Evaluation of an Automatic Electronic Device for Counting Bacterial Colonies

An automatic colony counter was tested extensively with colonies of two bacterial species, Serratia marcescens and Bacillus subtilis var. niger, grown on agar media. A stable relationship was established between machine counts and counts done visually by technicians. The calibration curve and estimates of the efficiency of the machine are presented and discussed. It is estimated that a 40% reduction in colony counting time is feasible through use of the machine.     

供光学显微镜观察的花粉样品制备的一种简单方法

传统的制备供光学显微镜观察的花粉样品的方法 (中国科学院植物研究所形态室孢粉组 ,1 960 )不但程序复杂 ,而且不同种类的花粉容易混杂。最近 ,我们在进行山茶属(Ｃａｍｅｌｌｉａ)花粉形态的系统研究中总结出一种制备供光镜观察的花粉材料的简单方法 ,现将其过程介绍如下 :( 1 )从标本或新鲜植株上取下花药 ,用冰醋酸浸软后 ,置洁净的凹玻片 (单凹玻片 )上 ,于解剖镜下将花药打开 ,滴上 95%酒精将花粉洗出。( 2 )滴上预先配制好的分解液 (醋酸酐 9份和浓硫酸 1份 ) ,于室温下或 50℃恒温箱里放置 5ｍｉｎ(具体温度和时间因花粉种类而异 ) …     

Technical Modification of the Method for Direct and Indirect Immunofluorescence of Unfixed Mycoplasma Colonies

1. A more reliable, time-saving and easily performed technical modification of the method for direct and indirect immunofluorescence of mycoplasma colonies, involving the use of steel rings to hold the agar-block specimens, is described.     

激光扫描共聚焦显微镜观察细菌生物膜形成的方法学研究

目的:建立激光扫描共聚焦显微镜观察生物膜形成过程的方法,为进一步研究生物膜的形成机制奠定基础。方法:以临床分离金葡菌X428为研究对象,在盖玻片上形成生物膜,分别于接种后的4、8、12、16、24和48h取出玻片,采用免疫荧光技术标记多糖和细菌,激光扫描共聚焦显微镜(CLSM)观察生物膜形成情况。结果:取得了生物膜形成过程的不同时间点的CLSM图像,4h时细菌在盖玻片上粘附形成小菌落,8h和12h细菌聚集成簇,多糖基质产生并逐渐增多,至16h形成成熟生物膜结构;24h和48h生物膜已经播散,其结构变小。结论:应用免疫荧光技术和激光扫描共聚焦显微镜技术研究生物膜形成过程是一种简便可行的方法。     

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.     

Improved Technique for Identification and Enumeration of Methanogenic Bacterial Colonies on Roll Tubes by Epifluorescence Microscopy

Methanogenic fluorescent colonies can be clearly identified on roll tubes by using an epifluorescence microscope equipped with a × 2 objective. Methanogenic and nonmethanogenic colonies could be counted in roll tubes prepared from methanogenic enrichment cultures. Late-developing colonies appearing after 25 days of incubation were mainly methanogenic.     

Repulsion and Metabolic Switches in the Collective Behavior of Bacterial Colonies

Bacteria inoculated on surfaces create colonies that spread out, forming patterns shaped by their mutual interactions. Here, by a combination of experiments and modeling, we address two striking phenomena observed when colonies spread out circularly, without dendritic instabilities. First, the velocity of spreading is generically found to decrease as levels of nutrients initially deposited on the surface increase. We demonstrate that the slowdown is due to phenomena of differentiation, leading to the coexistence of bacteria in different states of motility and we model their dynamics. Second, colonies spreading out from different inocula on the same surface are observed to merge or repel (halting at a finite distance), depending on experimental conditions. We identify the parameters that determine the fate of merging versus repulsion, and predict the profile of arrest in the cases of repulsion.     

Hoechst 33258 Staining for Detecting Mycoplasma Contamination in Cell Cultures: a Method for Reducing Fluorescence Photobleaching

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.     

Photoprotection by Dipicolinate Against Inactivation of Bacterial Spores with Ultraviolet Light

The resistance of three types of Bacillus cereus T spores to ultraviolet radiation corresponded to their dipicolinic acid (DPA) content. Photoprotection against ultraviolet light was observed in DPA-containing spores and in DPA-less spores irradiated through calcium dipicolinate.     

Imaging by Delayed Light Emission (Phytoluminography) as a Method for Detecting Damage to the Photosynthetic System

An improved apparatus for obtaining luminescence (delayed light emission) images of plants is described. It consists of a phosphoroscope equipped with an imaging lens and an electronic image intensifier. It is also equipped with light-sources for obtaining images with reflected light and fluorescence light. It is shown that damage to the photosynthetic system caused by virus, insects, high or low temperature, ultraviolet radiation, or herbicide, and also chioroplast senescence as part of a normal developmental process, can be followed by this non-destructive method. In many cases changes which are not visible in fluorescence images are clearly seen in luminescence images.     

Improved Method for Axenizing Blepharisma by Means of Antibiotics

Synopsis.
A simplified method for obtaining axenic cultures of Blepharisma involves the use of penicillin, streptomycin, and tetracycline. Cytologic examination disclosed no significant effect of these antibiotics on the structure of the ciliates.     

Three-Dimensional Apoptotic Nuclear Behavior Analyzed by Means of Field Emission in Lens Scanning Electron Microscope

Apoptosis is an essential biological function required during embryogenesis, tissue home-ostasis, organ development and immune system regulation. It is an active cell death pathway involved in a variety of pathological conditions. During this process cytoskeletal proteins appear damaged and undergo an enzymatic disassembling, leading to formation of apoptotic features. This study was designed to examine the three-dimensional chromatin behavior and cytoskeleton involvement, in particular actin re-modeling. HL-60 cells, exposed to hyperthermia, a known apoptotic trigger, were examined by means of a Field Emission in Lens Scanning Electron Microscope (FEISEM). Ultrastructural observations revealed in treated cells the presence of apoptotic patterns after hyperthermia trigger. In particular, three-dimensional apoptotic chromatin rearrangements appeared involving the translocation of filamentous actin from cytoplasm to the nucleus. FEISEM immunogold techniques showed actin labeling and its precise three-dimensional localization in the diffuse chromatin, well separated from the condensed one. The actin presence in dispersed chromatin inside the apoptotic nucleus can be considered an important feature, indispensable to permit the apoptotic machinery evolution.Key words: Chromatin, F-actin, immunogold, TEM, FEISEM     

Spindle Imaging in Living Mammalian Oocytes with Polarized Light Microscope and its Practical Use

Spindle Imaging in Living Mammalian Oocytes with Polarized Light Microscope and its Practical Use     

A Light Microscope Method for Following the Incorporation of Gluten Into Dough or Other Foodstuffs Containing Different Proteins

It has been shown that gluten powder stained for one minute and forty-five seconds in a 0.192% solution of fast green FCF and then washed repeatedly with distilled water can be detected both by visible light and fluorescence microscopy when incorporated into doughs. Evidence that the functional properties of the gluten are not affected by this treatment suggested that the technique may be useful for following the incorporation of other proteins into various food systems.     

Interplay between Intrinsic Noise and the Stochasticity of the Cell Cycle in Bacterial Colonies

Herein we report on the effects that different stochastic contributions induce in bacterial colonies in terms of protein concentration and production. In particular, we consider for what we believe to be the first time cell-to-cell diversity due to the unavoidable randomness of the cell-cycle duration and its interplay with other noise sources. To that end, we model a recent experimental setup that implements a protein dilution protocol by means of division events to characterize the gene regulatory function at the single cell level. This approach allows us to investigate the effect of different stochastic terms upon the total randomness experimentally reported for the gene regulatory function. In addition, we show that the interplay between intrinsic fluctuations and the stochasticity of the cell-cycle duration leads to different constructive roles. On the one hand, we show that there is an optimal value of protein concentration (alternatively an optimal value of the cell cycle phase) such that the noise in protein concentration attains a minimum. On the other hand, we reveal that there is an optimal value of the stochasticity of the cell cycle duration such that the coherence of the protein production with respect to the colony average production is maximized. The latter can be considered as a novel example of the recently reported phenomenon of diversity induced resonance.