JANUABY, 1960] HELMINTIIOLOGICAL SOCIETY 37

A Method for the Concentration of Nematodes for Mounting from the Baermann Apparatus* BERT M. ZUCKERMAN**

In the course of making routine nematode extractions from soil by Christie and Perry's (1951) modification of the Baermann technique, it became ap- parent that a rapid method for concentrating- the nematodes drawn from the Baermann funnel for mounting woxild be of great utility. A technique whereby a representative sample of the nematodes drawn from the Baermann extract is rapidly concentrated into a single drop of water on a slide prior to microscopic examination is described in this paper. The object of the method is to sample without bias rather than to get all of the nematodes concentrated. The sample is allowed to stand in the Baermann funnel for at least 24 hours to provide time for the less active nematodes to work themselves through the cloth. An aliquot of 10-15 ml is drawn from the Baermann funnel into a beaker and the nematodes relaxed by heating in an incubator at 60° C for five minutes. The contents of the beaker are then transferred to a 125 ml separatory funnel. A glass tube tapered to an opening of 1 mm is attached to the base of the funnel by means of rubber tubing. The liquid is passed slowly through an inverted 250 mesh sieve, the rate of flow being governed by the glass stopcock of the separatory funnel (Fig. 1,A). Follow- ing this operation the sieve is returned to its normal position and adjusted above a microscope slide until the spot where the water had passed through is centered over the slide. A drop of killing and fixing solution is passed through the mesh at the same spot and falls to the slide, carrying with it a large number of the nematodes collected on the screen. The addition of cover slip and sealing compound complete the making of the slide. The refinements described below aid in the application of this method. A ring of ZUT slide sealing compound! y2 incn in diameter applied to the upper and lower surfaces of the mesh helps to prevent the water from spread- ing as the liquid passes from the separatory funnel (Fig. 1,B). A drop of liquid detergent applied to the mesh in the center of this ring further aids the passage of the water through the sieve. Excess detergent is vigorously washed from the screen prior to passage of the Baermann extract. Any excess water hanging from the inverted sieve after this process is removed by applying suction with a medicine dropper. This step is particularly im- portant and is done prior to reinverting the sieve. The microscope slide which is to receive the nematodes is supported in a position a few millimeters below the mesh on which the nematodes are concentrated. If the drop of killing and fixing solution adheres to the screen, the mesh is lightly depressed until it comes into contact with the slide. Under this treatment the drop of fixative can be easily transferred to the slide. If it is desired to process several extracts in succession, the sieve must be thoroughly washed and then dried between samples. A lint-free cloth or filter paper is used to remove excess liquid from the pores of the screen. To obviate this difficulty a number of ZUT rings can be applied to the mesh,

"Contribution No. 1204 of the University of Massachusetts College of Agriculture Experi- ment Station, Amherst, Massachusetts. **Associate Professor, Department of Entomology and Plant Pathology, University of Massachusetts. tManufactured by Bennett Manufacturing Co., Salt Lake City, Utah.

Copyright © 2011, The Helminthological Society of Washington 38 PROCEEDINGS OF THE [VOL. 27, No. 1 then successive samples can be processed provided that the areas within the rings do not become wet or contaminated with nematodes from previous extracts. In other tests a piece of bolting cloth, of about 300 mesh to an inch, was used to concentrate the nematodes in lieu of a sieve. The cloth was mounted in a wooden frame and ZUT rings applied to the upper and lower surfaces of the cloth as previously described for the sieve (Fig. 1,C). An average of 61.2 percent of the nematodes from the Baermann extract were screened out when this method was applied in trials which involved 60 samples. These tests indicated that the sieve and the bolting cloth remove the nematodes from the Baermann extract with comparable efficiency. Some specimens are inevitably lost during the process of transferring the nema- todes to the microscope slide; however, extracts which had a total population of 400 nematodes yielded slides which contained at least 50 specimens. SUMMARY A technique is described which provides for rapid concentration and mount- ing of nematodes from Baermann extracts. The nematode population is sampled without bias by this method. Theoretically all forms are represented on the completed slide in numbers proportionate to their abundance in the Baermann extract. The method is suggested as an adjunct to other soil and

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Figure 1,A) Baermann extract in separatory funnel prior to passage through an inverted 250 mesh sieve. The sieve is supported by a beaker which receives the processed extract; B) Sieve returned to normal position and microscope slide supported beneath the sieve to receive the concentrated nematodes; C) Bolting cloth mounted in wooden frame showing rings of ZUT applied to surface of the cloth.

Copyright © 2011, The Helminthological Society of Washington JANUARY, 1960] HELMINTHOLOGICAL SOCIETY 39 plant tissue extraction techniques in which nematodes are separated from large quantities of liquid for microscopic examination. LITERATURE CITED CHRISTIE, J. B. and V. G. PERRY. 1951. Removing nematodes from soil. Proc. Hel. Soc. Wash. 18: 106-108.

Studies 011 Two Gorgoderid Cercariae from Oklahoma with the Description of a New Species* WILLIAM II. COIL During the summer of 1958, sphaeriid mollusks were collected from the Red River drainage in southeastern Oklahoma in order to study the trematodes which parasitize them. Both cercariae which are discussed here were collected from the Blue River in Johnston County. This stream is spring-fed and quite cool, supporting several species of fish and amphibians which could serve as definitive hosts for gorgoderid trematodes. The sphaeriids were removed from a sandy-mud bottom and placed in an ice cabinet for transportation to the laboratory. Living cercariae were studied with the aid of vital stains, and sections were stained with Harris' haema- toxylin and A. G. E. Pearse's stain (Hotchkiss PAS, Celestin Blue, Toluidine B]ue, and Orange G). This latter stain is especially good for unicellular glands. I am indebted to Dr. Carl D. Riggs and his staff at the University of Oklahoma Biological Station for their unstinting efforts to help with my summer research problems. This study was supported, in part, by a National Science Foundation Grant-in-Aid. Ccrcaria papillostoma n. sp. (Figures 1-3, 5) DIAGNOSIS : Body measurements in millimeters of specimens fixed in neu- tralized formalin and mounted in piccolyte are: length of stylet 0.008, Avidth of acetabulum 0.019-0.026, width of oral sucker 0.017-0.038, length of excre- tory bladder 0.034-0.039, distance between acetabulum and oral sucker 0.027- 0.031, length of body 0.11-0.15. Suckers well-developed, active, covered with long thin "hairs" or setae. Some setate papillae on oral sucker. Charac- teristic "double" papillae on acetabulum arranged in hexagonal pattern. Three to four pairs of penetration glands. Ducts of penetration glands slight- ly lateral to median, passing around edge of oral sucker, then through it, opening laterad to stylet, cytoplasm and secretion granular. Excretory bladder surrounded by large, coarsely granular, cystogenous glands. Tail elongate, very large, swollen at anterior end into cercarial chamber which completely encloses body of cercaria. Distal part of tail swollen and wrinkled, tapering to a terminal knob which is adhesive. In live, active specimens, bodv enclosed in cercarial chamber, but in moribund specimens, body is half out or completely detached from tail. Tail extremely active, but not natatory. Body swollen laterally, posterior to acetabulum giving a superficial resemblance to the rhopaloeercariae. Excretory system probably of the mesostomate type. Genital primordia present as undifferentiated clumps of cells. HOST : Pisidium compression Prime. SITE OF INFECTION : Gills. '•'•From the University of Oklahoma Biological Station, Willis, Oklahoma and Department of Zoology. University of Nebraska, Lincoln, Nebraska. Studies from the Department of Zoology, University of Nebraska No. 310.

Copyright © 2011, The Helminthological Society of Washington