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A disposable, heat-sealed packet has been developed for use in feeding and oviposition of the Debolt Lygus diet for laboratory rearing of Lygus hesperus Knight. An individual packet is formed by heat sealing the edges of a piece of Parafilm (10 by 10 cm) with a piece of polyethelene plastic (10 by 14 cm) film on three sides. The packet is partially filled with diet, and the remaining edge is then heat sealed to form a disposable packet of diet. The packets can be used for feeding nymphs and adults and also can be used for adult oviposition.

Females collectively presented warmed (31°—37°C), citrated beef blood from saturated Kimwipes imbibed more blood (x = 51.4 µl) than the other 3 methods tested. Grouped females presented warmed blood from Parafilm bags imbibed an avg of 40.3 µl, individual females in large (450 ml) containers imbibed 39.5 µl while individual females in small (35 ml) containers imbibed 4.3 µl. Twenty of 25 individually fed females in large containers and 1 of 25 individuals fed in small containers took blood meals exceeding their pre-fed weight. Based on an avg pre-fed weight of 25 females, 35 of the 50 grouped females tested took blood meals exceeding-the pre-fed avg weight.

A parafilm sachet is described for the collection and quantitative determination of honeydew excreted by a planthopper, Nilaparvata lugens (Stal), feeding on rice. The quantity of honeydew per insect per 24 h was used as a parameter in comparing the insect’s feeding activity on suscepttble and resistant rice varieties. The technique proved to be useful for determining the level of resistance of rice varieties to three N. lugens biotypes and is applicable to other sucking insects that feed on rice and other crop plants.

IN a recent report by Mittler and Dadd1 on the artificial feeding of Myzus persicae (Sulzer) through membranes of stretched ‘Parafilm’ it was noted that the aphids excreted numerous honey dew droplets when feeding on an 18 per cent sucrose solution with or without amino-acids, vitamins and salts.

Recent publications by the authors described a simple method for feeding the green peach aphid, Mysus persicae (Sulzer), on various fluid diets via a membrane of stretched Parafilm “M”® (Mittler and Dadd 1962, 1963a). Although the method proved satisfactory when relatively few cages were needed at any one time (Bragdon and Mittler 1963; Mittler and Dadd 1963b; Strong 1963), the initiation of routine experiments on a larger scale called for a number of improvements to be made in the system.

distilled water, plated on water agar in Krupinsky, J. M. 1986. Pyrenophora tritici-repentis, P. bromi, and Leptosphaeria nodorum on plastic petri dishes, sealed with Parafilm, Bromus inermis in the northern Great Plains. Plant Disease 70:61-64.

The newest laboratory products for spring include reagents for making probes for cellular oncogenes, a chamber for oxygen consumption experiments and a device for cutting Parafilm.

Various authors have studied hatching of cestode oncospheres infecting animals and man (Heath and Smyth, 1970, Parasitology 61: 329343). Silverman (1954, Ann. Trop. Med. Parasitol. 48: 207-215) reported up to 74% activation of oncospheres of Taenia saginata. Jones et al. (1960, J. Parasit. 46: 170-174) described the hatching of T. hydatigena; Berntzen and Voge (1962, J. Parasit. 48: 110-119) reported on the hatching in vitro of Hymenolepis citelli and H. nana. Berntzen and Voge (1965, J. Parasit. 51: 235-242) also described the requirements for hatching of oncospheres of four hymenolepid cestodes and noted that each of the four species of the genus Hymenolepis had slightly different requirements for hatching in vitro. Smyth (1963, J. Parasit. 51: 235-242) proposed the uniformity of requirements to be furnished, not only for the rupture of the membranes surrounding the eggs, but also for the activation of the oncospheres. Laws (1968, Exp. Parasitol. 23: 1-10) reported on the hatching in vitro of Taenia hydatigena, T. pisiformis, and Echinococcus granulosus. Hatching is here defined as the liberation of the oncosphere from all enclosing coats and membranes, with the activation of the embryo. There is no evidence from published papers that by our definition, eggs of T. solium have been hatched successfully in vitro. We report here the hatching in vitro of oncospheres of Taenia solium. Ten terminal gravid proglottids were obtained from a patient treated with niclosamide. The segments were passed by the patient 8 hr after treatment. They were washed in 0.85% NaCI and the uterine branches were counted for appropriate identification (Brown and Neva, 1983. Appleton-Century-Crofts, pp. 186187). The segments were placed in Petri dishes lined with filter paper moistened in saline, and were covered with moist cotton and filter paper. The dishes were sealed with Parafilm to prevent drying and kept for 1 wk at room temperature until experiments began. The following procedures were used as precautionary measures. Containers sufficiently large to accommodate Petri dishes, Pasteur pipettes, slides and other small glassware were filled with 95% ethyl alcohol. All material possibly contaminated with eggs was discarded into the container, taking care that the items were fully submerged in the liquid. The work area was swabbed periodically with 70% alcohol, and, at the end of the experiment, hands were rinsed in alcohol before washing with soap and water. Observations on eggs subjected to 50% ethyl alcohol (final concentration) or higher showed that, on a slide, embryophores fell apart within 5 min of addition of the alcohol and within 1 min when preparation was covered with a coverslip. The embryos appeared black and shrivelled. However, if the final alcohol concentration was 35%, the eggs remained intact during a 15min observation period under the microscope. Eggs were dissected from proglottids using a dissecting microscope and were transferred into Solution 1 consisting of: (a) 120 mg pepsin, 1: 10,000 (DIFCO Laboratories, Detroit, Michigan); (b) 2 ml HC1, 1 N; and (c) 20 ml distilled H20. The pH of this solution was 2.0. Ten ml of his solution was added to eggs in a Petri dish, and placed in an incubator at 41 C for 15 min. The eggs were then washed three times in 0.85% NaCl to prevent transfer of ingredients from the first solution into the second. Eggs were then pipetted into a 10-mm-diameter screw cap tube, containing Solution 2, consisting of: (a) 10 ml 0.85% NaCI; (b) 35 mg trypsin, crystalline (Type XII, Sigma Chemical Company, St. Louis, Missouri); (c) 0.5 ml whole bile from pig (kept frozen until use); and (d) 1 ml NaHCO3, 10% solution in distilled water. The pH of this solution was 7.2 to 7.4. The tube was then tightly capped and placed in an incubator for 15 min at 41 C, removed to room temperature (21-22 C) for observation and kept for another 15 min.

(...) Overall, there was no convincing evidence of significant effects on virus transmission efficiency due to differences in virus content. However, in further experiments in which aphids fed on virus preparations through a Parafilm membrane, correlation between virus concentration and efficiency of acquisition and transmission was evident for both efficient and inefficient vectors. Aphids also acquired virus more efficiently when feeding on virus through a Parafilm membrane than when feeding on leaves containing a similar concentration of virus as assessed by ELISA of extracts. The combined results indicated that overall virus content may not the primary factor limiting the acquisition of BYDV from plants by vectors, and that its effects may be overshadowed by other factors, including uneven distribution of virus within the leaf

Tetracotyles of Cotylurus lutzi were incubated to adults in a basic medium of 40% NCTC135 and 40% chicken serum, supplemented with 20% extract of chicken heart, liver, muscle, intestine wall, or mucosa. Ten per cent or more of eggs produced in media containing intestine extracts appeared normal, but miracidial development occurred only when upper small intestine mucosal extract was used. Five Biomphalaria glabrata snails became infected by such miracidia, shedding cercariae which encysted as tetracotyles and developed into normal adults in zebra finches. Dialysates of the effective mucosal extract failed to stimulate production of normal eggs when added to the basic medium. The strigeid trematode Cotylurus lutzi Basch, 1969, can be grown in vitro from tetracotyle to adult by the method of Voge and Jeong (1971). Their medium contains equal parts of NCTC-135 and chicken serum (CS), plus 100 units of penicillin and 100 tug of streptomycin per ml. Worms develop well in this but produce only fragile, shell-less eggs incapable of embryonation. We have modified this medium so that normal eggs, yielding infective miracidia, are produced by worms grown entirely in vitro. MATERIALS AND METHODS Young adult snails, Biomphalaria glabrata (Say), were exposed to large numbers of cercariae of C. lutzi. After 1 to 2 months the snails were washed and their shells gently crushed in 0.34% NaCl in distilled water. The ovotestes, containing encysted tetracotyles, were removed and teased apart in sterile 0.34% saline containing 10 ,ug gentamicin, 50'0 units of penicillin, and 500a ,g streptomycin per ml. Freed organisms were rinsed 4 times in this solution. Cultures were set up in plain-lipped 25by 150-mm glass test tubes containing 5 ml of medium. About 15 well-encysted tetracotyles were placed into each tube, which was closed by a silicone rubber stopper and then sealed by a tightly wound strip of parafilm. No special gas phase was used. Basic culture medium contained 40% NCTC-135 and 40% CS (Grand Island Biological Co.), plus 100 units of penicillin, 100 jug streptomycin, and 10 jug gentamicin per ml. This was supplemented Received for publication 29 August 1972. * Supported by Grant AI-10271 from the NIAID, NIH, Bethesda, Maryland. t At present student, School of Medicine, UCLA, Los Angeles, California. with various extracts of chicken origin. From 4 freshly killed 2to 3-kg adult white leghorn chickens, separate extracts of liver, breast muscle, heart, and intestinal wall muscle were prepared. About 15 g of tissue was finely minced, then ground with a mortar and pestle, and finally squeezed through silk bolting cloth. The resulting liquid was made up to 20 ml with normal saline and centrifuged for 60 min at 3,000 rpm at 5 C. The supernatant fluid was filtered twice through paper, then through 0.65and 0.45-Am Millipore filters for sterilization. For extracts of chicken intestinal mucosa, the small intestine was cut at the gizzard and at the beginning of the large intestine (as determined by texture changes and the appearance of fecal masses). This portion, approximately 70 cm, was divided into 2 equal lengths, referred to as "upper" and "lower" small intestine. Each half was cut open longitudinally and washed in several changes of cold (5 C) normal saline. A glass microscope slide was used to scrape off the mucosal cell layer, which was then collected and homogenized in a glass tissue grinder with teflon plunger. Mucosal homogenates from the upper and lower small intestine were then treated as described above. Dialysis of the upper small intestine mucosal extract was done through cellulose tubing (pore size 2.4 nm) overnight at 5 C against 1,000 ml of normal saline or of distilled water. In the former case, 6 ml of extract was used; in the latter, 7 ml. Following dialysis against distilled water, NaCl was added to restore the saline concentration of the dialysate, which in both cases was sterilized using 0.65and 0.45-utm Millipore filters. After filter sterilization, all extracts were stored at -10 C until used. Egg yolk medium was prepared as described by Berntzen and Macy (1969). Extracts were added to the basic medium as shown in Table I. Media were changed on alternate days. In any one tube, organisms were maintained in the same formulation throughout; when extract prepared from one chicken was used up, the homologous material from the next chicken

Lichen spores are discharged onto a parafilm sheet, thoroughly mixed in water, dried, stored, and returned at will into an aqueous suspension to be distributed homogeneously on a culture surface. The randomness of distribution has been verified, allowing the calculation of a confidence interval for the percentage of germinated spores according to the law of binomial distribution. Spores of Xanthoria parietina and Physcia pulverulenta stored on parafilm retain their germinative ability at least one month. The spores of lichen fungi have rarely been used in quantitative experiments on germination because it is difficult to obtain homogeneous samples of such spores. The classical method of collecting them aseptically on an agar nutrient medium has been described by Werner (1927): An agar plate is placed above moistened apothecia a short distance from the epithecium and the spores that are ejected upwards usually adhere to the agar. For monospore cultures, one spore can then be removed with a glass needle and transferred to another agar plate. But for statistical investigations, such as those concerning the average rate of germination, individual transfer of spores is not practical. Germination must be observed directly on the original plate and the drawbacks are the following: a) The spores discharged by one ascus usually remain packed together so that the beginning of germination is often difficult to detect. Later on, especially for spores giving rise to more than one filament, it is even more difficult to see to which spores the hyphae belong. b) Some areas are densely crowded with spores (above the apothecia) while others are nearly devoid of them. Up to now, "group effects," commonly described for other kinds of spores, have not been noticed in lichens but they may exist in some cases and it is better to avoid them if possible. c) Spores ejected from different apothecia, and most of all from different thalli (even if these have been collected near to each other), rarely have the same germinative capacity. Therefore, such a distribution of spores is far from random and it becomes necessary to deal with large numbers of spores and to make many replicate experiments to obtain significant results. An improvement of the method is thus required. Scattering spores by gently brushing the surface of the medium with a thin brush should overcome the first difficulties (a and b). However, this procedure is not entirely efficient because it does not suppress the heterogeneity (c) between populations from different culture dishes. The best solution is to mix the spores thoroughly before sowing them and then to scatter them as well as possible. Contrary to other kinds of spores (mosses, ferns), lichen spores are extremely I thank the Institute of Applied Mathematics of Grenoble for providing probability tables of the binomial distribution. I am also grateful to Dr. F. LeBlanc for revising the English text. 2 Laboratoire de Physiologie V~g6tale, Universit6 de Grenoble, Grenoble, France. This content downloaded from 207.46.13.163 on Fri, 08 Jul 2016 05:38:40 UTC All use subject to http://about.jstor.org/terms 1970] KOFLER: LICHEN SPORE GERMINATION 603 hydrophilic. They may adhere to glass surfaces, especially if they have been allowed to dry out. Water itself remains partly on such polar surfaces when drops are rolled across or when it is pipetted. Consequently a smooth hydrophobic surface was looked for, one onto which spores could be ejected and from which these could be easily collected in a suitable liquid. Parafilm, which is commonly used to cap culture vials, was found entirely suitable for this purpose.