A culture-dependent bacterial community structure analysis based on liquid cultivation and its application to a marine environment

A method for analyzing culture-dependent bacterial community structure by liquid cultivation was established using 96-well microplates. Using 96-well microplates, this method can easily provide accurate enumeration of viable microorganisms and simultaneous separation of bacteria, which allowed us to analyze the bacterial community. Bacteria in diluted surface seawater were separated using 96-well microplates and cultivated with 1/5 ZoBell 2216E liquid medium. The 98 cultures obtained were subsequently applied to phylogenetic analysis based on 16S rRNA gene sequences. The bacterial diversity, evaluated by the Shannon–Weaver index, was relatively small but comparable to previously reported bacterial communities of several environments. The most abundant group was the family Rhodobacteraceae , which has been frequently detected in marine environments. Most bacteria were phylogenetically related to bacteria or uncultured clones detected in marine environments, but distant from published species. The analysis of bacterial community structure by liquid cultivation would be useful as an alternative culture-dependent approach.     

培养法检测海滨浴场细菌多样性及耐药性研究

【目的】耐药基因细菌水平基因转移导致的耐药菌数量增加引发的公共安全问题日益引起人们关注,监测环境中耐药菌变得极为重要。【方法】采集湛江3处滨海浴场的水体、沙滩土样,通过平板稀释涂布和琼脂扩散法进行浴场微生物数量、多样性和抗生素耐药性分析。【结果】3处浴场水体无机氮含量偏高,浴场微生物数量随着客流量逐渐增加,沙滩中微生物数量显著高于水体。浴场细菌分布于3门12科18属,水体中变形菌门（Proteobacteria,49.64%）占优势,沙滩则是厚壁菌门（Firmicutes,54.74%）占优势。浴场细菌对β-内酰胺类耐药率较高,青霉素、万古霉素和头孢曲松耐药率分别达到23.25%、20.53%和17.42%,耐药菌株主要分布于芽孢杆菌属（Bacillus）、弧菌属（Vibrio）、假单胞菌属（Pseudomonas）、链霉菌属（Streptomyces）和肠杆菌属（Enterobacter）,水体中多重耐药细菌数量显著高于沙滩,集中于人流量多的浴场。【结论】滨海浴场环境中细菌耐药菌种类多,需持续监测以评估对当前地区公共卫生的潜在影响。     

Influence of moisture content and cultivation duration on Clostridium thermocellum 27405 end-product formation in solid substrate cultivation on Avicel

Avicel serves as a model microcrystalline cellulose substrate for investigations of cellulolytic microbial performance and cellulase enzyme systems in submerged liquid cultures. Clostridium thermocellum is a thermophilic, anaerobic bacterium capable of degrading lignocellulose and fermenting it to ethanol and other products, suggesting the native growth environment is similar to that supported by solid substrate cultivation. Few studies have examined the effects of process parameters on the metabolism of thermophilic anaerobes in solid substrate cultivation, however. The effects of solid substrate cultivation (SSC) substrate moisture content (30%, 50% and 70% wet-basis) and cultivation duration (2, 4 and 8 days) on the metabolic activity of C. thermocellum 27405 on Avicel was studied. The 70% substrate moisture content SSC culture yielded total end-product concentrations that were comparable to submerged liquid cultures. The SSC cultivation conditions with the highest end-product formation on Avicel were the combination of 70% substrate moisture content and cultivation duration period of 4 days, producing approximately 100mM of total end-products. The ethanol and lactate concentrations were fairly constant and did not change significantly over time in SSC. Acetate production was more dependent on the cultivation conditions in SSC and was significant for both the 70% substrate moisture content SSC and liquid cultivation experiments, making up on average 56% and 86% of total end-products, respectively. Performance of C. thermocellum 27405 in SSC was more dependent on the kinetic properties rather than the thermodynamic properties of substrate moisture content. High substrate loadings in C. thermocellum cultivation affected product ratios, resulting in the higher observed acetate production. In addition, cessation of metabolism was observed prior to complete Avicel conversion; the mechanisms involved need further investigation.     

Sunlight and the survival of enteric bacteria in natural waters

Escherichia coli and some salmonellas were exposed in seawater and freshwater to natural sunlight, visible light of comparable intensity, and light containing a similar proportion of u.v. as natural sunlight but of a much lower intensity. Direct viable bacterial counts and culturable counts on selective and non-selective media were made at intervals. The rate of decrease in numbers of culturable bacteria was significantly faster in seawater than in freshwater when exposed to natural sunlight. No significant difference was found between the rates of decrease in numbers of culturable bacteria in seawater and those in freshwater when bacteria were exposed to light with a small u.v. component of similar intensity. The effect of salinity on loss of culturability is, therefore, more significant in the presence of u.v. radiation. Direct counts by the acridine orange direct viable count method decreased much more slowly than the culturable counts in seawater but comparably with culturable counts in freshwater in natural sunlight. Direct viable counts and culturable counts decreased at a similar rate in seawater and in freshwater in visible light. This may signify the evolution of enteric bacteria towards a viable but non-culturable form in seawater when exposed to natural sunlight. The presence of humic acids significantly reduced loss of culturability but only in low salinity conditions. Salinity appears to be an important factor influencing culturability in bacteria exposed to sunlight.     

Sunlight and the survival of enteric bacteria in natural waters

Escherichia coli and some salmonellas were exposed in seawater and freshwater to natural sunlight, visible light of comparable intensity, and light containing a similar proportion of u.v. as natural sunlight but of a much lower intensity. Direct viable bacterial counts and culturable counts on selective and non-selective media were made at intervals. The rate of decrease in numbers of culturable bacteria was significantly faster in seawater than in freshwater when exposed to natural sunlight. No significant difference was found between the rates of decrease in numbers of culturable bacteria in seawater and those in freshwater when bacteria were exposed to light with a small u.v. component of similar intensity. The effect of salinity no loss of culturability is, therefore, more significant in the presence of u.v. radiation. Direct counts by the acridine orange direct viable count method decreased much more slowly than the culturable counts in seawater but comparably with culturable counts in freshwater in natural sunlight. Direct viable counts and culturable counts decreased at a similar rate in seawater and in freshwater in visible light. This may signify the evolution of enteric bacteria towards a viable but non-culturable form in seawater when exposed to natural sunlight. The presence of humic acids significantly reduced loss of culturability but only in low salinity conditions. Salinity appears to be an important factor influencing culturability in bacteria exposed to sunlight.     

A method of the selective isolation and enumeration of marine Vibrionaceae

A noninhibitory medium and GasPack anaerobic culture system were employed for the selective enumeration and isolation of Vibrionaceae in seawater and marine sediments.Vibrio counts obtained by the new method for seawater and sediment samples were compared with vibrio numbers in the heterotrophic bacterial population appearing on a medium routinely employed in the laboratory for such counts. The ratio of the former to the latter counts ranged from 0.5 to 1.3, the average being 0.96. The seawater and sediment bacteria that grew and produced visible colonies on the medium under anaerobic conditions for 3 days at 20°C were almost exclusively vibrios.From the results reported here it is concluded that most of the vibrios present in seawater and sediment samples can be recovered by the new method developed in this study.     

Isolation and distribution of oligotrophic marine bacteria.

A useful plate culture method for isolating oligotrophic bacteria found in the low-nutrient environment of the open sea has been developed. The method uses a glass-fiber filter substitute for agar. Nutritional requirements of oligotrophic bacteria consisted of a dilute mutrient solution containing 16.8 mg C/l total organic carbon aseptically added to the sterilized filter. Distribution of bacteria in oceanic and neritic seawater was determined using the membrane filter method. In the case of seawater containing less than 0.5 mg/l dissolved carbohydrates, plate counts of oligotrophic bacteria were found to be several- to 100-fold greater than the heterotrophic bacterial counts enumerated by standard methods routinely used for enumeration. However, in seawater containing approximately over 0.5 mg/l dissolved carbohydrates, heterotrophic bacterial counts were 10-fold greater than oligotrophic bacterial counts.     

Dynamics of oxygen evolution and biomass production during cultivation of Agardhiella subulata microplantlets in a bubble-column photobioreactor under medium perfusion

Cell and tissue cultures derived from macrophytic marine red algae are potential platforms for unique secondary metabolites. This work presents the first successful bioreactor cultivation study of an in vitro tissue culture derived from a macrophytic marine red alga. Specifically, the photosynthetic growth characteristics of a novel microplantlet suspension culture established from the macrophytic marine red alga Agardhiella subulata were studied. A bubble-column bioreactor with external illumination (43 microE m(-2) s(-1), 10:14 LD photoperiod), liquid medium perfusion, and 3800 ppm CO(2) in the aeration gas provided sufficient light and nutrient delivery for sustained growth of the microplantlet suspension at 24 degrees C and pH 8. Microplantlets, which consisted of shoot tissues of 3-5 mm length branching out from a common center, were not friable in a bubble-aerated suspension of about 1100 plantlets per liter. Since the microplantlet tissues were not friable, only batch and fed-batch cultivation modes were considered. Batch cultivation was phosphate-limited in ASP12 artificial seawater medium. However, cultivation at a medium perfusion rate of 20% per day avoided phosphate limitation and extended the growth phase to provide plantlet mass densities exceeding 14 g FW L(-1) (3.7 g DW L(-1)) after 50 days of cultivation if the suspension was not sampled. The specific oxygen evolution rate vs cultivation time profile possessed a significant pulse within the 14 days following inoculation and then leveled off at longer times. In recognition of this nonexponential growth pattern, a new photobioreactor growth model was developed that used the oxygen evolution rate vs time profile to predict the biomass growth curve in perfusion culture. Model predictions agreed reasonably with the measured growth curves.     

Screening of thermophilic anaerobic bacteria for solid substrate cultivation on lignocellulosic substrates

Interest in solid substrate cultivation (SSC) techniques is gaining for biochemical production from renewable resources; however, heat and mass transfer problems may limit application of this technique. The use of anaerobic thermophiles in SSC offers a unique solution to overcoming these challenges. The production potential of nine thermophilic anaerobic bacteria was examined on corn stover, sugar cane bagasse, paper pulp sludge, and wheat bran in submerged liquid cultivation (SmC) and SSC. Production of acetate, ethanol, and lactate was measured over a 10 day period, and total product concentrations were used to compare the performance of different organism-substrate combinations using the two cultivation methods. Overall microbial activity in SmC and SSC was dependent on the organism and growth substrate. Clostridium thermocellum strains JW20, LQRI, and 27405 performed significantly better in SSC when grown on sugar cane bagasse and paper pulp sludge, producing at least 70 and 170 mM of total products, respectively. Growth of C. thermocellum strains in SSC on paper pulp sludge proved to be most favorable, generating at least twice the concentration of total products produced in SmC (p-value < 0.05). Clostridium thermolacticum TC21 demonstrated growth on all substrates producing 30-80 and 60-116 mM of total product in SmC and SSC, respectively. Bacterial species with optimal growth temperatures of 70 degrees C grew best on wheat bran in SmC, producing total product concentrations of 45-75 mM. For some of the organism-substrate combinations total end product concentrations in SSC exceeded those in SmC, indicating that SSC may be a promising alternative for microbial activity and value-added biochemical production.     

Effects of continuous cucumber cropping and alternative rotations under protected cultivation on soil microbial community diversity

The diversity of soil microbial communities as affected by continuous cucumber cropping and alternative rotations under protected cultivation were evaluated using community level physiological profiles (CLPP) and random amplified polymorphic DNA (RAPD) analysis. The soils were selected from six cucumber cropping systems, which cover two cropping practices (rotation and continuous cropping) and a wide spectrum for cucumber cropping history under protected cultivation. Shannon–Weaver index and multivariate analysis were performed to characterize variations in soil microbial communities. Both CLPP and RAPD techniques demonstrated that cropping systems and plastic-greenhouse cultivation could considerably affect soil microbial functional diversity and DNA sequence diversity. The open-field soil had the highest Shannon–Weaver index (3.27 for CLPP and 1.50 for RAPD), whereas the lowest value occurred in the 7-year continuous protected cultivation soil (3.27 for CLPP and 1.50 for RAPD). The results demonstrated that continuous plastic-greenhouse cultivation and management can cause the reduction in the species diversity of the biota. Higher Shannon–Weaver index and coefficients of DNA sequence similarity were found in soils under rotation than those under continuous cropping. Cluster analysis also indicated that microbial community profiles of continuous cultivation soils were different from profiles of rotation soils. The reduction in diversity of microbial communities found in continuous cultivation soils as compared with rotation soils might be due to the differences in the quantity, quality and distribution of soil organic matter. Section Editor: D. E. Crowley     

Occurrence of tributyltin-tolerant bacteria in tributyltin- or cadmium-containing seawater.

Tributyltin chloride (TBTCl)-tolerant bacteria accounted for 90% of the flora in natural seawater to which TBTCl was added. These tolerant bacteria were insensitive to 250 nmol of TBTCl per disc, and all were Vibrio species. Total counts of viable bacteria did not decrease upon storage of the TBTCl-treated seawater, indicating that enrichment of tolerant strains took place. Addition of CdSO4 to seawater resulted in the occurrence of TBTCl-tolerant bacteria as well as Cd-tolerant bacteria, suggesting some correlation of Cd tolerance and TBTCl tolerance.     

Occurrence of tributyltin-tolerant bacteria in tributyltin- or cadmium-containing seawater.

Tributyltin chloride (TBTCl)-tolerant bacteria accounted for 90% of the flora in natural seawater to which TBTCl was added. These tolerant bacteria were insensitive to 250 nmol of TBTCl per disc, and all were Vibrio species. Total counts of viable bacteria did not decrease upon storage of the TBTCl-treated seawater, indicating that enrichment of tolerant strains took place. Addition of CdSO4 to seawater resulted in the occurrence of TBTCl-tolerant bacteria as well as Cd-tolerant bacteria, suggesting some correlation of Cd tolerance and TBTCl tolerance.     

Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.     

Model‐based high cell density cultivation of Rhodospirillum rubrum under respiratory dark conditions

The potential of facultative photosynthetic bacteria as producers of photosynthetic pigments, vitamins, coenzymes and other valuable products has been recognized for decades. However, mass cultivation under photosynthetic conditions is generally inefficient due to the inevitable limitation of light supply when cell densities become very high. The previous development of a new cultivation process for maximal expression of photosynthetic genes under semi‐aerobic dark conditions in common bioreactors offers a new perspective for utilizing the facultative photosynthetic bacterium Rhodospirillum rubrum for large‐scale applications. Based on this cultivation system, the present study aimed in determining the maximal achievable cell density of R. rubrum in a bioreactor, thereby providing a major milestone on the way to industrial bioprocesses. As a starting point, we focus on aerobic growth due to higher growth rates and more facile process control under this condition, with the option to extend the process by an anaerobic production phase. Process design and optimization were supported by an unstructured computational process model, based on mixed‐substrate kinetics. Key parameters for growth and process control were determined in shake‐flask experiments or estimated by simulation studies. For fed‐batch cultivation, a computer‐controlled exponential feed algorithm in combination with a pH‐stat element was implemented. As a result, a maximal cell density of 59 g cell dry weight (CDW) L^?1 was obtained, representing so far not attainable cell densities for photosynthetic bacteria. The applied exponential fed‐batch methodology therefore enters a range which is commonly employed for industrial applications with microbial cells. The biochemical analysis of high cell density cultures revealed metabolic imbalances, such as the accumulation and excretion of tetrapyrrole intermediates of the bacteriochlorophyll biosynthetic pathway. Biotechnol. Bioeng. 2010. 105: 729–739. © 2009 Wiley Periodicals, Inc.     

Effect of long-term lead exposure on the seawater and sediment bacteria from heterogeneous continuous flow cultures

Lead-influenced changes of the composition of seawater and sediment bacteria were studied in two flow cultures run with lead-contaminated artificial seawater (1 mg Pb²⁺1^–1) and one control culture. During the experiment viable counts of physiological groups of bacteria from the control culture were not significantly different from that of the lead-contaminated cultures. Lead tolerance of seawater and sediment bacteria strains was investigated. Comparisons of growth yields showed that lead tolerance of seawater and sediment bacteria was lost again if the bacteria were cultivated in a medium without lead. Lead tolerance could not be demonstrated for the sediment bacteria of one lead-contaminated culture. Heterotrophic uptake measurements with radioactive glucose indicated that seawater bacteria from the lead-contaminated cultures became adapted to lead pollution. The sediment bacteria, however, did not reveal lead tolerance by this method. Fluctuations in lead content of the sediment as well as of the overlying seawater gave indications of adsorption-desorption processes between seawater and sediment. Lead was not homogeneously distributed at the sediment surface.     

Enrichment and cultivation of prokaryotes associated with the sulphate-methane transition zone of diffusion-controlled sediments of Aarhus Bay, Denmark, under heterotrophic conditions

The prokaryotic activity, diversity and culturability of diffusion-controlled Aarhus Bay sediments, including the sulphate-methane transition zone (SMTZ), were determined using a combination of geochemical, molecular (16S rRNA and mcrA genes) and cultivation techniques. The SMTZ had elevated sulphate reduction and anaerobic oxidation of methane, and enhanced cell numbers, but no active methanogenesis. The prokaryotic population was similar to that in other SMTZs, with Deltaproteobacteria, Gammaproteobacteria, JS1, Planctomycetes, Chloroflexi, ANME-1, MBG-D and MCG. Many of these groups were maintained in a heterotrophic (10 mM glucose, acetate), sediment slurry with periodic low sulphate and acetate additions (~2 mM). Other prokaryotes were also enriched including methanogens, Firmicutes, Bacteroidetes, Synergistetes and TM6. This slurry was then inoculated into a matrix of substrate and sulphate concentrations for further selective enrichment. The results demonstrated that important SMTZ bacteria can be maintained in a long-term, anaerobic culture under specific conditions. For example, JS1 grew in a mixed culture with acetate or acetate/glucose plus sulphate. Chloroflexi occurred in a mixed culture, including in the presence of acetate, which had previously not been shown to be a Chloroflexi subphylum I substrate, and was more dominant in a medium with seawater salt concentrations. In contrast, archaeal diversity was reduced and limited to the orders Methanosarcinales and Methanomicrobiales. These results provide information about the physiology of a range of SMTZ prokaryotes and shows that many can be maintained and enriched under heterotrophic conditions, including those with few or no cultivated representatives.     

Factors influencing the cultivability of lake water bacteria

Counting bacteria in natural water samples by cultivation yields only low recovery efficiencies (ca. 1%), compared to total counts obtained after 4,6-diamidino-2-phenylindol (DAPI) staining. In order to optimize the cultivation of heterotrophic planktonic bacteria from Lake Constance (Germany), selected parameters of the medium composition were modified. The most important factor was the concentration of organic substrate (nutrient broth plus yeast extract), which significantly influenced the "most probable number" obtained in liquid growth medium. Reduced oxygen concentrations (3-12%) lowered the "most probable number". Addition of N-acyl homoserine lactones to the medium increased the cultivability slightly. Low substrate concentrations [0.03-0.06% (w/v)], an incubation atmosphere of 21% oxygen at 16 degrees C for 4 weeks were optimal and increased the cultivability ("most probable number" related to total bacterial counts) to an average cultivability of 18+/-11%, (n=8). The results indicate that cultivabilities of heterotrophic bacteria from lakewater samples can be significantly increased by modifying the cultivation methods.     

Membrane applications for microalgae cultivation and harvesting: a review

With renewed interest in microalgae due to their potential for biofuel and bioproducts production, efficient cultivation and harvesting mechanisms are needed to increase the economic competitiveness of microalgal products against traditional sources. With pore sizes ranging from microns to angstroms, membranes provide tailored functions for solid/liquid separation (cell retention, biomass concentration and dewatering), gas/liquid separation (gas delivery and removal), and solute/liquid separation (bioproduct recovery, feedstock preparation and effluent recycling) that are problematic or not possible with other technologies. Existing knowledge on membrane systems used in other disciplines, such as environmental engineering, marine science, and biomedicine, can be applied to algae production. Though membranes have great potential to facilitate cultivation and harvesting, challenges in energy reduction and fouling mitigation need to be overcome for long-term, cost-effective application.     

Influence of the RpoS (KatF) sigma factor on maintenance of viability and culturability of Escherichia coli and Salmonella typhimurium in seawater.

The sigma factor RpoS is essential for stationary-phase-specific, multiple-stress resistance. We compared the viabilities (direct viable counts) and culturabilities (colony counts) in seawater of Escherichia coli and Salmonella typhimurium strains and those in which rpoS was deleted or which were deficient in guanosine 3',5'-bispyrophosphate (ppGpp) synthesis (relA spoT). RpoS, possibly via ppGpp regulation, positively influenced the culturability of these bacteria in oligotrophic seawater. This influence closely depended, however, upon the growth state of the cells and the conditions under which they were grown prior to their transfer to seawater. The protective effect of RpoS was observed only in stationary-phase cells grown at low osmolarity. A previous exposure of cells to high osmolarity (0.5 M NaCl) also had a strong influence on the effect of RpoS on cell culturability in seawater. Both E. coli and S. typhimurium RpoS mutants lost the ability to acquire a high resistance to seawater, as observed in both logarithmic-phase and stationary-phase RpoS+ cells grown at high osmolarity. A previous growth of S. typhimurium cells under anoxic conditions also modulated the incidence of RpoS on their culturability. When grown anaerobically at high osmolarity, logarithmic-phase S. typhimurium RpoS+ cells partly lost their resistance to seawater through preadaptation to high osmolarity. When grown anaerobically at high osmolarity until stationary phase, both RpoS+ and RpoS- cells retained very high levels of both viability and culturability and then did not enter the viable but nonculturable state for over 8 days in seawater because of an RpoS-independent, unknown mechanism.     

Behavior of sulfate reducing bacteria under oligotrophic conditions and oxygen stress in particle-free systems related to drinking water

The response of sulfate reducing bacteria (SRB) to oxygen stress under oligotrophic conditions in particle-free systems was studied in (i) sterile Berlin drinking water; (ii) mineral medium; and (iii) in coculture experiments with aerobic bacteria. Using a polyphasic approach including anaerobic cultivation, fluorescent in situ hybridization (FISH) and digital image analysis, the behavior of the strains zt3l and zt10e, isolated from Berlin groundwater and affiliated to the family Desulfovibrionaceae, was compared to the type strains Desulfomicrobium baculatum and Desulfovibrio desulfuricans. Anaerobic deep agar dilution series were performed for the determination of cell culturability. FISH and subsequent digital image analysis of probe-conferred fluorescence intensities were used for the assessment of metabolic activity. For the in situ identification of both isolates in coculture tests, two strain-specific oligonucleotides were developed and evaluated. The total cell counts of stressed SRB in drinking water decreased during the course of the assay dependent on the strain. Both environmental isolates could be cultured for a longer period than cells of D. baculatum and D. desulfuricans, respectively. The FISH intensities showed a strain-specific behavior. When exposed to simultaneous oxygen stress and carbon limitation in mineral medium, total cell counts of all four strains remained constant throughout a period of 72 days. The rate of culturability differed between the investigated strains. The decrease of metabolic activity as assessed by FISH was a strain-specific property. Exposure of SRB to oxygen stress and carbon starvation in coculture experiments with Aquabacterium commune resulted in strain dependent prolonged culturability and a delayed decrease of the metabolic activity compared to pure culture tests for all strains tested. Total cell counts of SRB were constant throughout the whole experiment.