Method For Isolation Of Bacteria Research Articles

A method for quantitation of apoplast hydration in Arabidopsis leaves reveals water-soaking activity of effectors of Pseudomonas syringae during biotrophy

The plant apoplast has a crucial role in photosynthesis and respiration due to its vital function in gas exchange and transpiration. The apoplast is also a dynamic environment that participates in many ion and nutrient transport processes via plasma membrane-localized proteins. Furthermore, diverse microbes colonize the plant apoplast, including the hemibiotrophic bacterial pathogen, Pseudomonas syringae pv. tomato (Pto) strain DC3000. Pto DC3000 initiates pathogenesis upon moving through stomata into the apoplast and then proliferating to high levels. Here we developed a centrifugation-based method to isolate and quantify the apoplast fluid in Arabidopsis leaves, without significantly damaging the tissue. We applied the simple apoplast extraction method to demonstrate that the Pto DC3000 type III bacterial effectors AvrE1 and HopM1 induce hydration of the Arabidopsis apoplast in advance of macroscopic water-soaking, disruption of host cell integrity, and disease progression. Finally, we demonstrate the utility of the apoplast extraction method for isolation of bacteria proliferating in the apoplast.

Isolation of bacteria from artificial bronchoalveolar lavage fluid using density gradient centrifugation and their accessibility by Raman spectroscopy

Raman spectroscopy is an analytical method to identify medical samples of bacteria. Because Raman spectroscopy detects the biochemical properties of a cell, there are many factors that can influence and modify the Raman spectra of bacteria. One possible influence is a proper method for isolation of the bacteria. Medical samples in particular never occur in purified form, so a Raman-compatible isolation method is needed which does not affect the bacteria and thus the resulting spectra. In this study, we present a Raman-compatible method for isolation of bacteria from bronchoalveolar lavage (BAL) fluid using density gradient centrifugation. In addition to measuring the bacteria from a patient sample, the yield and the spectral influence of the isolation on the bacteria were investigated. Bacteria isolated from BAL fluid show additional peaks in comparison to pure culture bacteria, which can be attributed to components in the BAL sample. The isolation gradient itself has no effect on the spectra, and with a yield of 63% and 78%, the method is suitable for isolation of low concentrations of bacteria from a complex matrix.Graphical abstract

Microfluidic-Based Bacteria Isolation from Whole Blood for Diagnostics of Blood Stream Infection.

Bacterial blood stream infection (BSI) potentially leads to life-threatening clinical conditions and medical emergencies such as severe sepsis, septic shock, and multi organ failure syndrome. Blood culturing is currently the gold standard for the identification of microorganisms and, although it has been automated over the decade, the process still requires 24-72 h to complete. This long turnaround time, especially for the identification of antimicrobial resistance, is driving the development of rapid molecular diagnostic methods. Rapid detection of microbial pathogens in blood related to bloodstream infections will allow the clinician to decide on or adjust the antimicrobial therapy potentially reducing the morbidity, mortality, and economic burden associated with BSI. For molecular-based methods, there is a lot to gain from an improved and straightforward method for isolation of bacteria from whole blood for downstream processing.We describe a microfluidic-based sample-preparation approach that rapidly and selectively lyses all blood cells while it extracts intact bacteria for downstream analysis. Whole blood is exposed to a mild detergent, which lyses most blood cells, and then to osmotic shock using deionized water, which eliminates the remaining white blood cells. The recovered bacteria are 100 % viable, which opens up possibilities for performing drug susceptibility tests and for nucleic-acid-based molecular identification.

Microbial Quality of some Herbal Products in Benue State

The study was conducted to investigate the microbial qualities of some herbal products in Benue State, Nigeria. A total of twenty-one samples were randomly collected from herbal medicine sales outlets and retail pharmacy outlets and subjected to bacteriological examination using pour plate and modified pour plate method for isolation of bacteria and fungi colonies respectively. Gram reaction, Potassium Hydroxide string techniques and Biochemical analysis were used for identification of bacterial isolates. While fungi colonies were identified using simplified key to classes of fungi. Four microorganisms namely Escherichia coii, Salmonella typhi, Staphylococcus aureus and Fungi namely Aspergillus sp., Mucorsp., Fusarium sp. , and Rhizopus sp . were also found to be implicated in some of the samples examined. The total bacterial counts for Escherichia coii, Staphylococcus aureus, Salmonella typhi and Fungi ranged from 1.50 × 10 3 -13.0×10 3 , 1.0×10 3 -4.0×10 3 , 1.0×10 2 -1.5×10 2 and 1.2×10 4 -6.0×10 3 cuf/ml respectively. The microbial load of the products varied considerably. Six (28.6%) of the samples were without microbial contamination while fifteen (71.4%) were contaminated by one or more of the microorganisms isolated. 7(33.3 %) of the samples were contaminated by E. coli, 1 (4.8 %), 5(23.8%) Staphylococcus aureus , 3(27.3%) Aspergillus sp. , 4(36.4%) Mucorsp ., 2(18.2%) Fusarium sp . and 2(18.2%) Rhizopus sp. respectively. This research indicated that herbal medicinal products in Benue region of Nigeria required an urgent attention in process improvement to provide better quality products for consumers.

Autoradiographic method for isolation of diverse microbial species with unique catabolic traits

A novel autoradiographic method for isolation of bacteria with unique catabolic traits was developed to overcome many of the limitations of traditional selective enrichment techniques. The method consists of five steps. (i) An environmental sample is directly plated (without enrichment) on a microporous filter atop a solid medium that allows cultivation of diverse kinds of microorganisms. (ii) Once colonies form, two replicas of the filter are prepared and the colonies are regrown. (iii) The replica filters are starved 24 to 72 h to deplete intracellular carbon reserves and then (iv) placed on Na(inf2)(sup35)SO(inf4)-containing solid media with and without a test compound. (v) Following an incubation period, the replica filters are exposed to film in order to identify colonies that incorporate more (sup35)S into cell biomass in the presence of the test compound than in its absence, providing presumptive evidence for metabolism of the compound. The colonies identified in this manner can be recovered from the master filter. To demonstrate this technique, bacteria capable of degrading benzoate were isolated from a single soil slurry by traditional enrichment as well as by autoradiography. From the enrichment culture, a single isolate able to degrade benzoate was obtained. In contrast, 18 distinct strains were obtained by purifying 19 putative benzoate-degrading colonies identified by autoradiography. Each of the 18 strains was able to completely transform the substrate, as determined by high-performance liquid chromatography analyses. The doubling times of a subset of the isolates grown in benzoate medium ranged from 1.4 to 17.1 h, whereas the doubling time of the isolate obtained by enrichment was 2.0 h. These data demonstrate that the method described here can be used to obtain a collection of diverse organisms able to metabolize a specific compound.

An Ecological Study of Lactic Acid Bacteria: Isolation of New Strains of Lactococcus Including Lactococcus lactis subspecies cremoris

We previously described a colony hybridization method for isolation of bacteria from natural environments using rRNA probes and the successful use of the method for the isolation of dairy lactococci. Herein we report the use of the method for further isolation of dairy lactococci. Thirty-eight samples from US vegetables and other plant species; 24 individual raw milk and milk product samples from the US, People's Republic of China, Morocco, Yugoslavia, and Ukraine; and 12 other samples from US dairy farms were examined for lactic acid bacteria by the colony hybridization method. Lactococcus lactis ssp. lactis occurred on potato, cucumber, sweet pea, bean, cantaloupe, corn, body and tail of cows, colostrum, raw milk from goats and cows, cottage cheese, and cream. Lactococcus lactis ssp. lactis biovar diacetylactis was isolated from raw cow milk from Morocco and from raw goat milk and cottage cheese from Yugoslavia. Lactococcus lactis ssp. cremoris was isolated from raw milks from Morocco. People's Republic of China, and Ukraine and from cottage cheese and raw milk from Yugoslavia. Acid-producing properties of isolated strains were variable. Many strains were fast acid producers, most were acceptable for flavor when grown in milk, and a few produced a malty flavor, which is considered to be a defect.

Detection of bacteria in blood by centrifugation and filtration

Culture of blood is the most frequent means of diagnosing bacteremia. However, conventional blood culturing methods are slow in isolating bacteria. We developed a method for isolation of bacteria by centrifugation and filtration. Fresh human whole blood was inoculated with facultatively anaerobic and aerobic microorganisms (3 to 172 microorganisms per 5 ml). Seeded blood was then mixed with Ficoll-Hypaque (density, 1.149 +/- 0.002 g/ml) and centrifuged (386 x g) for 30 min at ambient temperature. The entire gradient (plasma, leukocytes, and Ficoll-Hypaque) was removed and filtered through a 0.22-micron membrane filter (Millipore). The filters were then placed on chocolate agar plates and incubated at 35 degrees C in a humidified atmosphere containing 5% CO2. For each bacterium tested, approximately 35 to 100% of the viable microorganisms were recovered when compared with control cultures (pour plates of seeded blood). All bacteria produced isolated colonies on filters after overnight incubation (18 h). This procedure may prove to be a more rapid method for isolating bacteria from clinical blood samples than the blood culture bottle technique.