THE NUMBERS AND GROWTH RATES OF THE BACTERIA IN HEVEA LATEX, AMMONIATED FIELD LATEX AND AMMONIATED LATEX CONCENTRATE

SUMMARY: The rates of growth of bacteria on Hevea latex systems in the presence and absence of ammonia have been derived from colony count data obtained on a modified Kligler's iron agar medium. For fresh latex, ammoniated field latex, and ammoniated latex concentrate, variations in the bacterial populations encountered are given for a period of about one year's testing, with some data on commercial samples of latex concentrate. Field latex at routine collection contained about 8 × 10⁶ bacteria/ml. This could be reduced by 62% by the use of sterile tapping cups. In the routine latex, the mixed population grew logarithmically after a lag phase of 2·1/2–3 hr. In clean latex the lag phase was extended to 4 hr. On ammoniation to 0.3% (w/w), the population was reduced over about 2 hr, but subsequently logarithmic growth recurred, without a lag phase. On concentration and further ammoniation to 0·7% (w/w), the count dropped still further, levels of 10/ml or less being achieved in clean conditions, but very slow logarithmic growth again occurred with a lag phase preceding it.     

A METHOD FOR COUNTING THE TOTAL BACTERIA IN AMMONIATED HEVEA LATEX SYSTEMS

SUMMARY: A method has been developed for obtaining the total count of bacteria in ammoniated Hevea latex systems. This involves ultracentrifugation of latex or latex concentrate diluted in isotonic saline and the modified use of a normal phase contrast microscope to give a dark ground effect. Examples are given of the degree of bacterial contamination of field latex and concentrate. In these inhibitory ammoniated systems the total count may be high even if the viable count is low: in fact, in all the commercial latex concentrates examined the total count exceeded 10⁸ cells/ml. The value of interference and fluorescence microscopy has also been studied.     

The feeding value of ammoniated flax straw,wheat straw and wheat chaff for beef cattle

Two experiments were conducted to assess the feeding value of ammoniated and untreated flax straw, wheat straw and wheat chaff in comparison to a mixed bromegrass/alfalfa hay. Anhydrous ammonia was applied to the crop residues at the rate of 35 kg t⁻¹ dry matter. In the first experiment, the effect of ammoniation on crude protein, acid detergent fibre (ADF), neutral detergent fibre (NDF) and acid detergent lignin (ADL), digestible organic matter in vitro and in vivo (DOM%), ADF and NDF digestibility of the crop residues was determined. In the second experiment, ammoniated flax straw, ammoniated wheat straw, ammoniated and untreated wheat chaff, each supplemented with barley, were compared to bromegrass/alfalfa hay as feed sources for wintering beef cows.Ammoniation increased the crude protein content of the crop residues ∼2-fold. Wheat straw DOM in vitro and in vivo was not increased by ammoniation. Ammoniation increased the DOM in vitro of wheat chaff from 36.3 to 46% and flax straw from 35.2 to 46.3%. The DOM in vivo increased from 53.3 to 63.4% (P < 0.05) for wheat chaff and from 33.9 to 58.4% (P < 0.05) for flax straw following ammoniation. Digestibility of ADF increased from 9.9 to 43.9% (P < 0.05) and of NDF from −0.6 to 37.9% (P < 0.05) in flax straw with ammoniation. Non-significant increases in ADF and NDF digestibility were observed for all other crop residues. Lignin content was not changed in the crop residues by ammoniation.In the winter feeding trial, young cows gained more weight than older cows (P < 0.05). Average daily gains of cows were greatest for hay followed by ammoniated flax straw, ammoniated chaff, untreated chaff and ammoniated wheat straw rations (P < 0.05). Increases in backfat in the younger cows was greatest with hay and ammoniated flax straw, followed by ammoniated chaff and ammoniated wheat straw (P < 0.05). Untreated chaff caused no increase in backfat thickness.Ammoniated flax straw (3.2 kg day⁻¹) given with barley (5.6 kg day⁻¹), is similar in feeding value to medium quality bromegrass/alfalfa hay. Furthermore, wheat chaff and ammoniated wheat chaff show good potential as alternatives to hay in winter feeding.     

Genotoxic activity of caramel on Salmonella and cultured mammalian cells

The genetic activity of 2 commercial caramel preparations, manufactured either by heating the malt sugar solution directly (non-ammoniated caramel) or by heating it with ammonia (ammoniated caramel) was studied in the Salmonella mutagenicity test and UDS assay in cultured mammalian cells. The non-ammoniated caramel was found to be mutagenic to S. typhimurium TA100, while the ammoniated one was genetically active in all the tester strains used, namely TA100, TA97 and TA98. It was also demonstrated that non-ammoniated caramel was capable of inducing UDS in cultured human amnion FL cells, but for the ammoniated one, no such activity was observed. Furthermore, based on the results obtained in the DNA synthesis inhibition assay, it was suggested that the DNA synthesis inhibition seen in our experiments with the ammoniated caramel was probably not of DNA damage in origin. These data indicate that the mutagenic fractions formed during ammoniated and non-ammoniated caramelization were quite different.     

Subchronic toxicological investigations of Fusarium moniliforme-contaminated corn,culture material,and ammoniated culture material

The fungus Fusarium moniliforme is ubiquitous on corn throughout the world and is a likely co-contaminant on corn infested with aflatoxin-producing Aspergillus flavus. Ammoniation has been used to detoxify aflatoxin-contaminated commodities. To determine the effect of ammoniation on the toxic potential of Fusarium moniliforme, male Sprague-Dawley rats were fed either diets containing 10% sound corn, ammoniated corn, corn culture material of hepatotoxic F. moniliforme strain MRC 826 (CM), or ammoniated CM for four weeks. They were observed for signs of toxicity and hematological, serum chemical and histopathological evaluations were made. Groups of male Balb/c mice were fed diets fortified with 10% sound corn or CM for four weeks and evaluated by serum chemical and histopathological means to determine the suitability of mice as a model species for investigation of F. moniliforme-induced hepatotoxicity. Ammoniation was ineffective for detoxification of the CM. Hepatotoxicity and renal toxicity of CM and ammoniated CM were qualitatively similar, although renal tubular lesions appeared more advanced in rats fed ammoniated CM. Adrenal cortical cellular vacuolation was also found in CM and ammoniated CM-fed rats, while focal seminiferous tubular degeneration and aspermia were found only in the testes of ammoniated CM-fed rats. Fumonisin B₁ concentrations of the CM and ammoniated CM diets averaged 99 and 75 ppm, respectively. CM containing 99 ppm fumonisin B₁ also produced hepatotoxicity in mice similar to that found in CM-fed rats. Thus, mice may be useful for investigations of F. moniliforme-induced hepatotoxicity.Mention of a trademark, proprietary name or vendor does not imply its approval by the US Department of Agriculture to the exclusion of others that may also be suitable.     

Fine structural aspects of phagocytotic activity in inverted follicle cells of cultured porcine thyroids

Inversion of thyroid follicles took place when they were isolated by collagenase and trypsin and cultured in suspension in Eagle's medium supplemented with 10% fetal calf serum without TSH. The apical surface facing the culture medium contained numerous microvilli and a central cilium, while the luminal surface became flattened. Phagocytotic activity by pseudopods was promoted after addition of TSH to the culture medium. When the inverted follicles were incubated in culture medium containing TSH (50 mU/ml) and human red blood cells, or TSH and polystyrene latex beads (2.02 micron in diameter) for 1-3 h, numerous red blood cells or latex beads respectively were observed to be taken up by the epithelial follicle cells by scanning electron microscopy, as well as conventional thin-section electron microscopy. These results show that the apical surface (culture medium side) of the epithelial cell of the cultured thyroid follicle whose polarity is reversed phagocytoses red blood cells and latex beads.     

Degradation of latex and of natural rubber by Streptomyces strain La 7

Streptomyces strain La 7 was isolated from the banquete of a city high way in Karlsruhe. According to partial 16S rRNA gene sequencing it was identical with Streptomyces albogriseolus and Streptomyces viridodiastaticus. DNA-DNA-similarity studies revealed 80.3-82.4% similarity between each of two of the three strains. Although phylogenetically closely related, Streptomyces strain La 7 differed from the two reference strains by morphological as well as physiological features and might represent a new species aside of S. albogriseolus and S. viridodiastaticus. The new Streptomyces strain La 7 was grown in a medium containing a latex emulsion or squares of natural rubber gloves as the only carbon source. On agar plates with a latex overlay agar, translucent halo formation around the colonies was observed. The unvulcanized latex was metabolized and the carbon from the isoprene units was apparently used for cell growth. In shake cultures with unlimited oxygen supply, during 60 days of incubation, 140 mg of the 175 mg totally emulgated latex were degraded exponentially. In sterile control flasks about 3% of the initial amount of latex could not be recovered after incubation on a shaker, presumably due to photochemical transformation. During static incubation of sterile medium, the latex formed a sticky layer at the surface of the medium and on the glass walls and recovery of the material was more difficult. Estimation of the protein content of cells from total nitrogen resulted in about 50% of the degraded latex being incorporated into cells, if a standard cell composition was assumed. Direct protein analysis according to Bradford (1976) gave much lower estimates, presumably due to a low content of aromatic amino acids. Stripes of natural rubber were degraded by Streptomyces strain La 7 during 70 days to an extent of about 30%. Scanning electron microscopy demonstrated, that hyphes of Streptomyces strain La 7 colonized and penetrated the latex surface with a concomitant deterioration of the latex material.     

Detection of aflatoxin D1 in ammoniated corn by mass spectrometry-mass spectrometry

Corn cultured with Aspergillus flavus to produce a high level of aflatoxin was ammoniated at 37 degrees C for 21 days. An extract of the ammoniated corn was separated by thin-layer chromatography, followed by reversed-phase high-pressure liquid chromatography. Examination of the fractions by tandem mass spectrometry led to detection of aflatoxin D1 as a product of the corn ammoniation process.     

Detection of aflatoxin D1 in ammoniated corn by mass spectrometry-mass spectrometry.

Corn cultured with Aspergillus flavus to produce a high level of aflatoxin was ammoniated at 37 degrees C for 21 days. An extract of the ammoniated corn was separated by thin-layer chromatography, followed by reversed-phase high-pressure liquid chromatography. Examination of the fractions by tandem mass spectrometry led to detection of aflatoxin D1 as a product of the corn ammoniation process.     

Ammoniated Forage Poisoning: Concentrations of Alkylimidazoles in Ammoniated Forage and in Milk,Plasma and Urine in Sheep and Cow

4-methylimidazolc (4-MeI) has until now been the only identified toxic compound in ammoniated forage considered to be of possible etiological significance in ammoniated forage poisoning. However, several authors have concluded that 4-MeI alone cannot explain the toxicity observed (Morgan & Edwards 1986, Nielsen et al 1986, Motoi et at. 1997). On this background, we have examined samples of ammoniated forage and of milk, plasma, and urine collected from ewes and lambs during a previous experimental poisoning study (Sivertsen et al. 1993). We have also studied similar samples from a Norwegian Red Cattle dairy cow fed ammoniated hay for the first 5 days after calving. We have, in addition to the previously known ingredients 4-MeI and 2-methylimida-zole (2-MeI), identified 5 new compounds in these samples, all of which were found to be di-alkylsubstituted imidazoles: 1,2-dimcthylimi-dazole (1,2-diMeI), 1,4-dimethyl imidazole (1,4-diMeI), 1,5-dimethyl imidazole (1,5-diMeI), 2,4-dimethylimidazole (2,4-diMeI) and 2-ethyl, 4-methylimidazole (2Et-4MeI) (Müller et al. 1998). In this paper we present quantitative measurements of these di- and monoalky 1 imidazoles in a selection of the collected samples.     

Methane fermentation of rubber (Hevea brasiliensis) latex effluent.

Four species of bacteria capable of CH4 fermentation of rubber latex effluent were isolated and identified as a Methanococcus, a strain of M. vannielii, a Methanobacterium and a strain of M. omelianskii. Auxanographic tests using the four strains showed growth and CH4 formation on a basal medium containing mineral salts or added H2 and Co2. Varied response was obtained when the basal medium was added to formate, acetate, butyrate, methanol, ethanol, and glucose. Previous work has established acid fermentation of Hevea latex arising from bacterial contamination and decomposition of the non-rubber constituents which consist of N-compounds, 2% quebrachitol, and smaller concentration of carbohydrates. This suggests that reduction of CO2 and fermentation of acids formed during metabolism of Hevea latex are possible pathways of CH4 production.     

Axenic Cultivation and Ultrastructural Study of a Phytomonas sp. Isolated from the Milkweed Plant Euphorbia hyssopifolia1

ABSTRACT. A large number of trypanosomatids was seen in the latex of the milkweed plant Euphorbia hyssopifolia found in Rio de Janeiro, Brazil. The parasites, which belong to the genus Phytomonas, were isolated and axenically cultivated in a biphasic culture medium. The form and the structural organization of the parasites as found in the intact plant, in the isolated latex, and in the culture medium was studied by scanning and transmission electron microscopy.     

The Bielschowsky Staining Technic a Study of the Factors Influencing Its Specificity for Nerve FIBERS

By comparing results obtained with adult mammalian tissue from introducing variables into each separate step in block-staining by the Bielschowsky silver method, the following conclusions were reached:

1. No specific means for inhibiting the staining of connective tissue and still permitting complete staining of nerve fibers was found, but the avoidance of overstaining was very helpful toward such differentiation.
2. Overstaining could be corrected by reducing the concentration of the silver nitrate bath or by adding an excess of ammonia to the ammoniated silver bath.
3. Staining of fine fibers was favored by adding acetic acid to the formaldehyde used for fixation or by adding pyridin to the silver nitrate bath.
4. Addition of protein-precipitating organic acids (trichloracetic or sulfosalicylic) to the fixative was disadvantageous.
5. Prolonged fixation favored an increase in intensity of the stain. Four days' time was sufficient.
6. Extraction of lipids with ammoniated alcohol gave results similar to those obtained after extraction with pyridin, but the stain was lighter.
7. Ammoniated silver carbonate without excess ammonia had an action similar to ammoniated silver hydroxide with excess ammonia.
8. An excess of ammonia in the ammoniated silver solution (Ag 0.1 N) was tolerated, without apparent impairment of nerve-fiber staining, up to 6 M NH₃, altho the use of more than 3 M excess (2 cc. concentrated ammonia water added to 100 cc. of balanced ammoniated silver hydroxide solution) seemed unnecessary.
9. Impregnation with 1.7% (0.1 N) silver nitrate solution was quite satisfactory and variations in the concentrations of this bath suggested that the practical limits of concentrations that would be generally satisfactory lay between 0.3 and 3.0%.
10. The writers' experiences agreed with Agduhr's relative to the advantage of washing in 2.5% acetic acid between the ammoniated silver bath and formaldehyde reduction.

     

Lactic acid fermentation of crude sorghum extract

Crude extract from sweet sorghum supplemented with vetch juice was utilized as the carbohydrate source for fermentative production of lactic acid. Fermentation of media containing 7%(w/v) total sugar was complex completed in 60–80 hr by Lactobacillus plantarum, product yield averaging 85%. Maximum acid production rates were dependent on pH, initial substrate distribution, and concentration, the rates varying from 2 to 5 g(liter·hr.) The lactic acid yield was lowered to 67% under limited medium supplementation. The fermented ammoniated product contained over eight times as much equivalent crude protein (N × 6.25) as the original medium. Unstructured kinetic models were developed for cell growth, lactic acid formation, and substrate consumption in batch fermentation. With the provision of experimentally determined kinetic parameters, the proposed models accurately the fermentation process.     

Effects of particulates and lipids on the hydraulic conductivity of Matrigel.

The hydraulic conductivity of a connective tissue is determined both by the fine ultrastructure of the extracellular matrix and the effects of larger particles in the interstitial space. In this study, we explored this relationship by examining the effects of 30- or 90-nm-diameter latex nanospheres or low-density lipoproteins (LDL) on the hydraulic conductivity of Matrigel, a basement membrane matrix. The hydraulic conductivity of Matrigel with latex nanospheres or LDL particles added at 4.8% weight fraction was measured and compared with the hydraulic conductivity of Matrigel alone. The LDL-derived lipids in the gel were visualized by transmission electron microscopy and were seen to have aggregated into particles up to 500 nm in size. The addition of these materials to the medium markedly decreased its hydraulic conductivity, with the LDL-derived lipids having a much larger effect than did the latex nanospheres. Debye-Brinkman theory was used to predict the effect of addition of particles to the hydraulic conductivity of the medium. The theoretical predictions matched well with the results from adding latex nanospheres to the medium. However, LDL decreased hydraulic conductivity much more than was predicted by the theory. The validation of the theoretical model for rigid particles embedded in extracellular matrix suggests that it could be used to make predictions about the influence of particulates (e.g., collagen, elastin, cells) on the hydraulic conductivity of the fine filamentous matrix (the proteoglycans) in connective tissues. In addition, the larger-than-predicted effects of lipidlike particles on hydraulic conductivity may magnify the pathology associated with lipid accumulation, such as in Bruch's membrane of the retina during macular degeneration and the blood vessel wall in atherosclerosis.     

The effect of ammoniation on the intake and nutritive value of straw

Studies were conducted to compare the increases in dry matter digestibility (DMD) in vitro and in vivo and to determine the metabolisable energy (ME) value of straw ammoniated at ambient temperature. Two stacks of straw sealed with polyethylene were allowed to react with 3% (w/w) anhydrous NH₃ for 30 and 56 days, respectively. Both DMD in vitro and nitrogen tests were carried out over an eight-week period subsequent to opening the stacks. Digestibility in vivo was measured with 12 wether lambs. The non-treated and ammoniated straws were given ad libitum, with a supplement of either ground barley or a lamb concentrate which contained 16% crude protein (CP).There was a mean increase of 15 percentage units in DMD in vitro for the ammoniated straw irrespective of whether it was treated for 30 or 56 days. The corresponding increase in mean DMD in vivo was 14.2 units. The CP content of the straw was increased from 3.1 to 7.6%. The increase in DMD in vitro and total N content was maintained throughout the sampling period. Approximately 58% of the anhydrous NH₃ added to the straw appeared to have been irreversibly “bound” to the straw. The ME values for the ammoniated straw were 6.78 and 7.49 MJ/kg when the straw was supplemented with either barley or the lamb concentrate, respectively. Straw ammoniation had a marked effect on intake. The overall increase in intake was 70% for the treated compared with the non-treated material.     

Effect ofCalotropis latex on laticifers differentiation in callus cultures ofCalotropis procera

Laticifers differentiation in callus cultures of Calotropis procera (Asclepiadaceae) as affected by own latex and its fractions incorporated in Murashige and Skoog (MS) medium is described. Callus cultures have been maintained on MS medium with 2.3 ΜM 6-furfurylaminopurine (FAP) and 3.0 ΜM 1-naphthylacetic acid (NAA). Marked increase in laticifers differentiation (from 10.1 to 28.4 %) was observed on this medium supplemented with 1 % (v/v) of latex. Latex fractions containing proteins + complex polysaccharides or inorganic salts also increased laticifers differentiation (by 21.8 % and 24.1 %, respectively). Other fractions (free amino acid + saccharides, phenols and terpenes + sterols) had no marked effect on laticifers differentiation while alkaloid fraction inhibited it. Effect of latex on laticifers differentiation was much more profound than the reported optimal concentration of plant growth regulators (4.6 ΜM FAP + 1 ΜM IAA). This research was supported by grant-in-aid for research from the University Grants Commission (No. F3-65/91SR II), New Delhi, to Dr. KG. Ramawat.     

Role of lutoid membrane transport and protein synthesis in the regeneration mechanism of latex in different <Emphasis Type="Italic">Hevea</Emphasis> clones

Natural rubber (cis-1,4 polyisoprene) is synthesised in the milky cytoplasm, the latex, of specialized cells called laticifers in the bark tissues of the rubber tree (Hevea brasiliensis). Regeneration mechanism of latex after each tapping (controlled wounding of the bark) was studied in relation to lutoid membrane enzymes and protein synthesis in twelve rubber clones with varying yield potentials during the peak rubber yielding season. High activity of membrane enzymes and better availability of biochemical energy [ATP] were observed in clones viz; RRII 105, RRIM 600, PB 260, RRII 422 and RRII 430. The highest protein biosynthetic capacity was noticed in clone PB 260 and RRIM 600. However, high ATP content, increased invertase activity and protein biosynthesis were observed in the medium yielding clone GT1 compared to clones with low rubber yield potential. Very low sugar content and increased invertase activity in the latex of clone PB 260 indicated intense latex metabolism with high protein turnover that implies fast recouping of the cellular metabolites lost during latex harvesting. Clone PB 217 was characterized by very high sucrose and low ATP concentration and ATPase activity in latex indicating slow metabolism and hence be suitable for inducing latex metabolism using ethylene stimulant. Low rubber yielding clones such as RRII 33 and RRII 38 were consistently recorded a high sucrose content but very low activity of membrane enzymes, reduced ATP concentration and low protein biosynthesis in latex. Among the recently released modern clones (RRII 400 series), latex regeneration capacity was higher in RRII 422 and RRII 430. The significance of lutoid membrane transport and protein synthesis is discussed in relation to general latex metabolism of these rubber clones. The outcome of this study would be helpful to design suitable latex harvesting systems and yield stimulation methods for optimizing latex production in each clone based on metabolic profiling.     

Phagocytic Activity of FRTL-5 Rat Thyroid Follicular Cells as Measured by Ingestion of Fluorescent Latex Beads

A rat thyroid cell line (FRTL-5) was used to study the phagocytic activity of thyroid follicular cells using fluorescent latex beads and flow cytometric analysis. Morphologic studies demonstrated that latex beads were engulfed and located within cytoplasmic vacuoles of thyrocytes. Flow cytometric evaluation of cell suspensions revealed high levels of fluorescence in cells engulfing latex beads. Using thyrotropin (TSH) as a stimulator of thyroid function and human interleukin-1β as an inhibitor, protocols were established for measuring the effects of these substances on either basal or TSH-induced phagocytosis. Cells exposed to latex beads over time in basal (0H) or TSH-containing medium had an increase in time-dependent phagocytic activity which was maximal after 24 or 8 h, respectively. Treatment of FRTL-5 cells with either a stimulator or an inhibitor revealed maximal change in phagocytic activity after 72 h as measured by the percentage of phagocytic cells as well as the mean fluorescence intensity. Phagocytic activity and iodide trapping by FRTL-5 cells were qualitatively similar in both sensitivity and magnitude of change in the assays used in this study. Phagocytosis of fluorescent latex beads represents a sensitive nonradioactive assay of thyrocyte function whose regulation is similar to iodide trapping.     

Latex test used for diagnosis of dysentery caused by Shigella sonnei]

The aim of this study was to evaluate the usefulness of latex reagent coated with immunoglobulins specific for antigens of phase I and II of S. sonnei for detection of these antigens in primary, mixed bacterial cultures. The study was performed on 919 fecal samples from individuals with clinical symptoms of dysentery, convalescents and from contact individuals. Material used for the test was bacterial suspension collected from McConkey or SS agars and a culture from selenite F broth heated at 100C. The results of the latex test were compared with the results of isolation of S. sonnei from the same cultures. S. sonnei was isolated from feces of 140 individuals (15.2%), while the latex test was positive in 215 cases (23.4%). The highest testing effectiveness , significantly higher than when isolation of pathogen was performed, was obtained only when 18-20 hr culture on Selenite F medium was used for latex test. The correlation between efficacy of testing for S. sonnei and phosphate content of Selenite F and a mode of its preparation was found. The latex test allows to eliminate from further bacteriological studies cultures free of S. sonnei thus it gives measurable economical profits and it shortens significantly time period of bacteriological examination.