Determination of Active Marine Bacterioplankton: a Comparison of Universal 16S rRNA Probes, Autoradiography, and Nucleoid Staining

We compared several currently discussed methods for the assessment of bacterial numbers and activity in marine waters, using samples from a variety of marine environments, from aged offshore seawater to rich harbor water. Samples were simultaneously tested for binding to a fluorescently labeled universal 16S rRNA probe; (sup3)H-labeled amino acid uptake via autoradiography; nucleoid-containing bacterial numbers by modified DAPI (4(prm1),6-diamidino-2-phenylindole) staining; staining with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), a compound supposed to indicate oxidative cell metabolism; and total bacterial counts (classical DAPI staining), taken as a reference. For the universal-probe counts, we used an image intensifying and processing system coupled to the epifluorescence microscope. All of the above-mentioned methods yielded lower cell counts than DAPI total counts. Universal-probe counts averaged about half of the corresponding DAPI count and were highly correlated to autoradiography counts (r(sup2) = 0.943; n = 7). Nucleoid-containing cell counts could be lower than DAPI counts by as much as 1 order of magnitude but sometimes matched autoradiography or probe counts. CTC counts were 2 orders of magnitude below DAPI counts. Universal 16S rRNA probe counts correlated well with autoradiography results, indicating a population with at least minimal metabolic activity. The greater variability of the nucleoid-containing cell counts calls for further investigation of the processes involved, and CTC counts were well below the range of the other methods tested.     

Enumeration of Viable Bacteria in the Marine Pelagic Environment

The low percentage of living bacteria commonly obtained when comparing viable counts with total direct counts in seawater could be due more to inappropriate techniques for appreciating the growth ability of living cells than to unadapted culture conditions. The most-probable-number counts in filtered seawater cultures and the microscopic counts of 4(prm1),6-diamidino-2-phenylindole (DAPI)-stained aggregate-forming units grown on black polycarbonate filters appeared significantly correlated to the direct counts. Both these techniques show that in the superficial and intermediate water masses, the living cells may constitute an important (frequently higher than 20%) but highly variable part of the total populations. These viable counts appear more realistic than the conventional CFU counts, which provide only 0.001 to 0.2% of the total counts.     

早期宫颈癌患者围手术期外周血淋巴细胞数与其预后的关系

目的:探讨早期宫颈癌患者围手术期外周血淋巴细胞数与其预后之间的关系。方法:回顾性分析2006年1月至2012年12年就诊于解放军总医院,根据2009年最新宫颈癌FIGO指南诊断为Ⅰb~ⅡA期宫颈癌进行阴式宫颈癌根治术患者的临床病历资料。分析患者的围手术期淋巴细胞数和其无瘤生存期、总生存期间的关系。结果:共143例阴式早期宫颈癌根治术患者纳入研究,随访时间为6~87个月,中位随访时间为53个月。术后,宫颈癌患者外周血淋巴细胞数显著下降。术前外周血淋巴细胞数较高的患者无瘤生存期及总生存期均较外周血淋巴细胞数低于平均水平的患者显著延长(P0.05);术后第三天淋巴细胞数恢复或高于术前水平的患者无瘤生存期较术后第三天淋巴细胞数低于术前水平的患者显著延长(P0.05),但总生存期无明显差异(P0.05)。COX回归分析显示术前淋巴细胞数高于平均水平及术后第三天淋巴细胞数高于或等于术前水平的宫颈癌患者的预后相对较好。结论:术前淋巴细胞数和术后第三天淋巴细胞数的变化均可作为评估宫颈癌术后患者预后的重要参考指标。     

COUNTING STELLER SEA LION PUPS IN ALASKA: AN EVALUATION OF MEDIUMFORMAT, COLOR AERIAL PHOTOGRAPHY

Estimates of Steller sea lion ( Eumetopias jubatus ) pup production are valuable for estimating population trend and size. Currently in Alaska, pups are counted by visiting rookeries, driving older animals into the water, then walking through the rookeries and counting the pups, a highly disruptive procedure. At smaller rookeries, with good vantage points, pups are occasionally counted from the periphery of rookeries without disturbing the sea lions. We evaluated counts made from medium-format, color, aerial photographs as an alternative to drive counts and peripheral counts. Neither the peripheral counts nor the aerial photographic counts disturbed animals on the rokeries. There were strong 1:1 linear relationships between photographic counts and drive counts ( r ²= 0.966, P < 0.001) and between photographic counts and peripheral counts ( r ²= 0.999, P < 0.001). Precision was similar for all three methods of counting. We suggest that medium-format, color, aerial photographs is appropriate for routine surveys of Steller sea lion pups in Alaska because it is not disruptive to the hauled-out sea lions and provides comparable estimates with similar precision to drive and peripheral counts. Large areas canbe rapidly surveyed during periods of good weather with a minimum of manpower.     

ROLL TUBES AND AGAR STRIP TUBES FOR THE BACTERIAL COLONY COUNT OF MILK

SUMMARY: Roll-tube colony counts, using the Astell equipment, were lower than the corresponding Petri dish counts with 27 out of 31 raw milks (87%). The difference between the counts by the two methods was greater than 25% of the plate count for 12 (39%) of the samples.
When the same dilution of milk was used for both strip-tube and plate colony counts, about equal numbers of samples gave counts from the strip tubes above and below about the colony count from plates. When, in order to obtain a more reasonable strip-tube count, the plates and strip tubes were prepared from different dilutions of the milk, the counts from the latter were, with only 3 exceptions out of 35 milks, below those from the former. The difference between the counts was greater than 25% of the plate count for 15 (43%) of the milks, a figure similar to that obtained in comparing roll-tube and plate colony counts.     

Quantification of tumor cellularity and mitotic index in invasive ductal carcinoma of the breast

OBJECTIVE: To evaluate the use of stereologically estimated tumor cell counts in the mitotic index as well as to investigate its correlation with the currently used method and test the reproducibility of the method. STUDY DESIGN: The stereologic method described by Simpson et al was used to estimate tumor cellularity in 50 invasive ductal carcinomas. Mitotic counts were also performed, and the mitotic index was calculated by the use of estimated tumor cell counts. Estimated cell counts and the mitotic index calculated were compared statistically with the actual cell counts and the traditional mitotic grades, respectively. Interobserver reproducibility of the method was also tested. RESULTS: Stereologically estimated tumor cell counts had a good correlation with actual cell counts (r = .891, P < .001). Besides, the mitotic indices calculated with tumor cell counts (calculated with both estimated and actual cell counts) in the denominator of the fraction of the mitotic index were in agreement with the currently used method (P < .01 for both). There was no statistically significant difference between the counts of two observers (P = .068). CONCLUSION: The suggested method, considering tumor cellularity as an influencing factor, was practical, reproducible and in agreement with the traditional method. This method should be studied in a large group of patients with follow-up data to determine the threshold values for different grades and determine its prognostic value during the disease course.     

Effect of vegetation type and environmental factors on European wild rabbitOryctolagus cuniculus counts in a southern Portuguese montado

This study assesses the effect of vegetation and variables related to weather and light conditions on the efficacy of rabbitOryctolagus cuniculus (Linnaeus, 1758) counts carried out in the south of Portugal. Counts were carried out in two years using driven line transects, and correlated with vegetation type and the variables using generalised linear models. The offset was a surveyed area estimated using Distance Sampling Theory as a means of correcting for detectability bias. More rabbits were observed in dense vegetation during day-time counts and in crops during night-time counts. In 1998, day-time counts were higher with higher average daily temperatures, whilst the night-time counts were higher with higher minimum daily temperatures. In 1999, day-time counts decreased with the amount of rainfall in the previous month, and the night-time counts decreased with the accumulated rainfall in the previous two months and with the higher wind speeds. In order to increase efficacy, counts should be carried out either at dawn or at dusk during the post-breeding season, and with greater intensity in dense scrub or open vegetation with high tree cover. During the breeding season and winter, counts should be carried out after dusk and with greater intensity in arable crops.     

Sites of attachment and density assessment of ixodid ticks (Acari: Ixodidae) on impala (Aepyceros melampus)

During 1994 and 1995, 45 impala ewes, Aepyceros melampus were examined at Letaba Ranch in the Northern Province. Tick counts were made from 15 animals on three occasions, in July, October and February. The objective was to determine whether the total body tick counts can be estimated from counts done on specific, selected sampling sites on the skin. Twelve sites were shaved within ten predilection sites and the parasite numbers counted from these samples. These counts were compared to the total body parasite count as determined by the scrub and digestion techniques. More than 80% of all the ticks were present on the muzzle, the head, the pinna and the legs. Larger numbers of ticks were collected in October than in July or February. Two mathematical models were tested for the tick counts. The first model was made up of tick counts from a single shaved sampling site, the pinna. The correlation between the tick counts on the pinna and the total tick counts was highly significant (p values ranging between 0.0208 and 0.0001). The second model was developed based on tick counts from four regions, namely the head, the pinna and the front and hind feet. A less significant correlation was obtained between the number of ticks counted on the four sites and the total tick count.     

THE FAILURE OF PHENOL TREATED ESCHERICHIA COLI TO GROW ON MEMBRANE FILTERS

SUMMARY: Counts of Escherichia coli were done on nutrient agar (control), on membrane filters on nutrient agar and on membrane filters on filter paper pads. With untreated bacteria counts were similar under all conditions, though membrane filters on nutrient agar tended to give slightly low counts. Phenol treated bacteria gave much lower counts when membrane filters were used: the mean counts for 3 strains of the test organism with filters on nutrient agar varied from 35–65% of the control, while counts with filters on filter paper pads were somewhat lower, varying from 30–47% of the control. The low counts on membrane filters on filter paper pads were not due to adsorption of phenol by the filters or to a low concentration of nutrients in the growth medium.     

Factors influencing the fecal egg and oocyst counts of parasites of wild European rabbits Oryctolagus cuniculus (L.) in Southern Western Australia

Abundance of intestinal parasites was monitored by fecal egg and oocyst counts for samples of wild rabbits Oryctolagus cuniculus with different levels of imposed female sterility from 12 populations in southwestern Australia. Differences in egg counts of Trichostrongylus retortaeformis between seasons and age groups were dependent on the sex of the host. Pregnancy may have been responsible for these differences because egg counts were consistently higher in intact females than in females surgically sterilized by tubal ligation. Egg counts for Passalurus ambiguus were influenced by season and host age but there were no differences between sexes or between intact and sterilized female rabbits. No differences were detected in the oocyst counts of the 8 species of Eimeria between male and female rabbits or between intact and sterilized females. Seasonal differences were detected in oocyst counts of Eimeria flavescens and Eimeria stiedai. The overwhelming determinant of coccidian oocyst counts was host age, with 6 species being much more abundant in rabbits up to 4 mo of age. There was a suggestion that egg counts of T. retortaeformis and oocyst counts of several species of Eimeria were reduced in populations where rabbit numbers had been depressed for at least 2 yr, but there was no evidence that short-term variations in rabbit numbers had a measurable effect on parasite abundance.     

Monitoring ungulates in steep non-forest habitat: a comparison of faecal pellet and helicopter counts

Faecal pellet counts have been widely used to monitor the abundances of introduced ungulates in New Zealand, but ground-based sampling cannot be conducted safely in the steep non-forest habitats that are common in New Zealand's Southern Alps. Helicopter counts may be an effective technique for monitoring ungulates in steep non-forest habitat. We evaluated the relationship between faecal pellet and helicopter counts of ungulates (primarily feral goat Capra hircus) at 12 non-forest sites in the Southern Alps. Within each site we counted the numbers of ungulates from a helicopter on three occasions and the number of intact faecal pellets along 30 transects. Mean observed densities of feral goats derived from helicopter counts ranged from 0.0 to 20.2 km^?2. There was a positive curvilinear (concave down) relationship between faecal pellet and helicopter counts. Compared with faecal pellet counts, helicopter counts were cheaper, could identify ungulate species and provided estimates of absolute density. Helicopter counts are a cost-effective method for monitoring ungulates in the steep non-forest habitats of New Zealand's Southern Alps.     

A comparison of methods used to count the elephant population of the Addo Elephant National Park, South Africa

Evaluation of alternative techniques used to estimate elephant population sizes is important in order to assess the accuracy of the results obtained, upon which management decisions may be based. Data from annual helicopter surveys carried out in the Addo Elephant National Park (1978–97) are compared with registration counts obtained from intensive ground surveys in which all elephants within the park are individually known. On average, total population size estimated in aerial surveys is 8.0% lower than registration counts (n₁₉₉₇ = 251), aerial calf (< 1 year) counts are 48.8% lower than registration counts, and aerial carcass counts are 50% below the total number of deaths documented in ground surveys. Registration counts provide more accurate demographic data than aerial surveys, the results of which are shown to vary widely and unpredictably, thus compromising their value. Where population estimates rely exclusively on aerial surveys, replicate counts are recommended in order to generate confidence intervals.     

LIVE/DEAD BacLight : application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water.

A rapid epifluorescence staining method using the LIVE/DEAD Bacterial Viability Kit (BacLight) was applied to estimate both viable and total counts of bacteria in drinking water. BacLight is composed of two nucleic acid-binding stains: SYTO 9 and propidium iodide. SYTO 9 penetrates all bacterial membranes and stains the cells green, while propidium iodide only penetrates cells with damaged membranes, and the combination of the two stains produces red fluorescing cells. Optimal incubation conditions were found to be 15 to 20 min, at room temperature in the dark. Total (red + green) and viable (green) cells can hence be counted simultaneously. Factors affecting the staining procedure were tested (addition of glutaraldehyde, staining time, chlorine impact). In the absence of stress, BacLight viable counts were comparable and to 5-cyano-2,3-ditolyl tetrazolium (CTC) counts. BacLight total counts were comparable to acridine orange counts (differing by <0.1 log/ml). However, the increase in environmental stresses (chlorine, growth rate or temperature) induced a decrease in viability that was more pronounced for CTC and plate counts than for BacLight viable counts.     

How reliable are dung counts for estimating elephant numbers?

Dung counts are the most commonly used techniques for estimating elephant numbers in forests, yet there is considerable scepticism concerning their accuracy. Published accounts of dung counts show that they give estimates similar to those from other methods for vertebrates ranging in size from lizards to elephants. For ungulates, macropods and elephants there are strong correlations between estimates from dung counts and other methods. Thus, dung counts are as accurate or inaccurate as other methods for estimating vertebrate numbers, including elephants. Dung counts for elephants give estimates that are as precise as, and sometimes more precise than, those from aerial surveys of elephants. This is because the variance in dung density is usually low and results in a lower than expected variance for the final elephant estimate when combined with the variances of defecation and decay rates. Dung counts may be more appropriate than aerial surveys for monitoring small or declining elephant populations.     

Methods of estimating ungulate populations in tropical forests

Pellet group counts, drive counts and track counts were used to estimate population abundances of one small and four large species of duiker (Cephalophus sp.) in a moist evergreen forest in northeastern Zaire. Procedures to develop estimates based on line transect counts of pellet groups are described. The Fourier Series estimator proved to be useful for estimating densities of pellet groups. Comparisons were made between population abundance in heavily and lightly hunted areas, and between small and large species. The three census methods were consistent in showing no significant difference between animal abundance in the hunted populations or between abundances of the smaller species relative to the larger species. All methods showed potential as population indices for assessing trends of rare and elusive species in forest environments. Drive counts and pellet group counts may also be useful for estimating densities.     

Comparison of bacterial counts obtained from naturally contaminated foods by means of Stomacher and blender

Four Regional Health Protection Branch laboratories each compared aerobic colony counts obtained after "stomaching" and blending, for a minimum of 10 samples in each of the seven food groups: dry pastas; chocolate and cocoa powders; frozen entrees (macaroni and cheese, chow mein, chop suey, fried rice, seafood casseroles, and Salisbury steak); nonfat dry milk; shrimp and crabmeats; spices; and breakfast sausages. Overall, counts obtained after using the Stomacher were equivalent to or higher than counts obtained after using the blender in 73% of the comparisons (alpha = 0.05). Where differences existed, counts obtained after using the Stomacher tended to be higher than counts obtained after using the blender from milk powder and lower from sausage. Aerobic colony counts from these foods are not unacceptably biased when obtained by Stomacher.     

Rapid quantification of planktonic ciliates: comparison of improved live counting with other methods

THE FOLLOWING EFFICIENT AND QUANTITATIVELY VALID METHOD TO FILTER CONCENTRATE AND COUNT LIVE PLANKTONIC CILIATES WAS DEVELOPED AND COMPARED WITH OTHER TREATMENTS: unconcentrated (raw) samples and centrifuged samples were counted live, and the effects of five different fixatives (HgCl(2), Lugol's iodine, formaldehyde, glutaraldehyde, and Champy-DaFano) on the counts were monitored. Samples originated from a eutrophic mountain lake (Lake Aydat, near Clermont-Ferrand, France). Overall, live filtered counts were similar to counts of raw samples, but they were significantly higher (2 to 2.3 fold, P < 0.05) by analysis of variance than counts from centrifuged samples. Nevertheless, some taxa, i.e., Halteria and Loxodes spp., were sensitive to filtration. The live filtered counts were also comparable to counts of raw HgCl(2)-fixed and settled samples. HgCl(2) and Lugol fixation consistently gave the highest total counts, while significantly lower counts were always obtained with Champy-DaFano-fixed samples. Losses due to fixation were insignificant for raw samples but were substantial and statistically significant in concentrated samples (15% after filtration and 71% after centrifugation, compared with counts from the corresponding live samples). Live counting of passively filter-concentrated ciliates has many advantages over other methods. It is two to four times quicker and more efficient. Ciliates are recognized with certainty, more species are identified, and enumeration of dead organisms (e.g., tintinnid loricas) is avoided. It should be recommended as a quantitatively valid alternative to classical methods for assessing planktonic ciliate populations.     

Estimating red deer Cervus elaphus populations: an analysis of variation and cost-effectiveness of counting methods

• 1 Different counting methods are currently used to estimate red deer populations in the open range in Scotland, but there are few data available to compare variation in estimates, or relative cost‐effectiveness.
• 2 While it is impossible to determine the accuracy of counts (as real numbers are unknown), variation within and between different methods can be measured by repeat counts of the same area within as short a period as possible.
• 3 This study aimed to quantify the variation observed from repeat counts using each of four methods (ground, helicopter, infrared helicopter and dung‐counting methods) at one of three study sites in late winters 2003, 2004 and 2005. Additional data from digital camera images of groups from counts in other areas of Scotland were also used to assess the accuracy of visual counts.
• 4 Coefficients of variation (CVs) within any method of between 5% and 16% were recorded, consistent with previous comparisons for red deer open range counts in Scotland. CVs were lowest for ground and helicopter counts. The infrequency of optimal conditions was likely to limit the applicability of infrared counts in Scotland.
• 5 In terms of cost‐effectiveness, helicopter counting was the least labour‐intensive, with costs of other techniques depending on the availability of existing manpower as an overhead cost.
• 6 It is concluded that helicopter counts are most likely to minimize errors while maximizing cost‐efficiency. Accuracy can be improved by the use of digital photography for counting larger deer groups. Estimates are likely to be improved further by increasing the frequency of counts and using the same methods, counters and routes for repeat counts.

     

Circannual rhythms in the blood picture of laboratory rats

Circannual rhythms of the erythrocyte, total leucocyte, absolute segmented neutrophil and lymphocyte counts in laboratory Wistar rats were found. The highest mean counts occurred in autumn and at the begining of winter. The lowest counts werte recorded in spring and at the begining of summer. A similar circannual rhythm of absolute eosinophil counts in females was also found, but it was not present in males. Circannual rhythms of the relative lymphocyte, segmented neutrophil and eosinophil were not found. The course of seasonal variations in erythrocyte, total leucocyte, absolute segmented neutrophil and lymphocyte counts of males was similar to those of females.     

Water quality and public health in northern Sudan: a study of rural and peri-urban communities

Access to adequate supplies of good quality drinking water continues to be limited among many rural and peri-urban communities in Africa, despite several decades of water improvement programmes. The present study investigated water quality at the source and point of consumption among rural and peri-urban communities in northern Sudan. Faecal coliform counts were determined by the membrane filtration technique and geometric mean counts compared in different seasons and among the different communities. Among nomadic pastoralists and riverine villages, both water sources and water stored for consumption had faecal coliform counts grossly in excess of WHO standards, with higher counts at the end of the rainy season. In the peri-urban community on the outskirts of Omdurman, while water quality from the distribution system had faecal coliform counts generally below 10 dl - 1, after storage, water was of considerably lower quality, with faecal coliform counts up to 1000 d1 - 1. The highest counts again occurred in the rainy season. Rates of diarrhoeal disease for Khartoum province were also greatest towards the end of the rainy season. The study has shown that poor quality water continues to be a major risk factor for public health in these communities.