Measurement of Light Absorption and Determination of the Specific Rate of Light Uptake in Cultures of Phototrophic Microorganisms

This article describes a novel method for the measurement of light absorption by cultures of phototrophic microorganisms. The rate of light absorption is calculated as the difference between the rate of light output from a culture containing cells and the rate of light output from a culture containing only growth medium. The specific rate of light uptake is calculated by dividing the rate of light absorption by the total biomass present in the culture. Application of the method to several case studies shows that light output from a culture varies widely depending on the absorption and scattering characteristics of the suspension.     

A screening model to predict microalgae biomass growth in photobioreactors and raceway ponds

A microalgae biomass growth model was developed for screening novel strains for their potential to exhibit high biomass productivities under nutrient‐replete conditions in photobioreactors or outdoor ponds. Growth is modeled by first estimating the light attenuation by biomass according to Beer‐Lambert's Law, and then calculating the specific growth rate in discretized culture volume slices that receive declining light intensities due to attenuation. The model uses only two physical and two species‐specific biological input parameters, all of which are relatively easy to determine: incident light intensity, culture depth, as well as the biomass light absorption coefficient and the specific growth rate as a function of light intensity. Roux bottle culture experiments were performed with Nannochloropsis salina at constant temperature (23°C) at six different incident light intensities (10, 25, 50, 100, 250, and 850 µmol/m² s) to determine both the specific growth rate under non‐shading conditions and the biomass light absorption coefficient as a function of light intensity. The model was successful in predicting the biomass growth rate in these Roux bottle batch cultures during the light‐limited linear phase at different incident light intensities. Model predictions were moderately sensitive to minor variations in the values of input parameters. The model was also successful in predicting the growth performance of Chlorella sp. cultured in LED‐lighted 800 L raceway ponds operated in batch mode at constant temperature (30°C) and constant light intensity (1,650 µmol/m² s). Measurements of oxygen concentrations as a function of time demonstrated that following exposure to darkness, it takes at least 5 s for cells to initiate dark respiration. As a result, biomass loss due to dark respiration in the aphotic zone of a culture is unlikely to occur in highly mixed small‐scale photobioreactors where cells move rapidly in and out of the light. By contrast, as supported also by the growth model, biomass loss due to dark respiration occurs in the dark zones of the relatively less well‐mixed pond cultures. In addition to screening novel microalgae strains for high biomass productivities, the model can also be used for optimizing the pond design and operation. Additional research is needed to validate the biomass growth model for other microalgae species and for the more realistic case of fluctuating temperatures and light intensities observed in outdoor pond cultures. Biotechnol. Bioeng. 2013; 110: 1583–1594. © 2012 Wiley Periodicals, Inc.     

红光下培养方式对雨生红球藻细胞增殖及其活力的影响

研究重点针对雨生红球藻绿色游动细胞的增殖培养阶段,分析了在利于细胞增殖的红光条件下,几种培养方式的调整对增殖过程和细胞活力的影响。结果显示:（1）在红光下,增殖平台期维持时间长,细胞活力稳定,细胞中性脂无累积,但进入平台期前,细胞中性脂有规律波动,进入平台期后相对稳定;通过更新率为20%的半连续培养,细胞数产出较批次培养提高57%;半连续培养中细胞呈现胁迫调节的时间较批次培养晚。随着培养时间增加,半连续培养下细胞营养盐吸收能力降低。（2）初始接种密度与细胞增殖速率及细胞光合活力呈负相关:初始密度低的细胞增殖速率较高,细胞光合作用活力高。（3）在培养过程中添加CO2时,最大密度均有提高,达6.0105 cells/mL,较无添加组提高54%;细胞分裂速率均有提高,但红光下较白光下增殖速率高（分别为0.223/d和0.198/d）;添加CO2降低培养液pH,利于维持适宜增殖的pH环境。叶绿素荧光参数以及细胞粒径在红光和白光下有显著差异:红光下,Fv/Fm显著高于白光下;红光下补充CO2显著减小细胞粒径,而白光下粒径无显著变化。研究结果显示,在红光下,采用间断式半连续培养补充CO2培养绿色游动细胞,有利于提升细胞活力与产出。     

Sulfated proteoglycan synthesis by confluent cultures of rabbit costal chondrocytes grown in the presence of fibroblast growth factor

We examined the effect of fibroblast growth factor (FGF) on proteoglycan synthesis by rabbit costal chondrocyte cultures maintained on plastic tissue culture dishes. Low density rabbit costal chondrocyte cultures grown in the absence of FGF gave rise at confluency to a heterogeneous cell population composed of fibroblastic cells and poorly differentiated chondrocytes. When similar cultures were grown in the presence of FGF, the confluent cultures organized into a homogenous cartilage-like tissue composed of rounded cells surrounded by a refractile matrix. The cell ultrastructure and that of the pericellular matrix were similar to those seen in vivo. The expression of the cartilage phenotype in confluent chondrocyte cultures grown from the sparse stage in the presence vs. absence of FGF was reflected by a fivefold increase in the rate of incorporation of [35S]sulfate into proteoglycans. These FGF effects were only observed when FGF was present during the cell logarithmic growth phase, but not when it was added after chondrocyte cultures became confluent. High molecular weight, chondroitin sulfate proteoglycans synthesized by confluent chondrocyte cultures grown in the presence of FGF were slightly larger in size than that produced by confluent cultures grown in the absence of FGF. The major sulfated glycosaminoglycans associated with low molecular weight proteoglycan in FGF-exposed cultures were chondroitin sulfate, while in cultures not exposed to FGF they were chondroitin sulfate and dermatan sulfate. Regardless of whether or not cells were grown in the presence or absence of FGF, the 6S/4S disaccharide ratio of chondroitin sulfate chains associated with high and low molecular weight proteoglycans synthesized by confluent cultures was the same. These results provide evidence that when low density chondrocyte cultures maintained on plastic tissue culture dishes are grown in the presence of FGF, it results in a stimulation of the expression and stabilization of the chondrocyte phenotype once cultures become confluent.     

The microaerophily and photosensitivity of Propionibacterium acnes

E. MARTIN GRIBBON, J.G. SHOESMITH, W.J. CUNLIFFE AND K.T. HOLLAND. 1994. The effect of oxygen on the in vitro propagation of Propionibacterium acnes was investigated under defined culture conditions. This micro-organism is the predominant bacterial resident within the pilosebaceous follicles of sebum-rich areas of human skin. The organism was grown in continuous culture in defined synthetic medium with glucose as the main carbon-energy source at various air saturation concentrations and in the presence and absence of light. Steady state continuous cultures were achieved at very low oxygen tensions in the presence of light, and at higher levels of oxygen when non-illuminated. Culture biomass yields were higher than those of anaerobic cultures. Bacterial cells were inactivated in the presence of light at high oxygen concentrations because of photosensitization reactions involving excess oxygen and microbial porphyrin species.     

细胞均一性对葡萄细胞生长和花青素合成的影响

通过色差筛选法建立了一个相对均一的葡萄细胞悬浮系E,其细胞团较小,在长期继代培养过程中花青素合成能力的变异系数为8.7%,重复摇瓶实验的变异系数为5%。以E为实验材料进行的各组前体饲喂、诱导子添加、光照等联合作用实验,其生物量和花青素合成的变异系数均可控制在12%以内,充分说明了培养体系的均一性对维持稳定生产的重要性;黑暗条件下添加30μmol/L苯丙氨酸(Phe)和218μmol/L茉莉酸甲酯(MeJA)可使单位细胞花青素含量达到对照组的5.89倍,花青素产量为对照组的4.30倍,且连续5次继代培养过程中生物量和花青素合成的变异系数均比对照组降低。     

Light-dependent induction of strongly increased microalgal growth by methanol

Low methanol concentrations (about 0.5% v/v) induce biomass production in cultures of the unicellular green alga Scenedesmus obliquus by more than 300%, compared to controls without this solvent. This effect on the microalgal growth was found to be dependent on the solvent concentration, the packed cell volume (PCV), light intensity and light quality. It could be shown that methanol addition leads to a decrease in size of the light harvesting complex (LHC) on the basis of chlorophylls and proteins, and thus to changes in structure and functioning of the photosynthetic apparatus. These alterations lead to enhanced photosynthesis and respiration rates. The action of methanol on the photosynthetic apparatus is thus comparable to the effect of enhanced CO(2) concentrations. These findings support the previously proposed pathway for methanol metabolization with CO(2) as the final product. We conclude that the subsequent assimilation of the increased CO(2) amounts by the Calvin-Benson cycle is a possible explanation for the methanol-mediated increase in biomass production in terms of PCV. The methanol effect is observed only in the light and in the presence of a functioning photosynthetic apparatus. Preliminary action spectra suggest that the primary photoreceptor is a chlorophyll-protein complex with two absorption maxima at 680 and 430 nm, which may possibly be attributed to the reaction center of photosystem II (PSII).     

A model for light distribution and average solar irradiance inside outdoor tubular photobioreactors for the microalgal mass culture

A mathematical model to estimate the solar irradiance profile and average light intensity inside a tubular photobioreactor under outdoor conditions is proposed, requiring only geographic, geometric, and solar position parameters. First, the length of the path into the culture traveled by any direct or disperse ray of light was calculated as the function of three variables: day of year, solar hour, and geographic latitude. Then, the phenomenon of light attenuation by biomass was studied considering Lambert-Beer's law (only considering absorption) and the monodimensional model of Cornet et al. (1900) (considering absorption and scattering phenomena). Due to the existence of differential wavelength absorption, none of the literature models are useful for explaining light attenuation by the biomass. Therefore, an empirical hyperbolic expression is proposed. The equations to calculate light path length were substituted in the proposed hyperbolic expression, reproducing light intensity data obtained in the center of the loop tubes. The proposed model was also likely to estimate the irradiance accurately at any point inside the culture. Calculation of the local intensity was thus extended to the full culture volume in order to obtain the average irradiance, showing how the higher biomass productivities in a Phaeodactylum tricornutum UTEX 640 outdoor chemostat culture could be maintained by delaying light limitation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 701-714, 1997.     

Elemental balancing of biomass and medium composition enhances growth capacity in high-density Chlorella vulgaris cultures

The basic requirements for high-density photoautotrophic microalgal cultures in enclosed photobioreactors are a powerful light source and proper distribution of light, efficient gas exchange, and suitable medium composition. This article introduces the concept of balancing the elemental composition of growth medium with biomass composition to obtain high-density cultures. N-8 medium, commonly used for culturing Chlorella vulgaris was evaluated for its capacity to support high-density cultures on the basis of elemental stoichiometric composition of C. vulgaris. This analysis showed that the N-8 medium is deficient in iron, magnesium, sulfur, and nitrogen at high cell densities. N-8 medium was redesigned to contain stoichiometrically balanced quantities of the four deficient elements to support a biomass concentration of 2% (v/v). The redesigned medium, called M-8 medium, resulted in up to three- to fivefold increase in total chlorophyll content per volume of culture as compared to N-8 medium. Further experiments showed that addition of each of the four elements separately to N-8 medium did not improve culture performance and that balanced supplementation of all four deficient elements was required to yield the improved performance. Long-term (24 d) C. vulgaris culture in M-8 medium showed continuous increase in chlorophyll content and biomass throughout the period of cultivation. In contrast, the increase in chlorophyll content and biomass ceased after 7 and 12 d, respectively in N-8 medium, demonstrating the higher capacity of M-8 medium to produce biomass. Thus, the performance of high cell density photobioreactors can be significantly enhanced by proper medium design. The elemental composition of the biomass generated is an appropriate basis for medium design.     

Nutrient Acquisition and Population Growth of a Mixotrophic Alga in Axenic and Bacterized Cultures

Axenic growth of a mixotrophic alga, Ochromonas sp., was compared in several inorganic and organic media, and in the presence of live bacteria under nutrient-replete and low-nutrient conditions. Axenic growth in the light was negligible in inorganic media with or without the addition of glucose. Addition of vitamins increased growth rate, but average cell size declined, resulting in no net increase in biomass. Supplementing axenic cultures with a more complex organic substrate resulted in moderate growth and higher maximal abundance (and biomass) than in the inorganic media with added vitamins. The absence of light did not greatly affect population growth rate in the presence of complex dissolved organic compounds, although cell size was significantly greater in the light than in the dark. The highest growth rates for the alga (up to 2.6 d-1) were measured in treatments containing live bacteria. Increases in cell number of Ochromonas sp. in the presence of bacterial prey were similar in the light and dark, although chloroplast and cell sizes differed. Bacterial abundance was reduced and dissolved phosphorus and ammonia were rapidly released in bacterized cultures in the light and dark, indicating high rates of bacterial ingestion and suggesting an inability of the alga to store or utilize N and P in excess of the quantities required for heterotrophic growth. Low-nutrient conditions in the presence of bacteria were promoted by adding glucose to stimulate bacterial growth and the uptake of N and P released by algal phagotrophy. Subsequent decreases in dissolved N and P following the addition of glucose corresponded to a second period of rapid growth of the alga in both light and dark. This result, combined with evidence for slow axenic growth of this strain, indicated that nutrient acquisition for this species in the presence of bacteria was accomplished primarily via ingestion of bacteria.     

Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L⁻¹ cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.     

New observations on first-generation merogony of Eimeria tuskegeensis in Sigmodon hispidus

First-generation development of Eimeria tuskegeensis was evaluated using light microscopy. Sporozoite-shaped meronts containing a prominent refractile body were observed in small intestinal cells of an experimentally infected cotton rat at 24 h post inoculation (PI). Mature spherical or subspherical meronts containing crescent-shaped merozoites were observed at 36 h PI. Refractile bodies were observed in some of these merozoites. Sporozoite-shaped meronts that were isolated from host intestinal cells and inoculated onto human fetal lung cell cultures penetrated the cultured cells by 2 h PI. A mature, subspherical, first-generation meront containing seven merozoites was observed at 9 h PI in cell culture, indicating that sporozoite-shaped meronts isolated from the host retained their infectivity.     

pH and Glucose Profiles in Aggregates of Bacillus laevolacticus

Size distributions and glucose and pH profiles of aggregates of the d-(-)-lactic acid-producing organism Bacillus laevolacticus were measured. The organisms were grown in continuous culture with a medium glucose concentration of either 280 or 110 mM. A maximal aggregate diameter of 2.2 mm, with a Sauter mean of 1.46 mm, was determined for the former culture condition, whereas aggregates from a culture with 110 mM glucose input had a maximal diameter of 1.9 mm (Sauter mean of 1.07 mm). A pH gradient of approximately 2 U was observed for large aggregates (above 1.5 mm). In smaller aggregates (0.75 mm), the pH value in the interior part was approximately 0.4 U lower than that in the culture fluid. It could be concluded that, in cultures with the high glucose input, lactic acid accumulated within the aggregates to such an extent that metabolism in the central region of the larger aggregates could not proceed further. In these cultures, approximately 90% of the total biomass was active. In aggregates from cultures with a low glucose input, glucose only partly penetrated the larger-sized aggregates, and the activity of this culture was reduced to approximately 70% of the biomass. These aggregates were found to decrease in size after prolonged periods of cultivation. It is suggested that this is caused by glucose depletion in the interior of the aggregates. It is concluded that the availability of glucose is an important factor in determining the size of aggregates of B. laevolacticus.     

Role of anaerobic spore-forming bacteria in the acidogenesis of glucose: Changes induced by discontinuous or low-rate feed supply

A mineral salts medium containing 1% (w/v) glucose providing carbon-limited growth conditions was subjected to anaerobic acidogenesis by mixed populations of bacteria in chemostat cultures. The formation of butyrate was shown to be dependent on the presence of saccharolytic anaerobic sporeformers in the acid-forming population. By the use of pasteurized activated sludge as an inoculum a culture was obtained consisting solely of anaerobic sporeformers that gave rise to the formation of butyrate, acetate, hydrogen and carbon dioxide as the main fermentation products. No formation of propionate could be detected. In this culture, the role of sporulation was investigated by applying periods of starvation and a single-step lowering of dilution rate (shift-down). In an experiment using a mineral salts medium supplemented with 1% (w/v) glucose and 0.5% (w/v) casein hydrolysate formation of refractile forespores as well as cell lysis could be demonstrated after 6 h starvation.In mixed cultures, initially inoculated with non-pasteurized activated sludge, a regular interruption of feed supply for 1 h per day resulted in selection of non-sporulatiog anaerobes. The fermentation pattern changed to a production of propionate and acetate, with a concomitant reduction of gas production. Similar results were obtained with shift-down in dilution rate.     

Real-time monitoring and automatic density control of large-scale microalgal cultures using near infrared (NIR) optical density sensors

Signals from near infrared (NIR) light transmittance sensors were used for both real-time monitoring of algal biomass density in growing mass cultures (200l tubular biofences), and also as feedback in a system that controlled the density of the culture by automatic injection of fresh growth medium. When operated in a semi-continuous production mode between predefined density values, diurnal growth patterns were recorded on-line that provided information on the dynamics of the microalgal cultures with respect to environmental conditions. The bioreactor system was also programmed to operate in constant biomass density mode, thereby maintaining the culture at the optimal population density (OPD), and sustaining high biomass production levels. The system has potential for operating a dynamic density set point for microalgal cultures where the optimal population density varies as a function of ambient growing conditions.     

Growth and respiration of Petunia hybrida cells in chemostat cultures: A comparison of glucose-limited and nitrate-limited cultures

Nitrate-limited and glucose-limited chemostat cultures of Petunia hybrida cells were compared at a specific biomass (+extracellular product) formation rate of 0.0042 C.mol/C.mol h. The composition of the biomass differed considerably in both culture types. The N/C (mol/mol) ratio in the biomass was almost four times lower in the nitrate-limited than in the glucose-limited cultures. On a dry weight basis (g/g DW) the biomass in the nitrate-limited cultures contained about 2.5 times less ions and protein N and about 2.5 times more carbohydrates than the biomass in the glucose-limited cultures. On a fresh weight basis (mmol/g FW) the biomass in nitrate-limited and glucose-limited cultures differed mainly in carbohydrate content. The yields of biomass on glucose and oxygen were generally higher in the nitrate-limited than in the glucose-limited cultures. Average values for these parameters were 0.27 C . mol biomass/C . mol glucose and 0.42 C . mol biomass/mol O(2) in the glucose-limited cultures and 0.34 C . mol biomass/C . mol glucose and 0.55 C . mol biomass/mol O(2) in the nitrate-limited cultures. On a C . mol basis the total respiration was about 25% and the maximally attainable cytochrome pathway activity (measured in the presence of hydroxamate) about 30% higher in the glucose-limited than in the nitrate-limited cultures. The maximally attainable activity of the alternative pathway (measured in the presence of KCN) was significantly lower in the glucose-limited cultures. On an organic N ( approximately protein) basis all respiratory parameters were significantly higher in the nitrate-limited cultures. In the presence of the respiratory uncoupler carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) and excess glucose, cellular respiratory activity shows its maximal activity; under these conditions the total respiration increased more than 150% in the glucose-limited and only 30% in the nitrate-limited cultures. It is suggested that glucose-limited cultures are able to react more flexibly to changes in the environmental conditions than nitrate-limited cultures. (c) 1996 John Wiley & Sons, Inc.     

Sand administration as an instrument for biofilm control of Pseudomonas putida ATCC 11172 in chemostat cultures

Pseudomonas putida ATCC 11172 was grown in chemostat on L-asparagine or phenol as the sole, limiting carbon and energy source. The growth characteristics of a culture where a biofilm was present, were compared with one where the biofilm was strongly reduced by the grinding and shearing effect of sand suspended in the culture. In the presence of the intact biofilm, the curve of steady-state biomass versus dilution rate diverged greatly from the theoretical pattern predicted by conventional chemostat models. The sand strongly retarded the biofilm formation and to a high degree restored the shape of the biomass versus dilution rate curve to a more conventional pattern. The maximum specific growth rate (mu(max)) could not be calculated from the biofilm cultures. However using the sand cultures, mu(max) was determined to 0.64 h(-1) with L-asparagine as the carbon source and 0.49 h(-1) with phenol which compare favorably with the respective mu(max) values calculated from batch cultures.Incorporation of sand into strongly agitated cultures is recommended as an efficient and simple means of controlling biofilm formation in continuous cultures. The method may enable the gathering of basic kinetic data difficult to obtain in the presence of biofilm.     

New Observations on First-Generation Merogony of Eimeria tuskegeensis in Sigmodon hispidus1

First-generation development of Eimeria tuskegeensis was evaluated using light microscopy. Sporozoite-shaped meronts containing a prominent refractile body were observed in small intestinal cells of an experimentally infected cotton rat at 24 h post inoculation (PI). Mature spherical or subspherical meronts containing crescent-shaped merozoites were observed at 36 h PI. Refractile bodies were observed in some of these merozoites. Sporozoite-shaped meronts that were isolated from host intestinal cells and inoculated onto human fetal lung cell cultures penetrated the cultured cells by 2 h PI. A mature, subspherical, first-generation meront containing seven merozoites was observed at 9 h PI in cell culture, indicating that sporozoite-shaped meronts isolated from the host retained their infectivity.     

Monitoring growth and lipid production of new isolated oleaginous yeast Cryptococcus aerius UIMC65 on glucose and xylose cultures

Process monitoring is one of the most important factors affecting production efficiency at industrial scale bioprocesses. In the present work, Flow-cytometric analysis has been employed to monitor and determine neutral lipid cell droplets, granularity and size of the cells of the new oleaginous yeast, Cryptococcus aerius UIMC65. It has been shown that, differences of fluorescent intensity as well as side and forward scatter light properties have close correlations with the differences in lipid production by these yeast cells. The lipid content-related fluorescent intensity versus forward scatter parameter has been used to monitor and compare different subpopulations during growth phases on both glucose and xylose in batch cultures. Flow cytometric results have revealed that the observed differences in the proportion of each subpopulation were related to the specific growth phase and lipid content of the cells. The highest lipid content and lipid productivity were attained at 82.62%, 4.47 g/L (at 72 h) and 78.41%, 6.21 g/L (at 60 h) on glucose and xylose growth cultures, respectively. The highest biomass, lipid yield and biomass yield were found to be 7.92 g/L (on glucose culture, at 60 h), 20.92% (on glucose culture, at 48 h) and 50.71% (on glucose culture, at 24 h), respectively.     

REPOPULATION OF THE POSTMITOTIC NUCLEOLUS BY PREFORMED RNA

This study is concerned with the fate of the nucleolar contents, particularly nucleolar RNA, during mitosis Mitotic cells harvested from monolayer cultures of Chinese hamster embryonal cells, KB6 (human) cells, or L929 (mouse) cells were allowed to proceed into interphase in the presence or absence (control) of 0.04–0 08 µg/ml of actinomycin D, a concentration which preferentially inhibits nucleolar (ribosomal) RNA synthesis 3 hr after mitosis, control cells had large, irregularly shaped nucleoli which stained intensely for RNA with azure B and for protein with fast green. In cells which had returned to interphase in the presence of actinomycin D, nucleoli were segregated into two components easily resolvable in the light microscope, and one of these components stained intensely for RNA with azure B. Both nucleolar components stained for protein with fast green In parallel experiments, cultures were incubated with 0.04–0 08 µg/ml actinomycin D for 3 hr before harvesting of mitotic cells, then mitotic cells were washed and allowed to return to interphase in the absence of actinomycin D. 3 hr after mitosis, nuclei of such cells were devoid of large RNA-containing structures, though small, refractile nucleolus-like bodies were observed by phase-contrast microscopy or in material stained for total protein. These experiments indicate that nucleolar RNA made several hours before mitosis persists in the mitotic cell and repopulates nucleoli when they reform after mitosis