Laboratory Culture Methods Research Articles

Preliminary Studies on Low-Cost Laboratory Culture Method for Daphnia

Preliminary Studies on Low-Cost Laboratory Culture Method for Daphnia

Bacterial Classification Using Deep Structured Convolutional Neural Network for Low Resource Data

Bacterial identification is an essential task in medical disciplines and food hygiene. The characteristics of bacteria can be examined under a microscope using culture techniques. However, traditional clinical laboratory culture methods require considerable work, primarily physical and manual effort. An automated process using deep learning technology has been widely used for increasing accuracy and decreasing working costs. In this paper, our research evaluates different types of existing deep CNN models for bacterial contamination classification when low-resource data are used. They are baseline CNN, GCNN, ResNet, and VGGNet. The performance of CNN models was also compared with the traditional machine learning method, including SIFT+SVM. The performance of the DIBaS dataset and our own collected dataset have been evaluated. The results show that VGGNet achieves the highest accuracy. In addition, data augmentation was performed to inflate the dataset. After fitting the model with augmented data, the results show that the accuracy increases significantly. This improvement is consistent in all models and both datasets.

Ten-year Review of Acute Pediatric Hematogenous Osteomyelitis at a New Zealand Tertiary Referral Center.

Acute hematogenous osteomyelitis (AHO) remains a cause of severe illness among children with the possibility of long-term consequences for growth and development. Recent research has highlighted an unusually high burden of disease in the New Zealand population compared with other Western regions. We have sought to identify trends in presentation, diagnosis, and management of AHO, with added focus on ethnicity and access to health care. A 10-year retrospective review of all patients <16 years with presumed AHO presenting to a tertiary referral center between 2008 and 2018 was performed. One hundred fifty-one cases met inclusion criteria. The median age was 8 years with a male predominance (69.5%). Staphylococcus aureus was the most common pathogen using traditional laboratory culture method (84%). The number of cases per year decreased from 2008 to 2018. Assessment using New Zealand deprivation scores showed Māori children were most likely to experience socioeconomic hardship ( P ≤0.01). Median distance traveled by families to first hospital consult was 26km (range 1 to 178km). Delayed presentation was associated with need for prolonged antibiotic therapy. Incidence of disease varied by ethnicity with 1:9000 cases per year for New Zealand European, 1:6500 for Pacific, and 1:4000 for Māori. Overall recurrence rate was 11%. The incidence of AHO in New Zealand is concerningly high within Māori and Pacific populations. Future health interventions should consider environmental, socioeconomic, and microbiological trends in the burden of disease. Retrospective study, Level III.

Differentiating individuals of Armillaria species in New Zealand forests

Background: Armillaria novae-zelandiae and A. limonea occur naturally as wood decay fungi in native forests in New Zealand. As pathogens they are responsible for significant root disease in trees and shrubs in plantations, crops and urban parks and gardens. A thorough understanding of their population dynamics entails knowledge of the spatial arrangement of their individual mycelia or genets. In previous work the distributions of vegetative compatibility groups (VCGs) of these fungi were mapped in an area of native forest prior to and after replacement by a young Pinus radiata plantation. With the advent of molecular technology, it has become possible to test species identities made earlier using culture techniques and to verify whether or not their VCGs, determined by incompatibility reactions between paired cultures, represent distinct individual genets. Methods: Stock subcultures of isolates representing each VCG were recovered from storage in order to obtain DNA. Extracted DNA was subjected to a polymerase chain reaction procedure (UP-PCR) using 11 universal primers to assess genetic variation between subcultures. Bands were scored as either present or absent for each primer-subculture combination and cluster analysis was undertaken by generating dendrogram trees to reveal genetic groupings among subcultures. Results: DNA cluster analysis divided subcultures of isolates into two species groups, A. novae-zelandiae and A. limonea, corresponding to identities determined through culture morphology. Within species, subcultures grouped into clusters that matched VCGs determined by earlier culture pairing. There was little indication of genetic variation within VCGs, except for one of A. limonea, which comprised two sub-clusters. Conclusions: The Armillaria species and VCGs identified by culture techniques in the laboratory were verified by independent molecular methodology. In general, the VCGs represent discrete individual genets or colonies in the field. Techniques that differentiate isolates based on differences in their DNA sequence provide a quick alternative to time-consuming laboratory culture methods for resolving population spatial structure. However, some complementary isolate pairing may be necessary when rationalising the significance of groupings in dendrogram trees.

STUDY OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS IN POSTSURGICAL SUTURE INFECTIONS IN A TERTIARY CARE CENTRE OF NORTH INDIA.

Introduction: MRSA has been recognised as an important nosocomial pathogen globally and is endemic in India. Its incidence varies from 25 % in 3 western part to 50 % in Southern part of India . Post operative wound infections continues to be a major source of morbidity and mortality for patients undergoing operative procures. To nd out the incidence of MRSA in post-surgic Aim and Objectives: al suture infections and to determine its susceptibility pattern by using a battery of antibiotics. Tota Material and method: l 100 pus samples were collected from surgical site infections of admitted patients applying inclusion and exclusion criteria of department of surgery and processed in department of microbiology of Darbhanga Medical College and Hospital. Isolation of MRSA is done by routine laboratory culture method by using Mannitol Salt Agar and Cefoxitin disc 30µg(Hi-media). Out of 100 samples collected from patients from 54, Result: Staph. aureus were isolated i.e., 54%. Whereas, out of 54 isolated Staph. aureus, 24 (44.4%) were MRSA, whereas it constitutes 24% of total study subjects. MRSA infected Male (62.5%) more than female (37.5%). During antibiotic sensitivity testing MRSA found sensitive against Vancomycin and Linezolid. Conclusion: This study shows that MRSA is one of the major infectant in post operative wound infection and this is worrisome as very limited antibiotic shows sensitivity against it.

Marine dinocysts, acritarchs and less well-known NPP: tintinnids, ostracod and foraminiferal linings, copepod and worm remains

Abstract Nine non-pollen palynomorph (NPP) groups occur in Quaternary marine and brackish-water sediments; these groups represent various planktonic or micro- to macrobenthic organisms. Some extant NPP were previously classified as fossil Acritarcha, Chitinozoa or scolecodonts. We refer to reviews of these fossils and their applications for Paleozoic–Mesozoic biostratigraphy and palaeoecology but focus on extant marine NPP that can be studied by laboratory culture, genetics or micro-geochemical methods. Marine NPP include resting cysts of planktonic dinoflagellates and prasinophytes, tintinnids and other cilates, copepod eggs and skeletal remains, and various microzoobenthos: microforaminiferal organic linings, ostracod mandibles and carapace linings, various worm egg capsules and mouthparts. New micro-Fourier Transform Infrared spectroscopy spectra suggest the probable affinities of the tintinnid cyst type P and Beringiella . Our applications in marine biodiversity and provincialism studies emphasize under-studied polar regions and neglected ice-algae nano-plankton and compare climate-based NPP distributions to Ocean Biogeographic Information System realms. Trophic relationships are outlined using sediment-trap studies. Seasonal to annual-scale investigations of palaeoproduction provide new perspectives on ocean carbon budgets during times of rapid climate change and atmospheric carbon increase. More taxonomic and source-linkage studies of non-dinocyst marine NPP are needed but we outline potentials for studies of hemispheric or global-scale shifts in marine food webs as driven by ocean warming.

Presence of Infection by Mycobacterium avium subsp. paratuberculosis in the Blood of Patients with Crohn's Disease and Control Subjects Shown by Multiple Laboratory Culture and Antibody Methods.

Mycobacterium avium subspecies paratuberculosis (MAP) has long been suspected to be involved in the etiology of Crohn’s disease (CD). An obligate intracellular pathogen, MAP persists and influences host macrophages. The primary goals of this study were to test new rapid culture methods for MAP in human subjects and to assess the degree of viable culturable MAP bacteremia in CD patients compared to controls. A secondary goal was to compare the efficacy of three culture methods plus a phage assay and four antibody assays performed in separate laboratories, to detect MAP from the parallel samples. Culture and serological MAP testing was performed blind on whole blood samples obtained from 201 subjects including 61 CD patients (two of the patients with CD had concurrent ulcerative colitis (UC)) and 140 non-CD controls (14 patients in this group had UC only). Viable MAP bacteremia was detected in a significant number of study subjects across all groups. This included Pozzato culture (124/201 or 62% of all subjects, 35/61 or 57% of CD patients), Phage assay (113/201 or 56% of all subjects, 28/61 or 46% of CD patients), TiKa culture (64/201 or 32% of all subjects, 22/61 or 36% of CD patients) and MGIT culture (36/201 or 18% of all subjects, 15/61 or 25% of CD patients). A link between MAP detection and CD was observed with MGIT culture and one of the antibody methods (Hsp65) confirming previous studies. Other detection methods showed no association between any of the groups tested. Nine subjects with a positive Phage assay (4/9) or MAP culture (5/9) were again positive with the Phage assay one year later. This study highlights viable MAP bacteremia is widespread in the study population including CD patients, those with other autoimmune conditions and asymptomatic healthy subjects.

Accuracy in clinical examinations for the diagnosis of vulvovaginitis by CANDIDA SPP. and IN VITRO susceptibility to the main antifungals

Vulvovaginal candidiasis (VVC) is a common infection. This work aims to determine the positive predictive value (PPV) of the clinical diagnosis of VVC and to characterize Candida species isolated from the vaginal mucosa. This cross-sectional study was conducted from February 2016 to February 2017 at the Gynecology and Obstetrics Outpatient Clinic of the Hospital das Clínicas, in Goiânia, Goiás State, Brazil. The study included samples of vaginal secretion from 55 women who complained of vaginal discharge and itching as their main symptoms. The PPV of the clinical diagnosis of VVC was estimated in comparison to the laboratory culture method. The phenotypic methods and molecular tests were performed to identify Candida spp. In vitro susceptibility of Candida spp. isolates to fluconazole, itraconazole, clotrimazole, nystatin, and amphotericin B was determined using the broth microdilution assay. Yeast growth using the enzymes protease, phospholipase, and hemolysin was carried out in media containing respectively bovine albumin, egg yolk, and sheep erythrocytes. A PPV of 61.8% (34/55) was determined. Among the 55 vulvovaginal samples collected, we identified 36 isolates in whichC. albicans was the most common species. High resistance to fluconazole and low minimal inhibitory concentration (MIC) values for clotrimazole, nystatin and amphotericin B were observed. All isolates were proteinase and hemolysin producers, while seven strains were phospholipase negative. The clinical diagnosis of VVC presented a moderate PPV, which meant that cultures had to be conducted in the laboratory to confirm infection. The high resistance to fluconazole and itraconazole indicated the importance of the in vitro susceptibility test.KEY WORDS: Vulvovaginal candidiasis; antifungal susceptibility; enzymatic activity

Ten-year retrospective review of paediatric septic arthritis in a New Zealand centre

Paediatric septic arthritis, although a rare diagnosis in the developed world, remains a significant challenge in the New Zealand population. In recent years, there has been effort to characterize the nature of paediatric bone and joint infection for New Zealand due to rising incidence of osteomyelitis and staphylococcal sepsis [1, 2]. We have sought to identify trends in presentation, diagnosis and management of paediatric septic arthritis, with added focus on ethnicity and access to healthcare. A ten year retrospective review of all cases < 16 years with presumed septic arthritis presenting to a tertiary referral centre between 2008 and 2018 was performed. A total of 103 cases met inclusion criteria. Mean age was 5.9 years (SD +/- 4.17) with a male predominance (64%). Traditional laboratory culture method isolated an organism in 66% of cases: Staphylococcus aureus was the most common pathogen identified (61%). Incidence varied greatly by ethnicity 1:16,000 for NZ European children; 1:8760 for Pacifica and 1:4300 for Māori. Mean distance travelled by patients to reach the nearest emergency department was 38.3 km, ranging from 2 to 188 km. Assessment using NZ deprivation scores showed the Māori paediatric population were likely to reside in areas of worse socioeconomic deprivation (p = 0.0005). The majority (66%) of cases were treated surgically with a low recurrence rate (2.9%). Delayed presentation was associated with worse outcomes and more likely in patients residing > 20 km away from the nearest emergency department. The incidence of paediatric septic arthritis in New Zealand is concerningly high within Māori and Pacific populations. Future health interventions should consider environmental, socioeconomic, and microbiological trends in the burden of disease.

Small Scale Laboratory Culture Method Production and Characterization of L-Glutamic Acid by E.Coli

Small Scale Laboratory Culture Method Production and Characterization of L-Glutamic Acid by E.Coli

Validation of the Minnesota Easy Culture System II

The objectives of this study were to perform an in-lab validation of the Minnesota Easy Culture System II tri-plate method as compared to standardized laboratory culture methods and to describe inter-reader agreement for tri-plate culture results.

Validation of the Minnesota Easy Culture System II

The objectives of this study were to perform an in-lab validation of the Minnesota Easy Culture System II bi-plate method as compared to standardized laboratory culture methods and to describeinter-reader agreement for bi-plate culture results.

From "the Worm" to "the Worms" and Back Again: The Evolutionary Developmental Biology of Nematodes.

Since the earliest days of research on nematodes, scientists have noted the developmental and morphological variation that exists within and between species. As various cellular and developmental processes were revealed through intense focus on Caenorhabditis elegans, these comparative studies have expanded. Within the genus Caenorhabditis, they include characterization of intraspecific polymorphisms and comparisons of distinct species, all generally amenable to the same laboratory culture methods and supported by robust genomic and experimental tools. The C. elegans paradigm has also motivated studies with more distantly related nematodes and animals. Combined with improved phylogenies, this work has led to important insights about the evolution of nematode development. First, while many aspects of C. elegans development are representative of Caenorhabditis, and of terrestrial nematodes more generally, others vary in ways both obvious and cryptic. Second, the system has revealed several clear examples of developmental flexibility in achieving a particular trait. This includes developmental system drift, in which the developmental control of homologous traits has diverged in different lineages, and cases of convergent evolution. Overall, the wealth of information and experimental techniques developed in C. elegans is being leveraged to make nematodes a powerful system for evolutionary cellular and developmental biology.

Benchmarking vector arthropod culture: an example using the African malaria mosquito, Anopheles gambiae (Diptera: Culicidae).

BackgroundNumerous important characteristics of adult arthropods are related to their size; this is influenced by conditions experienced as immatures. Arthropods cultured in the laboratory for research, or mass-reared for novel control methods, must therefore be of a standard size range and known quality so that results are reproducible.MethodsA simple two-step technique to assess laboratory culture methods was demonstrated using the mosquito Anopheles gambiae s.s. as a model. First, the ranges of key development outcomes were determined using various diet levels. The observed outcomes described the physiologically constrained limits. Secondly, the same outcomes were measured when using a standard operating procedure (SOP) for comparison with the determined ranges.ResultsThe standard method resulted in similar development rates to those of high and medium diets, wing length between those resulting from the high and medium diets, and larval survival exceeding all benchmark diet level values. The SOP used to produce experimental material was shown to produces high-quality material, relative to the biologically constrained limits.ConclusionsThe comparison between all possible phenotypic outcomes, as determined by biological constraints, with those outcomes obtained using a given rearing protocol is termed “benchmarking”. A method is here demonstrated which could be easily adapted to other arthropods, to objectively assess important characters obtained, and methods used, during routine culture that may affect outcomes of research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1288-4) contains supplementary material, which is available to authorized users.

Mouse models of amoebiasis and culture methods of amoeba

Entamoeba histolytica is the third leading parasitic cause of man mortality in the world. Infection occurs via ingestion of food or water contaminated with cysts of E. histolytica. Amoebae primarily colonize the intestine. The majority of amoebic infections are asymptomatic, but under some conditions, approximately 4–10% of infections progress to the invasive form of the disease. To better understand the pathogenesis of amoebiasis and the interaction between amoebae and their hosts, the development of suitable animal models is crucial. Pigs, gerbils, cats and mice are used as animal models for the study of amoebiasis in the laboratory. Among these, the most commonly used model is the mouse. In addition to intestinal amoebiasis, we developed a mouse model of liver abscess by inoculating amoeba through portal vein. However, the frequency of successful infection remains low, which is dependent on the conditions of amoebae in the laboratory. As the maintenance of virulent amoebae in the laboratory is unstable, it needs further refinement. This review summarizes mouse models of amoebiasis and the current state of laboratory culture method of amoebae.

Increased DNA Yield Following Enzymatic Release of Borrelia from a Collagen Matrix in Culture

Laboratory culture methods were developed to provide a means of harvesting greater amounts of DNA from Borrelia in culture. Borrelia burgdorferi B31 and Borrelia burgdorferi N40 were used in the initials experiments to develop optimal conditions. A collagenase enzyme preparation, Liberase TL, was used to release the organisms growing within a collagen matrix embedded in the culture. Optimal conditions of 2 U/mL of enzyme and 4 minutes of incubation at 37° C were obtained. Following the establishment of these parameters, 24 positive patient samples were treated with Liberase and 24 positive patient samples were left untreated. The DNA harvested from both sets was quantified and evaluated for purity. Approximately 3.3-fold more DNA was harvested from the samples treated with Liberase TL than from the untreated samples. A greater degree of DNA purity, with less residual protein remaining, was also achieved.

Evaluation of PetrifilmsTM as a diagnostic test to detect bovine mastitis organisms in Kenya

The study purpose was to validate PetrifilmsTM (3M Microbiology, 2005) against standard culture methods in the diagnosis of bovine mastitis organisms in Kenya. On 128 smallholder dairy cattle farms in Kenya, between June 21, 2010 and August 31, 2010, milk samples from 269 cows that were positive on California Mastitis Test (CMT) were cultured using standard laboratory culture methods and PetrifilmsTM (Aerobic Count and Coliform Count –3M Microbiology, 2005), and results were compared. Staphylococcus aureus was the most common bacterium isolated (73 % of samples). Clinical mastitis was found in only three cows, and there were only two Gram-negative isolates, making it impossible to examine the agreement between the two tests for Gram-negative- or clinical mastitis samples. The observed agreement between the standard culture and PetrifilmTM (3M Microbiology, 2005) results for Gram-positive isolates was 85 %, and there was fair agreement beyond that expected due to chance alone, with a kappa (κ) of 0.38. Using culture results as a gold standard, the PetrifilmsTM had a sensitivity of 90 % for Gram-positive samples and specificity of 51 %. With 87 % of CMT-positive samples resulting in Gram-positive pathogens cultured, there was a positive predictive value of 93 % and a negative predictive value of 43 %. PetrifilmsTM should be considered for culture of mastitis organisms in developing countries, especially when Gram-positive bacteria are expected.

Wound Healing Finally Enters the Age of Molecular Diagnostic Medicine

Many wounds are difficult to heal because of the large, complex community of microbes present within the wound. Classical laboratory culture methods do not provide an accurate picture of the microbial interactions or representation of microorganisms within a wound. There is an inherent bias in diagnosis based upon classical culture stemming from the ability of certain organisms to thrive in culture while others are underrepresented or fail to be identified in culture altogether. Chronic wounds also contain polymicrobial infections existing as a cooperative community that is resistant to antibiotic therapy. New methods in molecular diagnostic medicine allow the identification of nearly all organisms present in a wound irrespective of the ability of these organisms to be grown in culture. Advances in DNA analyses allow absolute identification of microorganisms from very small clinical specimens. These new methods also provide a quantitative representation of all microorganisms contributing to these polymicrobial infections. Technological advances in laboratory diagnostics can significantly shorten the time required to heal chronic wounds. Identification of the genetic signatures of organisms present within a wound allows clinicians to identify and treat the primary organisms responsible for nonhealing wounds. Advanced genetic technologies targeting the specific needs of wound care patients are now accessible to all wound care clinicians.

Identification of microbial volatile organic compounds (MVOCs) emitted from fungal isolates found on cinematographic film

We report the detection of microbial contamination and growth of cinematographic film utilising headspace solid phase micro-extraction coupled with Gas Chromatography–Mass Spectrometry. Microbial Volatile Organic Compounds (MVOCs) are produced only when the mould is actively growing on the cine film. Over 150 volatile compounds were detected from 16 fungal isolates, with over 40 being common to 2 or more isolates. It was found that 1-octen-3ol was produced from 13 of the isolates analysed, 3-octanone from 10 of the isolates and 3-octanol from 4 isolates. These three key chemical markers are indicative of viable fungal growth on cinematographic film precluding the need for traditional microbiology laboratory culture methods. Such an approach would prevent valuable historical footage from being discarded due to health and safety concerns regarding spore inhalation, and would enable safe handling.

해양생태독성 평가용 표준시험생물로서 섬모충류 Mesodinium rubrum에 대한 연구

The mixotrophic marine ciliate Mesodinium rubrum possesses a highly modified algal endosymbiont as a nutrition source for the species. Accordingly, we assumed that the species can reflect the ecotoxicity on marine producer (as phytoplankton) and consumer (as zooplankton) both. A series of experiments were conducted to identify the potential of the species as a standard test species for marine ecotoxicological study. The comparison of species sensitivity on reference toxic materials was made using potassium dichromate for phytoplankton and copper chloride for zooplankton. The ciliate revealed the highest sensitivity on both reference materials among the seven test species including phytoplankton, benthic copepod and rotifer species. The toxicity end point of the species was 72hr-EC50=1.52 mg/L (as potassium dichromate) estimated by population growth inhibition (PGI), which is more sensitive than the most sensitive phytoplankton Skeletonema costatum (72hr-EC50=3.05 mg/L). As comparison to rotifer, it also revealed higher sensitivity on copper chloride; 72hr-EC50=0.38 mg/L for ciliate and 48hr-EC50=0.48 mg/L for rotifer. Also, the elutriate toxicity test of various ocean disposal wastes were conducted to identify the potential of ciliate toxicity test application using industrial waste sludges. The toxicity of leather processing waste sludge was highest on the ciliate, followed by dyeing waste sludge and dye production waste sludge as an increasing order of toxicity. 72h-EC50 of ciliate PGI test was 1.83% and that of S. costatum 3.84% for leather waste sludge which showed highest toxicity. The toxicity test results also revealed that the highest sensitivity was observed on ciliate species on ocean disposed sludge wastes. Also, ciliate toxicity test well discriminated the degree of toxicity between sludge sources; 72h-EC50 values were 1.83% for leather processing waste sludge, 16.75% for dye production waste sludge and 27.75% for textile production waste sludge. Even the laboratory culture methods of the species were not generally established yet, the species has high potential as the standard test species for marine toxicity test in terms of the dual reflection of phyto- and zooplankton toxicity from single test, sensitivity and test replicability.