Production and stabilization of cells of Bacillus popilliae and Bacillus lentimorbus

Bacillus popilliae and B. lentimorbus grew most rapidly and to the greatest extent in aerated cultures at 30 to 32 C with oxygen absorption rates of 1 mmole of O₂ per min per liter, or above. The control of pH also increased the maximal populations attained. Media were developed which consistently produced cell populations of about 10⁹ within 24 to 48 hr in aerated cultures of these two species. The acetic acid produced in highly aerated cultures was shown not to be responsible for the rapid loss of cell viability in stationary phase cultures. However, H₂O₂ was very lethal to cells of B. popilliae, and this species is known to have the capacity to produce it. Stationary-phase cells were partially stabilized by reducing the availability of oxygen after 24 hr of incubation on a shaker, and the addition of low levels of glucose further stabilized the cells. The most stable cells were those produced in a medium in which 4% Trypticase (BBL) and 0.1% barbituric acid were incorporated. A high percentage of these cells contained refractile bodies visible under a phase microscope. Although these bodies were not heat-resistant and lacked other characteristics of endospores, cells in cultures containing them had reasonably high viability for extended periods, as compared with those in control cultures.     

Propagation of Bacillus popilliae in laboratory fermentors

The insect pathogen Bacillus popilliae Dutky causes a fatal milky disease of Japanese beetle larvae. Spores of the bacterium offer a biological means of controlling this insect. While satisfactory sporulation in vitro has not yet been accomplished, conditions have been developed for the proliferation of vegetative cells in shaken flasks and aerated fermentors. Vegetative cultures are maintained by frequent transfer or by lyophilization. Media based on yeast extract are used routinely, but corn steep liquor and casein hydrolyzates afford comparable yields of 5 × 10⁸ cells/ml. in 16–24 hr. Nutritional requirements have been established for growth in a synthetic medium. Oxygen availability affects the pathway of carbohydrate catabolism and is necessary for optimal growth. In rapidly growing cultures, a short period of maximum viability is characteristically followed by rapid death of the cells. When inoculum size and transfer time are suitably manipulated, viable cell yields reach 1–2 × 10⁹/ml. Alternative methods of propagation, including the addition of particulate carbon, and procedures designed either to neutralize acids or to remove metabolic products by dialysis, do not markedly enhance the yield of cells per volume of medium, although viability may be prolonged.     

Influence of Viable Cells on the Resuscitation of Dormant Cells in Micrococcus luteus Cultures Held in an Extended Stationary Phase: the Population Effect

A high proportion of Micrococcus luteus cells in cultures which had been starved for 3 to 6 months lost the ability to grow and form colonies on agar plates but could be resuscitated from their dormancy by incubation in an appropriate liquid medium (A. S. Kaprelyants and D. B. Kell, Appl. Environ. Microbiol. 59:3187-3196, 1993). In the present work, such cultures were studied by both flow cytometry and conventional microbiological methods and were found to contain various numbers of viable cells. Pretreatment of such cultures with penicillin G, and subsequent dilution, was used to vary this number. When the initial number of colony-forming cells per 30-ml flask was approximately nine (±five) or more, resuscitation of 10 to 40% of the cells, and thus culture growth, was observed. The lag period before the appearance of a population of cells showing significant accumulation of the fluorescent dye rhodamine 123 (i.e., of cells with measurable membrane energization) decreased from 70 to 27 h when the number of viable cells was increased from 30 to 10⁵ per flask, while the lag period before an observable increase in the number of colony-forming cells occurred was almost constant (at some 20 h). Provided there were more than nine (±five) initially viable cells per flask, the number of initially viable cells did not affect the final percentage of resuscitable cells in the culture. The lag period could be ascribed in part to the time taken to restore the membrane permeability barrier of starved cells during resuscitation, as revealed by flow cytometric assessment of the uptake of the normally membrane-impermeant fluorescent DNA stain PO-PRO-3 {4-[3-methyl-2, 3-dihydro-(benzo-1, 3-oxazole)-2-methylidene]-1-(3′-trimethylammonium propyl)-pyridinium diiodide}. Although cell populations which contained fewer than nine ±five viable cells per flask failed to grow, 4 to 20% of the cells (of 1.2 X 10⁶) were able to accumulate rhodamine 123 after 80 to 100 h of incubation, showing the ability of a significant number of the cells in the population at least to display “metabolic resuscitation.” Resuscitation and cell growth under such conditions were favored by the use of a 1:1 mixture of fresh lactate medium and supernatant from late-logarithmic-phase M. luteus cultures as the resuscitation medium. We conclude that the presence of a small fraction of viable cells at the onset of resuscitation facilitates the recovery of the majority of the remaining (dormant) cells. The cell density dependence of the kinetics, or population effect, suggests that this recovery is due to the excretion of some factor(s) which promoted the transition of cells from a state in which they are incapable of growth and division to one in which they are capable of colony formation.     

Effect of pH and Oxygen Tension on Staphylococcal Growth and Enterotoxin Formation in Fermented Sausage

Commercial fermented 0sausages that contained significant numbers of viable coagulase-positive staphylococci were found to have the growth localized in the outermost areas of the sausage where oxygen tension was highest. Staphylococci were found to be more acid-tolerant aerobically than anaerobically. With chemical acidulation of sausage, growth could be controlled both aerobically and anaerobically with approximately 1.5% glucono delta lactone. Biological acidulation with a high inoculum of Pediococcus cerevisiae inhibited anaerobic staphylococcal growth but failed to suppress aerobic growth completely. A staphylococcal count of approximately 4 × 10⁷ cells/g of sausage appeared to be necessary to produce detectable enterotoxin A within 24 hr in sausage. A minor difference existed in the relative rates of production of the different types of enterotoxin. Detectable enterotoxin A was produced in 24 hr in sausage held in atmospheres containing 10, 15, and 20% oxygen. In an atmosphere containing 5% oxygen, toxin was detected after 48 hr of incubation. No toxin was detected after 120 hr under anaerobic conditions. Most staphylococcal strains tested initiated growth and produced detectable enterotoxin aerobically at a pH of 5.1 in broth media. Anaerobically, however, most strains failed to produce detectable enterotoxin below pH 5.7.     

Use of Antibiotics in the Preparation of Canine Kidney Tissue Culture Vaccines to Eliminate Leptospiral Infection Hazards

The potential leptospiral infection hazard in the use of vaccines prepared from canine kidney monolayer cultures was studied. Cell cultures were prepared from kidneys of dogs experimentally infected with Leptospira serotype canicola. Viable leptospires were found in kidney cell suspensions at the time of seeding, surviving trypsinization either at room temperature for approximately 2 hr or overnight at 4 C, even in the presence of antibiotics. In tissue cultures maintained without antibiotics, leptospires were cultured up to the time of involution of cells at 25 to 34 days of incubation. Cytopathogenic effects of leptospires on cultured kidney cells were not noted; neither was growth of leptospires remarkable. Generally, the leptospire culture titer decreased to 10^-4 or 10^-5 at the 4th hr or 1st day of incubation to 10^-1 or negative by the 30th or 34th day of incubation. The addition of either a combination of penicillin (100 units per ml) plus streptomycin (100 μg/ml) or polymyxin B (50 units per ml) plus dihydrostreptomycin (100 μg/ml) to seeding cell suspensions resulted in the elimination of viable leptospires by the 4th hr of incubation. From cell cultures treated with neomycin (100 μg/ml) or chloramphenicol (100 μg/ml), leptospires were recovered, respectively, after 24 and 48 hr, but not thereafter. It was apparent that antibiotics, particularly the combination of polymyxin B and dihydrostreptomycin, could be effectively used to eliminate leptospires in tissue culture. Other antibiotics with known antileptospiral activities probably would be effective also. If antibiotics are not used in canine kidney tissue culture employed for viral vaccine preparations, rigid testing for the presence of leptospires in donor dogs and tissue-culture vaccine is indicated.     

The association of carbohydrate changes in the shoot tip of cauliflower with flowering

Changes in levels of sugars and starch in the shoot tip of cauliflower, Brassica oleracea L. var. botrytis D. C. cv. Main Crop were studied during periods of growth which were inductive or non-inductive to flowering. Flowering was induced by growing plants for 2 weeks under 16 hr of light at 5°. During this period of floral induction there was a significant increase in sugar and starch content compared to that in vegetative plants grown at 20 to 26°. Sugar and starch content did not increase and flowering was prevented when light and CO₂ were excluded during growth at 5°. A 3-day dark period at 20° or a high temperature treatment at 33° with light following growth at 5° reduced the carbohydrate level and prevented flowering.     

EXPERIMENTAL CONTROL OF DNA SYNTHESIZING AND DIVIDING CELLS IN EXCISED ROOT TIPS OF PISUM

The number of dividing and DNA-synthesizing cells in excised pea roots can be regulated by eliminating the carbohydrate normally supplied in the culture medium. When the excised roots were allowed to remain for 24 hr in a medium lacking carbohydrate, the number of mitotic figures and tritiated thymidine (H³-T) labeled cells was reduced almost to zero. After an additional 24 hr in the incomplete culture medium, 15% of the interphase cells were H³-T labeled, the percentage of the cells that were dividing never exceeded 1.4, and 30% of these were H³-T labeled. When the roots remained in the deficient medium for 72 hr, neither cell division nor cells synthesizing DNA were observed. Upon addition of 2% sucrose, cell division and DNA synthesis were resumed in the roots that were maintained for 24 or 72 hr without an exogenous carbohydrate supply. It has been hypothesized that some proliferative systems consist of two cellular subpopulations which selectively stop or remain in either the pre-DNA synthetic (G₁) or post-DNA synthetic (G₂) periods of the mitotic cycle. The addition of sucrose, H³-T, and 5-aminouracil to the medium, after the roots had been maintained for 24 hr without a carbohydrate, indicated that most of the proliferative cells in the roots had accumulated in either G₁, a quasi-G₁ condition, i.e., DNA synthesis stopped sometime before completion, or G₂ periods of interphase; the majority, however, were in G₁ or quasi-G₁ conditions. The results suggested that DNA synthesis (S period) and mitosis or the onset of these processes have the highest metabolic requirements in the mitotic cycle and that G₁ and G₂ were the most probable states for proliferative cells in a meristem with a low metabolic level.     

Acetylcholine synthesis by chick spinal cord neurons in dissociated cell culture

Acetylcholine (ACh) synthesis was examined in cultures of chick spinal cord cells to follow the development of the cholinergic neurons. The cells, prepared from 4-day-old embryonic chick spinal cords, were grown either alone in dissociated cell cultures (SC cultures) or with chick myotubes (SC-M cultures). ACh synthesis was measured by incubating the cultures in [³Hcholine and using high-voltage paper electrophoresis to quantitate the amount of [³H]ACh present in cell extracts prepared from the labeled cultures. The amount of [³H]ACh synthesized in SC-M cultures was strictly proportional to the number of spinal cord cells used to prepare the cultures, and was linear with the time of incubation in [³H]choline for periods up to 1 hr. Maximal rates of synthesis were observed with [³H]choline concentrations in excess of 100 μM. Such rates for 1-week-old SC-M cultures were approximately 10–20 pmoles of [³H]ACh/hr/10⁵ spinal cord cells. Studies on the stability of the intracellular [³H]ACh revealed the presence of a major pool with a half-time of 20–30 min. A second, small pool decayed more rapidly. No detectable [³H]ACh was spontaneously released from the cells, suggesting that most of the decay represented intracellular degradation. Development of cholinergic neurons as monitored by [³H]ACh synthesis continued over a 2-week period in SC-M cultures and paralleled general cell growth. When examined at 1 week, SC-M cultures had about a 50% greater capacity for [³H]ACh synthesis and 60% more choline acetyltransferase activity than did SC cultures. No difference was observed in the stability of the [³H]ACh formed for the two types of cultures at 1 week, and no further difference was observed in the rates of [³H]ACh synthesis at 2 weeks. Growth of SC cultures in medium containing different amounts of chick embryo extract (2–10%) or in medium with fetal calf serum (10%) instead of extract produced only small differences in the measured rates of [³H]ACh synthesis. Thus chick spinal cord cells can undergo some of the early stages of cholinergic development in cell culture without sustained contact with skeletal myotubes, one of the normal postsynaptic target cells for the cholinergic neuron population. No absolute requirement for muscle factors was revealed under these conditions, although such factors may have been provided by other cell types in the spinal cord population or may have been present in other additions to the culture medium.     

Responses of Ruminococcus flavefaciens, a Ruminal Cellulolytic Species, to Nutrient Starvation

Ruminococcus flavefaciens strain C94, a strictly anaerobic, cellulolytic ruminal bacterial species, was grown either in batch or continuous cultures (cellobiose limited or nitrogen limited) at various dilution rates. Washed cell suspensions were incubated anaerobically at 39°C without nutrients for various times up to 24 h. The effects of starvation on direct and viable cell counts, cell composition (DNA, RNA, protein, and carbohydrate), and endogenous production of volatile fatty acids by the cell suspensions were determined. In addition, the effect of the pH of the starvation buffer on direct and viable cell counts was determined. Survival of batch-grown cells during starvation was variable, with an average time for one-half the cells to lose viability (ST₅₀) of 10.9 h. We found with continuous cultures that viable cell counts declined faster when the initial cell suspensions had been grown at faster dilution rates; this effect was more pronounced for suspensions that had been limited by cellobiose (ST₅₀ = 6.6 h at a dilution rate of 0.33 h⁻¹) than for suspensions that had been limited by nitrogen (ST₅₀ = 9.5 h at a dilution rate of 0.33 h⁻¹). With continuous cultures, viable cell counts in all cases declined faster than direct cell counts did. The rates of disappearance of specific cell components during starvation varied with the initial growth conditions, but could not be correlated with the loss of viability. Volatile fatty acid production by starving cells was very low, and acetate was the main product. Starved cells survived longer at pH 7.0 than they did at pH 5.5, and this effect of pH was greater for cellobiose-limited cells (mean ST₅₀ = 7.1 h) than for nitrogen-limited cells (mean ST₅₀ = 12 h). Although it has relatively low ST₅₀ values, R. flavefaciens has sufficient survival abilities to maintain reasonable numbers in domestic animals having maintenance or greater feed intake.     

Effect of N Source on the Steady State Growth and N Assimilation of P-limited Anabaena flos-aquae

Phosphate-limited chemostat cultures were used to study cell growth and N assimilation in Anabaena flos-aquae under various N sources to determine the relative energetic costs associated with the assimilation of NH₃, NO₃⁻, or N₂. Expressed as a function of relative growth rate, steady state cellular P contents and PO₄ assimilation rates did not vary with N-source. However, N-source did alter the maximal PO₄-limited growth rate achieved by the cultures: the NO₃⁻ and N₂ cultures attained only 97 and 80%, respectively, of the maximal growth rate of the NH₃ grown cells. Cellular biomass and C contents did not vary with growth rate, but changed with N source. The NO₃⁻-grown cells were the smallest (627 ± 34 micromoles C · 10⁻⁹ cells), while NH₃-grown cells were largest (900 ± 44 micromoles C · 10⁻⁹ cells) and N₂-fixing cells were intermediate (726 ± 48 micromoles C · 10⁻⁹ cells) in size. In the NO₃⁻-and N₂-grown cultures, N content per cell was only 57 and 63%, respectively, of that in the NH₃-grown cells. Heterocysts were absent in NH₃-grown cultures but were present in both the N₂ and NO₃⁻ cultures. In the NO₃⁻-grown cultures C₂H₂ reduction was detected only at high growth rates, where it was estimated to account for a maximum of 6% of the N assimilated. In the N₂-fixing cultures the acetylene:N₂ ratio varied from 3.4:1 at lower growth rates to 3.0:1 at growth rates approaching maximal.

Compared with NH₃, the assimilation of NO₃⁻ and N₂ resulted either in a decrease in cellular C (NO₃⁻ and N₂ cultures) or in a lower maximal growth rate (N₂ culture only). The observed changes in cell C content were used to calculate the net cost (in electron pair equivalents) associated with growth on NO₃⁻ or N₂ compared with NH₃.

     

Cell yields of bacteria grown on methane

Several mixed cultures of methane-oxidizing bacteria have been isolated. Among them, culture HR (consisting of two gram-negative rods, one 0.5 × 1.0 μ, the other 0.8 × 2 to 3 μ) was found to be the fastest-growing and to give the highest yields. Optimal conditions for rapid growth and high cell yields from methane were found to be: 30 C, NH₄⁺ as nitrogen source, and pH 6.5. Requirements for CO₂ and Cu⁺⁺ were observed. Under these conditions, generation times of approximately 3 hr and cell yields from methane between 65 and 70% could be attained. Culture HR can utilize methane, methanol, ethyl alcohol, 1-propanol, n-butyl alcohol, and glucose, but not propane, for growth. Yeast and beef extracts are inhibitory. Carbon balances demonstrate that few if any products other than cells and CO₂ are produced from methane under the growth conditions used. Cell analyses for carbon, hydrogen, nitrogen, and amino acid content of culture HR were also made.     

The effects of cortisol on the primary response of mouse spleen cell cultures to heterologous erythrocytes

Cell viability and the production of direct PFC were studied in mouse spleen cell cultures after cortisol treatment in vivo or in vitro at various times relative to primary stimulation with SRBC in vitro.Cortisol treatment in vivo reduced spleen cell numbers by 88% after 48 hr, but cultures of the remaining cells produced as many PFC in vitro as did cultures of equal numbers of normal spleen cells.In normal spleen cell cultures incubated with cortisol for 4 hr prior to the addition of antigen, peak responses of PFC/culture and PFC/10⁶ cells occurred 24 hr later than in controls and averaged, respectively, 27% and 141% of control values. Minimum viable cell numbers were observed in cortisol-treated cultures after 3 days; thereafter cell numbers gradually increased. These results were not significantly altered when cultures were treated simultaneously with cortisol and antigen.The response was not suppressed if the addition of antigen preceded that of cortisol by more than 4 hr. Suppression was also considerably reduced if fetal calf serum was used when preparing cells for culture.     

Characterization of growth and Oryctes rhinoceros nudivirus production in attached cultures of the DSIR-HA-1179 coleopteran insect cell line

The DSIR-HA-1179 coleopteran cell line is a susceptible and permissive host to the Oryctesrhinoceros nudivirus (OrNV), which has been used as a biocontrol agent against the coconut rhinoceros beetle (Oryctes rhinoceros); a pest of palms in the Asia-Pacific region. However, little is known about growth and metabolism of this cell line, knowledge of which is necessary to develop an in vitro large-scale OrNV production process. The strong anchorage-dependent characteristics of the cell line, its particular fragility and its tendency to form dense clumps when manipulated, are the most likely reasons that have precluded further development of the cell line. In order to characterize DSIR-HA-1179 cells, there was first a need for a reliable technique to count the cells. A homogenous cell suspension suitable for enumeration could be produced by treatment with TrypLE Express™ with optimum mean time for cell release calculated as 30 min. The cell line was adapted to grow in four serum-supplemented culture media namely TC-100, IPL-41, Sf-900 II and Sf-900 III and cell growth, glucose consumption, lactate and ammonia production were assessed from static-batch cultures. The maximum viable cell density was reached in Sf-900 II (17.9 × 10⁵ cells/ml), with the maximum specific growth rate observed in this culture medium as well (0.0074 h⁻¹). Higher production of OrNV was observed in IPL-41 and TC-100 (4.1 × 10⁷ TCID₅₀/ml) than in cultures infected in Sf-900 III (2.0 × 10⁷ TCID₅₀/ml) and Sf-900 II (1.4 × 10⁷ TCID₅₀/ml). At the end of the growth period, glucose was completely consumed in cultures grown in TC-100, while remained in excess in the other three culture media. The cell line produced lactate and ammonia to very low levels in the TC-100 culture medium which is a promising aspect for its cultivation at large-scale.     

The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E₂ (PGE₂) on fibroblast proliferation was examined. The presence of PGE₂ for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [³H]thymidine occurred at concentrations greater than 10⁻⁷ M. The inhibitory effect of PGE₂ was less potent in exponentially growing cells and was not the result of conversion of PGE₂ to PGA₂ during incubation in growth medium. The G₁ phase was determined to be 12–14 h in untreated cultures. The extent of growth inhibition by PGE₂ was similar with addition of PGE₂ at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE₂-induced growth inhibition. Short-term exposure to PGE₂ (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE₂ affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE₂ for 24 h, indicated that release from PGE₂ exposure is associated with prolongation of the G₁ phase of the cell cycle.     

Degradation of Pure Aflatoxins by Tetrahymena pyriformis

Tetrahymena pyriformis W with nutrients, ca. 22 × 10⁶ cells, decreased the concentration of aflatoxin B₁ 58% in 24 hr and 67% in 48 hr. An unknown, bright-blue fluorescent substance was produced, with intensity about one-half that of the unchanged B₁, with an R_f of 0.52 compared with 0.59 for B₁ and 0.55 for B₂ on a thin-layer chromatography plate, and with an ultraviolet spectrum showing maxima of 253, 261, and 328 mμ. In a separate assay, the cells with nutrients did not degrade pure G₁. Starved, washed cells, ca. 11 × 10⁶, decreased the concentration of B₁ 50% in 10 hr, 70% in 22 hr, and 75% in 30 hr, producing the same unknown component. Ethyl alcohol, 1.96% (v/v), decreased cell populations and size, but the cells remained actively motile in broth plus the alcohol for 96 hr. In 72 hr, neither toxin (ca. 2 ppm) in combination with ethyl alcohol had more inhibitory effect on cell numbers, with or without nutrients, than was produced by alcohol alone. Aflatoxin B₁ had no observed effect on the viability of the starved cells for 30 hr or on the nourished cells for 72 hr. There was no noticeable effect of G₁ on the starved cells in 30 hr or on the nourished cells in 48 hr. After 72 hr with G₁ plus nutrients, many of the cells were round with blisters, nonmotile, and apparently dead or dying.     

Effects of the Metalloid Oxyanion Tellurite (TeO32−) on Growth Characteristics of the Phototrophic Bacterium Rhodobacter capsulatus

This work examines the effects of potassium tellurite (K₂TeO₃) on the cell viability of the facultative phototroph Rhodobacter capsulatus. There was a growth mode-dependent response in which cultures anaerobically grown in the light tolerate the presence of up to 250 to 300 μg of tellurite (TeO₃²⁻) per ml, while dark-grown aerobic cells were inhibited at tellurite levels as low as 2 μg/ml. The tellurite sensitivity of aerobic cultures was evident only for growth on minimal salt medium, whereas it was not seen during growth on complex medium. Notably, through the use of flow cytometry, we show that the cell membrane integrity was strongly affected by tellurite during the early growth phase (≤50% viable cells); however, at the end of the growth period and in parallel with massive tellurite intracellular accumulation as elemental Te⁰ crystallites, recovery of cytoplasmic membrane integrity was apparent (≥90% viable cells), which was supported by the development of a significant membrane potential (Δψ = 120 mV). These data are taken as evidence that in anaerobic aquatic habitats, the facultative phototroph R. capsulatus might act as a natural scavenger of the highly soluble and toxic oxyanion tellurite.     

Nitrogen Fixation Associated with Development and Localization of Mixed Populations of Cellulomonas sp. and Azospirillum brasilense Grown on Cellulose or Wheat Straw

Mixed cultures of Cellulomonas sp. and Azospirillum brasilense were grown with straw or cellulose as the carbon source under conditions favoring the fixation of atmospheric nitrogen. Rapid increases in cell numbers, up to 10⁹ cells per g of substrate, were evident after 4 and 5 days of incubation at 30°C for cellulose and straw, respectively. Nitrogen fixation (detected by acetylene reduction measured on parallel cultures) commenced after 2 and 4 days of incubation for straw and cellulose, respectively, and continued for the duration of the experiment. Pure cultures of Cellulomonas sp. showed an increase in cell numbers, but CO₂ production was low, and acetylene reduction was not detected on either cellulose or straw. Pure cultures of A. brasilense on cellulose showed an initial increase in cell numbers (10⁷ cells per g of substrate) over 4 days, followed by a decline presumably caused by the exhaustion of available carbon substrate. On straw, A. brasilense increased to 10⁹ cells per g of substrate over 5 days and then declined slowly; this growth was accompanied by acetylene reduction. Scanning electron micrographs of straw incubated with a mixed culture under the above conditions for 8 days showed cells of both species in close proximity to each other. Evidence was furnished that the close spatial relationship of cells from the two species facilitated the mutually beneficial association between them and thus increased the efficiency with which the products of straw breakdown were used for nitrogen fixation.     

Bacterial Culture Preservation in Frozen and Dry-Film Methylcellulose

Forty-seven of 61 bacterial cultures, including strains of Pseudomonas, Xanthomonas, Erwinia, Agrobacterium, Corynebacterium, Serratia, Klebsiella, and Escherichia, remained viable after storage in frozen methylcellulose or in dried methylcellulose for up to 38 months. Pathogenicity remained intact for those strains tested. Bacteria were grown on a solid medium and then removed and placed in 1.0% methylcellulose (cellulose methyl ether) to make a final suspension of 10⁸ colony-forming units (CFU) per ml. For storage in dried form, the bacteria-methylcellulose suspension was placed in a petri dish and dried in a forced-air incubator. After 24 h of storage at 25°C, viable populations of 10⁵ CFU/mg (equivalent to 10⁶ CFU/ml) were recovered. Populations of 10² to 10⁴ CFU/mg were recovered after storage of up to 38 months. Similar results were obtained in frozen methylcellulose. Survival was greatly enhanced when the growth medium for the bacteria was potato dextrose peptone rather than nutrient agar, yeast dextrose calcium carbonate peptone, or King's medium B. Addition of 0.1 M MgSO₄ to the methylcellulose suspension and to the resuspending liquid also increased survival and recovery from storage for some strains. Methylcellulose storage should be a simple, inexpensive, and reliable method of maintaining cultures for short or long periods of time.     

Toxic effects of resazurin on cell cultures

Resazurin, introduced as a cell viability indicator under the trade name alamarBlue^®, is generally regarded as nontoxic when used according to manufacturer’s suggested shorter-term incubation time specifications. However, problems arise when exposure times are extended to longer-term cultures on the order of days. To assess the effect of resazurin over longer incubation times, MCF7 (HTB-22), MCF10A (CRL-10317), 3T3-L1 (CL-173), and D1 (CRL-12424) cultures were tested with varying amounts of resazurin over 4- and 8-day periods. MCF7, 3T3-L1, and D1 cells cultured for 8 days with 20 % alamarBlue^® had significantly less cell survivability. Specifically, levels of metabolic activity, deoxyribonucleic acid (DNA) concentration, and glucose consumption of the cell lines cultured for 8 days in medium with 20 % alamarBlue^® were significantly lower (p < 0.05) than metabolic activity, DNA concentration, and glucose consumption of MCF7 cells cultured for 8 days in medium with no alamarBlue^®. MCF7, 3T3-L1, and D1 cells used less glucose at concentrations as low as 5 %. Data also suggests the toxic effects are more pronounced in the cancerous cell line as compared to the noncancerous cells.     

Sensitive rapid detection method for viable bacterial cells

A rapid sensitive method for the detection of viable bacterial cells is described in which P³² as inorganic orthophosphate is used to label the cells. Factors affecting the uptake of P³² by cells as well as the sensitivity of the method have been explored with suspensions of Aerobacter aerogenes. The uptake of P³²O₄ is dependent on several factors. Of various incubation media tested, one composed of 0.005 m KCl, 0.002 m MgSO₄ and 10 mg/ml of glucose was found to best stimulate the uptake of the tracer. Incubation time and temperature and level of isotope and of unlabeled P also affected uptake. Labeled cells were collected on a membrane filter for measurement of radioactivity. Under optimal conditions, as few as 23 viable cells per milliliter were detected in 1 hr with 95% confidence.