular tyoe microscooe is much more comfomtable. The use of a first surface mirror is recommended, but not neeeisary.. Ry utliring a second surface mirror, multinle images of the illuminator field ir's are formed which will detract from the erisoness of the final image. Ai mentioned oreviously, the microseooe nuat contain a reticle. Any reticle, such as the Porton or Patterson Globe and Circle, which orojects a constant counting area and has orovision for siring, will work. The Porton reticle which we use outMr.es a rectangle that fits easily within the nerishery c: focus. The rectangle is divided into two squares. The left square is divided into six rectangles which constitutes the counting area. Above ar.c below the large rectangles are a series of circles in which every other circl doubles in diameter. Hence,the third is twice the diameter of the first; the fourth is twice the si2e of the second, ete. The right half of the rec tangle contains a scale for extending the site above the number nine circle. The formu'e v/2^ describes the circles sites. The diameter D is four: from L which is the unit of ler.zth, and S which reoresen.es the number of :.-.e circle. L is determined by calibration with a stage micrometer. The entire DUP 1200015 DU 062543 3 length of the rectangle is 230 1 units. By measuring the length and dividing by 200, "1." is obtained and the circle sices may be calculated. Whenever a microscooe is disassembled or cleaned, the reticle calibration should be checked. Another fact to take into consideration is that changing the interouoillary distance of the binocular will change the calibration, so the aicrosconist nay want to cheek the calibration at both extremes of adjust ment. The counting area on the reticle should be keot as clean as sossible, as dirt on the reticle is in focus and may be counted. MTCROfcr^r s~t t? ait the proceeds ~o~ obtaining ko v.v.yr.nation By utilizing equipment that meets the requirements just mentioned, any counter may achieve conoarable counts after some oractice. This section vill deal with setting the microscooe uo and obtaining kohler illumination. It is extremely imnortant that every steo be followed exactly as indicated. 1. Place the microscooe on a flat, level surface at a height such that the eyeoieoes may be observed without strain or discomfort. 2. Place a moderate dust samole uncn the stage. 3. Place the illuminator directly in front of the eicroacooe. Sigh: across the two and be sure they are aligned. For coil filament bulbs the illuminator iris should be two inches from the center cf the microscooe mirror, "or ribbon filament bulbs, the front cf the filter holder should be seven inches from the center of the plane s,-de of the mirror. Never use the curved side. 4. Remove all diffusing filters from the system and insert a neutral density filter and a clear blue filter. Blue or green colored fil ters may be used at the discretion of the microscopist. NOTE: Where the microscooe includes a built-in illuminator to give Kohler illumination, the above steos will not all be necessary. mrv A. Cheek microscooe and illuminator alignment. B. Cheek Che distance between the microscooe and the illuminator. C. Be sure that you are using the flat side of the mirror. D. Make sure that there are only two filters in the system. 5. By meanj of the focusing knob on the side of the substage con denser assembly, raise it until the uooer ler.s nearly touches the bottom of the slide. Remember that the free working distant* of the condenser is in the order of 1.2 asn and that the s'.tce is about 1 mm thick. DUP 1200016 DU 062544 -4- 6 . Turn on the illuminetor. Using the tilt controls, direct the beam onto the center of the nierror. Then by tilting the mirror direct the beer, upward into the condenser. 7. Close the substage iris completely. Open the illumineror ;ris el 1 the wey. 8. By means of the focusing controls on the side of the il'uminator, focus the imege of the filament on the bottom of the subsesge ir's. This mey be done by leaning over the microscope and observing the aubstage iris in the nrcroseope mirror. (On such mode's the Bauseh & Lo.r.b PS-27, there is a f*ne focus knob at the rear ef the illuminator). In that case, move the condensing lens all the way forward and trim the focus with the fine focus knob. 9. Onen the substage iris about half way. arvrrv A. Check the distance between the slide and condenser. B. Be sure chat the beam is striking the center of the mirror, and that it is being deflected upward into the condenser. C. Please reneat step 7. D. Check aten 8. Tf the image is not in focus now, correct it. Do not continue until the filament la correctly focused. F. Please repeat seen 9. 10. Put the 10X objective in place ar.d focus on the sample. This is done as follows: First, looking from the side of the nicrsscsre, lower the objective until it gets very near to the slide. Then, by looking through the eyepieces, f?C"S un. Never focus down with coarse focus when looking through eyepieces. A word oK caution; Th objective lenses are very expensive, so be careful not to grind them through the slide. Always focus up. Now trim eh focus w-.th fine focus knob. Focus sharply on the samole. Secondly, close the fiels -r-.s fully. If the condenser is nearly in focus, you should set a bright snot of light. By using the condenaer focus knob, bring the field ir<s into sharp focus. New, both the field iris and the obiectiv* are in focus on the s*--'le. If there appear to be multiple images of the field iris, it is because of the second surface mirror. Some of the light waves art raflectad from the first aurfaee, resulting In secondary lmagea. 11. If the image of the field iris is not centered, re-center it by tilting the mirror. If the color around the field ir-'s is not uniform, re-check the eilting of the illuminator. DUP 1200017 DU 062545 Br7'rv - 5- A. Check End he sure vou its focused on rhe samole. 10X objectives have a relet'velv 'one foes1 length and it may be noss-ble to focus on: (a) the eon of the cover stio. (b) the samole. (c) ehe bottom of the filter, (d) the too of the sl'de. (e) the bottom of the sl'de. () the too of the condenser. So be sure of the focal olane. B. Be sure that the field iris is focused in the samole plane. Check this often when examining sanoles. As the samole focus changes, so does the condenser focus. C. Re-cheek the centering and tilt of the illuminator. DrTA*IrD INSTFVCTTOHS m? 3ACSCK AND LCV3 BINCCL'LA" MTCRCSCC?;; 1. Leave the mieroseooe phase ring on 0. Insert the telescooe that is Suonlied with the kit into the right eyeoieee tube. Focus on the dark phase ring. Onen end close the subscage iris to be sure that it is centered in relecion to the objective phase ring. If it is not in the center, loosen the set screw on the condenser mount and oroperly set the condenser. Then re-tighcen the sec screw. This should very seldom be neeesserv. Do not force enythir.g and be careful and patient, klner. you are satisfied that the condenser is aligned, open it all the way. 2. Turn the phase wheel to 10. This shows chat the 10X phase ring is in place. Look through the telescope again. By using the adjustment screw on the side of the condenser, move the rings, until they are perfectly concentric. When changing phase rings, move them carefully, so as not to knock the condenser out of alignment. 3. Turn to the MX objective. Carefully turn the wheel to the M ohase ring. Align the two rings in the same manner as performed in steo 2. M Remove the telescope and replace the eyepieces. Again, check the conden ser focus. You are now ready to examine the sample at MOX, assuming that you are using 10X eyeoicccs. FILTTR MPUMTTMC Set up Prior to Mounting Rave available at hand a quality sealotl and a suooly of No. 10 curved blades, a pair of tvatzers (fine pointed are oreferred), a snatula or fire polished glass rod and a box of lens tissue. Slides (1" x 3" or 25 x 75 on) with DUP 1200018 DU 062546 -6 - the dace, sample number, etc. It is advisable to use quality number 1-1/2" cover sites. Most dry objectives are corrected for this thickness cover glass. The mounting media should be kept in a Wheaton Balsam bottle. The edge around the lid should be coated with stooeock grease to keeo out the surrounding a'r. The mounting solution may ncv be oreoared. A one to one solution by volume of dimethyl ohthalate and diethyl oxalate is poured into the balsam bottle. Then add 0.05, gram of filter material for each milliliter'of solution. The added filters increase the viscosity of the solution. Before using the solution, all of the fitter material must be dissolved and the solution must be of uniform consistency. This may require a day or so with frequent stir ring, Refrigeration may add to the shelf life of the solution. Mounting p-ocedure Keen in mind that cleanliness is important. First, lay down two nieces of ler.s tissue; one directly in front of you end the other to the left or right for the mounting tools. Kext, wise the sceloel, tveerers and glass rod or soatula with the frosted end toward you. Even precleaned slides must be cleaned this vav. Hold a covtr s', is by the edges between the index finger and the thumb, and clean <t in the same manner as the sl'de. Place the cover sli.s so that one edge rests on the unfrosted end of the slide end the other edge rests on the lens tissue. Se sure that the lover surface does not cone into contact with the lens tissue. Using the glass rod, or dropper, disocr.se a small drop of the mounting media onto the eenter of the slide. Replace the rod or dropper into the solution bottle and cover it. Using the soatula, sorted the solution into a triangular shaoe. While the fluid is spreading, cut a wedge of eorresoonding sire from the filter. Piece it upon the mounting media, sample side up. Pick uo the cover si io end place it on too of the filter. Press lightly on the eover silo to be sure the mounting media has made contact with it. Label the slide before completing any other slide. Sometime, after an hour or so, the filter say not have cleared. Usually, this can be cured by pressing lightly or. the eover file with a pencil eraser that has been wrapped with lens tissue. If the filter has been used to filter liquids or is damp, it will require drying, for it will not clear unless completely dry. Use only enough solution to clear the filter. If too much solution is used the sample may spread and give an erronaous count per unit area. The filter should be counted as soon as possible because the solution may eventually form crystals that look like fibers and may be mistaken for pert of the sample. DUP 1200019 DU 062547 -7- After a little practice and experience, several samples may be mounted at the jane f'me with excellent result*. Nemember to keen the area `n whieh mounting is done as clean as possible to prevent contamination of the filter. K1CB0SC0"; PBErAlTirirS NAINTTNANCS Quality lens tissue is a must. The mlerseooist should use It for any kind of lens cleaning. Tt should be as l<nt free as possible. It may be wrapped around the blunt end of a small camels hair brush and used to clean the evenleees. Never wine the ins'de of the microscope body, Tf tt is necessary to remove dirt from the Inside of the tubes, blow it out with an asnlrator bulb. Whenever you are using imrerision oil, be sure that you wipe all surfaces that were in contact with the oil. Generally, solvents ehould net be used when cleaning lenses. Alcohol, like most solvents will dissolve the lens mounting cement, which may ruin the lens `system. Xylene should be used snaringly to remove immersion oil. Use a camels ha*r brush to clean first-surface mirrors and non-er<t-cal lens surfaces. Never wioe first-surface mirrors with lens tissue. Avo<d touching a lens surface with fingers as the finger prints has a ten dency to etch into the lens surface. COUNTTNf, ?gOCTTt*8r Plaee the samnle on the stage and focus on it. Since the samnte is retained ip the ton ten fifteen microns of the filter, be sure that you are focused In the prooer plane. The reticle in the left eyepiece is used to define the counting area. The reticle should be ea'ibrated prior to being used. The total fiber count (a fiber is anything three times as long as it is vide) should be at least 100 fibers, or twenty fields, whichever is less. ?t is advisable to make as large a eoune as practical to get an accurate average. If e fiber eorssed the limits of the counting field, only those crossing either or both of two adjtcent sides are counted. ?or example, a fiber crosing the top or right, or possibly both sides would be eountec. The tides ehosen are at the discretion of the counter, but anv counter must always u* the same sides. The fibers counted are estimated as to length, using the circles at the top of the reticle. Our practice has been te record all fibers seen, ail fibers longer than five microns and all f-'hers longer than 10 microns. However, for eomoarisen with the ACCIH T1.V (196S), only the longer than Ju fibers need be counted. DUP 1200020 DU 062548 - 8- Membrane fleers m*v be purchased -re-londed ir. plastic holders (Ae-oss' Analyses Monitors). The vcricors enns'st of three secfons. The f: ' : to if he'd beeveer rh* middle end bottom sections. 3etveen the membra-e fi'ter end the bottom seeC'on lies e sunort oed. The ec'ihrated ourp, designed to draw a known end constant sunolv of a'r, is always corrected to the bottom of the field monitor case. When many loaded eases *r taken into on* oartlcu'ar area for sann'ing, a blank or control filter (meaning a loaded fie'e monitor ease) should be taken out to the area, brought hack unused and counted. This will determine what degree of general contam<n*t!on is oresenc on the filters. We have found that hand'ng the fte'd monitor eases with ce--uiose bands greatly redunes the genera' contamination of the filters. The bands may be purchased from Waiter R. Jelly #n<j Company, Tnc. They are white, ooaque, sire 1*1 x ?5 and come necked fn solution S-132. Unon removal from the solution, the bands are s'inoed around the middle and bottom joints, or around all loints, depending uoon whether the samn'c is going to be drawn through the ton section plug hole, or the entire ton section removed for "onen" sampling. The latter procedure <s recommended for asbestos dust sa-olinc since ,-t results in a more even samnle distribution ove r the entire exnosed filter surface. Convenient personal samnlir.g pum-s, such as those distributed by Vine Safety An-iianoes Comnany, Vil'son Products Division or Union Industrial are excellent for short ten, low rate (arcur.d 2 lorn) samoling. A samn'ing numn is connected to a 3 foot length of nen-eo'laosahle. one quarter inch, rubber tubing with a male T.eur slio adancer (stock -umber l/-"!., avai'abie from Becton, Dickson and Comnany) inserted into the other enc. This enab'es one to connect the hose direet'y to the ease by simn'y insert ing the adapter into the olug hole at the bottom of the ease. Field monitor cases may aasi'y bt r-used. The olugs and all sections are separated and placed in warm detergent water. The parts should be scrubbed thoroughly and rinsed in tap water. However, if the f-' iters are to receive any chemical analysis, the cases should alo be r*'nsed in distilled water. The cases shou'd be allowed to dry in a clean are* (a clean room is preferable), to reduce contamination. The clean area is equally important when loading the filters into the cases. The filter o*d (the thicker, fibrous disc) is placed in the bottom section. The filter i* nlaced direocty on the nad. The middle and ten sections are added, the esse is nressed tightly together, and the ton nlug is inserted. Se-laee-e-.t of the bottom n'u* is not necessary. A cellulose band is then place arr-.nc the ease and after the band dries, the unit is ready for saonling. Sample code numbers, etc. may be written on the band or upon the filter ease, first making sure the markings can be removed when and if it is necessary. Additional in*o--nation concerning filters and field monitors mav be obtained frora Millinore Corporation. DUP 1200021 DU 062549 -9- CA*.cvi.ir:' V>ien a sard's is taken, oe-cinent ir.fcrmation about tue simn'e shou'd be recorded at the samrHns site. First, the samoie number shou'd be w'-tter on the fie'd nor.'tor cose and the sn-->'e ticket. Next, enter a deseriott.-r. of his onerntior. in the aooronriate snace. If the sarmle is a genera! a-.r semo'e and does net certain to any oarticular enoloyee, vrite "general air sareole" in the employee's name soace. Information about the area nay be written ir. the "descrintion of operation" soace. If you have several nur:s which are identical ''n aooearanee, but have different flew rates, it vcu'.d he a stood idea to assign then instrument numbers. This enables you to keen accurate records of maintenance and calibration. When instrument nur.be*s are keot it fs not necessary to enter the flow rate at the sanding s-te. It may be entered later from the record cards. Cthe'-v'se, enter the flew rate at the sampling site. When a number of filters are taken into a general area a control (or blank) is taken along. This should be assigned a number and entered <n the blank number soace. Do not assign blank fil ters dunHeate numbers. When the sand'ng nuno is turned en, enter the time <n the "time cr." soace. Whi'e the samn'e is being taken make notes of controls, i.e. exhaust hoods, fans, window venti1 at'on, etc. These notes shouldbe entered -n the "descrintion of cor.tro's". The samoie should thensign his name or initials at the bottom of the sheet. After sufficient time has nasse'*, the sanding instrument shou'd be turned off and the time recorded ''n the "tine off" soace. The difference bet-wee* "time on" and "time off" should be exoressec in minutes and should be en tered in the "total ti~e" soace. The "total air vo'ur.e" may b. found by multio'v-'ng the flow rate (exoressed liters oer minute) hy the total time and recorded in the aonrooriate snace. After the sanole has been taken, brought back into the lab and mounted, the counting procedure is next. The microscooe must be orocerly set un. lace the slide to be counted c*. the mechanical stage. Focus on the sande a short distance away from the edge. Never count close to the edges of the fi'ter because the filter will soread a little and lower Che count. Mow, enter the date, sar.ole number, blank number and fie'd area on the count sheet. The counter should in-ttal the form in the "initial" soace. The sized fibers are entered from r-t-t to left across the form, first entering the >lCu fibers, then the >3u f:be*s and finally, all fibers seen, called the total. After 100 fibers or 2C fields are counted and recorded, all of the verticil columns are added uo and the totals are entered under their eorresnondir.g columns. Divide the tota's at the bottom by the number of fie'ds counted to determine the averse* number of fibers oer size range oer field. 3y using information or. these two sheets the corcentrati on in fibers ner eub-'e milliliter may be ealtu' ?.:**' end entered -'n the "ce-.." snaee on the count sheet. DUP 1200022 DU 062550 Concentration (Av. Fiber Cnt.) (filter area) (field area) (Samole volume) (1CC0) Av. Fiber Count: Filter area: Counting area: Femnle volume: 1000 : Average fiber count ner size range 855 ran2 for 37 cm dia. membrane fi ters Reticle calibrated in total time x fl.ov rate, exoress in 1iters Converts liter to milliliters Tf blanks are used, subtract the average blank cout f the same size range rrnn the average f-'ber count. DU 062551 DUP 1200023 11 ACCESSES Mlllloore field Monitor Cases Cat. No. MAW? 037AO Mlllloore filter (''. Pads) Cat. No. AAWPC3700 MilHoore Ptlter Corooratlon Bedford, Massachusetts 07130 (complete) Porton Reticle Cat. No. 30084 Patterson Globe & Clrele Reticle Cat. No. 30033 Edmund Scientific Co. 701 Edseoro Building Barrington, New Jersey 08007 Wheaton Balsam Bottle Cat. No. 2244 Arthur H. Thomas Cotnoany Post Office Box 779 Philadelohla, Pennsylvania 19105 Adaoters, hose end for 1/4" tubing to male leur slip Cat. No. LK/L Becton, Dickinson and Company Rutherford, New Jersey White, ooaque, cellulose bands, 41 x 25 No. 28 Walter R. Jelly & Co., Inc. 2822 Birch Street Pranklin Park, Illinois 60131 Personal Sampler Kk. IT Willson Products Division Reading, Pennsylvania Monitaire Sampler Mine Safety Aooliances Co, Pittsburgh, Pennaylvania Dnico Telematic Model C-110 Dnlco Environmental Industries, Inc. Tall River, Massachusetts DUP 1200024 DU 062552 DU 062553 DUP 1200025 SAMPLE TICKET COUNT SHEET PHASE CONTRAST MICROSCOPE DUP 1200026 DU 062554 LAMP CONDENSER IMAGED KOEHLER ILLUMINATION DU 062555 DUP 1200027 OOOOOQooo. OOOooooooc PATTERSON GLOBE AND CIRCLE O O O O O O oooOOO PORTON RETICLE DUP 1200028 DU 062556