Inherent errors of the fixed-frame counting method for corneal endothelial cell density in eye banks

The aim of this work was to analyze the magnitude of inherent errors associated with the fixed-frame counting method for corneal endothelial cell density (ECD) measurements. This technique is common among most eye banks worldwide. Three types of mosaics were used: regular and irregular tessellated mosaics (eight increasing densities ranging from 800 to 3,600 cells/mm² by steps of 400 cells/mm²) generated by a computer, and real mosaics (four specimens) obtained from human corneal endothelium flat mounted and stained with Alizarin red. On the three mosaics, the fixed-frame counting method was applied using a computer program. The ECD was calculated for 3,000 successive random positions from calibrated grids which area ranged from 50 × 50 to 300 × 300 μm² (incremental steps of 25 μm). For each grid, the ECD was expressed either as a single count, a mean of five or a mean of 10 measures. The fixed-frame count was constantly associated with an inherent variability but repeatability increased with larger grid size and ECD. The mean calculated out of 10 measures was the most reliable, but still, we noted ±5 % of residual variability from the real ECD. The 100 × 100 μm² grid manual counts, performed in many eye banks, should be abandoned and upgraded to at least 200 × 200 μm² grid counts. Digital image analysis with a variable frame counting method would be the best alternative.     

Inter-animal radiation as potential heat stressor in lying animals

A model for predicting inter-animal radiant heat exchange in shaded animals is presented, with emphasis on mature cattle. When a cow’s surface temperature is 35 °C, as is common in warmer climates, it loses ～510 Watt m^?2 as radiant heat. Net radiant heat balance depends on radiation coming from bodies in the vicinity. In the 30 °C radiant temperature shaded environment typical of warm climates, net radiant loss from a lactating cow is ～60 Watt m^?2, i.e., 30 % of its ～173 Watt m^?2 heat production. Cows rest for 8–14 h day^?1. The heat exchange of a lying cow differs from that of a standing one: the body center is low and 20–30 % of its surface contacts a surface of relatively low heat conductance. Lying reduces the impact of the surrounding shaded area on heat exchange but increases that of heat radiating from neighboring cows. When a cow rests adjacent to other cows, with 1.25 m between body centers when in stalls, it occupies about 140° of the horizontal plane of view. Heat emitted from the animal’s surface reduces the net radiant heat loss of a resting cow by ～30 Watt m^?2. In contrast, the presence of cows at 5 and 10 m distance, e.g., cows resting on straw in loose yard housing, reduces the net radiant heat loss of the resting cow by 9 and 5 Watt m^?2, respectively. Radiant heat input increases with animal density, which is beneficial in cooler climates, but acts as a stressor in warm climates.     

Studies of the morphology of chromosomes of some selected species

Karyotypes of twelve species from twenty-four localities in southern Moravia and one locality in Slovakia were investigated. Their counts or karyotypic formulae are as follows:Chenopodium foliosum (Moench) Ascherson: K (2n)=18=16 A^m+2 B^sm;Astragalus austriacus Jacq.: K (2n)=16=8 A^m+8 B^sm;Astragalus exscapus L.: K (2n)=16=10 A^m+4 B^sm+2 C^st;Astragalus cicer L.: K (2n)=64;Astragalus onobrychis L.: K (2n=64 and K (2n)=64+1;Vicia dumetorum L.: K (2n=14=10 A^m+4 B^sm;Vicia sylvatica L.: K (2n)=14=2 A^m+10 B^sm+2 C^st;Vicia pisiformis L.: K (2n)=12=8 A^m+4 B^sm;Vicia cassubica L.: K (2n)=12=4 A^m+6 B^sm+2 C^st;Vicia cracca L. (from five localities in southern Moravia): K (2n)=28=4 A^m+12 B^sm+12 C^st and K (2n)=28+1=5 A^m+12 B^sm+12 C^st;Vicia cracca L. (from one locality in Slovakia): K (2n)=14=2 A^m+6 B^sm+6 C^st;Vicia tenuifolia Roth: K (2n)=24=4 A^m+16 B^sm+4 C^st;Serratula lycopifolia (Vill.) Kern.: K (2n)=60.     

Halimeda biomass,growth rates and sediment generation on reefs in the central great barrier reef province

The average biomass ofHalimeda per m² of solid substratum increased progressively on a series of reefs situated at increasing distances from the shore in the central Great Barrier Reef. There was none on a reef close inshore, increasing to nearly 500 g m^?2 total biomass (?90% calcium carbonate) on an oceanic atoll system in the Coral Sea. The biomass measured contained 13 species ofHalimeda but was dominated by only two species,H. copiosa andH. opuntia, except on the atoll whereH. minima was dominant. Three sand-dwelling species were also present but did not occur anywhere in substantial quantities. Growth rates of the dominant species were measured bv tagging individual branch tips. A mean value of 0.16 segments d^?1 was recorded but 41% of the branch tips did not grow any new segments whilst only 1% grew more than one per day. The number of branch tips per unit biomass was very constant and has been used in conjunction with growth rates and biomass to calculate productivity rates, and thence sedimentation, in the lagoon of one of the reefs. Biomass doubling time of 15 d and production of 6.9 g dry wt m^?2 d^?1 are considerably higher than previously reported values forHalimeda vegetation and there was little seasonal change detected over a whole year. Those values indicate annual accretion of 184.9 g m^?2 year^?1 ofHalimeda segment debris over the entire lagoon floor (5.9 km²) of Davies Reef, equivalent to 0.13 mm year^?1 due toHalimeda alone, or 1 m every 1,892 years when other contributions to that sediment are taken into account.     

The effects of temperature and light intensity on growth,reproduction and EPS synthesis of a thermophilic strain related to the genus Graesiella

Tunisian microalgae are diverse and rarely been studied. This study reports a first investigation of thermophile Chlorophyta isolated from mats community colonizing the geothermal springs in the north of Tunisia at water temperature 60 °C. In the study, the combined effect of temperature and light intensity was investigated on the cell growth, the mother and daughter cells abundance and the extracellular polymeric substances synthesis in batch culture of the isolated species. Three levels were tested for each factor, 20, 30, 40 °C for temperature; and 20, 70, 120 μmol photons m^?2 s^?1 for light intensity, using full factorial design and response surface methodology. The thermophile strain was identified as a genus Graesiella and showed 99.8 % similarity with two Graesiella species: Graesiella emersonii and Graesiella vacuolata based on the 18S rDNA molecular identification. The optimal growth condition was found at 30 °C and 120 µmol photons m^?2 s^?1 (7 MC mL^?1 day^?1), with the abundance of vegetative cells (daughter cells). In contrast, the number of mother cells increased significantly as the growth decreased; consequently, the highest ratio of auto spore mother cells versus daughter cells (19.4) was obtained at 20 °C and 20 µmol photons m^?2 s^?1. The highest yield of EPS production (11.7 mg L^?1 day^?1) was recorded at the highest temperature (40 °C) and lowest light intensity (20 µmol photons m^?2s^?1). These results revealed how the species respond to high and low temperatures and suggest that the species should be considered as facultative thermophile.     

Seasonal abundance of soil-surface arthropods in relation to some meteorological and edaphic variables of the grassland and tree-planted areas in a tropical semi-arid savanna

Seasonality of relative population abundance in different groups of soil-surface arthropods was investigated monthly by pit-fall traps during a 2-year period in the grassland and tree-planted areas of a tropical semi-arid savanna at Warangal (south India). Densities of most groups were lowest during summer and highest during the rainy season. They were less abundant during winter. Arthropods were recorded in higher numbers in tree-planted compared to grassland areas. Certain arthropods that were found only during part of the year were recorded for a longer period in the tree-planted area. Formicidae,Monomorium indicum Forel,Crematogaster sp. andPachycondyla? tesserinoda (Emery), and Coleoptera,Pachycera sp. reached maximum densities in the rainy season and minimum numbers during winter and summer in the grassland area. However, these species had lower densities during the rainy season and reached maximum densities during winter and summer in the tree-planted area. The seasonal abundance of arthropods showed significant linear correlations with different abiotic environmental variables such as rainfall, soil moisture, organic matter, soil and air temperatures, soil pH, relative humidity at the soil surface, and potassium and phosphorus of surface soil. Soil moisture and rainfall were generally the strongest correlates with densities, particularly in the grassland area.     

Effects of Bone Marrow Mesenchymal Stem Cells on Hematopoietic Recovery and Acute Graft-Versus-Host Disease in Murine Allogeneic Umbilical Cord Blood Transplantation Model

To investigate the effect of bone marrow mesenchymal stem cells (MSC) on hematopoietic recovery and acute graft-versus-host disease (GVHD) in a murine allogeneic umbilical cord blood transplantation (allo-UCBT) model. MSCs were obtained from C57/BL mouse bone marrow. The MSC phenotypes were identified by flow cytometry (FCM), and their ability to differentiate into osteoblasts and adipocytes was tested. Once murine allo-UCBT and aGVHD models were established, mice were divided into five groups: (1) total body irradiation (TBI) group, each mouse receiving 0.3 ml sterile saline infusion after TBI and used as control; (2) UCB group, receiving 2 × 10⁶ umbilical cord blood mononuclear cells (UCB-MNC) after TBI; (3) UCB+MSC group, receiving 2 × 10⁶ UCB-MNC and 2 × 10⁷ MSC after TBI; (4) UCB+SC group, receiving 2 × 10⁶ UCB-MNC and 2 × 10⁶ spleen cells after TBI; and (5) UCB+SC+MSC group, receiving 2 × 10⁶ UCB-MNC, 2 × 10⁷ MSC and 2 × 10⁶ spleen cells after TBI. To evaluate the engraftment of HSC, the white blood cells, red blood cells, and platelets counts were tested at different time points after transplantation, and the ratio of chimerism was identified by FCM. The acute GVHD clinical scores, recipient mice survival, and the histopathological analyses were used to evaluate the effect of MSC on acute GVHD. MSCs were successfully obtained in vitro and FCM analysis showed that these cells are highly positive for CD90.2, CD44, and negative for CD34, CD45, and they are capable to differentiate into osteoblasts and adipocytes after being induced. Compared to UCB group, the UCB+MSC mice had shorter duration of myelosuppression and higher percentage of donor-derived cells which was up to 22.87 ± 4.3 % and the white blood cell (WBC), red blood cell (RBC), and platelet counts started to increase by day 6 after transplantation. Moreover, the average survival time for UCB+MSC mice was 25.0 ± 10.55 days, while for the UCB group it was 15.5 ± 12.50 days. The UCB+SC mice showed fatigue, loss of appetite, weight loss, arched back, and hair ruffling on day 13 post transplantation. Approximately 50 % of mice showed skin ulcers, had diarrhea and other manifestations of acute GVHD, and all mice were died within 20 days. Histopathological analysis confirmed grade II–IV GVHD manifestation. In addition to transient weight loss, some UCB+SC+MSC mice developed arched back, hair ruffling, diarrhea and other manifestations of acute GVHD. The clinical scores in UCB+SC+MSC mice with acute GVHD (grade I–II or without GVHD) were lower than UCB+SC group (P < 0.05). Bone marrow MSCs can promote hematopoietic recovery and decrease the incidence of acute GVHD in murine allo-UCBT model.     

Combined Effects of Lead and Acid Rain on Photosynthesis in Soybean Seedlings

To explore how lead (Pb) and acid rain simultaneously affect plants, the combined effects of Pb and acid rain on the chlorophyll content, chlorophyll fluorescence reaction, Hill reaction rate, and Mg²⁺-ATPase activity in soybean seedlings were investigated. The results indicated that, when soybean seedlings were treated with Pb or acid rain alone, the chlorophyll content, Hill reaction rate, Mg²⁺-ATPase activity, and maximal photochemical efficiency (F _v/F _m) were decreased, while the initial fluorescence (F ₀) and maximum quantum yield (Y) were increased, compared with those of the control. The combined treatment with Pb and acid rain decreased the chlorophyll content, Hill reaction rate, Mg²⁺-ATPase activity, F _v/F _m, and Y and increased F ₀ in soybean seedlings. Under the combined treatment with Pb and acid rain, the two factors showed additive effects on the chlorophyll content in soybean seedlings and exhibited antagonistic effects on the Hill reaction rate. Under the combined treatment with high-concentration Pb and acid rain, the two factors exhibited synergistic effects on the Mg²⁺-ATPase activity, F ₀, F _v/F _m, as well as Y. In summary, the inhibition of the photosynthetic process is an important physiological basis for the simultaneous actions of Pb and acid rain in soybean seedlings.     

Composition and species diversity of pine-wiregrass savannas of the Green Swamp,North Carolina

Fire-maintained, species-rich pine-wiregrass savannas in the Green Swamp, North Carolina were sampled over their natural range of environmental conditions and fire frequencies. Species composition, species richness, diversity (Exp H′, 1/C), and aboveground production were documented and fertilization experiments conducted to assess possible mechanisms for the maintenance of high species diversity in these communities. Although savanna composition varies continuously, DECORANA ordination and TWINSPAN classification of 21 sites facilitated recognition of 3 community types: dry, mesic, and wet savannas. These savannas are remarkably species-rich with up to 42 species/0.25 m² and 84 species/625 m². Maximum richness occurred on mesic, annually burned sites. Aboveground production, reported as peak standing crop, was only 293 g · m^?2 on a frequently burned mesic savanna but was significantly higher (375 g · m^?2) on an infrequently burned mesic site. Production values from fertilized high and low fire frequency sites were equivalent. Monthly harvest samples showed that savanna biomass composition by species groups did not vary seasonally, but within groups the relative importance of species showed clear phenological progressions. The variation in species richness with fire frequency is consistent with non-equilibrium theories of species diversity, while phenological variation in production among similar species and the changing species composition across the moisture gradient suggest the importance of equilibrium processes for maintenance of savanna diversity.     

Some properties of pea cholinesterase and its activity in plant parts at different growth stages

Cholinesterase activity was studied in 2 to 10-week-old pea plants cultivated under artificial illumination. Free and membrane-bound forms of the enzyme were separated by extracting the enzyme from pea shoots with buffers differing in ionic strength. The ratio of the free cholinesterase to the membrane-bound one fluctuated between 1 : 1 and 1 : 2.5. The free cholinesterase was inhibited by neostigmine (0.1mmoll^-1) by 50%, the membrane-bound enzyme by 90%. The pH optimum of cholinesterase activity was 8.5, the temperature optimum 37 °C. The enzyme activity was increased by some cations in this order: Mg²⁺ < < K⁺. The K^m value for the substrate S-acetylthiocholine iodide was 250 μmoll^-1, the enzyme activity being inhibited by concentrations higher than 3 mmoll^-1 of this substrate. The activity of the membrane-bound enzyme was demonstrated in the roots, leaves, stems, fruits, seeds and carpels, but could not be reliably detected in the blossoms. The highest activity expressed per fresh matter was found in older leaves and in the fruits, the lowest in the roots and stems. Cholinesterase activity in plant parts markedly varied during the investigated growth period.     

Expression of the NAD-dependent FDH1 β-subunit from Methylobacterium extorquens AM1 in Escherichia coli and its characterization

The efficient regeneration of nicotinamide cofactors is an important process for industrial applications because of their high cost and stoichiometric requirements. In this study, the FDH1 β-subunit of NAD-dependent formate dehydrogenase from Methylobacterium extorquens AM1 was heterologously expressed in Escherichia coli. It showed water-forming NADH oxidase (NOX-2) activity in the absence of its α-subunit. The β-subunit oxidized NADH and generated NAD⁺. The enzyme showed a low NADH oxidation activity (0.28 U/mg enzyme). To accelerate electron transfer from the enzyme to oxygen, four electron mediators were tested; flavin mononucleotide, flavin adenine dinucleotide, benzyl viologen (BV), and methyl viologen. All tested electron mediators increased enzyme activity; addition of 250 μM BV resulted in the largest increase in enzyme activity (9.98 U/mg enzyme; a 35.6-fold increase compared with that in the absence of an electron mediator). Without the aid of an electron mediator, the enzyme had a substrate-binding affinity for NADH (K _m) of 5.87 μM, a turnover rate (k _cat) of 0.24/sec, and a catalytic efficiency (k _cat/K _m) of 41.31/mM/sec. The addition of 50 μM BV resulted in a 22.75-fold higher turnover rate (k _cat, 5.46/sec) and a 2.64-fold higher catalytic efficiency (k _cat/K _m, 107.75/mM/sec).     

Physiological activities of ergoline alkaloids in submerged cultures ofClaviceps paspali andClaviceps purpurea

Ergoline alkaloids do not belong to a group of physio ogically inert secondary metabolites. Ergometrine as well as ergotamine increased the acetyl-CoA carboxylase activity and inhibited the activities of citrate synthase and isocitrate lyase. The slightly inhibitory effect of ergotamine and stimulatory effect of ergometrine on malate synthase was established. Supplied ergometrine brought about a change in the culture growth rate,i.e. inhibition (10^?4 m) or stimulation (10^?3 m). Addition of 10^?4 m ergometrine to the culture ofClaviceps paspali MG-6 increased the total alkaloid yield by 120%. The stimulatory influence was inversely proportional to the concentration of added alkaloid. It is assumed that accumulating alkaloids suppress attainment of the yield that would be achieved by continuous control of the alkaloid level. A positive relationship is assumed between the process of alkaloid synthesis and the viability of the culture.     

Effects of colony size and polyp position on polyp fecundity in the scleractinian coral genus Acropora

This study examined the effects of colony size and polyp position on six variables of polyp fecundity [egg number, egg size, total egg volume, total testis volume, total gonad volume, and gonad ratio (egg volume/testis volume)] in three tabular Acropora corals (Scleractinia), A. hyacinthus, A. japonica, and A. solitaryensis. Samples were collected from various colony sizes (n = 21–30 colonies species^?1), just before the predicted spawning at Kochi, Japan, in 2009. Five replicate polyps were sampled at three positions (center, middle, and outer) from the center to the marginal area in each tabular colony. Results indicated effects of colony size and polyp position on both male and female gonads polyp^?1. A positive effect of colony size was observed on variables of female gonads polyp^?1 (egg number, total egg volume) in A. hyacinthus only, while the positive effect on the variable of male gonads polyp^?1 (total testis volume) was common in all Acropora species, with total testis volume polyp^?1 increasing 2–4-fold from the small (200–400 cm²) to the large size class (5,000–9,000 cm²). Among the polyp positions, lower values were observed mostly in center polyps in A. hyacinthus, while lower values were observed only in outer polyps in the other Acropora species. The distinct patterns between A. hyacinthus and the other two Acropora species suggest different reproductive strategies at the species level. Further studies are needed to confirm the prevalence of these effects in scleractinian corals, which will broaden our understanding of reproductive life history strategies and improve the estimation of reproductive performance.     

Genetic regulation of the expression of catalase activity in murine red blood cells

The specific activity (k′₁) and concentration of red blood cell catalase from four inbred strains of mice (BALB/c, C57BL, C57BL/6, and NBL) were measured to determine the mechanisms responsible for interstrain variations in enzyme activity. The specific activities of RBC catalase in NBL and the C57BL sublines are equal (2.5×10⁷ m ^?1 sec^?1), while that of BALB/c (4.0×10⁷ m ^?1 sec^?1) is 67% greater. The relative concentration of catalase is approximately 30% lower in NBL erythrocytes compared to the other three strains. The activity of BALB/c RBC catalase is due to a high k′₁ coupled with a high intracellular concentration; RBC catalase activity in the C57BL sublines is the result of a low k′₁ and high concentration. A low k′₁ and a low concentration are responsible for the low catalase activity levels found in NBL erythrocytes.     

On the Horizontal Distribution of the Profundal Bottom Fauna in a Limited Area of Central Lake Malaren,Sweden

The spatial distribution of bottom animals in a large, flat, and unexposed bottom area (about 8 km²) in central Lake Mälaren was investigated. As far as could be seen from preliminary tests the area was homogeneous in most respects. Working from the ice in all 42 tube samples and 6 Ekman samples were obtained from randomly distributed quadrats in a grid measuring 100 × 50 m². A 0.3 mm gauge net was used for sieving. The mud surface area covered with the tube samples was about 40 cm². Different methods of illustration were applied to demonstrate how many samples were actually required to show certain characteristics in the bottom fauna. For most purposes the accumulated mean value (cf. Kajak, 1963) was close enough to the stable mean value already after two or three tube samples for both chironomids and tubificids. In order to get a good idea of the five major constituents amongst tubificids (about 96% of all tubificids) two tube samples were found to be quite enough in this particular test. For the seven major constituents (about 99% of all tubificids) four tube samples were sufficient — or one single Ekman sample. The main constituents were well represented after two tube samples or one Ekman sample. Very little further information about the major constituents was obtained after the minimum number of samples mentioned above had been taken. A statistical study based on the same material was performed by a group of statisticians at the Institute of Statistics, University of Stockholm. Different statistical tests were applied to the material. Due to the relatively small number of observations nothing definitely could be said about the actual distribution of tubificids or chironomids in the area. The chironomids seemed to conform better to the Poisson distribution than to the Negative Binomial distribution, while the tubificids showed greater agreement with the Negative Binomial distribution.     

Purification of an amide hydrolase DamH from Delftia sp. T3-6 and its gene cloning,expression, and biochemical characterization

A highly active amide hydrolase (DamH) was purified from Delftia sp. T3-6 using ammonium sulfate precipitation, diethylaminoethyl anion exchange, hydrophobic interaction chromatography, and Sephadex G-200 gel filtration. The molecular mass of the purified enzyme was estimated to be 32 kDa by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The sequence of the N-terminal 15 amino acid residues was determined to be Gly-Thr-Ser-Pro-Gln-Ser-Asp-Phe-Leu-Arg-Ala-Leu-Phe-Gln-Ser. Based on the N-terminal sequence and results of peptide mass fingerprints, the gene (damH) was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). DamH was a bifunctional hydrolase showing activity to amide and ester bonds. The specific activities of recombinant DamH were 5,036 U/mg for 2′-methyl-6′-ethyl-2- chloroacetanilide (CMEPA) (amide hydrolase function) and 612 U/mg for 4-nitrophenyl acetate (esterase function). The optimum substrate of DamH was CMEPA, with K _m and k _cat values of 0.197 mM and 2,804.32 s^?1, respectively. DamH could also hydrolyze esters such as 4-nitrophenyl acetate, glycerol tributyrate, and caprolactone. The optimal pH and temperature for recombinant DamH were 6.5 and 35 °C, respectively; the enzyme was activated by Mn²⁺ and inhibited by Cu²⁺, Zn²⁺, Ni²⁺, and Fe²⁺. DamH was inhibited strongly by phenylmethylsulfonyl and SDS and weakly by ethylenediaminetetraacetic acid and dimethyl sulfoxide.     

Plasma membrane H+-ATPase in sorghum roots as affected by potassium deficiency and nitrogen sources

We studied the influence of inorganic nitrogen sources (NO₃ ^? or NH₄ ⁺) and potassium deficiency on expression and activity of plasma membrane (PM) H⁺-ATPase in sorghum roots. After 15 d of cultivation at 0.2 mM K⁺, the plants were transferred to solutions lacking K⁺ for 2 d. Then, K⁺ depletion assays were performed in the presence or absence of vanadate. Further, PMs from K⁺-starved roots were extracted and used for the kinetic characterization of ATP hydrolytic activity and the immunodetection of PM H⁺-ATPase. Two major genes coding PM H⁺-ATPase (SBA1 and SBA2) were analyzed by real-time PCR. PM H⁺-ATPase exhibited a higher V_max and K_m in NH₄ ⁺-fed roots compared with NO₃ ^? -fed roots. The optimum pH of the enzyme was slightly lower in NO₃ ^? -fed roots than in NH₄ ⁺-fed roots. The vanadate sensitivity was similar. The expressions of SBA1 and SBA2 increased in roots grown under NH₄ ⁺. Concomitantly, an increased content of the enzyme in PM was observed. The initial rate of K⁺ uptake did not differ between plants grown with NO₃ ^? or NH₄ ⁺, but it was significantly reduced by vanadate in NH₄ ⁺-grown plants.     

Counting viable Azotobacter in vertisols

In counting Azotobacter in vertisols by the soil dilution and spread-plating method, mean colony counts/plate did not decrease in proportion to the dilution factor and consequently derived counts of Azobacter cells/g soil decreased with increasing dilution of the soil suspension. This non-proportionality phenomenon was analysed in several experiments with six soils. Conformity to the Poisson distribution for counts on parallel plates was measured by Fisher's index of dispersion (χ²), which proved too high (P<.05) at lower dilutions, indicating inaccurately low mean colony counts. At highest dilutions, proportional errors increased resulting in less precise estimates of means, because with a Poisson distribution the standard deviation is equal to the square root of the mean, and the multiplication factor for derived counts/g soil is greatest at highest dilutions. By varying both plate surface area and dilution factor, results indicated that the non-proportionality phenomenon is caused by crowding at lower dilutions increasing the probability of colony coincidence on the plates. Graphical analysis of results of several dilution series indicated that derived counts are best based on dilutions giving 10–40 colonies/9 cm diameter plate, which is best achieved from two-fold dilution series.