Survival of Bacteria in Lyophilized Plasma

Although lyophilization reduces the bacterial population in plasma, some species survive for several months. Sensitivity or resistance to lyophilization in plasma is a characteristic of each morphological type, with gram-negative species being more sensitive to destruction than cocci or sporulating gram-positive species. Reconstituted preparations of lyophilized plasma must be incubated for 24 hr to prevent false-negative sterility tests. The killing process in lyophilized plasma continues with time if the preparations are stored at room temperature. Strict asepsis must be realized during processing of plasma because lyophilization alone does not destroy the microorganisms present.     

Targeted Proteomics for Studying Pathogenic Bacteria

Mass spectrometry‐based proteomics has been extensively used to map bacterial proteomes, which has led to a better understanding of the molecular mechanisms underlying bacterial infection and bacteria–host interactions. Quantitative proteomics using selected or parallel reaction monitoring is considered one of the most sensitive and specific quantitative MS‐based approaches and has significantly advanced proteome studies of pathogenic bacteria. Here, recent applications of targeted proteomics for bacteria identification, biomarker discovery, and the characterization of bacterial virulence and antimicrobial resistance are reviewed among others. Results of such studies are expected to further contribute to improve the fight against the most common human pathogenic bacteria.     

Correlations Between Free Radical Production and Viability of Lyophilized Bacteria

When Serratia marcescens cultures were treated with dilute solutions of phenol or hydrogen peroxide before drying or by lyophilization at suboptimal pH, the log of the number of cells surviving lyophilization was correlated with subsequent free radical production by the dried cells. Since the rate of free radical production and rate of death were similarly affected by temperature, the log of the number of cells surviving after 6 days was inversely related to the free radical concentration at that time. Free radicals were produced in proportion to the log of oxygen pressure, and viability was inversely related to oxygen tension; again, free radical concentration appeared to be correlated with the death of organisms.     

Smear Technique for Studying Lepidopteran Chromosomes

There is scant information on the chromosomes of Lepidoptera, the largest order of insects with more than 100,000 described species (Imms 1965), despite the abundance of the material and cosmopolitan distribution of many species. the lack of information on the chromosomes (the haploid number being known for about 1% of the total number of species) and detailed analysis of the karyotype in this order may be partly due to the small size and isodiametric nature of the chromosomes and partly due to the difficulty in obtaining satisfactory squash preparations of the adult testes, which are enclosed together in a thick-walled scrotum. This difficulty of the interference of the scrotum in obtaining stages satisfactory for study (fixation of the material worsens the situation, for the scrotal wall hardens on fixation) seems to have discouraged the cytological study of this group, especially in the adults. Some workers have tried to circumvent this difficulty by squashing larval testes, which are not enclosed in a common scrotum. This method, however, calls for rearing larvae through the pupal stages for correct identification of the adults.     

Method for Studying Particle Size and Infective Potential of Infectious Bovine Rhinotracheitis Virus Aerosols

A technique is described for estimating the number of potential respiratory infectious loci of aerosolized infectious bovine rhinotracheitis virus.     

Dialysis Membrane Technique for Studying Microbial Interaction

A dialysis membrane method is described which allows (i) cultivation of fungi on an agar support, (ii) observation of growth and development by direct light microscopy, (iii) transfer of cultures from agar surfaces for subsequent treatments or for biochemical analysis, and (iv) preparation for scanning electron microscopy. The method is used routinely in studies of fungus-nematode and fungus-fungus interactions.     

A Flow-Cytometric Gram-Staining Technique for Milk-Associated Bacteria

A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50°C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at −18°C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5°C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.     

An Unsterilized Soil-agar Technique for Studying Rhizobium-plant Relationships

The value of unsterilized soil-agar slopes for studying the effect of chemical and biological characteristics of soil on the rhizobia-plant relationships has been demonstrated by examining the characteristics of 4 strains of Rhizobium meliloti isolated from salt-affected soils of Greece. The technique enabled information to be obtained on: the survival of indigenous rhizobia and bacteria; the survival of rhizobia added to soil; the antagonistic-symbiotic effects, and the effectiveness of inoculation on nitrogen fixation to be closely followed.     

Joint Immobilization of Plant Growth-Promoting Bacteria and Green Microalgae in Alginate Beads as an Experimental Model for Studying Plant-Bacterium Interactions

A simple, quantitative experimental model, offering a convenient and basic approach to studies of plant-bacterium interactions, is proposed. This involves immobilizing a unicellular, freshwater microalga, a Chlorella species, serving as the plant, with a plant growth-promoting bacterium, an Azospirillum species, in small alginate beads to allow close interaction and to avoid external interference from bacterial contaminants.     

Ultrasonication—An Enrichment Technique for Microcyst-forming Bacteria

Brief ultrasonic treatment of samples of bark, soil or water containing fruiting myxobacteria yields higher isolation rates than conventional procedures when aliquots are applied to solid media. Many normal contaminants, including nematodes, are destroyed and representative strains of most genera of Myxobacteria and Sporocytophaga have been isolated in this way. The technique can also be applied to the isolation of cyst-forming Azotobacter spp. and may be suitable for cyst-forming Methanobacterium spp. A further application is in the comparison of bacillary and resting forms in natural environments.     

Improved Filtration Technique for Concentrating and Harvesting Bacteria

An improved technique is described for the filtrative concentration and harvesting of bacterial cultures. A pleated tangential flow filtration unit containing 1,000 cm² of 0.2-μm-pore-size microporous membrane was used to rapidly (30 to 50 min) reduce the volume of 5 liters of bacterial culture of approximately 10⁹ cells per ml to 0.2 to 0.5 liters of concentrated bacterial suspension. The effects of cell concentration, filtration pressure, and tangential flow rate were examined with respect to the rate of concentration and cell viability. Recovery efficiencies were between 60 and 75%, with no apparent impairment of organism viability. Cell concentration exerted the predominant effect on the filtration rate.     

Use of Real-Time PCR Technique in Studying Rumen Cellulolytic Bacteria Population as Affected by Level of Roughage in Swamp Buffalo

A real-time polymerase chain reaction approach was used in this study to determine the population of major ruminal bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) in digesta and rumen fluid of swamp buffalo (Bubalus bubalis). Four rumen-fistulated, male swamp buffalo were randomly assigned according to a 4 × 4 Latin square design to evaluate the effect of the urea-treated rice straw (roughage source)-to-concentrate ratio on cellulolytic bacterial distribution. Animals were fed roughage-to-concentrate (R:C) ratios of 100:0, 75:25, 50:50, and 25:75, respectively. At the end of each period, rumen fluid and digesta were collected at 0 h and 4 h post-morning-feeding. It was found that feeding urea-treated rice straw solely increased these three cellulolytic bacteria numbers up to 2.65 × 10⁹ and 3.54 × 10⁹ copies per milliliter for F. succinogenes, 5.10 × 10⁷ and 7.40 × 10⁷ copies per millilter for R. Flavefaciens, and 4.00 × 10⁶ and 6.00 × 10⁶ copies per milliliter for R. albus in rumen fluid and digesta, respectively. The distribution of the three cellulolytic bacteria species in digesta were highest at 3.21 × 10⁹, 4.55 × 10⁷, and 4.56 × 10⁶ copies per milliliter for F. succinogenes, R. flavefaciens, and R. albus, respectively. Moreover, at 4 h post-morning-feeding, the populations of the three cellulolytic bacteria were higher than found at 0 h post-morning-feeding. It is most notable that F. succinogenes were the highest in population in the rumen of swamp buffalo and cellulolytic bacteria mostly adhered to feed digesta in the rumen.     

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a ‘phloem sandwich’ and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.     

Technique and Apparatus for Rapid and Inexpensive Enumeration of Bacteria

A foot-operated diluter/dispenser and a projection viewer were developed for use with a rapid bacterial counting technique employing agar droplets. The equipment allows the advantages of the technique to be properly realized and assists many conventional bacteriological tests which may be made at the same time. Significant cost and labor savings are made, with reductions in incubation time, incubator space, and preparative work.     

生物滤塔体系好氧反硝化菌的分离鉴定与特性研究

从实验室生物滤塔填料的生物膜上, 经选择性培养基筛选, 分离出4株好氧反硝化菌。好氧状态下4种菌的40 h反硝化率均大于80%, 其中菌种A1反硝化率可达到99.05%。跟踪菌种反硝化过程中氮元素24 h变化过程, 发现4株菌除A1外都有亚硝酸根积累。菌种A1为短杆菌, 革兰氏阴性。生理生化特性研究与16S rDNA 序列测定(GenBank接受号DQ836052.1)初步判定菌种A1为假单胞菌Pseudomonas putida。适于菌A1生长的初始pH值是7.0左右, 温度30℃左右, 当DO大于2.0 mg/L时, DO的变化对菌种A1的反硝化效果影响很小。     

生物滤塔体系好氧反硝化菌的分离鉴定与特性研究

从实验室生物滤塔填料的生物膜上,经选择性培养基筛选,分离出4株好氧反硝化茵.好氧状态下4种菌的40h反硝化率均大于80%,其中菌种A1反硝化率可达到99.05%.跟踪菌种反硝化过程中氮元素24h变化过程,发现4株菌除A1外都有亚硝酸根积累.茵种A1为短杆菌,革兰氏阴性.生理生化特性研究与16S rDNA序列测定(GenBank接受号DQ836052.1)初步判定菌种A1为假单胞菌Pseudomonas putida.适于茵Al生长的初始pH值是7.0左右,温度30℃左右,当DO大于2.0mg/L时,DO的变化对菌种Al的反硝化效果影响很小.     

An Experimental Method for Studying the Differentiation of Vessel Endings

A simple experimental method for studying the differentiationof vessel endings is described. Vessel endings are induced andtheir location is determined experimentally along an internodeby a complete horizontal cut. The frequency of vessel endingsis very high in the first 5 mm immediately below the cut andtheir number declined sharply in the basipetal direction. Patternsof the secondary wall of complete and partly dissolved vesselendings are demonstrated. Hydraulic safety zone, Luffa cylindrica, vessel differentiation, vessel endings, xylem anatomy     

A Modified Bradley's Squash Technique for Studying Embryo Sacs in Gramineae

This procedure is especially suited for studying the embroyology of sexual and apomictic grasses. Material is fixed in a 2:2:1 alcohol-chloroform-propionic acid mixture for a minimum period of 2 days, soaked in 4% iron alum at 75 C for 7 min, and 2 min each in 2 changes of distilled water, also at 75 C. After 2-3 min in cold water, it is macerated in 50% HCI for 10 min at about 22-25 C, washed and mordanted for 12-16 hr in 50% alcohol saturated with ferric acetate. Ovules are then dissected out and squashed in 1% carmine in 45% propionic acid. Squashing should be firm enough to separate and flatten the embryo sacs but not to burst them. The slides are set aside for 12-24 hr for intensification of the stain.