Immunocytochemical identification of murine and human megakaryocyte colonies in soft-agar cultures

Summary Murine megakaryocyte (MK) colonies in soft-agar cultures were immunocytochemically stained with platelet antiserum and an immuno-alkaline phosphatase procedure. Subsequently, cytochemical staining for acetylcholinesterase was used to confirm the specificity of the immunolabelling technique. The correlation of numbers of megakaryocyte colonies enumerated by independent observers was excellent. A comparable platelet antiserum directed against human platelet epitopes was utilized to identify human MK colonies in soft-agar cultures of human bone marrow. Using this method, we determined that the frequency of detectable human MK colonies in our agar culture system was maximal between days 10 and 12. The immunocytochemical staining technique we have developed for identification of MK colonies in soft-agar cultures yielded good cellular morphology and produced an intensely specific label against a clear background; it therefore facilitated accurate enumeration of MK colonies. This non-fluorescent method avoids dependence upon a non-permanent marker, and allows the simultaneous enumeration of positive and negative colonies.     

A microcytofluorometric method for quantitative and qualitative evaluation of CFU-E and BFU-E colonies

Summary A new method has been developed for the precise identification of human bone marrow colony forming unit erythroid (CFU-E) and burst forming unit erythroid (BFU-E) colonies, and for determination of the hemoglobin contents using microcytofluorometry. The method relies on a photochemical reaction in which intracellular hemoglobin is converted into fluorescent porphyrin under violet light (=405 nm) in the presence of an SH-donor (mercaptoethylamine hydrochloride). The CFU-E and BFU-E colonies showed red fluorescence with two spectrum peaks at 600 and 650 nm when illuminated by violet light. These two peaks are consistent with those of porphyrin fluorescence. The porphyrin fluorescence was not inducible in colony forming unit granulocyte-macrophage (CFU-GM) colonies, while 20% of the CFU-GM colonies were false positive with respect to the conventional benzidine reaction. The photochemically inducible fluorescence began to appear in BFU-E colonies on the 4th day of culture, while the same colonies started to be positive for the benzidine reaction on the 9th day. Therefore, the photochemical reaction was more specific and sensitive than the benzidine reaction for the identification of CFU-E and BFU-E colonies. In addition, this method enabled us to measure the hemoglobin level in the cells forming the colonies because the intensity of the fluorescence was proportional to the amount of hemoglobin when the photochemical reaction was carried out for 50 min. As a result of qualitative and quantitative analysis of CFU-E colonies by this method, it was possible to detect the hemoglobin levels in the colonies from 1 of 4 cases of untreated acute nonlymphocytic leukemia and from 2 of 4 cases of myelodysplastic syndrome in which the hemoglobin levels were too low to be detected by the benzidine reaction. These cases, where the CFU-E colonies showed very low levels of hemoglobin, were associated with poor prognosis. Thus, our method is useful for identifying CFU-E colonies, determining their hemoglobin synthesis, and as a cue to predict the clinical course of the patients.     

A Membrane Filtration Technique for the Enumeration of Escherichia coli in Seawater

S ummary . Membrane filtration has become an accepted method for enumerating Escherichia coli in water, but little published evidence could be found to judge the specificity of the method to assess faecal contamination in either fresh or saline waters. The method is used in our laboratory to monitor the extent and degree of sewage pollution in coastal areas, but there is need for information on what proportion of lactose-fermenting colonies from seawater, developing at 44° on a 4% enriched Teepol medium, are E. coli type I. A total of 1352 colonies from seawater was tested for production of indole and for gas from lactose at 44°. In addition, 46% of the colonies were screened by the IMVEC series of tests. The proportion of colonies tested ranged from 10–100%, depending on the number of colonies on the membrane. Many of the colonies (81.9%) to which IMVEC tests were applied were E. coli type I; a further 10.9% were Irregular type I. The practical implications of these findings are discussed.     

A microcytofluorometric method for quantitative and qualitative evaluation of CFU-E and BFU-E colonies.

A new method has been developed for the precise identification of human bone marrow colony forming unit erythroid (CFU-E) and burst forming unit erythroid (BFU-E) colonies, and for determination of the hemoglobin contents using microcytofluorometry. The method relies on a photochemical reaction in which intracellular hemoglobin is converted into fluorescent porphyrin under violet light (lambda = 405 nm) in the presence of an SH-donor (mercaptoethylamine hydrochloride). The CFU-E and BFU-E colonies showed red fluorescence with two spectrum peaks at 600 and 650 nm when illuminated by violet light. These two peaks are consistent with those of porphyrin fluorescence. The porphyrin fluorescence was not inducible in colony forming unit granulocyte-macrophage (CFU-GM) colonies, while 20% of the CFU-GM colonies were false positive with respect to the conventional benzidine reaction. The photochemically inducible fluorescence began to appear in BFU-E colonies on the 4th day of culture, while the same colonies started to be positive for the benzidine reaction on the 9th day. Therefore, the photochemical reaction was more specific and sensitive than the benzidine reaction for the identification of CFU-E and BFU-E colonies. In addition, this method enabled us to measure the hemoglobin level in the cells forming the colonies because the intensity of the fluorescence was proportional to the amount of hemoglobin when the photochemical reaction was carried out for 50 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

一种从培养皿上挑取阳性噬菌斑的改进方法

从密集噬菌斑的培养皿上挑选少数几个阳性噬菌斑是一件繁琐而又容易出错的工作。以往的做法是在阳性噬菌斑周围多挑选几个 ,然后通过重复杂交筛选 ,直到准确筛选出阳性噬菌斑为止 ,但该方法对于大规模筛选显得非常笨拙。这里介绍一种改进方法 ,只需一次杂交筛选即可准确挑出阳性噬斑 ,做到省时、省力、省材 ,而且准确性非常高。     

A spreading colony formed method for rapid evaluation of dicarboximide fungicides resistance level of field tobacco brown spot disease

A new method (spreading colony formed method) for rapidly identifying and evaluating the dicarboximide fungicides resistance level of field tobacco spot brown disease caused byAlternaria longipes was developed. Two typical colonies with distinct differences in colony morphology on media containing 5 μg/ml dicarboximide fungicides dimethachlon (CAS registration number: 24096-53-5) were discovered by using this method. The two typical colonies were named spreading colony and dense pad colony, respectively. Isolates (250) ofA. longipes were quickly separated by this method, and their growth properties (including the sensitivity to dimethachlon, the cross-resistance to phenylpyrroles fludioxonil and hyphal development) were examined. Our results indicated that (1) monospore isolates from spreading colonies and dense pad colonies were respectively resistant and sensitive to dimethachlon; (2) resistant and sensitive isolates formed respectively spreading colonies and dense pad colonies on dimethachlon media. Furthermore, molecular experiments confirmed the spreading colony formed method reliable. In conclusion, field resistant isolates and resistant situation in population level of field tobacco spot brown disease could be exactly and timely determined and evaluated by spreading colony formed method.     

单菌落PCR法直接快速鉴定重组克隆

利用单菌落PCR法直接筛选含有GFP、LTB-ST外源基因的重组克隆,阳性克隆可以扩增出目的条带,和质粒PCR扩增的结果一致。同时,单菌落PCR法也可应用于重组质粒转化后的农杆菌的筛选,单菌落PCR法的扩增结果和农杆菌液扩增的结果一致。结果表明,单菌落PCR法是一个有效简便的鉴定重组阳性克隆的方法。     

Novel assay for screening rifamycin B-producing mutants.

A simple method that allows easy identification of rifamycin B-producing strains is described. This method involves the use of an enzyme, rifamycin oxidase, which converts inactive rifamycin B to active rifamycin S. In this method, colonies to be tested are grown in pairs. The two colonies are then transferred to two plates seeded with a sensitive strain of Staphylococcus aureus, one plate of which contains the enzyme rifamycin oxidase. All paired colonies which show a larger inhibition zone diameter on the enzyme-containing plate are identified as rifamycin B producers.     

Novel assay for screening rifamycin B-producing mutants.

A simple method that allows easy identification of rifamycin B-producing strains is described. This method involves the use of an enzyme, rifamycin oxidase, which converts inactive rifamycin B to active rifamycin S. In this method, colonies to be tested are grown in pairs. The two colonies are then transferred to two plates seeded with a sensitive strain of Staphylococcus aureus, one plate of which contains the enzyme rifamycin oxidase. All paired colonies which show a larger inhibition zone diameter on the enzyme-containing plate are identified as rifamycin B producers.     

A note on colony-blot double-stain method for isolation of Vibrio cholerae serotype 01 from specimens heavily contaminated with non-01 Vibrio cholerae

A colony-blot double-stain method was developed to identify individual colonies of Vibrio cholerae serotype 01 (pandemic strain) in mixed bacterial cultures on solid media. The colonies are transferred from agar to nitrocellulose membranes for an enzyme-linked immunosorbent assay (ELISA). Colonies of 01 vibrios bind the enzyme-linked antibodies and appear as brown dots on the membranes; pale black dots develop at the site of replicated colonies of other bacteria as a result of the activity of endogenous oxidase-like enzymes and serve as reference points. The results indicate that the colony-blot double-stain method is useful for the isolation of colonies of V. cholerae serotype 01 in specimens that are heavily contaminated with non-01 vibrios.     

Quantification of transformation efficiency using a new method for clonal growth and selection of axenic Dictyostelium cells

A new method for clonal growth of Dictyostelium axenic amoebae has been developed. Cells are plated in growth medium containing 1% ultra-low gelling temperature agarose. Cells grow normally in the agarose and form colonies up to several millimeters in diameter. When the colonies have grown to a sufficient size, they begin multicellular development. Pseudoplasmodia are formed, migrate to the surface of the agar, and then undergo fruiting body formation. Cells can be removed from the soft agarose colonies with a toothpick or by picking spores from the fruiting bodies. This method should be useful for drug, auxotrophic, and temperature selections where clonal maintenance of axenic colonies is important. This method has been used in combination with a selection for resistance to G418 to isolate independent colonies following DNA-mediated transformation. Several parameters in the calcium phosphate and electroporation transformation protocols have been optimized and the transformation frequency quantified. Independent transformed colonies are obtained at a frequency of 1 in 10(4) to 1 in 10(5) cells when integrating plasmids are introduced using calcium phosphate coprecipitation. The frequency is about tenfold higher when extrachromosomal shuttle vectors are introduced into cells.     

入侵性害虫——苹果绵蚜田间种群数量的调查方法

2007年6月中旬到10底在山东省莱阳市对苹果绵蚜Eriosoma lanigerum(Hausmann)的田间发生数量进行调查研究。在田间定点调查5株苹果树上苹果绵蚜虫落数量和虫落面积,并在其它苹果树上随机选择20个虫落,计数每个虫落中苹果绵蚜的活体数,从而计算单位面积虫落中苹果绵蚜的平均数量。分析指出,以计算法求得的虫落中苹果绵蚜的实际数量为指标,既考虑了苹果绵蚜虫落的数量和大小,也考虑了单位面积虫落中苹果绵蚜活体数,并且把被苹果绵蚜蚜小蜂寄生的僵蚜排除在外,能较客观地反映出田间实际情况,是一种比较准确的表示苹果绵蚜田间数量的方法。     

Radioimmunological screening method for specific membrane proteins.

A simple and sensitive radioimmunoassay is described which can detect insoluble membrane proteins in single colonies of Escherichia coli K-12. The method involves transfer of colonies onto filter paper, extraction with organic solvents, exposure to radioiodinated specific immunoglobulin, and autoradiography. Some 30,000 colonies can easily be screened within a week. The method should be applicable for shot-gun-type cloning experiments aiming at genes for insoluble membrane proteins and when selection for a corresponding wild-type allelc is not possible.     

Detection of multiple-strain carriers: the value of re-sampling

Bacteria may colonize a carrier with more than one strain of a species at any one time. Attempts to determine multiple colonization are labour intensive because of the large number of colonies per carrier which need to be tested. A possible solution -- in which only 3 colonies per carrier are initially tested and only multiple-strain carriers are re-sampled -- was recently described. We evaluated the accuracy of the "re-sampling method" devised by Cespedes et al. with 500,000 stochastic simulations per scenario. Re-sampling is acceptable where > or = 5 colonies are initially tested or where one strain predominates over others in colony counts. The method introduces bias towards overestimation which decreases with the number of available carriers, increases with the proportion of truly multiple carriers, with decreasing number of colonies available for testing, and with decreasing number of colonies tested per carrier. Initial testing of 5-8 colonies tested with re-sampling are adequate for a large study (>100 carriers), or a small study where it is suspected that no strain predominates over the other in colony counts. Testing 9-20 colonies with re-sampling is necessary for small studies where one strain predominates over others. Re-sampling is unnecessary where >20 colonies are tested.     

A Replica Plating Method for Plant Cells

A replica plating method is described for plant cells growing in Petri dishes. The method involved a uniform application of plant cells (Morinda citrifolia L.) by spraying cells evenly on agar plates containing 60% conditioned medium. Subsequently the cells were allowed to grow through a nylon net. The net was removed from the master plate and placed upside down on replica plates. Cells from colonies adhering to the threads of the net were thus transferred to the replica plate and yielded colonies that, after a growth period of about 10–20 days, corresponded in position to the colonies on the master plate. An 80% transfer of colonies from the master plate to the copy plate was possible.     

Development of a Method for Detection of Lactic Acid Bacteria Producing Exclusively the l-(+)- Isomer of Lactic Acid

A method was developed for the detection and isolation, within a population of lactic acid bacteria, of strains producing exclusively the l-(+)- isomer of lactic acid; the visual detection of colonies of these particular strains can be carried out directly on agar plates (50 to 70 colonies per plate). The method is based on an enzymatic stereospecific reaction involving d-(-)-lactate dehydrogenase and linked to a staining reaction; the diffusion area of the d-(-)- isomer stains red around the d-(-)- and the dl-lactic acid-producing colonies, while the colonies producing exclusively l-(+)-lactic acid are detected by the absence of the colored halo. The intensity of staining was increased when cellulose powder and Tween 20 were added to the agar medium.     

COVASIAM: an Image Analysis Method That Allows Detection of Confluent Microbial Colonies and Colonies of Various Sizes for Automated Counting

In this work we introduce the confluent and various sizes image analysis method (COVASIAM), an automated colony count technique that uses digital imaging technology for detection and separation of confluent microbial colonies and colonies of various sizes growing on petri dishes. The proposed method takes advantage of the optical properties of the surfaces of most microbial colonies. Colonies in the petri dish are epi-illuminated in order to direct the reflection of concentrated light coming from a halogen lamp towards an image-sensing device. In conjunction, a multilevel threshold algorithm is proposed for colony separation and counting. These procedures improved the quantification of colonies showing confluence or differences in size. We tested COVASIAM with a sample set of microorganisms that form colonies with contrasting physical properties: Saccharomyces cerevisiae, Aspergillus nidulans, Escherichia coli, Azotobacter vinelandii, Pseudomonas aeruginosa, and Rhizobium etli. These physical properties range from smooth to hairy, from bright to opaque, and from high to low convexities. COVASIAM estimated an average of 95.47% (ς = 8.55%) of the manually counted colonies, while an automated method based on a single-threshold segmentation procedure estimated an average of 76% (ς = 16.27) of the manually counted colonies. This method can be easily transposed to almost every image-processing analyzer since the procedures to compile it are generically standard.     

Method of application of tylosin,an antibiotic for American foulbrood control,with effects on small hive beetle (Coleoptera: Nitidulidae) populations

The method of application of the antibiotic tylosin (Tylan) for control of oxytetracycline-resistant American foulbrood (Paenibacillus larvae White) was tested in honeybee (Apis mellifera L.) colonies. A powdered sugar mixture with tylosin, applied as a dust, was efficacious in eliminating American foulbrood symptoms at a rate of 200-mg Tylan per 20 g of powdered sugar, applied at weekly intervals for 3 weeks. A second method of treatment consisting of Tylan mixed with granulated sugar and vegetable shortening and applied once as a patty, at an equivalent total dose as the dust method, to diseased colonies also effectively eliminated symptoms of disease. In all colonies treated with patties, however, small hive beetle (Aethina tumida Murray) populations significantly increased, compared with the powder sugar method or untreated controls. Bee populations in patty-treated colonies also were significantly reduced, most likely the result of the invasion and proliferation of adult and larval small hive beetles. Such reduction in colony strength was not seen in dust-treated colonies. Because of the obvious damaging populations of small hive beetles, concerns about development of disease resistance, unknown risks of residues, and lack of support by regulatory agencies for the use of the patty method, the use of the dust method of tylosin is greatly favored over the patty method.     

Blastocystis hominis: A simplified, high-efficiency method for clonal growth on solid agar

Colony growth of protozoan parasites in agar can be useful for axenization, cloning, and viability studies. This is usually achieved with the pour plate method, for which the parasite colonies are situated within the agar. This technique has been described for Giardia intestinalis, Trichomonas vaginalis, and Entamoeba and Blastocystis species. Extracting such colonies can be laborious. It would be especially useful if parasites could be grown on agar as colonies. These colonies, being exposed on the agar surface, could be conveniently isolated for further investigation. In this study, we report the successful culture of B. hominis cells as colonies on solid agar. Colonies were enumerated and the efficiency of plating was determined. It was observed that B. hominis could be easily cultured on agar as clones. The colonies were dome-shaped and mucoid and could grow to 3 mm in diameter. Flow cytometric analyses revealed that parasite colonies remained viable for up to 2 weeks. Viable colonies were conveniently expanded in liquid or solid media. Scanning electron microscopy revealed that each colony consists of two regions; a dome-shaped, central core region and a flattened, peripheral region. Older colonies possessed numerous strand-like surface coat projections. This study provides the first report of clonal growth of B. hominis on agar and a simple, effective method for cloning and expansion of B. hominis cells.     

Application of the complex of DNA with the congo red anionic diazo dye for detection of nuclease-producing colonies of marine bacteria

This study was aimed at the development of a method for detection of colonies of nuclease-secreting marine bacteria. The BAL nuclease-producing marine bacterium Pseudoalteromonas espejiana BAL-31 was used as the test object. A new method was developed involving the congo red (CR) anionic dye. The P. espejiana culture was plated on nutrient agar with CR and denatured DNA. In such media. CR was found to form complexes with DNA. After two days of incubation at 30 degrees C, halos were found around the P. espejiana colonies. No halos appeared when DNA was not introduced, when BAL nuclease was inactivated, or when the medium was inoculated with Escherichia coli. It was concluded that the halos around the colonies indicated nuclease excretion. The halos were shown to result from the coagulation of CR released after digestion of the CR-DNA complex by the nuclease. This method for detection of nuclease-producing colonies can probably be used for all marine bacteria and possibly for halophilic bacteria as well.