Rapid Method For Isolation Research Articles

A Modified Combination of Two Methods for Isolation of Polymorphonuclear Granulocytes from Human Blood for Polymerase Chain Reaction (PCR).

The aim of the study was to determine a rapid and simple method for isolation of polymorphonuclear (PMN) granulocytes from human blood appropriate for PCR of extracted DNA. A modification of the combined dextran plus Ficoll-Hypaque methods was the final method used in 41 samples of normal white cells from humans. The changes encompassed a lower dextran-blood ratio (2:1), a shorter centrifugation time of leukocytes, a small volume of Ficoll-Hypaque suspension, and a decreased speed in the last centrifugation. The final key point was skipping the lysis of erythrocytes to avoid interference by salt solutions. These modifications rendered a mean PMN granulocyte recovery and purity of over 95% with a very narrow 2 - 8% coefficient of variation and did not alter the purity of DNA extraction. The combined dextran plus Ficoll-Hypaque method changes offer a fast and simple method to obtain an adequate purity of granulocytes for PCR.

Cytotoxic effects of ergone, a compound isolated from Fulviformes fastuosus.

BackgroundMushrooms inspired the cuisines of many cultures and conventional medicaments for cancer. However, a substantial number of mushroom species are yet unexplored, possessing an unknown chemical, biological and pharmacological profiles. Fulviformes fastuosus is a terrestrial mushroom, which is commonly found in Sri Lankan woodlands. The current study was aimed at isolation and characterization of a potent cytotoxic compound from F. fastuosus and investigating the apoptotic effect induced by the active principle against cancer and normal cell lines.MethodsBioactivity guided isolation of active principles from the methanol extract of F. fastuosus was performed by a rapid extraction and isolation method using different chromatographic techniques. Potential cytotoxic compound was identified using one and two dimensional nuclear magnetic resonance spectroscopy and mass spectrometry. Isolated compound was screened for in vitro cytotoxicity against Hepatocellular carcinoma (HepG-2), Muscle rhabdomyosarcoma (RD) and Rat Wistar liver normal (CC-1) cell lines using 3 4, 5-(dimethylthiazol-2-yl) 2-5-diphenyl tetrazolium bromide (MTT) cell viability assay. Apoptotic features of cells were observed via microscopic examination and ethidium bromide/acridine orange fluorescent staining.ResultsThe interpretation of spectral data resulted in the identification of the chemical structure as ergosta-4,6,8 (14),22-tetraen-3-one (ergone). Ergone exhibited promising cytotoxic properties against RD cells with less cytotoxicity effect on CC-1 cells. In addition, ergone also possesses a strong cytotoxic effect against HepG-2 cells showing low toxic level for CC-1 cells. Apoptotic features of treated cells were detected via morphological characterization and ethidium bromide/acridine orange staining.ConclusionThe present study elaborates the isolation of a potent cytotoxic compound; ergone, from F. fastuosus via a rapid and efficient isolation method. Importantly, ergone has exhibited greater cytotoxic activity against RD cells with high selectivity index compared to cytotoxicity against HepG-2 cells. Ergone can be used in the development of therapeutic strategies for curbing rhabdomyosarcoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1471-8) contains supplementary material, which is available to authorized users.

Invert biopanning: A novel method for efficient and rapid isolation of scFvs by phage display technology

Phage display is a prominent screening technique for development of novel high affinity antibodies against almost any antigen. However, removing false positive clones in screening process remains a challenge. The aim of this study was to develop an efficient and rapid method for isolation of high affinity scFvs by removing NSBs without losing rare specific clones. Therefore, a novel two rounds strategy called invert biopanning was developed for isolating high affinity scFvs against EGFRvIII antigen from human scFv library. The efficiency of invert biopanning method (procedure III) was analyzed by comparing with results of conventional biopanning methods (procedures I and II). According to the results of polyclonal ELISA, the second round of procedure III displayed highest binding affinity against EGFRvIII peptide accompanied by lowest NSB comparing to other two procedures. Several positive clones were identified among output phages of procedure III by monoclonal phage ELISA which displayed high affinity to EGFRvIII antigen. In conclusion, results of our study indicate that invert biopanning is an efficient method for avoiding NSBs and conservation of rare specific clones during screening of a scFv phage library. Novel anti EGFRvIII scFv isolated could be a promising candidate for potential use in treatment of EGFRvIII expressing cancers.

Abstract 3149: Enumeration and mutational profiling of CTCs and comparison to ctDNA and colorectal cancer liver metastases

Abstract Background Colorectal cancer (CRC) is the 3rd most common cancer diagnosed worldwide in both men and women. Only 39% of cancers are diagnosed at a localized stage, and 5-year survival rates decrease rapidly for patients with advanced and metastasized disease (stage III 61%, stage IV 8%). Better markers for detection of disease progression, therapeutic resistance and minimal residual disease are still needed. Liquid biopsies, such as CTCs and ctDNA, are emerging biomarkers shed by the tumor into the blood stream. Both markers currently are attracting growing interest for their use in disease prognosis, early detection of recurrence and are promising candidates for guiding cancer therapy in real-time. Method For rapid label-free isolation of CTCs from peripheral blood we used the Vortex technology, a microfluidic device using inertia and laminar microvortices. From 15 patients with metastatic CRC to the liver that underwent liver metastatectomy with curative intent, we collected CTCs preoperatively, at the 5th postoperative day and during follow-up visits. Cells collected were immunostained for EpCAM, CD45 and DAPI, enumerated using standardized classification criteria, and subjected to Sanger sequencing. CTC enumeration and mutational patterns were compared to the primary tumor, liver metastases and ctDNA (detected by a multiplexed PCR and enrichment technology; Kidess E et al., 2015) as well as CEA levels when available. Results 41 blood samples from 15 patients were collected at different time points prior to and after surgical resection of liver metastases. More CTCs were found in preoperatively collected CRC patient samples (2.4 CTCs/mL, 0.1 - 5.5/mL) than in age-matched healthy controls (0.1 CTCs/mL, 0 - 0.4/mL). 80% of all CRC samples were identified as positive for CTCs (based on a calculated threshold from healthy controls), with varying levels of EpCAM expression (81.4% of CTCs being EpCAM+). The number of CTCs for each patient, showed a close correlation to clinical parameters and ctDNA levels: detection of CTCs, CTC mutational profiles as well as ctDNA revealed minimal residual disease and anticipated tumor recurrence earlier than carcinoembryonic antigen (CEA) value or imaging. For example, for P006, postoperative imaging surveillance revealed progressive disease, which was accompanied by rising levels of CTCs (up to 29 CTCs/mL at the last time point) and PIK3CA mutant DNA in both plasma ctDNA and CTC DNA, while CEA remained in the normal range. Conclusion Our data illustrate that CTCs as well as ctDNA can efficiently reveal disease recurrence as well as disease progression earlier than imaging and far more reliable compared to CEA, the currently standard biomarker for CRC. Beyond enumeration, CTC molecular analysis gives additional information and will potentially help to promote the development of tailored therapies for every individual patient. Citation Format: Evelyn Kidess-Sigal, Haiyan E. Liu, Melanie Triboulet, James Che, Georges A. Poultsides, Brendan C. Visser, Andre Marziali, Marc Lee, Valentina Vysotskaia, Matthew Wiggin, Vishnu C. Ramani, Ulrich Keilholz, Ingeborg Tinhofer, Amin Zia, John Coller, Jeffrey A. Norton, Elodie Sollier, Stefanie S. Jeffrey. Enumeration and mutational profiling of CTCs and comparison to ctDNA and colorectal cancer liver metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3149.

A rapid method for isolation of genomic DNA from food-borne fungal pathogens.

Food contaminated with fungal pathogens can cause extremely harmful effects to human even when present in low concentrations. Researchers now pay more attention towards rapid DNA extraction for the quick screening, which is highly demanded in diverse research field. Molecular description of many fungal species is identified by different molecular characteristics. Hence, the efficient isolation of genomic DNA and amplification using PCR is a prerequisite for molecular characterization. Here, we used an improved Sodium dodecyl sulfate-Cetyltrimethyl ammonium bromide-Chloroform-isoamyl alcohol method by combining Sodium dodecyl sulfate with cetyl methylammonium bromide without addition of proteinase K, RNase K, and β-mercaptoethanol. To analyze the quality of recovered DNA, this method was compared with the other four routine methods. The present method has been chosen in the study as a preferred method because of easy adaptation to routine laboratories/food industries considering its rapid, sensitivit,y and cost effectiveness involved in the method.

Rapid method for DNA isolation from a tough cell wall green alga Tetraspora sp. CU2551.

Genetic studies are important to understand the complex biological system of various organisms. Some eukaryotic green organisms have tough cell wall which precludes the efficient extraction of the genetic materials. Here, we developed the method for simple and rapid isolation of high quality DNA from a green alga Tetraspora sp. CU2551. The cell homogenization procedures were combined with physical force plus heat treatment to disrupt the cell envelope of Tetraspora sp. CU2551. Without protease treatment, vortexing with glass bead for 30-105s at 70°C led to the isolation of a high purity DNA which was suitable for downstream process. The improved method was successfully developed and could be applied for the rapid isolation of DNA from other unicellular and filamentous green microalgal strains.

Complement C1Q has a novel tetrameric structure in Nile tilapia (Oreochromis niloticus)

Complement C1q is the first subcomponent of the classical complement pathway, and a major molecule connecting link between innate and adaptive immunity. Mammalian C1q is a hexamer of a molecular weight about 410 kDa; however, little is known about its structural characteristics in teleost. In this study, a rapid method for isolation of the C1q (OnC1q) was developed to investigate its biochemical characterization in Nile tilapia (Oreochromis niloticus). Using a rabbit IgG-Sepharose affinity column, complement OnC1q was isolated from fish sera in a single step, and the C1q further purified by gel filtration through a Sephacryl-S300 column. OnC1q on reduction by electrophoresis on SDS-PAGE yields equimolar amounts of C1qA, C1qB and C1qC chains with molecular weights about 23, 25, 21 kDa, respectively. Unreduced OnC1q was shown to be composed of two non-covalently linked subunits of molecular weights about 48 (A-B dimers) and 42 kDa (C-C dimers), with a ratio of 2:1. Further, Gel filtration on Sephacryl-S300 showed that native OnC1q was eluted as a single peak of molecular weight about 280 kDa, which was confirmed by SDS-CAGE analysis. Thus, in comparison to the hexameric form in mammals, OnC1q may possess a unique tetrameric structure with a mammalian-like heterotrimeric organization composed of C1qA, C1qB and C1qC. The revealing of C1q novel structural in teleost may provide insight into the structural and functional evolutionary history of the C1q family and the classical pathway in early vertebrates.

New Rapid Method of DNA Isolation from Milk Somatic Cells

ABSTRACTIsolation of genomic DNA is one of the basic steps in many different molecular analyses. There are a few reports on methods of DNA isolation from milk, but many of them are time consuming and expensive, and require relatively large volumes of raw milk. In this study a rapid, sensitive, and efficient method of DNA extraction from milk somatic cells of various mammals (cattle, sheep, goats, horses) is presented. It was found that milk is a good source of genomic DNA, and to obtain a sufficient amount and quality of DNA, suitable for molecular analysis such as PCR, 10 mL of raw milk is sufficient. Thanks to this method, stress in animals can be reduced during collection of researched material. Therefore, this method could be widely used in molecular analyses.

A simple and rapid method for separation and isolation of marine algal species from naturally evolved populations

ABSTRACTTo improve the study of mixed microalgal populations, three naturally evolved marine microalgal cultures were subjected to a light crushing mechanical treatment using a silicon spatula coupled with zymolyase treatment at four concentrations: 5, 10, 20 and 25 U/ml, for 15, 30, 45 and 60 min before being observed under a microscope. The enzyme concentration of 20 U/ml after 45 min reduces the size of macroscopic microalgal aggregates and improves the microscopic observation of the different microalgal species comprising the population. There was no improvement using the higher enzyme concentration. This paper proposes a new approach to the study of naturally evolved microalgal populations which is useful for distinguishing the morphology of the different species present in the population and allowing for the identification by classical keys, and also to obtain a pure culture from an inoculum of mixed species by using a micromanipulator for cell counting.

Rapid, Effective DNA Isolation from Osmanthus via Modified Alkaline Lysis.

Variability of leaf structure and presence of secondary metabolites in mature leaf tissue present a challenge for reliable DNA extraction from Osmanthus species and cultivars. The objective of this study was to develop a universal rapid, effective, and cost-efficient method of DNA isolation for Osmanthus mature leaf tissue. Four different methods were used to isolate DNA from 8 cultivars of Osmanthus. Absorbance spectra, DNA concentration, appearance on agarose gel, and performance in PCR were used to analyze quality, quantity, and integrity of isolated DNA. Methods were ranked in order, based on total quantity, quality, and performance points as the following: 1) solid-phase extraction (SPE), 2) modified alkaline lysis (SDS), 3) cetyltrimethylammonium bromide (CTAB) with chloroform (CHL), and 4) CTAB with phenol/chloroform (PHE). Total DNA, isolated via SPE, showed the least contamination but the lowest mean quantity (9.6 ± 3.4 μg) and highest cost. The highest quantity of DNA was isolated via SDS (117 ± 54.1 μg). SPE and SDS resolved the most individuals on agarose gel, whereas the 2 CTAB methods had poorly resolved gels. All methods except PHE performed well in PCR. Additions to the modified alkaline lysis method increased A260:A230 by up to 59% without affecting yield. With the use of SDS, an average of 1000 μg/g DNA was isolated from fresh leaf tissue of 18 samples in ∼1.5 h at a cost of 0.74 U.S. dollars (USD)/sample. We recommend improved alkaline lysis as a rapid, effective, and cost-efficient method of isolating DNA from Osmanthus species.

Rapid Methods for Isolation and Screening of Methane Degrading Bacteria

There are only few microbial strains available world over having capacity to degrade methane due to lack of rapid methodology for their isolation and efficiency studies. In present study we have isolated five methane degrading bacterial strains by enrichment of soil samples in the flasks containing methane as sole source of carbon in its head space and water as basal medium. Moreover their survival in the evacuated tubes containing methane and presence of soluble methane monooxygenase enzyme by colorimetric plate assay was studied which represents a simplest method of screening of methane utilizing microbial strains as compared to conventional gas liquid chromatographic technique as well as enzymatic assay and molecular detection of the genes encoding methane monooxygenase and methanol dehydrogenase genes. In addition to this presence of a key methane metabolism enzyme methane monooxygenase and ethanol dehydrogenase enzyme was confirmed by detection of genes encoding the enzymes as well as qualitative detection of the enzyme activities in the isolates to provide strong support to the methane degrading capacity of the isolates and to validate the method used in this study to isolate methanotrophs.

A Rapid Isolation Method for Bacillus coagulans from Rice Straw

A Rapid Isolation Method for Bacillus coagulans from Rice Straw

Development of Ultra-Rapid Reverse Transcription Real-Time PCR for Detection against Black Queen Cell Virus in Honeybee

Black Queen Cell Virus (BQCV) is one of the most prevalent viruses in honeybee, and could not be recognized by any infected symptoms in the adult bees. Thus, a newly developed method for rapid RNA isolation and cDNA generation from honeybee samples will be useful and easy to evaluate the presence of BQCV. A significant improvement by only one-step RNA isolation for 10 minutes made possible to detect BQCV RNAs at high quantity. cDNAs were also generated directly from isolated BQCV RNAs by different primer sets for 1 minutes and were subsequently applied to Ultra-Rapid Real-Time PCR by using microchip of 6μl reaction volume with extremely short time in each step of PCR. This system provided the ultra-high speed reaction (30 cycles in less than 10 min) including melting temperature analysis for amplified BQCV products. These results suggest that BQCV detection can be completed within 6 min 42 sec, and the Ultra-Rapid Real-time PCR is sensitive, reliable and time-saving for monitoring BQCV in honeybee.

A Rapid, low cost, and efficient method for isolation of high quality mitochondrial DNA from Oryza sativa

A rapid and inexpensive protocol for isolation of mitochondrial DNA from Oryza sativa with negligible genomic DNA contamination is developed without use of density-gradients materials. Mitochondria were isolated from rice seedlings in an in-house lysis buffer containing sucrose followed by DNase I treatment to remove nuclear DNA. Modified CTAB method was used to isolate mitochondrial DNA from isolated mitochondria. The presence of mitochondrial DNA was confirmed by using selective amplification of mtDNA specific genes. PCR amplification was observed in all genes except β-actin gene. In addition, Sanger sequencing and gene mapping to reference gene sequences in public database was performed to confirm the presence of mitochondrial DNA. The mapping analysis showed 99.71% similarity with mitochondrial DNA. The protocol demonstrated high specificity and yielded high purity mitochondrial DNA. It is concluded that the protocol described here will be beneficial for scientific communities by providing a cheap and robust mitochondrial DNA isolation protocol for potential applications.

귤피 유래 면역활성 람노갈락투로난류의 신속 분리방법

다당의 생물활성은 다당의 구조적인 특징과 분자량 분포에 의해 중요한 영향을 미치기 때문에, 특정 다당의 정제는 다당 연구를 위해 필수적이다. 따라서 본 연구에서는 서로 다른 특성을 소유한 다당의 분획을 위한 신속 분리 방법을 개발하고 대표 화합물로 한국산 귤피로부터 조제한 다당 혼합물을 이용, 본 분리법을 최적화 하였다. 귤피는 펙티나아제 처리 후 에탄올 침전법을 통해 조다당 획분인 CPE로 조제되었으며, CPE는 재차 농도별로 연속 희석된 에탄올 용액(EtOH:DIW=8:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 및 0.5:1)을 이용하여 7가지 획분(CPE8-CPE0.5)으로 분획되었다. CPE8-CPE1획분은 구성당 분석 결과 람노갈락투로난-I과 람노갈락투로난-II 다당의 지표 구성당인 총 11종의 서로 다른 당으로 구성되어 있었으며, arabino-<TEX>${\beta}$</TEX>-3,6-galactan 잔기를 함유하고 있는 것으로 확인되었다. 그러나 CPE0.5 획분에서는 람노갈락투로난-II 및 arabino-<TEX>${\beta}$</TEX>-3,6-galactan 잔기를 함유하고 있지 않았다. 한편, CPE8-CPE1 획분을 처리한 mouse 복강 대식세포에서는 농도의존적으로 IL-6의 생산 증가가 관찰된 반면, CPE1 및 CPE0.5 획분에서는 활성이 급격히 감소됨을 확인 할 수 있었다. 따라서 이상의 결과로부터 분리방법이 다양한 특성을 갖는 다당의 혼합물로부터 생물활성을 갖는 람노갈락투로난류를 신속히 분리하는데 매우 유용한 것으로 판단되었다. We developed a rapid isolation method for fractionation of polysaccharides with different characteristics, and optimized it for the polysaccharide mixture from Korean citrus peels. A crude polysaccharide mixture, citrus-peel-enzyme (CPE) fraction was isolated from the citrus peels digested with pectinase and ethanol precipitation. CPE was further fractionated with serially diluted ethanol solution (ethanol:deionized water=8:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, and 0.5:1) to produce seven fractions labeled from CPE8 to CPE0.5. Fraction from CPE8 to CPE1 were mostly composed of 11 different sugars, including rhamnogalacturonan (RG) I and II, and the sugars contained arabino-<TEX>${\beta}$</TEX>-3,6-galactan moiety. However, CPE0.5 did not contain RG-II and arabino-<TEX>${\beta}$</TEX>-3,6-galactans. Treatment of macrophages with fractions CPE8-CPE1 led to a dose-dependent increase in interleukin-6 production (IL-6), while treatment with CPE1 and CPE0.5 fractions resulted in decreased levels of IL-6. These results indicate that this isolation method may be useful for the rapid fractionation of bioactive RGs from polysaccharide mixtures.

A Rapid and Effective Method for Isolation of Genomic DNA from Small Amount of Silica-Dried Leaf Tissues

Isolation of high molecular weight DNA is the prerequisite for molecular systematic studies. The tradi- tional lengthy plant DNA isolation protocol by using CTAB detergent has been modified to a simple, rapid and efficient protocol so as to isolate large number of plant samples within a short period of time. This modified pro- tocol has been tested successfully on Chenopodium album, Calotropis procera and Ziziphus mauritiana that signifi- cantly minimizes the time and use of laboratory materials and provides a high yield of DNA in the range of 142.5 lg (Calo1) and 49.5 lg (Zizy2) from 60 mg of silica dried leaves. The yields and quality of genomic DNA are con- siderably affected when the common protocol for DNA isolation is applied to the Chenopodium, Calotropis and

Application of zinc chloride precipitation method for rapid isolation and concentration of infectious Pectobacterium spp. and Dickeya spp. lytic bacteriophages from surface water and plant and soil extracts.

This is the first report describing precipitation of bacteriophage particles with zinc chloride as a method of choice to isolate infectious lytic bacteriophages against Pectobacterium spp. and Dickeya spp. from environmental samples. The isolated bacteriophages are ready to use to study various (ecological) aspects of bacteria-bacteriophage interactions. The method comprises the well-known precipitation of phages from aqueous extracts of the test material by addition of ZnCl2, resuscitation of bacteriophage particles in Ringer’s buffer to remove the ZnCl2 excess and a soft agar overlay assay with the host bacterium to isolate infectious individual phage plaques. The method requires neither an enrichment step nor other steps (e. g., PEG precipitation, ultrafiltration, or ultracentrifugation) commonly used in other procedures and results in isolation of active viable bacteriophage particles.

Enrichment of cardiomyocytes in primary cultures of murine neonatal hearts.

In vitro culture of neonatal murine cardiomyocytes is vital for understanding the functions of the heart. Cardiomyocyte cultures are difficult to maintain because they do not proliferate after birth. The maintenance of primary cultures of viable and functional cardiomyocytes is considerably affected by the yield from initial steps of isolation procedures. This protocol describes an efficient and rapid method for isolation and maintenance of long-term cultures of neonatal murine cardiomyocytes by effectively shortening the trypsin enzyme digestion period and the cardiomyocyte enrichment step.

Development and extraction optimization of baicalein and pinostrobin from Scutellaria violacea through response surface methodology.

Objective:To develop a process that involves optimization of the amount of baicalein and pinostrobin from the hydro-methanolic extract of the leaves of Scutellaria violacea by response surface methodology (RSM).Materials and Methods:The combinatorial influence of various extraction parameters on the extraction yield was investigated by adopting Box–Behnken experimental design. Preliminary experiments carried out based on the traditional one variable at a time optimization revealed four such operational parameters to play a crucial role by influencing the yield. These four process parameters at three levels were considered to obtain the Box–Behnken experimental design.Results:RSM based model fitted to the resulting experimental data suggested that 52.3% methanol/water, 12.46:1 solvent-solid ratio, 285 rpm agitation and 6.07 h of extraction time are the optimal conditions which yielded a maximized amount of baicalein and pinostrobin of 2.9 and 4.05 mg/g DM. Analysis of variance revealed a high correlation coefficient (R2 = 0.999 for baicalein and 0.994 for pinostrobin), signifying a good fit between the regression model (second order) and the experimental observations.Conclusion:The present study signifies that both the metabolites have been extracted from S. violacea for the first time. Further, this study developed an optimized extraction procedure to obtain maximum yield of the metabolites, which is unique and better than conventional extraction methodology. The operational parameters under optimized conditions accounts for the lowest cost in extraction process thus, providing an efficient, rapid and cost-effective method for isolation and scale up of these commercially vital flavonoids.

Bacteriological profile and antibiogram of blood culture isolates done by automated culture and sensitivity method in a neonatal intensive care unit in a tertiary care hospital in Odisha, India

Background: Sepsis is one of the commonest cause of neonatal mortality. The aetiology of neonatal sepsis has variations according to the various customs and practices in the perinatal and neonatal period and geographical area. This study was designed to analyse the magnitude and aetiological characteristics of neonatal sepsis & also the antibiotic sensitivity pattern of the isolates. Methods: This prospective study was conducted in the Department of Microbiology including 250 neonates with suspected neonatal sepsis admitted to Dept. of Paediatrics & NICU, of Kalinga Institute of Medical Sciences (KIMS), Bhubaneswar, during the period from Nov. 2012 - April 2014. Two ml of venous blood was taken from each neonate & cultured by automated BacT/Alert & VITEK2 method for rapid isolation & sensitivity test. A structured proforma was used to collect the information for the baseline characteristics like age, gender, birth weight, gestational age, mode of delivery of the neonate and age of onset of illness. Results: Amongst the total samples processed, 82(32.8%) became culture positive among which gram positive bacteria were the most predominant (65.8%). Out of all the gram positive pathogens, CoNS were the most common isolates (88.5%). Among the total culture positive pathogenic isolates (n=82), S. haemolyticus was the predominant bacteria 28 (34.1%) followed by S. epidermidis 12 (14.6%) and Esch coli . 8 (9.8%). Staph. aureus was isolated from 6 (7.3%) cases whereas candida spp. were isolated from only 8 cases (9.6%). Conclusions: Present study revealed that both gram positive and gram negative bacteria were responsible for blood stream infections and most of the strains were multidrug resistant. Among gram positive bacterial pathogens, most common were Coagulase Negative Staphylococcus (CoNS) (88.5%) followed by Staphylococcus aureus (7.3%).