Cotton Stopper Research Articles

Screening and characterization of bacteria that can utilize ammonium and nitrate ions simultaneously under controlled cultural conditions

Eighteen bacterial stock cultures were examined for their ability to utilize NH(4)(+) and NO(3)(-) simultaneously in a medium containing NH(4)NO(3) with shaking using a test tube capped with a cotton stopper. Pseudomonas aeruginosa NBRC 12689 utilized 1 mg/ml of NH(4)NO(3) most rapidly of the cultures tested. The bacterium could completely utilize 5 mg/ml of NH(4)NO(3) within 3 d, 6 mg/ml of NH(4)Cl within 3 d, and 20 mg/ml of NaNO(3) within 2 d under optimum conditions. The addition of Fe(2+) to the NH(4)NO(3) medium markedly promoted the utilization of the two ions. When the Pseudomonas strain utilized 5 mg/ml of NH(4)NO(3) completely, the total nitrogen in the culture including its cells decreased to 41% of that of the NH(4)NO(3) originally provided. GC-MS analysis showed that the removed nitrogen was probably denitrified. When the bacterium was incubated in the NH(4)NO(3) medium with shaking in a vial sealed with a rubber stopper, N(2) accumulated, but not N(2)O at the final phase of cultivation. On the other hand, both N(2) and N(2)O were detected in the NaNO(3) medium. We concluded that the bacterium removed NH(4)(+) from NH(4)NO(3) as a nitrogen source for its cell components, together with the denitrification of NO(3)(-) under controlled shaking conditions. In addition, NH(4)(+) promoted the cell growth of the bacterium and denitrification to N(2), preventing the accumulation of N(2)O.

Horse resistance to natural infestations of Anocentor nitens and Amblyomma cajennense (Acari: Ixodidae)

The objective of this study was to investigate some aspects of horse resistance to natural infestations of Anocentor nitens and Amblyomma cajennense over a 2-year period. Free-living stages of A. nitens were used to evaluate the influence of season of the year on horse resistance. Every 2 weeks, 10 selected engorged females, weighing 150–329 mg, were removed from naturally infested horses and were individually placed into glass tubes closed with a cotton stopper, and maintained in an incubator. The biological parameters evaluated varied according to the season of the year, i.e., reproductive efficiency, maximum larval survival (MLS) and egg-hatching rates were significantly lower in spring/summer than in fall/winter. At 14-day intervals, females of A. nitens and A. cajennense were counted on each side of the body of five mares. A comparison between the animal with the lowest infestation and the animal with the highest infestation showed greater differences in animals infested with A. nitens than those infested with A. cajennense. Ten animals were found to carry 64.9% of the total parasite burden of A. cajennense and 88.6% of the highest infestations of A. nitens females. A seasonal variation on intensity of horse resistance to A. nitens was observed during the study. However, this was not observed for A. cajennense. Cross-resistance was observed in animals with the highest total number of A. cajennense and the highest number of A. cajennense adults ( r=0.817), larvae ( r=0.756), and A. nitens females ( r=0.799). These results led to the conclusion that changes in horse resistance to A. nitens occur seasonally. As far as the total population of A. cajennense and the female stage of A. nitens are concerned, horses develop cross-resistance.

Studies on the egg parasitism inThaumetopoea pityocampa over a period of four years (1991–1994) at marikostino/bulgaria

In Bulgaria near Marikostino, in a forest ofPinus nigra Arn. 239 egg batches were sampled over four generations ofThaumetopoea pityocampa (Den. & Schiff.) (Lep., Thaumetopoeidae) in various seasons. Directly after collection the batches were singled in test tubes, closed with cotton stoppers and stored under laboratory conditions at 20–22°C.

Parasitism of egg‐batches of the pine processionary moth Thaumetopoea pityocampa (Den. & Schiff.) (Lep., Thaumetopoeidae) in various regions of Bulgaria1

Abstract: In forests of Pinus nigra Arn. 147 egg‐batches of T. pityocampa were collected at different locations in Bulgaria in September‐October 1991 and March and June 1992. Directly after collection all batches were put singly into test tubes with cotton stoppers and stored under laboratory conditions at 20–22°C. Oviposition always started from the base towards the top of the needle. The emergence of egg parasitoids were controlled almost daily and they were counted and identified. The eggs from which parasitoids emerged were marked for further studies. After removing the scales and opening the eggs, the impact of the parasitoid species was evaluated by their meconia and remains.Ooencyrtus pityocampae (Mercet) was observed as the most abundant egg parasitoid, followed by Anastatus bifasciatus (Fonsc.) and Baryscapus servadeii (Dom.). The hyperparasitoid, Baryscapus transversalis Graham was recorded for the first time in Bulgaria and the polyphagous species Macroneura vesicularis (Retzius) was collected in Bulgaria for the first time from an egg‐batch of T. pityocampa. Trichogramma sp. was obtained in small amounts, too. Total mortality of host eggs varied from 34% to 48%; the mortality rate due to parasitism was 24.5% to 38.9%.The emergence dynamics of the egg parasitoids was studied under laboratory conditions, and the distribution of the egg parasitoids in the egg‐batches was recorded in detail. Additionally, the structure of the meconia of A. bifasciatus and M. vesicularis was described.

Effect of Oxygen on Upgradation of Wheat Straw withPleurotus sajor cajuat High Partial Pressure of Carbondioxide

Abstract Zadrazil, F. and Puniya, A.K. 1993. Effector oxygen on upgradation of wheat straw with Pleurotus sajor caju at high partial pressure of carbondioxide. J. Appl. Anim. Res., 4: 73–82. The aim of this experiment was to study the efficiency of fungal upgrading of wheat straw with Pleurotus sajor caju. The hypothesis, that fungal mineralization can be lowered and efficiency of enzymatic degradation increased, was tested. The influence of different gas concentrations (carbondioxide, 50%; oxygen, 0–50%; nitrogen, 50-0%; v/v) on primordia formation and degradation pattern of wheat straw by P. sajor caju were studied. The cultures were gassed in three different ways i.e. A: for 20 days, B: for 40 days and C: incubated by passive air exchange for first 20 days and exposed to gas mixtures for the next 20 days. Lignin degradation increased in all variants with increasing oxygen concentrations. The highest loss of lignin (39.7%) was in cultures grown with passive air exchange through cotton stopper. The in vi...

Convenient and effective methods for in vitro cultivation of mycelium and fruiting bodies of Lentinus edodes

Three strains were selected from 18 commercial strains of the shiitake mushroom, Lentinus edodes , on the basis of rate and density of mycelial growth. Comparison of biomass production between stationary and shake flask cultures was strain-dependent, strain Le-465 producing higher yields from shaken than stationary cultures while no differences were recorded in Le-11 and Le-103. Among 5 media tested for mycelial growth yield, a molasses medium gave the highest biomass after 14 d, but by day 28 yield from that medium was the second lowest. By day 28, a chemically defined medium was the best for biomass yield. Homogenization of mycelia provided an excellent uniform source of inoculum for replicate trials. Homogenization of the inoculum increased the yield of mycelium by day 28, when fruiting started. Chemically-defined media were best for fruiting of L. edodes . There was not much difference in fruiting performance between cultures incubated at a uniform 25 °C and those incubated in a 14/22° cycle. Supplementation of flask cultures with vermiculite promoted growth and fruiting. In flask cultures kept in a normal incubator a cotton stopper, as opposed to one made of plastic foam, was most suitable for growth and fruiting. Inclusion of a carbon dioxide trap was not necessary. The best fruiting in vitro was obtained by inoculation on to solid supports, like a wire mesh or agar, in crystallizing dishes which were kept within a plastic chamber whose air-flow and humidity could be regulated with air-vents and an air-pump. Under such conditions, vermiculite addition to the media did not enhance fruiting.

Growth of Bacteria in Inorganic Medium at Different Levels of Airborne Organic Substances

Invasion rates of airborne organic substances into sterile mineral medium were compared by using flasks closed with cotton stoppers, silicone stoppers, and screw caps with Teflon gaskets. The resulting increases of dissolved organic carbon were 0.5, 0.2, and 0 mg/liter per week, respectively. The compounds supported the growth of lake water bacteria and a strain of Pseudomonas fluorescens. Growth rates were correlated to the permeability of the stoppers used. The measured input of organic carbon in the sterile mineral medium is considered to be a minimum value for the actual contribution of organic compounds by the air. Multiplication rates of the bacteria suggest that the organisms prevent the escape of volatile organic substances from the medium by rapid utilization. The steady nutrient supply through the air should be considered in growth experiments with bacteria at low concentrations of nutrients.

Technique for Mass Rearing of the Rice Stem Borer and Development Studies of New Insecticides

1. A simple technique for mass rearing of the rice stem borer, Chilo suppressalis on rice seedlingsThe rice seedlings were germinated from 50g of rice seeds in the glass jar at a insectary room (28°C, 15hr of light, 2000 Lux). A hole of 2cm diameter which sealed with a cotton stopper was made on a screw metal cap for ventilation. The cotton stopper also offered pupation site of the mature larvae. Several mature egg-masses were placed on the seedlings in the jar. The larvae were allowed to feed the seedlings for about 10 days (3rd instar) and the seedlings were consumed by feeding. At this time, the larvae were transferred to fresh seedling jar at a rate 100 larvae per jar. Most of the mature larvae bored into the cotton stopper and pupated. The pupae were taken off together with the stopper from caps and placed a plastic tray in an oviposition box. The emerged moths were allowed to copulation in the box and oviposited on rice plants.2. Insecticidal action of CartapCartap hydrochloride, S, S [2-(dimethylamino) trimethylene] bis (thiocarbamate) hydrochloride is an insecticide developed from derivating a natural toxin, nereistoxin.Insecticidal activity against Chilo sappressalisWhen cartap was applied by topical application or dipping method, the larvae were rapidly paralysed without convulsive or agonizing syptom. The paralysed larvae, however, recovered slowly from the intoxication with the elapse of time. If the larvae fed continously on an artificial diet with a small amount of cartap, they were suppressed in growth, and the final death of the larvae occurred by the starvation. Furthermore, when the moths were directly sprayed or contacted on the leaves of the rice plants treated with cartap, they dropped rapidly by the paralysis in the water and died.Mode of action and metabolismThe distribution of 35S-labeled cartap concentrated at the central nervous system of the paralysed Periplaneta americana or Mamestra brassicae and approximated 2/3 of an amount of the compounds uptaken by the nervous cord were detected in the ganglions. Metabolites of cartap in the larvae of M. brassicae, Spodoptera litura and in the rice plants were investigated in experiments of thin layer chromatography. In every chromatograms, cartap was not identified from the Rf values, whereas three kinds of radioactive or sulfurated substances were observed. One of the substances was identified with nereistoxin or dihydronereistoxin, but others were unidentified. On the other hand, the results of electrophysiological examination demonstrated that cartap blocked the ganglonic transmission in P. americana, and that the action was almost similar to nereistoxin.3. Insecticidal action of phytoecdysonesAssay of phytoecdysonesThe ligated larvae of Chilo suppressalis were dipped into test solutions containing phytoecdysones and examined on the effect of the larval stage, the kind of the solvent of test solution, time of dipping and time of application after the ligation. The standard method in which the final instar larvae were dipped into the methanolic solution for 10sec was sensitive enough to approximately 70-80ppm of ponasterone A and ecdysterone, and appliable even for the assay of phytoecdysones in crude plant extracts.Insecticidal activity for various insectsThe high insecticidal activity was found when ponasterone A, inokosterone, ecdysterone and cyasterone were acted on the body surface of the larvae of C. suppressalis, Pluttella xylostella and Culex pipiens molestas. However, the larvae of Musca domestrica vicina and Pieris rapae crucivora were tolerant against the phytoecdysones. The insecticidal activity of C. suppressalis varied with the developmental stage. That is, the final instar larvae was more sensitive against the phytoecdysone

組織培養によるNewcastle Disease Virusの培養

A method of propagating Newcastle disease virus in a static culture of adult chicken tissue (spleen) was described in the previous paper. Influence of various conditions for this type of culture upon the virus propagation was further investigated in the present study. The important results are summarized as follows.1. There was no remarkable difference in final virus titers among cultures using different material, such as Japanese rice paper, filter paper, perforated cellophane, glass surface and agar bed, to fix tissue fragments. Of there materials, Japanese rice paper was the most convenient for practical use.2. There was no difference in final titers between cultures under anaerobic conditions (using nitrogen) and those under aerobic condition (using air or oxygen). This fact seems to indicate that no oxygen is necessary at least practically, for the multiplication of virus in tissue culture.3. Rubber stopper or carbon dioxide gas is used usually in culture with a low concentration (below 1%) of tissue. In a culture with a higher concentration (1 to 3%) of tissue, however, the multiplication of virus was more conspicuous when cotton stopper was used. It seemed that the cotton plug served for the discharge of excess carbon dioxide gas which was usually produced in the culture with such a high concentration of tissue and that consequently, it also served to prevent the pH of the medium from unfavorable lowering.4. The virus propagated in tissue cultures with spleen from a highly immune chicken to almost the same level of virus titer as in those with spleen from a normal chicken.

THE EFFECT OF NITROGEN ON THE GERMINATION OF VANILLA PLANIFOLIA

UP TO 1937, when Bouriquet and Boiteau presented their paper on the asymbiotic germination of the seeds of Vanila planifolia, the seeds of this plant were considered to be nonviable. They obtained some germination on a glucose-agar medium containing a decoction of soil and parts of the mature vanilla plant. Later, Bouriquet (1947) reported an improvement on this medium through the addition of fresh yeast. The Federal Experimental Station became interested in the possibility of developing a vanilla industry on the Island when McClelland demonstrated that the plant could be grown commercially in Puerto Rico and trials were undertaken. A Fusariuwn root disease (Tucker, 1926) shortly after assumed serious proportions however, and threatened to wipe out the plantations. It was then decided to explore the possibility of developing disease-resistant hybrids of vanilla of equal or better quality than Vanitla pklnifoJli. With this end in view, the Federal Experimental Station at Mayaguez, Puerto Rico invited Dr. Lewis Knudson to do research on the project. Knudson (1950) performed all his experiments with his Solution B as the basic nutrient medium and must be credited as being the first to produce hybrid seedlings of Vanilla. After Knudson's success, the desirability of further investigation of vanilla seed dormancy and germination became apparent, and the study reported here was undertaken. MATERIALS AND METHODS-In this study, seeds of Vanilla planifoia were used in all experiments. The pods were grown in Puerto Rico and were provided by the Pathology section of the Federal Experiment Station at Mayaguez. The seeds were sterilized for about 20 mmn. in filtered calcium hypochlorite solution prepared by shaking 10 g. of calcium hypochlorite with 140 ml. of distilled water. Knudson's Solution B2 was used as the culture medium for germinating the Vanilla seeds. Acid-cleaned, soft glass test tubes, 200 mm. X 25 mm., were used exclusively except in the pH experiments, where Pyrex test tubes of the same size were used. From 25 to 30 ml. of culture solution were added to each tube. The tubes were provided with tightly-fitted cotton stoppers, autoclaved for 15-20 min., and allowed to cool in a slanted posl-

Use of thymol as a preservative for fungi.

It is often of value to preserve the culture of a fungus for a long period of time. Such preservation not only furnishes the investigator with a permanent mount of the growth, but also allows the instructor to have access to cultures as life-like exhibits for use in demonstrating to his students. Almost all present-day textbooks 1 suggest that a formaldehyde solution be placed on the cotton stopper of the test tube, in order that the fumes of formaldehyde may kill the growth which has been placed within. The cotton stopper is then pressed slightly down into the tube, and the tube is sealed with paraffin wax. This commonly used method invariably results in a quick cessation of the fungous growth, but fails as an effective preservative in that, after a few months, the formaldehyde is instrumental in destroying the color of the culture. Inasmuch as the color of the

Studies on oxgen reguirement of crop roots. : 1. Biochemical reduction of nitrate by crop roots and varietal difference,

It has already been reported that oxygen requitement of crop roots in the soil varied with the difference of crops or varieties. The author found a close relation between reducing power and oxygen requirement of roots during the water-culture of various plants, and then cultured young plants in a solution containing nitrate as nitrogen source (cf. table I) and investigated the amount of nitrous acid formed in the solution. Soybean (12 varieties), barley (14 varieties), wheat (7 varieties), sweet potato (12 varieties), rape (14 varieties), other two or three crops and weeds were employed in this experiments. All these plants excluding sweet potato were sown in the quartz sands until they bears one or two leaves, then the seedlings were water-cultured in the 300cc flasks covered by blackpapers, and the plants were supported with cotton stoppers. On sweet potato, the top of vine was cut in 15 cm and then water-cultured. The solution was changed every 3-4 days and nitrous acid in the residual solution was measured, and the total amount of nitrous acid formed during the term of water-culture (generally one month) was calculated. The results of the experiments are as follows : Nitrous acid was not formed in the culture solutions of paddy rice, Alopeculus aequalis, and Beckmannia erucaeformis, but it was found in those of barley, wheat, soybean, corn and rape having strong reducing power in their roots (cf. table 2). Considering from these facts upland plants generally have the strong reducing power of nitrate in the culture solution, but marsh plants have the very weak reducing power. Further the varietal difference of nitrate reducing power was investigated with soybean, sweet potato, barley, wheat and rape, and the following results were obtained (cf. table 3-6): Drought-resistant varieties form more nitrous acid than drought-susceptible varieties in the same species, but dump-resistant varieties form less nitrous acid on the contrary, i. e., have less reducing power, than the susceptible varieties. According to the results, it may certainly be concluded that the reducing power is closely correlated with oxygen requirement in roots, i. e., species or varieties having the stronger reducing power in their roots require more oxygen in the soil.

Effect of Aeration and Lack of CO2 on Growth of Bacteria-Free Cultures of Protozoa.

It has been known for some time that CO2 is necessary for the growth of bacteria, yeast, and molds on solid media, and recent investigations have shown that it is also necessary for bacteria when synthetic liquid media are used., In liquid media containing hydrolyzed protein growth is not completely inhibited, but it may be delayed considerably by the absence of CO2., It has also been suggested that lack of sufficient CO2 might be a cause of the lag period in growth of cultures of free-living protozoa and a cause of the failure of various investigators to obtain bacteria-free cultures of intestinal species. For these reasons information concerning the effect of lack of CO2 on protozoa seems highly desirable. In the present experiments bacteria-free cultures of Glaucoma piriformis and Chilomonas paramecium were grown in a heavily buffered medium of 0.5% hydrolyzed casein and 0.25% KH2PO4 at pH 6.0 under the following conditions: (a) unaerated, in flasks with cotton stoppers, (b) aerated, in Drechsel gas washing bottles with ordinary air which had been washed through 2 bottles of water and an indicator solution (brom thymol blue), (c) aerated, in Drechsel bottles with CO2,-free air prepared by passing air through 2 Milligan spiral gas washing bottles of strong KOH and then through 2 bottles of water and an indicator solution. These conditions are similar to those used by Walker for bacteria. The rate of bubbling was a but 5 liters per hour per flask. The cultures were immersed in a stirred water bath, and the temperature was thereby maintained equal in all cultures and approximately that of the room (2O°-25°C.).

THE PREPARATION OF PETROLATUM DRAINAGE GAUZE

Petrolatum gauze as a drainage material is less irritating than other gauze. Secretions do not clot so readily, and it does not stick to the edges of the wound, but it should be prepared in such a manner that plenty of capillary space is available for drainage. The following method of preparation has been found to give a satisfactory product: Take a large test tube, pack cotton in the bottom to the height of 1 inch (2.5 cm.), pack dry drainage gauze over this to within 2 inches (5 cm.) of the top, place on top of this a small dab of petrolatum, place cotton stopper in mouth of the tube, set upright in the autoclave and sterilize. The heated petrolatum, in making its way to the bottom of the tube, impregnates the threads of gauze but does not fill the interstices between the threads. Any excess petrolatum is absorbed by the

Department of Radiodontia: The Radiographic Diagnosis of Periapical Dental Infection in the Light of Bacteriologic Findings

THE dental radiograph is widely used as a means of detecting infection in the periapical tissues and alveolar process. Certain criteria, such as rarefaction or condensation of bone, changes in the peridental membrane and lamella, and absorption of root tip are taken as diagnostic of infection. What the physician or dentist interested in correlating systemic disease and dental infection really wishes to know is how many and what kind of bacteria are present in a given area or areas. Active infection necessitates the presence of bacteria, and only by reason of the presence of bacteria are pulpless teeth of importance from the standpoint of systemic disease. The radiographic criteria of infection, if dependable, must be a means of determining the presence or absence of bacteria in a suspected area. Such considerations bring up the question of how far one can translate radiographic evidence of infection into terms of bacteria. Does the absence of radiographic evidence of infection rule out the presence of bacteria? Does the finding of certain radiographic evidence of infection always mean the presence of bacteria? The problem is entirely a bacteriologic one, yet it is surprising how slight dependable data are available concerning the bacteriologic status of the pulpless tooth. In fact there is not available in the literature a single comprehensive bacteriologic study of dental infection by modern methods. I have endeavored by cultures of pulpless teeth to obtain further facts concerning periapical dental infection. Numerous difficulties enter into bacteriologic work about the teeth which are not encountered in similar work elsewhere. The chances of contamination are always great. If broth is used as the culture medium, one never knows just how to interpret positive results. One bacterium in a tube of liquid medium gives rise after twenty-four hours of incubation to as profuse a growth as if a large number were inoculated. Dependable data in this field then must give, first of all, some idea of the number of bacteria in the material cultured. It is imperative also that proper control be available by which the chances of contamination can be estimated numerically. A final requirement in a study such as this is use of a culture medium suitable for the growth of the bacteria dealt with here. The organisms found in chronic foci of infection are often difficult to cultivate. Rosenow has emphasized especially the need of employing a medium which supplies all gradations of oxygen tension between complete anaerobiosis and full atmospheric pressure. This is a fundamental requirement both f or proper growth and for preserving during the incubation period the specific affinity of the organism for different types of tissue. To meet these several requirements I have made all cultures in deep tubes of glucose brain agar. The tubes used are 6×¾, inches and filled as close as possible to the top without allowing the medium to touch the cotton stopper.

A Study of the Effect of Cotton Stoppers Used in Dilution Blanks on the Numerical Bacterial Count, and of Other Practices in the Technique of Bacteriological Laboratories

A Study of the Effect of Cotton Stoppers Used in Dilution Blanks on the Numerical Bacterial Count, and of Other Practices in the Technique of Bacteriological Laboratories