Revisiting with a Relative-Density Calibration Approach the Determination of Growth Rates of Microorganisms by Use of Optical Density Data from Liquid Cultures

To solve the problems of measuring the growth rates of microorganisms from optical density (OD)-growth time plots, we used relative-density (RD) plots. The relationship of OD and RD was built from the diluted grown cultures. This method was satisfactorily applied to study the growth of Escherichia coli and the cyanobacterium Anabaena spiroides.In spite of the popularity of the optical density (OD) method, the direct use of OD records of liquid cultures of microorganisms to study their growth kinetics may yield problematic results. For instance, for an Escherichia coli culture, the cell doubling time as derived from incremental OD rates varies from 46 to 38 min, depending on the wavelength of light used for measurement (Fig. ​(Fig.1A).1A). Here, we report an approach to obtain more reliable results (Fig. ​(Fig.1B).1B). Briefly, the OD of the liquid cell culture is recorded frequently throughout the growth period. At or near the end of cultivation, the cell density of the culture is arbitrarily defined as a relative density (RD) of 1.0, and aliquots of the culture are diluted to prepare reference samples of various RD values. For example, a reference sample of 0.3 RD is prepared by diluting 0.3 ml of grown cell culture with 0.7 ml of fresh growth medium. The ODs of the reference samples are also determined and plotted against the RD values to construct an OD-RD calibration curve using the equation OD = m × RD/(n + RD), where m and n are empirical constants (Fig. ​(Fig.1B).1B). The recorded ODs of the cell culture are converted into RD values, and the cell doubling time is determined from the RD-growth time plot (Fig. ​(Fig.1C).1C). We determined the growth rates of E. coli bacteria and Anabaena spiroides cyanobacteria (Fig. ​(Fig.2)2) using this method. We found that a common laboratory E. coli strain, BL21(DE3), doubled every 31 ± 3 min (mean ± 1 standard deviation [SD]) (Fig. ​(Fig.1C),1C), irrespective of the light wavelength used for measurement, consistent with the incremental rate of the absolute density (AD; CFU/ml) of the E. coli cells (32 ± 4 min) (Fig. ​(Fig.1C).1C). Furthermore, the growth rates obtained from RD-time plots and OD-time plots were statistically different (t test P values were ≪0.05 for RDs versus ODs with wavelengths used). We also found that E. coli DH5α, another common E. coli laboratory strain, doubled every 46 ± 2 min (data not shown). For Anabaena spiroides, the cells doubled every 18.5 ± 1.5 h, agreeing with the result of 17.6 ± 0.8 h obtained from the incremental AD rate (Fig. ​(Fig.2B).2B). Again, the results were not affected by the wavelength of light used for OD measurements.Open in a separate windowFIG. 1.The use of OD readings to determine growth rates of E. coli bacteria. (A) OD-time response of an E. coli culture monitored with light of different wavelengths. The OD of an LB broth-grown E. coli BL21(DE3) culture at 37°C was monitored with light of the indicated wavelengths (e.g., OD600 for measurements with 600-nm light). The doubling times of E. coli cells in the logarithmic growth phase were estimated to be 46 ± 3 min, 43 ± 2 min, 40 ± 2 min, and 38 ± 2 min (mean ± 1 SD) for measurements with 450-nm, 500-nm, 600-nm, and 700-nm light, respectively. The differences among these values are significant (analysis of variance P value, 0.006). (B) RD-OD calibration curves of the bacterial culture whose OD results are shown in panel A. Aliquots of the grown culture were diluted with various amounts of fresh LB broth to prepare reference samples, and their ODs were recorded with light of different wavelengths. (C) RD and AD as a function of growth time of the E. coli culture whose OD results are shown in panel A. The RD data were derived from OD-RD calibration curves and plotted against the growth time. The AD of the cell culture at each indicated time was determined by plating the serially diluted bacterial cultures on LB plates and counting the colonies. An OD of 1.09 at 600 nm corresponds to (3.0 ± 0.5) × 10⁸ CFU/ml. The growth rates are expressed as cell doubling times, which were inferred from the slopes of the regression lines. The error bars represent three standard deviations of the OD600, RD600, and AD measurements (three repeats). For clarity, only the SDs of measurements with 600-nm light are shown. The SDs of measurements with light of other wavelengths were very close to those with the 600-nm light. The reported growth rates are the results of four growth experiments. KaleidaGraph (version 4.0; Synergy Software) was used for curve fitting. Note that in the experiment, we added pregrown cells into prewarmed (37°C) LB medium to avoid the initial slow growth of the cells. The details of E. coli strains and culture conditions are given in the supplemental material.Open in a separate windowFIG. 2.Determination of growth rates of the algal cyanobacterium Anabaena spiroides. (A) OD-RD calibration of an A. spiroides algal culture. The grown algal culture was diluted with fresh medium as described in the text, and light of the indicated wavelengths was used to measure the ODs of the reference samples. (B) RD and AD as functions of growth time of the algal culture shown in panel A. The algal cells were grown in an aerated plain medium for ∼100 h (see the supplemental material for details). The growth of the algal cells was monitored by measuring the OD of the culture with light of the indicated wavelengths. Absolute cell density (cells/ml) was determined by counting the observed cell number under a bright-field microscope. The error bars represent three standard deviations. For clarity, only SDs of measurements (three repeats) with the 720-nm light are shown. The SDs of the measurements with light of other wavelengths were similar to those with the 720-nm light. The growth rates are the results of three growth experiments.The problems from using OD-time plots to determine the growth rates of microorganisms are in the misuse of the Beer-Lambert law, which is only applicable to light-absorbing molecules (3, 6). However, when light hits microorganisms, the light may be scattered and/or absorbed, and the OD of a liquid culture of microorganisms is the combination of light scattering and light absorption (3, 6). Generally, light scattering will be prominent when the particle sizes are close to the wavelength of the light (e.g., the size of E. coli cells and the visible light wavelengths). Also, the intensity of light scattering is not linear with particle concentrations (3). Thus, it is not surprising that measurements with lights of different wavelengths in OD-time plots yield wavelength-dependent growth rates.We propose the use of RD-time plots to obtain the growth rates of microorganisms. In fact, the relationship among OD, RD, and AD can be expressed mathematically, and the use of RD-time plots to derive the growth rates of microorganisms can be justified (see the supplemental material). Our idea was inspired by the use of standard curves in biochemical studies. This method has two features. First, it employs serial dilutions of grown cultures. Second, it uses OD-RD calibration curves to infer RD values from OD records. There are precedents for using dilution methods to study the growth of microorganisms. For example, in the method of Baranyi and Pin (1, 2, 5), the microorganisms are serially diluted into several flasks before cell culture, and the growth rates can be inferred from the delayed time intervals for the diluted cultures to reach a given OD. By using the method of Baranyi and Pin (1, 2, 5), we obtained a similar averaged growth rate for our E. coli strain [BL21(DE3)], with a larger standard deviation (34 ± 5 min) (unpublished data of T.-C. Kuo). On the other hand, Lawrence and Maier also noticed the problems of using OD to determine the actual cell density of bacteria (4). They proposed the use of the OD of the diluted grown cultures to establish OD-dry weight calibration curves for bacteria. However, they did not extend their idea to determine the growth rates of bacteria.In this study, we used bacteria with different physical characteristics to test the applicability of our method, because the OD of the microorganism culture is the result of light absorption and light scattering by the cells. For E. coli cells, which are colorless, relatively small (∼0.5 μm by 1 μm), and single celled in liquid culture, the OD is mainly the result of light scattering (3), as suggested by the observation that at a given RD, the OD decreased as the light wavelength increased (Fig. ​(Fig.1B).1B). On the other hand, the cells of the photosynthetic algal cyanobacterium Anabaena spiroides are pigment rich, large (∼3 μm by 5 μm), and filament forming. The visible light spectrum of the algal culture is similar to that of the purified phytochromes of the algae (data not shown), suggesting that light absorption is the major factor in the OD measurements of the algal culture. Moreover, in the OD-RD calibration curves of the A. spiroides algae (Fig. ​(Fig.2A),2A), at a given RD, the OD reading did not decrease as the light wavelength increased, again indicating that light scattering was not the main factor in OD measurements. For both organisms, the growth rates obtained from the RD-time plots and the AD-time plots were very close, and the results were independent of light wavelength, as predicted by the theory (see the supplemental material).Despite the promising results, we caution that our method is applicable only if the morphology (e.g., color, size, shape, etc.) of the microorganism of interest and the optical properties (e.g., color) of the culture medium both do not vary with culture time.Finally, for routine cultures of the same microorganism (i.e., E. coli DH5α) under conditions that are identical except for culture dates and growth periods, it is not necessary to prepare the OD-RD calibration curves each time. The OD records of new cultures can be converted to RD by using the RD-OD calibration curves of previous cultures, as long as the identical instrument is used for recording ODs (see the supplemental material). This eliminates the need for dilution work. The reason for this shortcut is discussed in the supplemental material.      

The combination of ADI-PEG20 and TRAIL effectively increases cell death in melanoma cell lines

Current treatment for advanced, metastatic melanoma is not very effective, and new modalities are needed. ADI-PEG20 is a drug that specifically targets ASS-negative malignant melanomas while sparing the ASS-expressing normal cells. Although laboratory research and clinical trials showed promising results, there are some ASS-negative cell lines and patients that can develop resistance to this drug. In this report, we combined ADI-PEG20 with another antitumor drug TRAIL to increase the killing of malignant melanoma cells. This combination can greatly inhibit cell growth (to over 80%) and also enhanced cell death (to over 60%) in four melanoma cell lines tested compared with control. We found that ADI-PEG20 could increase the cell surface receptors DR4/5 for TRAIL and that caspase activity correlated with the increased cell death. These two drugs could also increase the level of Noxa while decrease that of survivin. We propose that these two drugs can complement each other by activating the intrinsic and extrinsic apoptosis pathways, thus enhance the killing of melanoma cells.     

Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation

Osteogenesis is a complex series of events involving the differentiation of mesenchymal stem cells to generate new bone. In this study, we examined the effect of pulsed electromagnetic fields (PEMFs) on cell proliferation, alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and gene expression in bone marrow mesenchymal stem cells (BMMSCs) during osteogenic differentiation. Exposure of BMMSCs to PEMFs increased cell proliferation by 29.6% compared to untreated cells at day 1 of differentiation. Semi‐quantitative RT‐PCR indicated that PEMFs significantly altered temporal expression of osteogenesis‐related genes, including a 2.7‐fold increase in expression of the key osteogenesis regulatory gene cbfa1, compared to untreated controls. In addition, exposure to PEMFs significantly increased ALP expression during the early stages of osteogenesis and substantially enhanced mineralization near the midpoint of osteogenesis. These results suggest that PEMFs enhance early cell proliferation in BMMSC‐mediated osteogenesis, and accelerate the osteogenesis. Bioelectromagnetics 31:209–219, 2010. © 2009 Wiley‐Liss, Inc.     

The Chlorella variabilis NC64A Genome Reveals Adaptation to Photosymbiosis,Coevolution with Viruses,and Cryptic Sex

Chlorella variabilis NC64A, a unicellular photosynthetic green alga (Trebouxiophyceae), is an intracellular photobiont of Paramecium bursaria and a model system for studying virus/algal interactions. We sequenced its 46-Mb nuclear genome, revealing an expansion of protein families that could have participated in adaptation to symbiosis. NC64A exhibits variations in GC content across its genome that correlate with global expression level, average intron size, and codon usage bias. Although Chlorella species have been assumed to be asexual and nonmotile, the NC64A genome encodes all the known meiosis-specific proteins and a subset of proteins found in flagella. We hypothesize that Chlorella might have retained a flagella-derived structure that could be involved in sexual reproduction. Furthermore, a survey of phytohormone pathways in chlorophyte algae identified algal orthologs of Arabidopsis thaliana genes involved in hormone biosynthesis and signaling, suggesting that these functions were established prior to the evolution of land plants. We show that the ability of Chlorella to produce chitinous cell walls likely resulted from the capture of metabolic genes by horizontal gene transfer from algal viruses, prokaryotes, or fungi. Analysis of the NC64A genome substantially advances our understanding of the green lineage evolution, including the genomic interplay with viruses and symbiosis between eukaryotes.     

Cloning of an orange-spotted grouper Epinephelus coioides heat shock protein 90AB (HSP90AB) and characterization of its expression in response to nodavirus

The heat shock proteins (HSPs) family which consists of HSP90, HSP70, and low molecular mass HSPs are involved in chaperone activity. Here, we report the cloning and characterization of HSP90AB gene from orange-spotted grouper, Epinephelus coioides. The full-length of grouper HSP90AB was 727 amino acids and possessed an ATPase domain as well as an evolutionarily conserved molecular chaperone. The HSP90AB-green fluorescent protein fusion protein was evenly distributed in the cytoplasm. Immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR) analyses indicated that the expression of grouper HSP90AB was marginally increased following nodavirus infection. Grouper E. coioides that received HSP90 inhibitor geldanamycin (GA) showed an increase in HSP90AB expression and growth of nodavirus supporting nodavirus replication.     

Apoptosis in chondrogenesis of human mesenchymal stem cells: effect of serum and medium supplements

Apoptosis is an inevitable process during development and is evident in the formation of articular cartilage and endochondral ossification of growth plate. Mesenchymal stem cells (MSCs) can serve as alternative sources for cell therapy in focal chondral lesions or diffuse osteoarthritis. But there are few, if any, studies investigating apoptosis during chondrogenesis by MSCs. The aim of this study was to find the better condition to prevent apoptosis during chondrogenesis by MSCs. Apoptosis were evaluated in MSCs induced in different chondrogenic media by the use of Annexin V, TUNEL staining, lysosomal labeling with lysotracker and immunostaining of apoptotic markers. We found apparent apoptosis was demonstrated by Annexin V, TUNEL staining and lysosomal labeling during chondrogenesis. Meanwhile, the degree of apoptosis was related to the reagents of the defined chondrogenic medium. Adding serum in medium increased apoptosis, however, TGF-β1 inhibited apoptosis. The apoptosis was associated with the activation of caspase-3, the increase in the Bax/Bcl-2 ratio, the loss of lysosomal integrity, and the increase of PARP-cleavage. Pro-inflammatory cytokines, IL-1α, IL-1β and TNFα did not induce any increase in apoptosis. Interestingly, the inhibition of apoptosis by serum free medium supplemented with ITS was also associated with an increase in the expression of type II collagen, and a decrease in the expression of type X collagen, Runx2, and other osteogenic genes, while TGF-β1 increased the expression of Sox9, type II and type X collagen and decreased the expression of osteogenic genes. These data suggest apoptosis occurs during chondrogenesis by MSCs by cell death intrinsic pathway activation and this process may be modulated by culture conditions.     

Evolutionary divergence in the fungal response to fluconazole revealed by soft clustering

Background  

Fungal infections are an emerging health risk, especially those involving yeast that are resistant to antifungal agents. To understand the range of mechanisms by which yeasts can respond to anti-fungals, we compared gene expression patterns across three evolutionarily distant species - Saccharomyces cerevisiae, Candida glabrata and Kluyveromyces lactis - over time following fluconazole exposure.