Activated charcoal does not catalyze sucrose hydrolysis in tissue culture media during autoclaving

Tissue culture media or aqueous sucrose solutions containing activated charcoal buffered to pH 5.5 and autoclaved did not undergo appreciable sucrose hydrolysis as reported. Rather, the extent of sucrose hydrolysis in media containing activated charcoal was found to be directly proportional to the hydrogen ion concentration (pH). This finding is consistent with the known mechanism of acid-catalyzed hydrolysis of acetals such as sucrose. Several types of charcoal were identified that acidified culture media to the extent that considerable acid-catalyzed sucrose hydrolysis occurred under autoclave conditions, making it appear as though activated charcoal was responsible for catalyzing sucrose hydrolysis. A simple mathematical expression was empirically derived that can be used to predict the extent of sucrose hydrolysis based on the post-autoclave pH of the media. This revised version was published online in June 2006 with corrections to the Cover Date.     

Polygalacturonase,biomass and ascospore production by Byssochlamys fulva. II. Effects of sugars found in fruits

Polygalacturonase, biomass, and ascospore production by four strains of Byssochlamys fulva cultured in laboratory media supplemented with glucose, sucrose, or fructose was studied over a 20-day incubation period at 30° C. The production of polygalacturonase was variable, but most activity was detected between 4 and 8 days in 1% sugar media at an initial pH of 4 or 5. The rate of biomass production was retarded early in the incubation period in media initially at pH 3 or 4 as compared to pH 5, but the amount of growth was about the same in media containing the test sugars after 20 days. Large numbers of ascospores were produced between 8 and 10 days in media containing 5% sugar initially at pH 5 and 4. Production of ascospores was retarded at pH 3 in media containing 5% sugar as compared to media initially at pH 5 and 4.     

pH variations during growth of Acidithiobacillus thiooxidans in buffered media designed for an assay to evaluate concrete biodeterioration

The pH of two buffered media having their initial pH ranging between 3.5 and 8.5 was monitored during growth of Acidithiobacillus thiooxidans. The first media was buffered with tricyclic phosphate whereas the second one contained phosphate ions and thus exhibited a stronger buffer capacity. Bacterial growth was not observed in any of the two media when the initial pH was higher than 5.5. On the other hand, for initial pH lower than 5.5, bacterial growth induced pH drops in both media. This drop was preceded by a lag phase during which the pH remained unchanged. However, in the medium buffered with phosphate ions, the lag periods were longer. As these media were developed for designing a bioleaching test to evaluate concrete biodeterioration caused by A. thiooxidans, the medium containing tricyclic phosphate appeared to be the most appropriate.     

Inverse pH-dependent shape changes of erythrocytes in the presence of albumin

A comparative study on the shape of human erythrocytes suspended in 7 different media showed, contrary to the well-known albumin-free case, an enhancement of the number of discocytes and stomatocytes for pH rising in all HSA containing media applied. At the same time, the transmembrane potential as determined by extra- and intracellular pH was lowered in all of 6 media tested. Consequently, there is no simple relationship between the pH-dependent behaviour of cell shape and corresponding changes of transmembrane potential.     

Culture medium pH is influenced by basal medium,carbohydrate source,gelling agent,activated charcoal,and medium storage method

Summary When four carbohydrates were tested against six commonly cited inorganic basal media, post-autoclave pH was highest for carbohydrate-free and sucrose containing media, and progressively lower for maltoseglucose and fructose-containing media, respectively. Post-autoclave pH for these media without carbohydrates was related to medium buffering capacity. Addition of gelling agents (10 of 11 tested) increased the postautoclave pH of MS medium containing sucrose. Neutralized and acid-washed activated charcoal also increased the post-autoclave pH of liquid and agarsolidified MS medium, and the pH changed further during 8 weeks of storage. Changes in medium pH caused by gelling agents, but not charcoal, could be alleviated by adjusting the pH after their addition but prior to autoclaving.     

The etiolated cucumber hypocotyl weight-growth test: A sensitive,easy and ultrafast bioassay for IAA

A sensitive, easy and fast bioassay is described for the detection of IAA. The bioassay consists of measuring the weight increase of hypocotyl sections from etiolated cucumber seedlings incubated in a simple medium containing 2 mM KCl, 0.1 mM CaCl and 10 mg/l chloramphenicol (pH 7). The sensitivity of the test is comparable to that of the Avena first internode test, exhibiting a significant response at an IAA concentration as low as 1 ng/ml. The bioassay requires only 3 h, and is easy to perform. The slope of the log-linear concentration-response curve is moderately steep giving good accuracy. The assay is insensitive to the pH of the media between 4 to 7 and can be performed in the presence or absence of buffer. The growth of the etiolated hypocotyl sections is insensitive to GA and to kinetin except at high concentrations.     

ESR study of proton transport across phospholipid vesicle membranes

A new method for measuring the rates of proton transfer through bilayer phospholipid membranes using pH-sensitive nitroxyl radicals is suggested. The pH-sensitive alkylating radical was covalently bound to glutathione. This modified glutathione is pH sensitive at pH 1.5-4.5 and does not penetrate across phospholipid membranes. Using ESR this probe was applied to register the kinetics of pH variations inside large unilamellar phospholipid vesicles after creation of a transmembrane proton gradient. In the acidic region (pH approximately 3) the main mechanism of transmembrane proton transfer is that via transport of a proton in the form of an undissociated acid. The membrane permeability coefficients have been determined for a series of acids (HCl, HClO4, HNO3, upper estimate for H2SO4). Taking into account that imidazoline and imidazolidine nitroxyl radicals can be used as pH probes in a wide range of pH, the present method can be developed for measuring the rates of transmembrane proton transfer in neutral and alkaline media.     

Some effects of monovalent anion replacement on the volume and composition of cells in incubated slices of rat renal cortex

Steady-state cellular water content and cation content and concentration have been studied in slices of rat renal cortex incubated in media in which Cl- was replaced by various monovalent anions at pH 7.35. Anions derived from low molecular weight aliphatic acids caused net uptake of cell water and K+. In the presence of larger anions cell water content fell in a manner related to anionic equivalent weight, but water content in media containing anions from weak aliphatic or alicyclic acids was slightly but consistently higher than that in media containing anions from strong acids. Cell K+ content did not decrease in these media. Aromatic anions caused enhanced cell water loss. When external pH adjusted to 6.8 or 7.8 it was found that in media containing anions from weak acids, but not from strong acids, increasing pH was associated with significantly decreased cell water content.     

An adenosine triphosphate-dependent stabilization of proteolytic activity in heterolysosomes. Evidence for a proton pump

1. Homogenization of mouse kidneys or livers in 0.25m-sucrose buffered with tris-acetate, pH7.3, resulted in a decreased rate of proteolysis within isolated heterolysosomes containing injected (125)I-labelled albumin when these particles were incubated at 35 degrees C. Proteolysis in mouse kidney or liver heterolysosomes isolated from homogenates made in 0.25m-sucrose buffered at pH7.3 was stimulated by pH5 buffer or by additions of ATP. 2. A greater inhibition of proteolysis was produced by including bicarbonate or pH8 borate buffers in the incubation media, and this inhibition was also reversed by ATP. 3. Other nucleoside triphosphates were not as effective as ATP, but GTP and ITP were more effective than CTP or UTP. ADP, AMP, or adenosine 3':5'-cyclic monophosphate were completely without effect. 4. Although ATP prevented some heterolysosome breakage in media containing bicarbonate, the primary effect appeared to be to promote proteolytic activity. 5. These observations are consistent with the presence of a proton pump in the heterolysosome membrane, which functions to maintain intralysosomal pH in alkaline media.     

Influence of CO2 and pH on the attachment response of the cercaria of Diplostomum spathaceum (trematoda) (author's transl)]

1. The attachment of the cercaria to artificial substrates (offered via dialyzing membranes) in definite media was investigated under conditions of variable pH and [CO2]. 2. A decrease of the pH of the substrate releases only attachments in CO2 containing media and consequently acts via CO2 systems of the medium. 3. As effective components of CO2 systems, dissolved CO2 + H2CO3 are confirmed. 4. The sensitivity of the reaction on gradients of the CO2 partial pressure (in solution) could be established by offering substrates with lowered pH in CO2 containing media. Thus, by raising the CO2 partial pressure from ca. 0,04% to 0,15% maximal fixation rates were obtained (Fig. 3). 5. The carboanhydrase inhibitor acetazolamide, when added to the medium, had no direct influence on the CO2 receptors.     

Effects of glycose on NADP and NAD glutamate dehydrogenase activities and their relation to ammonium and pH levels in cultures of Bipolaris maydis race T

NADP-glutamate dehydrogenase (NADP-GDH) and NAD-glutamate dehydrogenase (NAD-GDH) activities from Bipolaris maydis race T (ATCC 36180) were determined by measuring the change in absorbance at 340 nm of either reduced NADP or NAD in a reaction mixture of NH₄C1, -ketoglutarate and a cell free extract of the fungus. NADP-GDH activity was high at 48 h, but low at 72 and 96 h when the fungus was incubated on a reciprocal shaker at 28 °C in a mineral salts medium containing 2 g/l glucose and 4 g/l Lasparagine. In contrast, in these cultures NAD-GDH activity was low at 48 h, but high at 72 and 96 h. At 72 and 96 h glucose was not detected in the culture medium. In addition, levels of ammonium and pH increased from 0.0 moles/ml and pH 5.8 at 48 h to 10.6 moles/ml and pH 7.2 at 72 h, and to 23.0 moles/ml and pH 8.4 at 96 h. Fungal mycelia were transferred after 48 h of incubation on media containing 2 g/l glucose and 4 g/l L-asparagine to fresh media containing 0, 2 or 5 g/l glucose with and without 4 g/l L-asparagine. Twenty-four h after transfer to fresh media containing 5 g/l glucose with L-asparagine or 2 or 5 g/l glucose without L-asparagine, NADP-GDH activity was high and NAD-GDH activity was low. Glucose was detected in the culture medium, ammonium was not detected and the pH remained unchanged or decreased. In contrast, 24 h after transfer to fresh media with 0 or 2 g/l glucose with L-asparagine and on media lacking glucose or L-asparagine, NADP-GDH activity was low and NAD-GDH activity was high. Glucose was not detected in the culture medium, ammonium levels were high and the pH increased. Thus, accumulation of ammonium and pH increases accompanying depletion of glucose in a L-asparagine medium could be related to a change in the capacity of B. maydis race T to assimilate and produce ammonium via pathways involving glutamate dehydrogenases.     

PH and growth of Torulopsis pintolopesii in media containing various sugars as carbon and energy sources.

Growth within the pH range 2 to 8 of a strain of the yeast Torulopsis pintolopesii was tested in media containing various sugars as carbon and energy sources. Of the sugars tested, only D-glucose, D-fructose, and D-mannose supported growth of the yeast. In media containing those sugars, the organism grew over the entire pH range tested.     

Choice of extraction procedure for estimation of anterior pituitary hormone content

Searching for the best procedure for simultaneous estimation of the anterior pituitary hormones, extraction efficiencies of various media, additives such as urea and triton X-100, and physical treatments such as freezing-thawing (F-T) and sonication, were examined by measuring prolactin (PRL), growth hormone (GH), lutropin (LH), follitropin (FSH), and thyrotropin (TSH) in the extracts. Ethanolic media (60% EtOH) gave high yields of PRL at neutral to alkaline pH, but poor extraction of GH accompanied by a marked loss of its immunoreactivity during storage. Ethanolic media also gave a poor yield of LH even at high pH. Aqueous media like PBS at various pH, 0.1 M acetic acid and distilled water were considerably effective in the extraction of GH, LH, FSH and TSH if they were coupled with F-T and sonication. However, high yields of PRL could not be obtained with these aqueous media even with F-T and sonication. Hartree's 40% EtOH-6% ammonium acetate, pH 5.1, solubilized considerable amounts of glycoprotein hormones, but yielded almost no GH and only a small amount of PRL. The addition of triton X-100 to PBS (pH 7) at 0.1% resulted in the maximum extraction of glycoprotein hormones with homogenization and F-T, but further sonication was necessary for GH and PRL. When the anterior pituitaries were homogenized and frozen-thawed in PBS (pH 7) containing 1 M urea, yields of PRL, GH, LH, FSH, and TSH were maximum, and sonication did not cause any additional extraction, indicating that this procedure, i.e. homogenization and F-T in 1 M urea-PBS, would be the best for the simultaneous estimation of these anterior pituitary hormones.     

Combined effect of growth medium,age of cells and phase of sporulation on heat resistance and recovery of Hansenula anomala

Effects of two growth media, age of cells and phase of sporulation on heat resistance of Hansenula anomala were determined. Cells were grown on two solid media, McClary's acetate and V8 juice agars, at 21 ° C for 16 days. Heat resistance of cells was determined in 0.06 M potassium phosphate buffer at 48 ° C. Heat-stressed cells were plated on four recovery media: yeast extract-malt extract-peptone-glucose (YMPG), pH 7.0; YMPG, pH 3.5; YMPG containing 6% NaCl, pH 7.0; and YMPG containing 20% sucrose, pH 7.0. The composition of sporulation medium influenced the extent of sporulation and the relative heat resistance of sporulating cells. One-day-old cells were the most sensitive to heat. The heat resistance of cells was generally increased as the incubation time was extended to 16 days. Heat treatment caused a greater increase in sensitivity to NaCl than to sucrose or acid pH in recovery media. Young cells were more sensitive to NaCl than were older cells.     

Effect of Ca2+ and K+ on the intracellular pH of an Escherichia coli L-form.

The L-form NC7, derived from Escherichia coli K12, grew in a complex medium containing 0.2 M-CaCl2 as osmotic stabilizer, but not at pH values above 7.8. The cessation of growth at alkaline pH was not due to cell death. In complex media containing K+ or Na+, the L-form grew ove a wide pH range. Growth at alkaline pH was inhibited by 1 mM-amiloride, indicating that Na+/H+ antiport activity was required for growth at alkaline pH. The internal pH (pHi) of the L-form in media containing K+, Na+ or Ca2+ was constant at about 7.8 to 8.0 at external pH (pHo) values of 7.2 and 8.2. The rates of O2 consumption by intact cells, lactate oxidation by membrane vesicles from cells grown in Ca(2+)-containing medium, and cell division were all strongly repressed under alkaline conditions.     

Conformational change of influenza virus hemagglutinin is sensitive to ionic concentration

The homotrimeric spike glycoprotein hemagglutinin (HA) of influenza virus undergoes a low pH-mediated conformational change which mediates the fusion of the viral envelope with the target membrane. Previous approaches predict that the interplay of electrostatic interactions between and within HA subunits, HA 1 and HA2, are essential for the metastability of the HA ectodomain. Here, we show that suspension media of low ionic concentration promote fusion of fluorescent labelled influenza virus X31 with erythrocyte ghosts and with ganglioside containing liposomes. By measuring the low pH mediated inactivation of the fusion competence of HA and the Proteinase K sensitivity of low pH incubated HA we show that the conformational change is promoted by low ionic concentration. We surmise that electrostatic attraction within the HA ectodomain is weakened by lowering the ionic concentration facilitating the conformational change at low pH. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Novel selective and non-selective optical detection of micro-organisms

A new instrument, capable of detecting metabolic changes due to microbiological activity, is described. Optical changes in growth media are monitored in a semi-fluid zone that separates the liquid medium containing the sample. Data demonstrate that common media can be utilized in conjunction with this rapid automated technology. Nutrient broth with the pH dye indicator bromocresol purple was suitable for total counts. Selective media containing dyes were utilized to assess the presence or absence of specific groups of organisms. Biochemical reactions, such as lysine decarboxylase activity, were identified by the unique generated patterns, and specific enzymatic cleavage reactions with chromogenic substrates, such as 5-bromo-4 chloro-3 indolyl-β- D -glucuronic acid (X-GLUC), were monitored.     

Cellulase production by Trichoderma reesei when cultured on xylose-based media supplemented with sorbose

The ability of L-sorbose to stimulate cellulase production In shake flask culture of Trichoderma reesei was examined in mineral salts media (initial pH 5.0) containing either 1.0% D-xylose, 1.0% cellulose, and/or 0.1, 0.3, or 0.5% L-sorbose. When sorbose was the only carbon source, growth was limited, little substrate was utilized, pH increased, and cellulase activity was not apparent. The other carbon sources promoted good growth, pH dropped sharply to 2.5-3.0, substrate was utilized rapidly, and cellulase activity was detected. After three weeks of fermentation, twice as much cellulase activity was detected in the medium containing only cellulose as the carbon source, as compared to xylose as the carbon source. Cellulase activity was higher when media contained xylose supplemented with sorbose compared to xylose as the only carbon source. At 0.3 and 0.5% levels of sorbose supplementation of xylose-based media, cellulase activity was similar to that in cellulose-based media.