Issued April 20, 1911. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. BULLETIN 135. A. D. MELVIN, CHIEF OH BUREAU. _ . COMPARATIVE STUDY OF METHODS OF I B' EXAMINING FECES FOR EVIDENCES ' 5 OF PARASITISM. BY MAURICE C. HALL, Assistant Zoologist, Zoological Division. *TUA1WLM JAN 4 1943 Ul 5KA.RY WASHINGTON : GOVERNMENT PRINTING OFFICE. 1911. V April 2U, 1'Jll. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. BULLETIN 135. A. D. MELVIN, CHIEF OK BUREAU. A COMPARATIVE STUDY OF METHODS OF EXAMINING FECES FOR EVIDENCES OF PARASITISM. BY MAURICE C. HALL, Aftxititant Zoologist, Zoological Dirixwn. WASHINGTON : GOVKRNMHNT PRINTING OI-KICE. 1911. THE BUREAU OF ANIMAL INDUSTRY. Chief: A. D. MELVIN. Assistant Chief: A. M. FARRINGTON. Chief Clerk: CHARLES C. CARROLL. Animal Husbandry Division: GEORGE M. ROMMEL, chief. Biochcmic Division: M. DORSET, chief. Dairy Division: B. H. RAWL, chief. Inspection Division: RICE P. STEDDOM, chief; MORRIS WOODEN, R. A. RAMSAY, and ALBERT E. BEHNKE, associate chiefs. Pathological Division: JOHN R. MOHLER, chief. Quarantine Division: RICHARD W. HICKMAN, chief. Zoological Division: B. H. RANSOM, chief. Experiment Station: E. C. SCHROEDER, superintendent. Editor: JAMES M. PICKENS. ZOOLOGICAL DIVISION. Chief: B. H. RANSOM. Assistant Zoologists: ALBERT HASSALL, HARRY W. GRAYBILL, and MAURICE C. HALL. Junior Zoologists: HOWARD CRAWLEY and WINTHROP D. FOSTER. 2 LETTER OF TRANSMITTAL. UNITED STATES DEPARTMENT 01 ACJRHTLTIRK, BIKEAI OK ANIMAL IMHSTKY, Washington, I). ('.. January 17. 1!)1 1. SIR: I have the honor to transmit the accompanying manuscript of an article entitled "A Comparative Study of Methods of Examin- ing Feces for Evidences of Parasitism," by Mr. Maurice C. Hall. Assistant Zoologist in the Zoological Division of this bureau, with the recommendation that it be published as a bulletin of this bureau. Mr. Hall has devised a method of examining feces by means of which the presence or absence of parasites infesting the alimentary tract may be rapidly and accurately determined. Various methods of fecal examination are in vogue among medical and zoological investigators, and these are all critically compared with Mr. Hall's method, which is shown to yield superior results. Respectfully, A. I). MKLVIX, ('kief of Bureau. Hon. JAMES WILSON, Secretary of Agriculture. CONTENTS. Page Introduction Methods of microscopic examination 7 Smear method 7 Sedimentation method Burette method Centrifuge methods Sieve methods ( Jasteiger's filter method !> Bass's salt solution method Bass's calcium chlorid centrifuge method Garrison's calcium chlorid sedimentation method Wellman's sodium acetate centrifuge method Pepper's adhesion method Telemanu's ether hydrochloric acid method Culture methods Purpose of methods Advantages of concentration Properties used in concentration Physical proper! ies Chemical properties 1 Biological properties 11 The theoretical ideal 15 Practical objections 15 Gross examination 1 "> The writer's method I (> Comminution of feces I (5 Sieving IS Sediment ing and centrifuging _1 Preparat ion of slides '2 1 Concentration obtained by the use of sieves '2'2 Summary of method '2'2 ( 'ollection index '2:\ Tests of unpublished methods :M ( lomparative tests of the writer's method and other methods ~2~> Smear method L'5 Sedimentation method L'5 Burette method L'5 Gasteiger's filter method 2(1 Bass's calcium chlorid centrifuge method L'(i Garrison's calcium chlorid sedimentation method l'S Telemann's ether hydrochloric acid method L'S Culture methods :',<) Objections to certain methods :;| Injury to parasites :]\ Limited application or incommensurate results :V2 Disinfection of feces :V2 Summary ;];] Adaptability of method :w Economy of method :>,\ Conclusion :! 1 Bibliography :',") ILLUSTRATION. Page FIG. 1. Apparatus used in the writer's method of examining feces 17 G A COMPARATIVE STUDY OF METHODS OF EXAMINING FECES FOR EVIDENCES OF PARASITISM. INTRODUCTION. Having occasion to make numerous examinations of feces for evi- dences of parasitic infection, the writer lias devoted some time to developing a satisfactoiy method which would be capable of general application in routine examinations and which would give the surest results in the shortest time, one which would detect any existing in- fection, even though light, in any kind of feces, so far as the existing limitations of fecal examinations permit. The different methods which have been used and advocated, most of them being methods used in examining human feces, were tested on feces of various kinds. This paper deals with the results of these tests. The method which gave the most satisfactory results and which is hereafter referred to as the writer's method is a modification of previous methods. Fecal examinations are of two sorts first, examination with the naked eye for gross evidence of infection in the shape of entire' para- sites or fragments, and, second, microscopic examination for para- site eggs, embryos, etc. The first is very commonly, perhaps wrongly, neglected; the second is commonly used and will be considered first here. METHODS OF MICROSCOPIC EXAMINATION. SMEAR METHOD, The microscopic method which is commonly used and advocated is what may be called the smear method. A small particle of feces is taken up with a toothpick or stirring rod, smeared on a glass slide in a drop of water or salt solution, covered with a cover glass, and examined under the miscroscope. This method is the oldest and the simplest, can be used under almost any conditions, and, in that it calls for no equipment beyond a toothpick, a slide and cover glass, and a microscope, is also the most inexpensive. It is usually considered satisfactoiy. providing a numl)er of slide preparations, commonly 10, are examined. This method has been repeatedly commended by 8 METHODS OF EXAMINING FECES FOR PARASITISM. Stiles (1902hh, 19031, lOOCa), is one commonly used by physicians (Jones, 1907, et al.), and was used by Garrison, Ransom, and Steven- son (1903a), and Stiles and Garrison (1906a) in Washington, and by Garrison (1908) in Manila. SEDIMENTATION METHOD. The simplest modification of this method consists in allowing the feces, if sufficiently fluid, to settle, and then examining the sediment; if the feces are too solid for this, large or small amounts are washed in a sufficient quantity of water, decanted as long as any matter will float, and the sediment finally examined. Stiles, in the papers just cited, Braun (Braun and Liihe, 1909), Garrison (1910), Letulle (1905), Jones (1907), and others have advocated this sedimentation method. BURETTE METHOD. I am informed by Dr. Ransom that one writer, in an article to which I have not a reference at present, has varied the preceding method by taking the sediment from the bottom, through a stopcock, a method designated in this paper as the burette method. CENTRIFUGE METHODS. Another variation of the sedimentation method is to use a centri- fuge for the purpose of giving a more rapid and certain concentra- tion of material. Pepper (1908) states that he has found repeated centrifuging, in which the sediment is shaken up each time with some fresh water, very useful. This is the process commonly known as washing. Bass (1909) uses the same device as part of his method. Braun (Braun and Liihe, 1909) notes that under certain circum- stances the sediment may be centrifuged after sedimenting and decanting. He does not state under what circumstances this is to be done. Letulle (1905) admits the use of the centrifuge if needed, but objects to it on the ground that certain eggs suffer mechanical injury from its use. Stiles (190'2hh. 19031) states: "The centrifuge does not appear to be of any special value in fecal examinations." SIEVE METHODS. Another variation of technique consists in the use of one or more sieves or screens to take out coarse particles of undigested food and similar objects. Stiles (1902hh, 19031) gives a qualified indorse- ment of this method in the following terms: In case an unusually large amount of large, coarse material is present in the feces, it is sometimes convenient to pour the entire mass through a sieve, re- GASTEIUER'S AND BASS'S METHODS. 9 jecting the portion left in the sieve; or to \vsish the feres in a sieve, holding the latter under water. As a rule, however, the sieve is not very useful in fecal examinations. On the other hand, the use of the sieve receives unqualified indorse- ment from Cobb (1904). In examining sheep feces for fluke eggs he boils a pellet or part of a pellet of sheep dung in water for a few minutes; puts it in a hemispherical brass sieve of 80 to 100 meshes to the inch, the sieve being in a watch glass full of water, and itself standing partly full of water; brushes the pellet through the sieve by means of a sable brush; sediments the fluid containing the fecal matter which passes through the sieve, and repeatedly removes the supernatant muddy fluid with a pipette until water poured on the sediment remains clear: and then brushes this sediment through a sieve of miller's silk bolting cloth of five meshes to the millimeter. The water which passes through the silk sieve is then examined for fluke eggs. Bass (190!)), Telemann (1908). and Garrison (1910) use a sieve of some sort in making examinations of feces. GASTEIGER'S FILTER METHOD. Gasteiger's (1904) method is an inversion of the methods where a centrifuge and sieve are used. Whereas with the latter the object is to screen out foreign particles larger than parasites and eggs and seek for the parasitic material at the bottom of the centrifuge tube. Gasteiger, in his search for eggs of Ascaris in the stalls of cattle, soaks the manure, straw, and mud in water; filters this water through some unspecified sort of filter, and examines the residue, not the fil- trate, for eggs. This accomplishes to a lesser degree the same thing that is accomplished by sedimentation, sieving, and centrifuging it gives a concentration of material. BASS'S SALT soi.t TION METHOD. Bass (1906) proposed a method which consisted in putting a quan- tity of feces in a vial three-fourths full of a nine-tenths saturated so- lution of sodium chlorid, shaking this well, allowing it to settle, and then examining a drop from the surface of the fluid. This method was proposed for the examination of human feces for hookworm eggs. According to Bass, all the eggs rise to the surface and large numbers may be found in one drop. Of course this method depends on the specific gravity of the parasite egg being less than that of the solu- tion employed. AVaddell (1910) states that if an examination of three slides, presumably smear preparations, did not disclose human hookworm eggs, this method of Bass's was used, and says: " By this method from 30 to 40 minutes could be saved per stool examined." 78989 Bull. 13511 1 10 METHODS OF EXAMINING FECES FOR PARASITISM. BASS'S CALCIUM CHLORID CENTRIFUGE METHOD. Afterwards Bass (1909) modified the above method as follows: A quantity of feces is diluted with water, 1 in 10, and strained through gauze to get rid of coarse particles. What comes through is centrifuged, the fluid poured off, the centrifuge tube refilled and the fresh material and the old sediment centrifuged again, thereby constantly adding to the total sediment, until all the diluted feces have been used. The sediment is rewashed several times until all matter that can be washed out in this manner is removed. Then a calcium chlorid solution of a specific gravity of 1.050 is substituted for the water. This disposes of everything having a specific gravity below 1.050, and the sediment may be examined at this point. If much sediment remains, the heavier matter may be removed by cen- trifuging with a calcium chlorid solution having a specific gravity of 1.250. In this solution the eggs come to the top and a few drops from the surface may be removed and examined, or, better, some of the top fluid may be poured off, diluted with water sufficiently to bring the specific gravity below 1.050, and centrifuged. The sediment will now contain most of the eggs that Avere in the original amount of feces and may all be put on a slide and examined. GARRISON'S CALCIUM CHLORID SEDIMENTATION METHOD. Garrison (1910) has outlined a method which is essentially a modi- fication of Bass's (1909) method. According to Garrison The specific gravity of the ova is from 1.050 to 1.100 (old eggs sometimes higher, according to Bass). If the stool be liberally diluted with tap water the mixture has a considerably lower specific gravity, which varies, of course, with the character of the stool, but is usually about 1.005, so that the ova, to- gether with the heavier sediment, sink to the bottom. [Allow to stand for an hour or more ; decant and add fresh water repeatedly until soluble matter is washed out.] 'At any time during the sedimentation, but preferably after the specimen has been washed a few times, the coarser material may be removed by straining and washing the sediment through a fine wire gauze, using a small, strong jet of water. To completely wash a specimen until the super- natant water is clear may require quite a number of sedimentations, and it may be desirable to continue the process throughout one or more days. * * * Sometimes the feces contain heavy, gritty, solid material, which is particularly annoying in making slide preparations. The specific gravity of much of this material is sufficiently higher than that of the eggs to allow the use of a solu- tion with a specific gravity between the two which will float the eggs to the surface and allow the heavy sediment to sink. This may be done by suspend- ing the specimen, preferably after it has been well washed, in a solution of cal- cium chlorid containing 350 grams to the liter of water, which gives a specific gravity of about 1.200. (A saturated solution of the commercial salt has about the same specific gravity.) The top layer, containing eggs and the lighter debris, is decanted, leaving the heavy sediment behind. WELLMAN S, PEPPER S, AND TELEMANN S METHODS. 11 WELLMAX's SOD It M ACETATK CEXTRIFl 36 Ancylostoma. 2 Toxocara. 299 Txnia. Dog No. 2 \8 Ancylostoma (28 A ncylostoma 5 A ncylostoma DogNo.3 4 Toxocara Dog No. 4 13 Ancylostoma flO Ancylostoma Dog No. 5 (3 Txnia Dog No. 6 Dog No. 7 4 Tnxocara 258 Txnia \10 Ancylostoma 1 Toxocara 209 Txnia An examination of the above table shows that the results obtained by the use of the waiter's method and of Bass's method without the step involving the use of the solution of 1.050 specific gravity, are on the whole superior to those obtained by the use of Bass's method. Dock and Bass (1910) state of Bass's method: Gage and Bass conclude, after this extensive experience [the examination of the feces of 315 students by the smear method, by sedimentation and centrifug- ing, and by Bass's method], that for all practical purposes washing with water alone is all that is necessary and that the washing with calciuni-chlorid solution is unnecessary except for special purposes. The fact that the use of the strong calcium chlorid solution gives at times better results than the use of water alone is the writer's warrant for making use of it in one centrifuge tube to check the findings from the other tube. GARRISON'S CALCIUM CHLORID SEDIMENTATION METHOD. Garrison's method of using calcium chlorid and sedimenting instead of centrifuging has come to the writer's attention too recently to permit of adequate tests. A test made by sedimenting in a centrifuge tube instead of centrifuging did not give as good results as cen- trifuging. Sedimenting has the disadvantage of being slower than centrifuging. TELEMANN'S ETHER HYDROCHLORIC ACID METHOD. A number of tests were made to determine the applicability of Telemann's (1908) chemical methods to the examination of feces. Telemann himself states that he has found his method satisfactory in a large series of human and animal feces. He does not specify what animals are included in the series, but his statement may be believed in any case. At the same time, the tests made in this laboratory indicate that his success was due more to the use of the hair sieve than to the use of chemicals. The latter probably did COMPARATIVE TESTS OF METHODS. 29 more in mechanically breaking up the feces by a slight amount of chemical action than in actually concentrating parasitic material by eliminating nonparasitic matter soluble in ether and hydrochloric acid. As previously stated, the tests of the writer's method on sheep feces gave a constant theoretical concentration of 4: 1. The addition of ether and hydrochloric acid to a given 0..") cubic centimeter of sediment so obtained gave a reduction to 0.4 cubic centimeter, or an additional theoretical concentration of only f> : 4. When the ether and hydrochloric acid were added to the fresh feces a somewhat smaller sediment was obtained. This does not indicate a greater con- centration, as it appears to, but a lesser breaking up of feces by these reagents than by water, so that more agglomerations of material that should be broken up to allow the finer matter to pass the sieve are left unbroken, and the small as well as the large particles are held by the sieve. This is evident from the result obtained by adding these reagents last to determine the action due to them alone. Another result sometimes obtained by using Telemann's method on sheep feces was that a plug of plant material formed at the top of the centrifuge tube and held in its mesh almost all the fecal mate- rial. The feces of herbivora are composed largely of plant matter and hence largely of cellulose. This is removable to a great extent by sieving but is not at all soluble in Telemann's reagents. This is also true of the feces of such birds as chickens and pigeons, as tests with such feces indicate. On the other hand, the feces of man and of the carnivora have less plant matter of the sort and more matter that is soluble in ether and hydrochloric acid. Nevertheless, comparative tests indicate that the writer's method is practically as good as Telemann's for these feces also. In tests with human feces where the writer's method gave a theoretical concentration of 4:1. Telemann's method gave the same result. In one case the sediment obtained by the writer's method was treated by Telemann's method, and after shaking up and stirring was again centrifuged. The resultant sediment showed no reduction in volume as a result of the action of the chemicals. In another ca>e where the writer's method gave a sediment of 0.8."> cubic centimeter.au application of Telemann's method to this sediment reduced it to 0.05 cubic centimeter. Even more surprising result > were obtained with dog feces, used as representative of feces of the carnivora. In test cases sieving in water gave a concentration of 5: 1. In one case the application of Telemann's method to the sediment so obtained reduced it to half of its volume, thereby doubling the concentration. Although the concentration was doubled, the fact that the writer's method had reduced the original 1 cubic centimeter to O.i2 cubic centi- meter, thereby eliminating 0.8 cubic centimeter, while the chemicals had onlv reduced it bv 0.1 cubic centimeter more, indicate- that the 30 METHODS OP EXAMINING FECES FOR PARASITISM. additional solution of material accomplished by the use of these chemicals was but slightly more than the solution accomplished by water alone. In two other cases where dog feces had been allowed to stand a long time in water and had been shaken up for four or five minutes before being sieved and centrifuged, the resultant sediment of 1.4 cubic centimeters was actually increased after the application of Telemann's reagents followed by centrifuging to a flocculent sedi- ment of 1.9 cubic centimeters. The results of comparative tests of Telemann's and the writer's method were quite unexpected, as the writer had used Telemann's method for a year and only abandoned it when its inapplicability in the case of sheep feces became to.o evident to overlook. As has been stated previously in this paper, the success of Telemann's method ap- pears to be due more to the efficiency of a slight solvent action in mechanically breaking up feces and putting them in condition to sieve, than to an extensive solvent action. Much that might be dis- solved in the chemicals is soluble in water, much that might be dis- solved is perhaps taken out by the screen where water alone is used, and the fact that the slight solvent action breaks up the feces and makes them easy to sieve seems to be the feature to which the results obtained by this method are to be attributed. The feces of herbivora have little material that is soluble in Tele- mann's reagents, and the breaking up secured by them is inferior to that secured by the use of the writer's method. Human feces break up rapidly in Telemann's reagents, but the method advocated by the writer takes about the same time and gives as good results. Of Tele- malm's method, Dock and Bass (1910) say: We have found the method admirable with some stools, but in others not enough solution occurs to permit a concentrated layer of eggs to be thrown down. Telemann's method has one disadvantage which neither Telemann (1908), Pfister (1909), nor Quadflieg (1909) mentions in connection with it it injures the microscope. The fumes of pure hydrochloric acid attack the lens mountings and also the stage and the lenses themselves. The use of vaseline to seal the edges of the cover glass affords an uncertain protection, and the use of high powers under such circumstances is decidedly unsafe. In the writer's opinion the injury to the microscope would of itself be sufficient reason for dis- carding this method. CULTURE METHODS. Quadflieg (1909) has recommended using culture methods as an aid in detecting infection. Tests of this method did not indicate that it added to the certainty of detecting infection, though live embryo nematodes could be detected by the use of a dissecting microscope OBJECTIONS TO CERTAIN METHODS. 31 with low-power lenses. As the determination of nematode genera and species is in general more difficult from the embryos than from the eggs, the usefulness of the culture method is limited. Emerson (1910) uses the following method in searching for Strongyloides intestinalix : The stool should be placed in a dish, a small depression made on its surface, this filled with water, and the stool left in a thermostat overnight. If embryos are present, they are easily found * * * actively swimming in this water. Culture methods have been used by Ransom (190Gi) in studying Hcemonchus contortus in sheep feces. Dr. Garrison tells me that he has seen the culture method used successfully in Arnn r work in exam- ining a company for hookworm infection. It is evident that such methods are suitable only for special pur- poses and not for general work. OBJECTIONS TO CEETAIN METHODS. The reason for rejecting some of the methods outlined in the hypothetical ideal treatment previously mentioned is, as was stated, that in actual practice objections of two general kinds may be urged against them. These objections are: (1) The injury to parasites or eggs; (2) the unsuitability of the methods for certain kinds of feces or the fact that the concentration resulting from the application of the method may be too insignificant to warrant the time and effort ex- pended. It will be fairly evident from the discussion of methods already given just how those methods fail in these two respects, but a brief summary will indicate some general truths. INJURY TO PARASITES. In regard to the first point, it is evident that if parasite eggs or embryos are to be used in feeding experiments to produce infection, or are desired alive for study, or if the movement of embryos is de- pended upon to indicate viability or to aid the eye in detecting or locating the parasites, certain methods, such as boiling (Cobb, 1904) or the use of chemicals which will kill by poisoning or by rapid osmosis (Telemann, 1908 ; Bass, 1909) , can not be used. Bass's ( 190!) ) method has the further objection that it not only kills, but distorts. Letulle (1905) urges that certain eggs, such as those of BilJiarzni and Uncinaria, are mechanically injured by the use of the centrifuge. I have not found this true of the eggs of the human, sheep, or dog hookworm, or any other parasites encountered, but there might be cases where the objection would hold good. The writer's use of shot would work occasional injury to parasite material and it should l>e avoided when possible. In almost all cases, the feces can be broken up in a little longer time by shaking in water alone, as experiments show. 32 METHODS OF EXAMINING FECES FOR PARASITISM. LIMITED APPLICATION OR INCOMMENSURATE RESULTS. In regard to the second point, experience shows that in using specific gravity as an aid in concentration it is safe to assume that the specific gravity of a parasite is greater than water, though all eggs will not go to the bottom, as some are held up by lighter material, bubbles, etc., but it is not safe to rely on a parasite floating on a solu- tion of specific gravity greater than itself for any length of time, owing to the action of endosmosis. An additional objection to this method advocated by Bass (1909) is that experience shows that all the steps are not warranted by the results. In most cases the simpler methods used by the writer give as good or better results. The use of the 1.050 solution appears to be a defect of the method. The concen- tration attained by it seems trifling in any case and at times it apparently results in actual loss of parasite material. Finally, under the same class of objections, experience shows that the concentra- tion obtained by the use of chemicals instead of water (Telemann, 1908) is no greater than that obtained by the use of water alone, and hence there appears to be no reason for using the more expensive chemicals, especially in view of the injury to the microscope which one of them occasions. It might also be urged that the odor of these chemicals is such that one would prefer to avoid them, though it is also true that they deodorize the feces after a fashion, and that while they kill the parasites they also disinfect the feces in cases where the killing is immaterial. DISINFECTION OF FECES. Disinfection and deodorization of feces can be more easily accom- plished, if desired, by the use of formol solution instead of water, as suggested by Letulle (1905). Jones (1907), and Garrison (1910). In a discussion of this point before the Helminthological Society of Washington, December 1, 1910, Dr. Stiles advocated the use of coal- tar disinfectants on the ground that protracted work with formol material would cause headaches. The writer has never experienced any inconvenience from the use of formol. Dr. Stiles also pointed out the great desirability of using some disinfectant, for the reason that the greater number of fecal examinations now being made in this country are for evidence of hookworm infection, and the localities with the greatest amount of hookworm infection have a high typhoid index. In using a disinfectant, the waiter's method of comminution has the advantage of bringing the disinfectant into intimate contact with all parts of the fecal mass. The thorough breaking up of the feces and the use of large amounts of water reduces the, odor to a point where it is almost imperceptible in most cases. SUMMARY. 33 SUMMARY. After testing the various methods as above indicated, the writer finds that the best results in routine examinations of feres of all kinds are obtained from the simple method already given. Briefly, the method consists in breaking up the feres very thoroughly by shak- ing in water, adding a quantity of small shot if necessary or desir- able; sieving through a set of brass sieves and then through a silk bolting-cloth sieve or a sieve made with a jeweler's fine-meshed brass screen, examining the material left on the sieve for parasites: sedi- ment ing (and washing) : centrifuging (and washing) one tube being filled with calcium chlorid solution of l.'2.">0 specific grav- ity, centrifuged, and if desired the top cubic centimeter removed with a pipette, shaken up in a tube with 14 cubic centimeters of water and centrifuged and then making a microscopic examination of a drop of sediment from the bottom of the tube centrifuged with water, and one from the top when the calcium chlorid solution alone was used or from the bottom in case water was added to the top cubic centimeter. The material is washed at either or both of the points indicated. ADAPTABILITY ()!' METHOD. The writer does not claim that the method advocated here is the best possible method. It is. however, the method which his experi- ence shows to be the best for routine examination of various kinds of feces after comparative tests with other methods. It serves very well for the feces of man. and of the carnivora. herbivora, and birds, so far as fecal examinations for representatives of the last three groups indicate. It is not only of service in examining feces for worm para- sites, but also for coccidia. It has not been tested for other protozoa. Presumably the writer's comminution method would damage flagel- lates, ciliates. or amelne. It is often useful in detecting parasitic in- fection of stomach and intestinal contents. It has the advantage of speed and certainty over the smear method or sedimentation methods. It takes longer to make the microscopic preparation than in the smear method, but the resulting concentration justifies it. Nor is it a long or difficult process. The time required for each step is slight a minute to shake up the feces. another to sieve them, and another to centrifuge them, leaving out the sedimentation after sieving, which needs no at- tention, and the time spent in examining material on the screens. which examination is incidental to the technique, not part of it. As everyone who has used laboratory methods knows, the total time necessary to perform three one-minute operations is not just three minutes or. as a rule, even six minutes, as preparation, intermediate steps, cleaning up. etc.. add considerably to the time necessary. At the same time, the method outlined here is reasonably rapid and takes less time than is required to examine the additional slides 34 METHODS OF EXAMINING FECES FOR PARASITISM. which the use of the smear method would demand. I find that the careful examination of a slide not uncommonly takes 9 or 10 minutes. Garrison, Ransom, and Stevenson (1903a) state that the examination of 100 preparations is an average day's work for one person. This is a little over four minutes for each preparation. Stiles (1909) states that a thorough examination of 10 slides will take 40 to 60 minutes, or 4 to G minutes for each preparation. Dock and Bass (1910) claim to look over an ordinary slide thoroughly in 2 to 4 minutes. If one slide prepared by the writers method be considered as equivalent to 4 smear preparations, and experi- ence shows that it is much better than this, then it will save time to spend 8 minutes in obtaining a sediment and 8 more in examin- ing 2 preparations made from this sediment, rather than spend 82 minutes examining 8 smear preparations. Where positive infor- mation as to parasitic infection is desired, the best methods and the time necessary for these methods are abundantly warranted. Dock and Bass (1910) state that Gage and Bass, in the examination of the feces of 315 students, found only 47 per cent of the cases of intestinal parasitism by the smear method, the remaining 53 per cent being found by the use of centrifuge methods. They note cases where the examination of 25 smear preparations failed to show infection, although the existence of infection in these cases was demonstrated by preparations made after sedimenting and centrifuging. Only in those cases where the necessary apparatus for better meth- ods is not available, or where evidence of heavy infection is sought for with a view to immediate medical treatment, would the writer consider the routine use of the crude smear method warranted. In exceptional cases its use might be warranted in examining one or two slides for specific infection where there is likelihood of the infection being heavy enough to be promptly discovered by this method. ECONOMY OF METHOD. If the time of a physician, veterinarian, or scientist is of any value, then the smear method is not even more economical than the writer's method. The centrifuge is a thing which the workers just mentioned should have for purposes other than fecal examinations, and the only additional pieces of apparatus required the screens are inexpensive and durable. CONCLUSION. The method of examining feces for evidences of parasitism which consists in putting the feces through a process of thorough shaking. sieving, sedimenting, and centrifuging appears, from a theoretical standpoint and in actual experience, to give the best results in the shortest time and with a minimum financial expenditure when the value of time saved is considered. It is therefore advocated as a practical routine method of examining feces of all sorts. BIBLIOGRAPHY. 35 BIBLIOGRAPHY. 1 BASS, C. C. [M. D.] 1906. Uncinariasis in Mississippi