scholarly journals New Strategies for Cultivation and Detection of Previously Uncultured Microbes

2004 ◽  
Vol 70 (8) ◽  
pp. 4748-4755 ◽  
Author(s):  
Bradley S. Stevenson ◽  
Stephanie A. Eichorst ◽  
John T. Wertz ◽  
Thomas M. Schmidt ◽  
John A. Breznak
Keyword(s):  
Molecular Techniques ◽  
Integrative Approach ◽  
Growth Media ◽  
Phylogenetic Groups ◽  
Culture Methods ◽  
Acid Analogue ◽  
Pure Cultures ◽  
Agar Media ◽  
Surveillance Method ◽  
New Strategies

ABSTRACT An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites. Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorum-signaling compounds (acyl homoserine lactones) in growth media. The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O2 [vol/vol]) and anoxic atmospheres. Some bacteria were incubated with elevated concentrations of CO2 (5% [vol/vol]). Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO2. A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed. This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates. Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture.

scholarly journals Dissemination of Fluoroquinolone-Resistant Campylobacter spp. within an Integrated Commercial Poultry Production System

2006 ◽  
Vol 72 (5) ◽  
pp. 3441-3447 ◽  
Author(s):  
Umelaalim Idris ◽  
Jingrang Lu ◽  
Marie Maier ◽  
Susan Sanchez ◽  
Charles L. Hofacre ◽  
...  
Keyword(s):  
Broiler Chickens ◽  
Molecular Techniques ◽  
Broiler Chicks ◽  
Campylobacter Coli ◽  
Poultry Production ◽  
Broiler Breeders ◽  
Culture Methods ◽  
Specific Pcr ◽  
Commercial Broiler ◽  
Multiple Levels

ABSTRACTWhile characterizing the intestinal bacterial community of broiler chickens, we detected ε-proteobacterial DNA in the ilea of 3-day-old commercial broiler chicks (J. Lu, U. Idris, B. Harmon, C. Hofacre, J. J. Maurer, and M. D. Lee, Appl. Environ. Microbiol.69:6816-6824, 2003). The sequences exhibited high levels of similarity toCampylobacter jejuniandCampylobacter colisequences, suggesting that chickens can carryCampylobacterat a very young age.Campylobactersp. was detected by PCR in all samples collected from the ilea of chicks that were 3 to 49 days old; however, it was detected only in the cecal contents of chickens that were at least 21 days old. In order to determine whether the presence ofCampylobacterDNA in young chicks was due to ingestion of the bacteria in food or water, we obtained commercial broiler hatching eggs, which were incubated in a research facility until the chicks hatched. DNA sequencing of the amplicons resulting fromCampylobacter-specific 16S PCR performed with the ileal, cecal, and yolk contents of the day-of-hatching chicks revealed thatCampylobacterDNA was present before the chicks consumed food or water. The 16S rRNA sequences exhibited 99% similarity toC. jejuniandC. colisequences and 95 to 98% similarity to sequences of other thermophilicCampylobacterspecies, such asC. lariandC. upsaliensis. The presence ofC. coliDNA was detected by specific PCR in the samples from chicks obtained from a commercial hatchery; however, noCampylobacterwas detected by culturing. In order to determine whether the same strains of bacteria were present in multiple levels of the integrator, we culturedCampylobactersp. from a flock of broiler breeders and their 6-week-old progeny that resided on a commercial broiler farm. The broiler breeders had been given fluoroquinolone antibiotics, and we sought to determine whether the same fluoroquinolone-resistant strain was present in their progeny. The isolates were typed by pulsed-field gel electrophoresis, which confirmed that the parental and progeny flocks contained the same strain of fluoroquinolone-resistantC. coli. These data indicate that resistantC. colican be present in multiple levels of an integrated poultry system and demonstrated that molecular techniques or more sensitive culture methods may be necessary to detect early colonization byCampylobacterin broiler chicks.

scholarly journals Effective medium for in vitro sprouting of the buds and multiplication of the plantlets, and identification of the fungal contaminant associated with the explants of Gnetum

2021 ◽  
Vol 16 (2) ◽  
pp. 001-013
Author(s):  
Abwe Mercy Ngone ◽  
Lawrence Monah Ndam ◽  
Rita Mungfu Njilar ◽  
Doungous Oumar ◽  
Thomas Eku Njock
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Fungal Growth ◽  
Growth Media ◽  
Fungal Contamination ◽  
Surface Sterilization ◽  
Pure Cultures ◽  
Nodal Cuttings ◽  
Baseline Information

Plant tissue culture requires the optimization of growth media. Gnetum, known locally in Cameroon as “Eru” is an indigenous gymnospermous vegetable with diverse medicinal, nutritional, cultural and socio-economic values. This resource is over-exploited and expected to neighboring countries, resulting to increased scarcity in the forest. Preliminary work on the in vitro culture of nodal cuttings was faced by the problem of fungal contamination. It was therefore necessary to isolate and identify the fungal contaminant, optimize the surface sterilization of field material and compose an appropriate medium for sprouting. Pure cultures of the fungus were obtained and grown on Potato Dextrose Agar (PDA) and Sabouraud Dextrose Agar (SDA). The identification was based on the appearance of the fungal growth on plates and also on the microscopic view. This was affected by the use of keys. Gnetum explants were disinfected with the various concentrations of disinfectants, preceded in some instances by pre-treatments, as well as incorporating fungicides in the culture medium. Two different culture media were employed: the Woody Plant Medium (WPM) and the Murashige and Skoog (MS) based establishment medium (Y-1). Gnetum was found to live in association with a complex of Microsporum species. The level of contamination of cultures was reduced from 100% to 40% when pre-treated before disinfection and even lower to 10% by incorporating fungicides in the medium. Sprouting was observed in WPM. This study provides baseline information on the in vitro propagation of Gnetum and thus opens up avenues for more research to be carried out in this field.

Direct detection ofCampylobacter jejuniin human stool samples by real-time PCR

2008 ◽  
Vol 54 (9) ◽  
pp. 742-747 ◽  
Author(s):  
Shiyong Lin ◽  
Xinying Wang ◽  
Haoxuan Zheng ◽  
Zhengguo Mao ◽  
Yong Sun ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Culture Method ◽  
Turnaround Time ◽  
Culture Methods ◽  
Time Saving ◽  
Whole Process ◽  
Pure Cultures ◽  
Stool Samples

Our purpose was to establish a quick and accurate real-time PCR (rtPCR) method to detect Campylobacter jejuni directly from human diarrheal stool as an alternative to traditional culture methods. To determine the consistency of rtPCR and culture method, 256 clinical diarrheal stool samples and 50 normal stool samples from healthy individuals were examined, and the whole process was double-blinded. Our data showed that the sensitivity of rtPCR in pure cultures and stool was 102CFU·mL–1and 103CFU·g–1, respectively. Of the 256 diarrheal samples, 10 specimens were successfully detected by both methods, whereas two specimens were PCR positive but culture negative. No positive results were found by these two methods in 50 normal specimens. Our data suggested that rtPCR was convenient in operation and time-saving (turnaround time 3.5–4 h), so it could be used for clinical diagnostic and epidemiological purposes.

scholarly journals Natural Compounds as Elicitors of Plant Resistance Against Diseases and New Biocontrol Strategies

Agronomy ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 173 ◽  
Author(s):  
Agnieszka Jamiołkowska
Keyword(s):  
Plant Resistance ◽  
Organic Agriculture ◽  
Plant Protection ◽  
Self Regulation ◽  
Natural Compounds ◽  
Molecular Techniques ◽  
Plant Production ◽  
Animal Origin ◽  
Chitin And Chitosan ◽  
New Strategies

The goal of sustainable and organic agriculture is to optimize the health and productivity of interdependent communities of soil life, plants, animals, and people. Organic plant production uses natural products and natural self-regulation processes occurring in the ecosystem. The availability of innovative applications and molecular techniques opens up new possibilities in the approach to plant protection for sustainable and organic agriculture. New strategies not only directly protect plants against pathogens but can also induce enhanced immunity that permanently protects against pathogenic strains. This review focuses on the bioactive properties of selected natural compounds (of plant and animal origin), their action on pathogens, and their roles in the mechanism of inducing plant resistance. The author presents selected activities of organic bioactive compounds, such as allicin, naringin, terpenes, laminarin, carrageenans, chitin and chitosan, and outlines the possibilities for their application in protecting crop plants against diseases. In addition, this mini review describes the mechanism of action of the above compounds as elicitors of defense reactions in the plant and the possibility of their utilization in the production of biological preparations as elements of a new plant protection strategy.

scholarly journals Rapid Diagnosis of Blood Stream Infections in ICU: Recent Advances

2019 ◽  
Vol 2 (1) ◽  
pp. 177-180
Author(s):  
Raju Prasad Pangeni
Keyword(s):  
Intensive Care ◽  
Diagnostic Tests ◽  
Rapid Detection ◽  
Blood Stream ◽  
Turnover Time ◽  
Rapid Diagnosis ◽  
Molecular Techniques ◽  
Culture Methods ◽  
Blood Stream Infections

Blood stream infections and sepsis are major causes of hospitalization in most intensive care units around the globe, especially in developing countries like Nepal. Although the sepsis guidelines emphasize the role of early institution of appropriate antibiotics, it is practically challenging due to delayed turnover time of currently available diagnostic tests. Modifications in traditional blood culture methods, use of molecular techniques and availability of biomarkers have raised hope in rapid detection of blood stream infections.

scholarly journals mcrA-Targeted Real-Time Quantitative PCR Method To Examine Methanogen Communities

2009 ◽  
Vol 75 (13) ◽  
pp. 4435-4442 ◽  
Author(s):  
Lisa M. Steinberg ◽  
John M. Regan
Keyword(s):  
Quantitative Pcr ◽  
Alternative Energy ◽  
Molecular Techniques ◽  
Pig Manure ◽  
Phylogenetic Groups ◽  
Anaerobic Digesters ◽  
Α Subunit ◽  
Real Time Quantitative Pcr ◽  
Three Samples ◽  
Culture Independent

ABSTRACT Methanogens are of great importance in carbon cycling and alternative energy production, but quantitation with culture-based methods is time-consuming and biased against methanogen groups that are difficult to cultivate in a laboratory. For these reasons, methanogens are typically studied through culture-independent molecular techniques. We developed a SYBR green I quantitative PCR (qPCR) assay to quantify total numbers of methyl coenzyme M reductase α-subunit (mcrA) genes. TaqMan probes were also designed to target nine different phylogenetic groups of methanogens in qPCR assays. Total mcrA and mcrA levels of different methanogen phylogenetic groups were determined from six samples: four samples from anaerobic digesters used to treat either primarily cow or pig manure and two aliquots from an acidic peat sample stored at 4°C or 20°C. Only members of the Methanosaetaceae, Methanosarcina, Methanobacteriaceae, and Methanocorpusculaceae and Fen cluster were detected in the environmental samples. The three samples obtained from cow manure digesters were dominated by members of the genus Methanosarcina, whereas the sample from the pig manure digester contained detectable levels of only members of the Methanobacteriaceae. The acidic peat samples were dominated by both Methanosarcina spp. and members of the Fen cluster. In two of the manure digester samples only one methanogen group was detected, but in both of the acidic peat samples and two of the manure digester samples, multiple methanogen groups were detected. The TaqMan qPCR assays were successfully able to determine the environmental abundance of different phylogenetic groups of methanogens, including several groups with few or no cultivated members.

scholarly journals EKSTRAKSI RUMPUT LAUT Gelidium sp. MENJADI BAKTO AGAR SEBAGAI PEMADAT MEDIA PERTUMBUHAN MIKROBA

2019 ◽  
Vol 9 (2) ◽  
pp. 71
Author(s):  
Zachra Resha Shantika ◽  
Srikandi Srikandi ◽  
RTM Sutamihardja
Keyword(s):  
Trace Elements ◽  
Bacillus Subtilis ◽  
Culture Media ◽  
Economic Value ◽  
Growth Media ◽  
Microbial Culture ◽  
Metal Levels ◽  
Bacterial Morphology ◽  
Microbiological Testing ◽  
Agar Media

Extraction of Seaweed Grass Gelidium Sp. Become A Bakto For A Microbic Growth Media Competition      Red seaweed is seaweed that produces gelatin one example is Gelidium sp. The product that has high economic value is bakto agar which is widely used in the field of microbiology, namely as a microbial culture media compactor. The use of bacto so that it will increase domestically, so as to meet the needs of bakto so that in the country with the expected quality classification with bakto so that the commercial is carried out this research. Agar yield value of seaweed type Gelidium sp. produced an average of 26.69%. The proximate test results for the parameters of water content, levels of ash, insoluble ash and sulfate in bakto so that the research is 6.89%, 1.67%, 0.0235%, and 1.31% respectively. The results of testing the levels of protein, fat, carbohydrate and gel strength at bacto so that respectively are 1.72%, 0.05%, 32.87% and 214.45 (g / cm2). The results of testing the mineral metal levels of calcium, magnesium and potassium were obtained at bakto so that they were 1806.06 mg / kg, 680 mg / kg and 338.22 mg / kg respectively. The results of testing the levels of trace elements of iron and copper were obtained at 8.75 mg / kg and 2.10 mg / kg. The results of microbiological testing using the total plate number method using Bacillus subtilis and Eschericia coli bacteria were found to be 8.6 x 106 colonies / mL and 2.81 x 107 colonies / mL. Based on the observation of bacterial morphology by comparing it with bakto so that the commercial standard in general is not much different. So based on the results of proximate and microbiological testing, bakto so that this research can be used as a medium for bacterial growth.Keywords: Gelidium sp., Agar, media and bacteriaABSTRAK      Rumput laut merah merupakan rumput laut yang menghasilkan agar-agar salah satu contohnya adalah Gelidium sp. Produk agar yang memiliki nilai ekonomis tinggi adalah bakto agar yang banyak digunakan dalam bidang mikrobiologi yaitu sebagai pemadat media kultur mikroorganisme. Pemanfaatan bakto agar di dalam negeri semakin meningkat, sehingga untuk mencukupi kebutuhan bakto agar di dalam negeri dengan klasifikasi mutu yang diharapkan sama dengan bakto agar komersial maka dilakukan penelitian ini. Nilai rendemen agar dari rumput laut jenis Gelidium sp. dihasilkan rata-rata sebesar 26.69%. Hasil pengujian proksimat untuk parameter kadar air, kadar abu, abu tak larut asam dan sulfat pada bakto agar penelitian ini berturut-turut adalah sebesar 6,89%, 1,67%, 0,0235%, dan 1.31%. Hasil pengujian kadar protein, lemak, karbohidrat dan kekuatan gel pada bakto agar berturut-turut adalah sebesar 1,72%, 0,05%, 32,87% dan 214,45 (g/cm2). Hasil pengujian kadar logam mineral kalsium, magnesium dan kalium didapatkan pada bakto agar berturut-turut adalah sebesar 1806,06 mg/kg, 680 mg/kg dan 338,22 mg/kg. Hasil pengujian kadar logam trace elements besi dan tembaga didapatkan sebesar 8.75 mg/kg dan 2,10 mg/kg. Hasil pengujian mikrobiologi dengan metode angka lempeng total dengan menggunakan bakteri Bacillus subtilis dan Eschericia coli didapatakan sebesar 8,6 x 106 koloni/mL dan 2,81 x 107 koloni/mL. Berdasarkan hasil pengamatan morfologi bakteri dengan membandingkannya dengan bakto agar standar komersial secara garis besar tidak jauh berbeda. Sehingga berdasarkan hasil pengujian proksimat dan mikrobiologi, bakto agar penelitian ini dapat digunakan sebagai media pertumbuhan bakteri.Kata kunci: Gelidium sp., Agar, media dan bakteri

scholarly journals A Hidden Pitfall in the Preparation of Agar Media Undermines Microorganism Cultivability

2014 ◽  
Vol 80 (24) ◽  
pp. 7659-7666 ◽  
Author(s):  
Tomohiro Tanaka ◽  
Kosei Kawasaki ◽  
Serina Daimon ◽  
Wataru Kitagawa ◽  
Kyosuke Yamamoto ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Environmental Samples ◽  
Agar Medium ◽  
Total Cell ◽  
Plate Count ◽  
Growth Media ◽  
Content Type ◽  
Cell Counts ◽  
Soil Sediment ◽  
Agar Media

ABSTRACTMicrobiologists have been using agar growth medium for over 120 years. It revolutionized microbiology in the 1890s when microbiologists were seeking effective methods to isolate microorganisms, which led to the successful cultivation of microorganisms as single clones. But there has been a disparity between total cell counts and cultivable cell counts on plates, often referred to as the “great plate count anomaly,” that has long been a phenomenon that still remains unsolved. Here, we report that a common practice microbiologists have employed to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower than those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture ofGemmatimonas aurantiacaT-27Tand three representative sources of environmental samples, soil, sediment, and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of CFU on PS medium than on PT medium usingG. aurantiacaor any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen peroxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated microbes grew on PS than on PT medium.

scholarly journals Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques

2001 ◽  
Vol 67 (7) ◽  
pp. 2942-2951 ◽  
Author(s):  
Beatriz Dı́ez ◽  
Carlos Pedr�s-Ali� ◽  
Terence L. Marsh ◽  
Ramon Massana
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
18S Rrna Gene ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Phylogenetic Groups ◽  
Gradient Gel Electrophoresis ◽  
Phylogenetic Identification ◽  
Phylogenetic Composition ◽  
Primer Sets

ABSTRACT We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea. Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked. The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample. The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses. DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes. In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members.

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