Document YG2EavOqvw552O5VkXz2ekBeN

1/3/7?. Asbestos. Standard: 29 CFR 1910.93a(a) of December 7. 1971 8-hour time-weighted average*: 5 fibers* per milliliter greater than 5 microns in length. Excursion limit*: 10 fibers per milliliter, up to 15 minutes in an hour for up to 5 hours per 8-hour day. -'Unprotected or improperly protected worker exposures Analytical Method: Fibers counted at AGQ-A50 magnification, using phase contrast illumination, with sample mounted in high-viscosity solution of membrane filter material. ( * Sampling Equipment: Personal sampling pump plus Millipore type AA filter (37mm, 0.8 }.\ pore size). Face cap is removed and filter used open face during sampling. Sampling rate: 2 L/n. Sample Size: . Minimum period of 15 minutes for evaluation of excursion limit. Several samples of up to A hours for evaluation of 8-hour average. Samples with a visible deposit may be too heavy to count. Compare with a clean filter. Heavy concentrations of visible dust in the air (100 to 500 fibers/ml) may require short sampling periods of only 5 minutes, or less. Blanks: With each batch of samples submit two filters which are subjected to exactly the same handling 3S for the samples except that no air is drawn through them. Label these as blanks. 1 of 7 EQUIPMENT ANT) PROCEDURES FOR MOUNTING MI LIT PORE FILTERS AND COUNTING AS3ES70S FIBERS BY PHASE CONTRAST MICROSCOPY Stephen G. 3ayer Ralph D. Ztsrvalda Thomas A. Brcvn Bureau of Occupational Safety and Health 1014 Broadway Cincinnati, Chio 45202 JULY 1969 U S. DEPARTMENT OP HEALTH, EDUCATION, AND WELFARE Public Health Service Consumer Protection and Environmental 'health Service Environmental Control Administration TARLE OF CONTENTS Introduction--------------------------------------------------------------------------------------------------------------------- j Equipment - List-------------------------------------------------------------------------------------------------- l Laboratory Set-uo---------------------------------------------------------------------------------- ---------- ---___ i 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 nounting------5 Mounting nrocedure------------------ --------------------------- - -- ---------- ----------------------------- $ Microscope Precautions and Maintenance---------- 7 Counting Procedure----------------------------------------- ------------------------- -------- -- 7 Field Monitor Discussion---------------- ---------------- ------------------------------------------------- 8 Calculation of Concentration-------------- --------- ----- ---------- --------- -------------------------- -- 9 Addresses-------------------------------------- ------ ------- --------- ------------------------------- ----- ------------------- 11 FOREWORD To determine concentrations of asbestos dust in air for comparison with established hyeiene limits, a simple reproducible method is required. A simole and inexpensive sampling method uses disDOsable (reloadable if desired) clastic membrane filter holders, pre-loaded with membrane filters. These membrane filters nay be rendered transparent and the asbestos fibers counted with a nhase-ccntrast microscope at 430X (or 400X). This method has been used by the Asbestosis Research Council in Creat 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. INTRODUCTION For several years, a set method for counting and sizing airborne particulate matter, which has been dcoosited uoon membrane filters, has been used. The filters used in this technique are 37 mm diameter cellu lose ester membrane Iters manufactured by Millipore Corporation, (cat. no. AAWP03700). The slide mounting technique refers only to these filters, and will not work for any others. The filters may be turehased oremounted in convenient field monitor cases, and are ready for samoling. The filters are nearly 100Z efficient for any asbestos dust, even for fibers with diameters much smaller than the 0.8 micron (p) pore size. Sample oreparation for electron and optical examination are both relatively slmole. Slide mounts of the filters are semi-permanent. The following procedures and recommendations will produce'accurate results, if each step is performed exactly as indicated. FQUIPMF.NT T.TST 1. Table 2. Adjustable chair 3. Phase 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 Tt is important to provide a suitable counting area, for the room in which the counting is to bj done has a great bearing upon the microscopist. It should be out of the way of normal traffic, and be keot as free from dust and smoke as possible. The tables 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 tha comfort of the counter. PRFJ.TJ-JTNARY MICROSCOPE SET UP 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 *n the plane of the substage condenser dianhragm. The illuminator must also have an iris diaphragm located as near as possi ble to the condensing lens. The iris serves as a field diaphragm and is focused in the nlana of the specimen. The microscope used must have a substage condenser fitted vith 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 eyepieces are used, essentially the en tire field will be in focus). The microscopes must be equipped with phase contrast accessories. Using phase contrast, even objects with almost the same refractive index as the mounting media can be sean. Light waves which travel through such a specimen are retarded by a fraction of a wave length 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 Lcmb phase-equiooed microscopes, so that microscope set-uo procedures correspond with these. However, any good 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 surface 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 reticl such as the Porton or Patterson Globe and Circle, which projects a constant counting area and his provision for sizing, 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 constitutes the counting area. Above and below the large rectangles are a series of circles in which every other cir doubles in diameter. Hence,the third Is twice the diameter of the first; the fourth is twice the size of the second, etc. The right half of the rec tangle contains a scale for extending the size above the number nine circle The formula D=L v/?'*' describes the circles sizes. The diameter D is found from L which is the unit of length, and N which represents the number or th circle. L is determined by calibration with a stage micrometer. The encir 3- length of the rectangle is 200 L units. By measuring the length and dividin by 200, "L" Is obtained and the circle sizes 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 inter pupillary distance of the binocular will change the calibration, so the microscooist 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. PROCEDURE FOR OBTAINING KOHLER ILLUMINATION By utilizing equipment that meets the requirements just mentioned, any counter may achieve comparable counts after some oractice. This section vill deal with setting the microscooe up and obtaining kohler illumination. Tt 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 eyepieces may be observed without strain or discomfort. 2. Place a moderate dust sample upon the stage. 3. Place the illuminator directly in front of the microscope. Sight across the two and be sure they are aligned. For coil filament bulbs the illuminator iris should be two inches from the center c the microscope mirror. For ribbon filament bulbs, the front of 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 fi 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 steps will not all be necessary. REVIEW 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 filters in the system. 5. By means of the focusing knob on the side of the substage 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. -4- 6. Turn on the illuminator. Using the tilt controls, direct the beam onto the center of the micrror. Then by tilting the mirror . direct the bean unward into the condenser. 7. Close the substage iris completely. Open the illuminator iris all 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 iri This may be done by leaning over the microscope and observing the substage iris in the microscope mirror. (On such mode1*: as the . Bausch & Lom'o PR-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. RKVTEW 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 step 7. D. Check sten 8. If the image is not in focus now, correct it. -Do not continue until the filament is correctly focused. Please repeat step 9. 10. Put the 10X objective in dace 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 eyeoieces,fccu3 ud. Never focus down wit coarse focus when looking through eyepieces. A word o^ caution: The objective lenses are very expensive, so be careful r to grind them through the slide. Always focus up. Now trim the focus with fine focus knob. Fc-cus s'naroly on the sample. Secondly, close the field ir fully. Tf the condenser is nearly in focus, you should see a bright snot of light. By using the condenser focus knob, bring the field ids into sham focus. Now, both the field iris and the-objective are in focus on the sannl Tf there appear to te multiple images of the field iris, it is because of th second surface mirror. Some of the light waves are reflected from the first surface, resulting in secondary images. 11. Tf the image of the field iris is not centered, re-center it by tilting the mirror. If the color around the field iris is not uniform, re-check the tilting of the illuminator. - 5- HP.VTW A. Check and be sure you are focused on the sample. 10X objectives have a relatively Ions focal length and it may he possible to focus on: (a) the ton of the cover slit>. (b) the sample. (c) the bottom of the filter. (d) the top of the slide. (e) the bottom of the sl'de. (f) the too 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 when examining samples. As the sample focus changes, so does the condenser focus. C. Fe-check the centering and tilt of the illuminator. DETAILED INSTRUCTIONS EOF BAUSCH AND LOMB BINOCULAR NTCROSCOPES 1. Leave the microscope phase ring on 0. Insert the telescope that is supplied 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 oblective 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 rot force anything and be careful and patient. When you are satisfied that the condenser is aligned, open it all the way. 2. Turn the phase wheel to 10. This shews that 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 43X objective. Carefully turn the wheel to the 43 phase 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. You are now ready to examine the sample at 430X, assuming that you are using 1CX eyepieces. FILTER MOUNTING Set up Prior to Mounting Have available at hand a quality scalpel and a supply of No. 10 curved blade 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 -6 - the date, samnle number, etc. It is advisable to use quality number 1-1/2" cover si ins. Most dry objectives are corrected for this thickness cover glass. The mounting media should be kent in a Wheaton Balsam bottle. The cage 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 rhthalace 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 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 inoortant. First, lay down two pieces of lens tissue; one 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 spatula with the frosted end toward you. Ever, precleaned slides must be cleaned this wav. Hold a cover slip by the edges between the index finger and the thumb, and clean it in the sama 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 lower surface does not come into contact with the lens tissue. Using the glass rod, or dropper, dispense a small drop of the mounting nedir 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 triangu": shape. While the fluid is spreading, cut a wedge of corresponding size fror the filter. Place it upon the mounting media, sample side up. Pick up the cover slip and place it on too of the filter. Press lightly on the cover slip to he sure the mounting media has made contact with it. Label the slic before completing any other slide. Sometime, after an hour or so, the filtc 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 dryir 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. After a little practice and experience, several samples may be mounted at the sane time with excellent results. Pemember to keen the area in which mounting is dene as clean as possible to prevent contamination of the filter. MTCrnSCOPE PFFCAUTynNS AND MATNTFN'ANCE Quality lens tissue is a must. The micrscopist should use it for any kind of lens cleaning. It should be as lint free as possible. It may be wrapped around the blunt end of a small camels hair brush and used to clean the eyenieces. 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 are using immerision oil, be sure that you vioe 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. Xylen? should be used sparingly to remove immersion oil. Use a camels hair brush to clean first-surface mirrors and non-crit->cal lens surfaces. Never wise first-surface mirrors with lens tissue. Avoid touching a lens surface with fingers as the finger prints has a ten dency to etch into the lens surface. COUNTING PROCEDURE Place the sannle on the stage and focus on it. Since the sannle is retainec in the ton ten fifteen microns of the filter, be sure that you are focused in the proper plane. The reticle in the left eyepiece is used to define the counting area. The reticle should be calibrated prior to being used. The total fiber count (a fiber is anything three times as long as it is wide) should be at least 1.00 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 corssed th*e 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 possibly 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 too of the reticle. Our practice has been to record all fibers seen, all fibers longer than five microns and all fibers longer than 10 microns. However, for comparison with the ACGIH TLV (1S68) , only the longer than 5u fibers need be counted. . F7FLD MOIMTOP. dtscusston Membrane ffters may be purchased pre-loaded in plastic holders (Aerosol Analysis Monitors). The Monitors consist of three sections. The filter is held betveer the middie and bottom sections. Betveen the membrane filter and the bottom section lies a support pad. The calibrated pump, designed to draw a known and constant sunoly of a-'r, is alvays connected to the bottom of the field monitor case. When 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 degree of general contamination is present on the filters. We have found that bonding tbe field monitor cases with cellulose bands greatly reduces the general contamination of tbe filters. The bands may be purchased from Walter H. Jelly and Company, Inc. They are white, opaque, size 41 x 25 and come packed in solution S--132. Upon removal from the solution, the bands are slipped around the middle and bottom joints, or around all joints, depending upon 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 fo" asbestos dust sampling since it results in a more even sample distribution over the entire ex posed filter surface. Convenient persona1 sampling pumns, such as those distributed by Mine Safety Appliances Ccmnany, Willson Products Division or Union Industrial are excellent for short term, low rate (around 2 Ipra) sampling. A sampling rumn is connected to a 3 foot length of non-collansable, one quarter inch, rubber tubing with a male l.eur slip adapter (stock number LH/L available from Beetc-n, Dickson and Company) inserted into the other end. This enables one to connect the hose directly to the case by simply insert ing the adapter into the plug hole at the bottom of the case. 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 tap 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 placed directly cn the nad. The middle and ton sections are added, the ease is nressed tightly together, and the too plug is inserted. Replacement of the bottom n'ue is not necessary. A cellulose band is then place around the case and after the hand dries, the unit is ready for sampling. Sample code numbers, etc. may be written on the band or uoon the filter case, first making sure the markings can be removed when and if it is necessary. Addition.-,1 information concerning filters and field monitors'may be obtained from Mlllinore Corporation. CALCUT-\T7 ON OF CONCFNTP.ATT0N When a sample is taken, pertinent information about the sample should be recorded at the sampling site. First, the sample r.unber should be written on the field monitor case and the sample ticket. Next, enter a description of his oncration in the appropriate snace. If the samnle is a general air sample and does not pertain to any particular emoloyee, write ''general air sample" in the employee's name space. Information about the area nay be written in the "description of operation" space. If you have several numos which are identical 'n appearance, but have different flew 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. Tt may be entered later from the record cards. Otherwise, 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 in the blank number space. Do not assign blank fil ters duplicate numbers. When the sampling nunp is turned on, 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. t` i After sufficient time has passed, the sampling instrument should be turned off and the tine recorded in the "time off" space. The difference between "time on" and "tine 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 tine 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 he properly set up. Place the slide to be counted on the mechanical staae. 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 'nwer the count. Now, enter the date, sample number, blank number and fie'd 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 >10u 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 sizt: 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. - TO - Concentration = (Av. Fiber Cnt.) (filter area) (field area) (Sar.oie volume) (1000) Av. Fiber Count: Filter area: Counting area: Famnle vo'une: 1000: Average fiber count ner size range 855 rm2 for 37 m dia. membrane filters Reticle calibrated in total tine x flow rate, exnress in liters Converts liter to milliliters Tf blanks are used, subtract the average blank cout of the same sire range crnm the average f'ber count. 11 - addresses Milllpore Field Monitor Cases Cat. Ko. MART 037AO Milllpore filter (W. Pads) Cat. No. AAUT03700 Milllpore Filter Corporation Bedford, Massachusetts 07130 (complete) Porton Reticle Cat. No. 30084 Patterson filobe & Circle Reticle Cat. No. 30083 Edmund Scientific Co. 701 Edscoro Building Barrington, New Jersey 08007 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. LH/L Becton, Dickinson and Company Rutherford, New Jersey White, opaque, cellulose bands, 41 ;c 25 No. 28 Walter H. Jelly & Co., Inc. 2322 Birch Street Franklin Park, Illinois 60131 Personal Sampler Mk. IT Willson Products Division Reading, Pennsylvania Monitalre Sampler Mine Safety Appliances Co. Pittsburgh, Pennsylvania Unlco Telematic Model C-110 Unlco Environmental Industries, Inc. Fall River, Massachusetts cn *5 i-- m --t C"> z mH --i > cro* c-> GO INST, TIME JOB o m ron rn x <o/i 33 k c~ -< -< -o H O "0 6 --< r* m mrn V) o z z zz O*Tt o o o n O u m > 2 m o > n -4 O-->< r~ -n a> r-- X o r</> ->i o z a < m2: o i a r~ c 2 5 m mH Ate m o-nti CD r~ > z 3* r. ) IMAGE PLANE PHASE CONTRAST MICROSCOPE LAMP CONDENSER IMAGED IN SPECIMEN PLANE ty./' FIELD DIAPHRAGM KOEHLER ILLUMINATION