Document BRq913ezryyakbMQkZXGoR4ZL

EQUIPMENT AND PROCEDURES FOR MOUNTING MILLTPORE FILTERS AND COUNTING ASBESTOS FIBERS BY PHASE CONTRAST MICROSCOPY I PLAINTIFFS EXHIBIT CHV-104 Stephen G. Bayer Ralph D. Zumvalde Thomas A. Brown Bureau of Occupational Safety and Health 1014 Broadway _ Cincinnati, Ohio 4S202 JULY 1969 U. S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service Consumer Protection and Environmental Health Service Environmental Control Administration . -HEVBB 003137 table OF CONTEXTS Introduction------------------------------------------ ------------ ----------------- ------------------------------- 1, Equipment List------------------ ------------------------- 1 Laboratory Set-up----------------------------------------------------------------------- 1 Preliminary Microscope Set-uo------------------------------- 2 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----------------------- ------------g Microscope Precautions and Maintenance---------------------------------------- 7 Counting Procedure------------------------ -------- ------------------------- ---------------- ---------- ----- 7 Field Monitor Discussion-- -------------- ------ ---------------------- R Calculation of Concentration------------------------------------- -- -- ---------- ----- 9 Addresses---------------------------------- -------- ---------------------------------------------------------------------11 CHEV BB 0003138 FOREWORD To determine concentrations of asbestos dust in air for comoarison with established hygiene limits, a simole reproducible method is required. A simnle and inexpensive samnling method uses disposable (reloadable if desired) plastic membrane filter holders, pre-loaded with membrane filters. These membrane filters may be rendered transparent and the asbestos fibers counted with a phase-contrast mieroscooe at 430X (or 400X). This method has been used by the Asbestosis Research Council in Great Britain and the U.S, Public Health Service in the United 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 procedures must be used for carrying out these counts is reproducible results are to be achieved. Detailed equipment and procedures used by the U.S. Public Health Service are described here. CHEV BB 0003139 - 1- INTRODUCTION* For several years, a set method for counting and siring airborne particulate matter, which has been deposited unon membrane filters, has been used. The filters used in this technique are 37 mm diameter cellu lose ester membrane f Iters manufactured by Killipore Corporation, (cat. no. AAWPO3700). The slide mounting technique refers only to these filters, and will not work for any others. The filters nay be. purchased nrenounted in convenient field monitor cases, and are ready for sampling. The filters are nearly 100% efficient for any asbestos dust, even for fibers with diameters much smaller than the 0.8 micron (u) nore si::e. Sample preparation for electron and optical examination are both relative!}* simple. Slide mounts of the filters are s emi-permanent. The following procedures and rcco~r.Kr.datior.s will produce accurate results, if each step is performed exactly as indicated. . equip? nu?7 list 1. Table 2. Adjustable chair '. 3. Pharr contrast microscope 4. Ribbon filament illuminator or (built-in illuminator) 5. Rye piece reticle (Porton type) 6. Lens tissue 7. Small camels hair brush 8. Slides 9. Cover slips 10. Spatula 11. Tweezers 12. Scalpel 13. Wheaton Balsam bottle LABORATORY SET-UP . It is important to provide a suitable counting area, for the room in which the counting is to be done has a great bearing upon the taicroscopist. It should he out of the way of normal traffic, and be kept as free from dust and smoke as possible. The rabies on which the microscopes are place should be as free from vibrations and shocks as possible. An adjustable chair will do much to add to the comfort of the counter. CHEVBB 0003140 ' 2 ~ T'RF.LT'rNArv microscope rrr it? Before any dust sizing or counting can be done, proper equipment must be at hand. This section describes the required equipment. The illuminator should be small and reasonably compact. Ribbon filament- illuminators are generally much easier to use. The illuminator must in corporate a condensing lens, so that the enlarged image of the lamp fila ment may be focused in the plane of the substsgc condenser diaphragm. The illuminator must also'have an iris diaphragm located as near as possi ble to the condensing lens. The iris serves an a field diaphragm and is focused in the nlane of the specimen. ' The microscope used must have a nubstage condenser fitted with an iris diaphragm. This iris serves as an aperture diaphragm. the microscope should have a calibrated reticle inside the non-adjustable eyepiece. The reticle should be small enough that all the counting field is in focus (if the flat field objective and cyeoie.ces are used, essentially the en tire field will be in focus). The microscopes must he equipped with phase contrast accessories. Using phase contrast, even objects with almost the same refractive index as the mounting media can be seen. Light waves which travel through such a specimen are retarded by .a fraction of r. vravclenpth to produce a change in phase. The phase contrast optical system reveals these phase changes as light or dark contrast against the back ground. This procedure was written by a user of Bausch and Lomb phase-equinoe*d microscopes, so that micros.cone set-up procedures correspond with these. However, any pood microscope with phase contrast accessories will be satisfactory. ' For continual routine examination of filters, the binocular type microscope is much more comfortable. The use of a first su:.aco mirror is recommended, but not necessary.. By utlizing.a second surface mirror, multiple images of the illuminator field iris are formed which will detract from the crisp ness of the final image. ' As mentioned previously, the microscope must contain a reticle. Any reticle, sucli as the Porton or Patterson Globe and Circle, which projects a constant counting area end has provision for sizinp, will work. The Porton reticle which we use outlines a rectangle that fits easily within the periphery of focus. The rectangle is divided into two squares. The left square is divided into six rectangles which constitutor, the counting area. Above and below the large rectangles are a scries of cit*cles in which every other eirel doubles in diameter. Hence,the third is twice the diameter of the first; the fourth is twice the size of tho second, etc. The right half of the rec tangle contains a scale for extending the size above the number nine circle. The formule D=I. describes the circles sizes. The diameter D is found * . . r .11-1. .u,, f and TJ which represents the number of the :hev bb tnrr%i/ii -3- length of the rectangle Is 200 t units. Ey measuring the length and dividing by 200, "L" is obtained and the circle sixes may be calculated. Whenever a microscope. is disassembled or cleaned., the reticle calibration should be checked. Another fact to taka into consideration is that changing the inter pupillary distance of the binocular will change the calibration, so the microsconist may want to check the calibration at both extremes of adjust ment. The counting area on the reticle should be kept as clean as possible, as dirt on the reticle is in focus and may be counted. microscope set up and the ppo^rrups fop quaint kg rjrrr.np illumtkattok By utilising equipment that meets the requirements just mentioned, ar.y counter may achieve comparable counts after some practice. This section will deal with sotting the microscope up and obtaining kohler illumination. It is extremely important that every step be followed exactly ns indicated. ` 1. 2. 3. 4. Place the microscope on a flat, level surface at a height such that the eyepieces uvsy be observed without strain or discomfort. Place a moderate dust sample upon the stage. Place the illuminator directly in front cf the microscope. Sight across the two be sure they are aligned. For coil filament bulbs the illuminator iris should be two inches from the center of the microscope mirror. For ribbon filament bulbs, the front of the filter holder should be seven inches from the center of the plane s'dc af the mirror. Never use the curved side. Fcmove 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. ROTE: Where the microscope includes a built-in illuminator to give Kohler illumination, the above steps will not all be necessary. FEVTEW ' A. Check microscope and illuminator alignment. B. Check the distance between the microscope and the illuminator. C. Be sure that you are using the flat -side of the mirror- D. Make sure that there are only two filtersin the system. . 5. By means of the focusing knob on the side of the suhstage con denser assembly, raise it until the upper lens nearly touches the bottom of the slide. Remember that the free working distance ' of the condenser is in the order of 1.2 mm and that the slide is about 1 mm.thick. CHEV BB 0003142 -4- 6. Turn on the illuminator. Using the tilt controls, direct the bears onto the center of the mierror. Then by tilting the mirror direct the beam upward into the condenser. 7. Close the substngc Iris completely. Open the illuminator iris nil the way. 8. By means of the focusing controls on the side of the illuminator, focus the image of the filament on the bottom of the substage iris. This may be done by loaning over the microscope and observing the substage iris in the microscope mirror. (On such node's rs the Bsusch & bomb PP.-27, there is a fine focus knob at the rear of the illuminator), in that case, move the condensing lens all the way forward and trim the focus with the fine focus knob. 9. Open the substage iris about half way. ' ireVTKff' A. Check the distance between the slide and condenser. B. Be sure that the beam is striking the center of the mirror, and that it is being deflected upward into the condenser. C. Please reneat sten 7. D. Check sten 8. Tf the image is not in focus now, correct it. Do not continue until the filament is correctly focutecl. ` F. Please repeat step 9. 10. Put the 10X objective in place and focus on the sample. This is done as follows: First, looking from the side of the microscope, lower the objective until it gets very near to the slide. Then, . by looking through the eyepieces, fccus un. Never focus down with coarse focus when looking through eyepieces. A word caution: The objective lenses are very expensive, so be careful not to grind them through the slide. Always focus up. New trim the focus with fine focus knob. Focus sharply on the sample. Secondly, close the field iris fully. Tf the condenser is nearly In focus, you should cc-o a bright snot of light. Pjy using the condenser focus knob, bring the field iris into shern focus. Now, both the field iris and the- objective are in focus on the samnlc. If there appear to be multiple images of the field irin, it is because of the second surface mirror. Some of the light waves are reflected from the first surface, resulting in secondary imngta. * 11. ' Tf the image of the field iris is not centered, re-center it by tilting the mirror. Tf the color around the field iris is not uniform, re-check the tilting of the illuminator. CHEV BB 0003143 PFVTFP A. Check and he sure you are focused on the sample. 10X objectives have a relativelv long focal lennth and it may be possible to focus on: (a) the ton of the cover slio. (b) the sample, (c) the bottom of the filter, (d) the ton of the slide, (e) the bottom of the slide. (f) the ton of the condenser. So be sure of the focal plane. B. Be sure that the field iris is focused in the sample plane. Check this often vhen examining samples. As the sample focus changes, so does the condenser focus. . C. Be-check the centering and tilt of the illuminator. DETATLFD INSTRUCTIONS FOB BATTSCP AND LOMB BINOCULAR MICROSCOPES 1. Leave the microscone phase ring on 0. Insert the telcscooe that is suonlied vith the kit into the right eyepiece tube. Focus on the dark phase ring. Open and close the substage iris to be sure that it is centered in relation to the objective phase ring. If it is not in the center, loosen the set screw on the condenser mount and properly set ' the condenser. Then re-tighten the set screw. This should very seldom be necessary. Do not force anything and be careful and patient. When you are satisfied that the condenser is aligned, open it all the way. 2. Turn the nhase wheel to 10. This shows that the 10X phase ring is in place. Look through the telescope epain. 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 43X objective. Carefully turn the wheel to the 43 nhase ring. Align the two rings in the same manner as performed in step 2. 4. Remove the telescope and replace the eyepieces. Again, check the conden ser focus. Tou are now ready to examine the sample at 430X, assuming . that you are using 10X eyepieces. FILTER MOUNTING Set up Prior to Mounting . Have available at hand a quality scalpel and a supply of No. 10 curved blades, a pair of tweezers (fine pointed are preferred), a snatula or fire polished glass rod and a box of lens tissue. Slides (1" x 3" or 25 x 75 mm) with CHEV BB 0003144 V7 6 the date, sample number, etc. It is advisable to use quality number 1-1/2" cover slins. 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 stopcock grease to keep out the surrounding a^r. The mounting solution may now be prepared.. A one to one solution by volume of dimethyl phthalate and diethyl oxalate is poured into the balsam bottle. Then add 0.05 pram of filter material for each milliliter of solution. The added filters increase the viscosity of the solution. Before using thr solution, all of the filter 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 Procedure Keep in mind that cleanliness is important. First, lay down two pieces of lens tissue; ovie directly in front of you and the other to the left or right for the mounting tools. Next, wipe the scalpel, tweezers and glass rod or soatula with the frosted end toward you. Even precleaned slides must he cleaned this vav. Hold a cover slip by the edges bctv.'een the index finger and the thumb, and clean it in the sar.se manner as the slide. Place the cover slip so that one edge rests on the unfrosted end of the slide and the other edge rests on the lens tissue. Be sure that the lever surface does not come into contact with the lens 'tissue., . Using the glass rod, or dropper, dispense a small drop of the mounting media onto the center of the slide. Replace the rod or dropper into the solution bottle and cover it. Using the spatula, spread the solution into a triangular shape. While the fluid is spreading, cut a wedge of corresponding sire fron the filter. Place it upon the mounting media, sample side up. Pick up the cover slip and place it on top of the filter. Press lightly on the cover slip to- he sure the mounting media has made contact with it. Label the slide before completing any ether slide. Sometime, after an hour or so, the filter may not have cleared. Usually, this can be cured by pressing lightly on the cover slip 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 erroneous 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 part of the sample. ' , :hev bb >003145 7 After a little practice and exnerience, several samples'may be mounted at the same time with excellent results. v Demember to keen the area in which mounting is done as clean as possible to prevent contamination of the filter. MICROSCOPE PRECAUTIONS ASP MAINTENANCE Quality lens tissue is a must. The micrscopist should use it for any kind of lens cleaning. Tt should be as lint free as possible. It may be wrauped around the blunt end of a small camels hair brush end used to clean the eye pieces. Never wine the inside of the microscope body. Tf it is necessary to remove dirt from the inside of the tubes, blow it out with an aspirator bulb. Whenever you arc using immerision oil, be sure that you wipe all surfaces that were in contact with the oil. Generally, solvents should not 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 sparingly to remove immersion oil. Use a camels hair brush to clean first-surface miri*ors and noii-critical lens surfaces. Never vice first-surface mirrors with lens tissue. Avoid touching a lens surface with fingers os the finger prints has n ten dency to etch into the lens surface. COUNTING PROCEDURE Place the sample on the stage and focus on it. Since the samnle is retained in the ton ten fifteen microns of the filter, be sure that you are focused in the proner plane. The reticle in the left eyepiece is used to define the counting prea. The reticle should be calibrated prior to being used. The tota.1 fiber count (a fiber is anything three times as long as it is . wide) should be at 'least 100 fibers, or twenty fields, whichever is less. Tt is advisable to make as large"a "count as practical to get an accurate average. If a fiber corsced the limits of the counting field, only those crossing either or both of two adjacent sides are counted. For example., a fiber crosing the top or right, or pespibly both sides would be counted. The sides chosen are at the discretion of the counter, but any counter must always use the same sides. The fibers counted are estimated as to length, using the circles at' the ton of the reticle. Our practice has bfcen to record all fibers seen, all fibers longer than five micro;.- and all fibers longer than 10 microns. However, for comparison with the ACGIH TLV (1968), only the longer than 5p fibers need be counted. CHEV BB 0003146 8 FTT.D MOTJTTOR DTSCPSSTON Membrane filters may be purchased pre-loaded in plastic holders (Aerosol Analysis Monitors). The Monitors consist of three sections. The filter is held between rh middle and bottom sections. Between the membrane filter and the bottom section lies a support oad. The calibrated pump, designed to draw a known and constant sunoly of air, is always connected to the bottom of the field monitor case. Wien many loaded cases "re taken into one particular area for sampling, a blank or control filter (meaning a loaded field monitor case) .should be taken out to the area, brought back unused and counted. This will determine what decree of general contamination is present on the filters. We have found that banding the field, monitor cases vi th cellulose bands greatly reduces the general contamination of the filters. The bands may be purchased from Walter H. .Telly and Company, 7nc. They are white, opaque, size 41 x 25 and cone nacked in solution S-132. Upon removal from the solution, the bands are slinned around the middle and bottom joints, or around all joints, depending unen whether, the sample is going to be drawn through the ton section plug hole, or the entire ton section removed for "onen" sampling. The latter procedure is recommended for asbestos du.et sampling since, it results in a more even sample distribution over the entire ex posed filter surface. Convenient nersonai sampling pnmns, such as those distributed by Mine Safety Appliances Company, Willson Products Division or Union Industrial are excellent for short term, low rate (around 2 1pm) sampling. A ' sampling pump is connected to a 3 foot length of non-collansaMe, one quarter inch, rubbe* tubing vi th a male T.eur slip adanter (stock number LH/T. available from Beeton, Dickson and Comnany) inserted into the other end. This enables one to connect the hose directly to the case by simply insert ing the adanter into the plug hole at the bottom of the ease. Field monitor cases may easily be re-used. The plugs'and all sections are separated and placed in warm detergent water. The parts should be scrubbed thoroughly and rinsed in tao water. However, if the filters are to receive any chemical analysis, the cases should also be rinsed in distilled water. The cases should be allowed to dry in a clean area (a clean room is preferable), to reduce contamination. The clean area is equally important when loading the filters into the cases.' The filter pad (the thicker, fibrous disc) is placed in the bottom section. The filter is nlaced directly on the pad. The middle and ton sections are added, the case is pressed tightly together, and the ton plug is inserted. Replacement of the bottom plug is not necessary. A cellulose, band is then nlace around the case and after the band dries, the unit is ready for sampling. Sample code numbers, etc. may be written on the band or tiaon the filter case, first making sure the markings can be removed when and if it is necessary. Additional information concerning filters and field monitors may be obtained from Millinore Corporation. . CHEV BB 0003147 9 CALCULATION OF CONCENTRATION . Wien a sample is taken, pertinent information about the sample should be recorded at the sampling site. First, the sample number should be written on the field monitor case and the sample ticket. Next, enter a description of his operation in the appropriate snace. If the sample is a general air sample and docs not uertain to any particular emnloyee, write ''general air sample" in the employee's name space. Information about the area may be written in the "description of operation" space. If you have several pumps which are identical in appearance, but. have different flow rates, it would be a good idea to assign them instrument numbers. This enables you to keen accurate records of maintenance and calibration. When instrument numbers are kept it is not necessary to enter the flow rate at the sampling site. It may be entered later from the record cards. Otherwise, enter the flow 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 in the blank number space. Do not assign blank fil ters duplicate numbers. . When the sampling pump is turned cn, enter the time in the "time on" space. While the sample is being taken make notes of controls, i.e. exhaust hoods, fans, window ventilation, etc. These notes should be entered in the "description of controls". The sample should then sign his name or initials at the bottom of the sheet. - After sufficient time has passed, the sampling instrument should be turned eff and the time recorded in the "time off" space. The difference between "time on" and "time off" should be expressed in minutes and should be en tered in the "total time" space. The "total air volume" may be found by multiplying the flow rate (expressed liters per minute) by the total time and recorded in the appropriate space. After the sample has been taken, brought back into the lab and mounted, the counting procedure is next. The microscope must be properly sot un. Place the slide to be counted on the mechanical stage. Focus on the sample a short distance away from the edge. Never count close to the edges of the filter because the filter will spread a little and lower the count. Nov;, enter the date, sample number, blank number and field area on the count sheet. The.counter should initial the form in the "initial" space. The sized fibers are entered from right to left across the form, first entering the >10p fibers, then the >5u fibers and finally, all fibers seen, called the total. After 100 fibers or 20 fields are counted and recorded, all of the vertical columns are added up and the totals are entered under their corresponding .columns. Divide the * totals at the bottom by the number of fields counted to determine the average number of fibers per size range per field. By using information on these two sheets the concentration in fibers per cubic milliliter may be calculated and entered in the "con." space on the count sheet. CHEV BB 0003148 - 10 - Concentration * (Av. Fiber Cnt.) (filter area) (field area) (Sample volume) (1000) Av. Fiber Count: Filter area: Counting area: Sample volume: 1000: Average fiber count ner sixe range 855 mm2 for 37 mm din. membrane filters Reticle calibrated in tir2 total time x flow rate, express in liters Converts liter to milliliters 7f blanks are used, subtract the average blank cout of the same sire range *rnm the average f*ber count. "HEV BB >003149 11 ADDRESSES ' Mlllipore Field Monitor Cases (complete) Cat. No. MART 037AO . Mlllipore filter (W. Pads) Cat. No. AAWPO3700 Mlllipore Filter Corporation Bedford, Massachusetts 07130 Porton Reticle Cat. No. 30054 Patterson Riche & Circle Reticle Cat. No. SCOS3 Edmund Scientific Co. 701 Edscorp Building Barrington, Nt.v Jersey 0SC07 Wheaton Balsam Bottle Cat. No. 2244 Arthur H. Thomas Company Post Office box 779 Philadelphia, Pennsylvania 19105 Adapters, hose end for 1/4" tubing'to male leur slip Cat. No. -T.K/L ' Becton, Dickinson end Company Rutherford, New Jersey ` White, opaque, cellulose bands, 41 x 25 No. 28 Walter H. Jelly & Co., Inc. 2822 Birch Street . Franklin Park, Illinois.60131 Personal Sampler Mk. IT Willson Products Division. Reading, Pennsylvania Monitaire Sampler . . Mine Safety Appliances Co. Pittsburgh, Pennsylvania Unico Telematic Model C-110 Unico Environmental Industries, Ine.^ Fail River, Massachusetts CHEV BB 0003150 PM WMOO L%. C P Is c. CHEV BB 0003151 IMAGE PLANE PHASE CONTRAST MICROSCOPE CHEV BB 0003152 LAMP CONDENSER IMAGED KOEHLER ILLUMINATION :hev bb 1003153 OOOoooooo. PATTERSON GLOBE AND CIRCLE oooOOO PORTON RETICLE . CHEV BB 0003154