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Results for “Microdroplet”

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12 recordsLinked to original sources
PubMed2026

Microdroplet-based high-throughput screening for antagonistic bacteria targeting penaeid shrimp pathogenic Vibrio harveyi.

Antagonistic bacteria that suppress the growth of specific bacteria have attracted attention as an antibiotics-independent strategy for infectious disease control in aquaculture. Microfluidics-based water-in-oil droplets (microdroplets) enable high-throughput screening of antagonistic bacteria in the field of medicine or agriculture. However, the use of this screening system in aquaculture has not yet been reported. In particular, penaeid shrimp aquaculture, one of the largest sectors of global aquaculture, has a strong demand for alternative disease control strategies because vaccination is ineffective. Here, we demonstrated a proof-of-concept study of microdroplet-based high-throughput screening system for antagonistic bacteria targeting penaeid shrimp pathogenic Vibrio harveyi. Using this screening system, we successfully isolated 18 bacterial candidates with potential growth-inhibitory activity, representing three genera (Pseudoalteromonas, Shewanella, and Tenacibaculum), from the bacterial community of kuruma shrimp Penaeus japonicus rearing water. 16S rRNA gene-based bacterial community analysis revealed that these isolates included several low-abundance, rare taxa. Although these isolates showed no inhibitory activity on agar plates, one out of four tested strains showed a trend toward improved survival during co-infection tests using kuruma shrimp. Overall, our study highlights both the potential and limitation of microdroplet-based antagonistic bacterial screening to accelerate the development of biological control strategies in shrimp aquaculture.

Animals↗
PubMed1978

Counts on viable Bacteroides fragilis: a modification of the microdroplet technique.

A modification of Sharpe's microdroplet counting technique is presented which permits viable counting of Bacteroides fragilis. Lysed horse blood was added to 0.1 soy digest agar droplets and 2% was found to be the optimal concentration which combined adequate intradroplet colony size with acceptable visibility on the counting screen of the Colworth Droplette machine.

Bacteriological Techniques↗
PubMed1977

Migration inhibition by an agarose microdroplet assay: monitoring of tumor-associated antigens on a simian virus 40-induced sarcoma.

Macrophage migration inhibition assays, with a direct agarose microdroplet method, were used to monitor TAA activity of preparations of SV-40-induced mKSA cells. These preparations included cell-free crude membranes, papain-solubilized and NP40 detergent-solubilized membrane extracts from mKSA tumor cells. The assay was extremely sensitive and could detect migration inhibition reactivity with all three types of antigenic preparations with concentrations as low at 250 ng protein/ml. The reactivities were quite reproducible from experiment to experiment using the same or different lots of these antigen preparations, and the reactivities were specific in that peritoneal exudate cells from BALB/c mice, immunized with antigenically unrelated but syngeneic plasmacytomas, were not inhibited by these antigens. The results demonstrated the usefulness of this assay in rapidly detecting small concentrations of partially purified TAA preparations by using small number of immune cells.

Animals↗
PubMed2025

Ultrahigh-throughput screening assay for PET-degrading enzymes.

In recent years, several PET-degrading enzymes have been identified from both known microorganisms and metagenomic sources in response to the growing environmental issue of polyethylene terephthalate (PET) accumulation. Despite this progress, there is a limited number of (ultra)high-throughput screening methods for assessing PET-hydrolyzing activity without relying on surrogate substrates. This method utilizes the coupled activity of ketoreductases (KREDs) and diaphorase to produce a fluorescent compound (resorufin) in the presence of PET degradation products, offering a more direct and efficient screening approach. A metagenomic KRED was coupled with the diaphorase from Clostridium kluyveri to enable the detection of the hydrolysis of PET degradation products catalyzed by the Bacillus subtilis BS2 esterase. The coupled reaction was established in water-in-oil microdroplets, encapsulating a single E. coli cell per droplet, demonstrating its potential for use in the ultrahigh-throughput screening of metagenomic libraries or randomized libraries for directed evolution campaigns.

High-Throughput Screening Assays↗
PubMed1978

An in-vitro immunological assay for diagnosis of coeliac disease.

Production of leucocyte-migration-inhibition factor (L.I.F.) by peripheral-blood lymphocytes in response to challenge with gluten fractions was studied in 55 patients with coeliac disease and in 32 controls. 96% of the patients with coeliac disease demonstrated significant L.I.F. reaction in response to gluten fractions irrespective of their dietary status. Only 2 out of 32 controls had a positive reaction. This was in response to the B2 or B3 fraction, but never to both. The agarose microdroplet method of L.I.F. assay is reliable and technically simple enough for use in most clinical laboratories. The assay of L.I.F. production by peripheral-blood lymphocytes in response to gluten fractions, would be a useful adjunct in the diagnosis of gluten-sensitive enteropathy.

Adolescent↗
PubMed1978

Leukocyte migration inhibition of tumor antigen and purified protein derivative reactivity in guinea pigs sensitized to line 10 hepatocarcinoma and BCG.

Agarose microdroplet leukocyte migration inhibition (LMI) assays were performed to measure reactivity against line 10 hepatocarcinoma antigens and purified protein derivative (PPD) with the use of peripheral blood leukocytes from line 10 and/or BCG-sensitized syngeneic guinea pigs. The assay was quite sensitive and detected leukocyte migration inhibition with concentrations as low as 12.6 ng protein/ml of the crude sonicate of the line 10 tumor and 0.1 pg PPD. Specificity was shown by lack of reactivity in leukocytes of line 10 and/or BCG-sensitized animals with antigen preparations of L2C leukemia cells or normal syngeneic liver. Furthermore, leukocytes from normal control guinea pigs failed to react with any antigen. The results also suggested antigen cross-reactivity between line 10 tumor and BCG. Leukocytes from guinea pigs sensitized to only BCG became LMI reactive to the line 10 sonicate as well as PPD. No reactivity was observed with leukocytes of the animals in simultaneous tests with a sonicate of guinea pig L2C leukemia cells. The results demonstrated the usefulness of this microassay in detection of LMI reactivity with low antigen concentrations and small volumes of whole blood.

Animals↗
PubMed2025

Genes required for Mycobacterium tuberculosis to survive the transition from aerosol to pulmonary alveolar lining fluid and early infection in a model of transmission.

Mycobacterium tuberculosis (Mtb) must withstand physical and chemical stresses during airborne transmission, including during the desiccation of aerosols small enough to reach pulmonary alveoli in a new host. There, Mtb encounters an antimicrobial pulmonary alveolar lining fluid (ALF) before it is engulfed by macrophages. To study the genes involved in Mtb's ability to survive the transition from desiccated droplet to pulmonary alveolus in an in vitro model, we formulated a model alveolar lining fluid (MALF) that mimics the composition of ALF as inferred from human bronchoalveolar lavage fluid (BALF). We compared the transcriptome of log-phase Mtb in MALF to the transcriptome of Mtb in BALF as BALF from the lungs of healthy adults was reconstituted to compensate for the dilution of ALF by lavage (rcBALF). Mtb from log-phase culture in a standard laboratory medium survived quantitatively in MALF and rcBALF for at least 24 hours. In contrast, Mtb that had passed through earlier stages of transmission began to succumb after 3 hours in MALF, past the time when particles have been observed to be phagocytized by alveolar macrophages. Screening of a genome-wide CRISPRi library of Mtb identified 35 genes as uniquely required by Mtb to survive the transition from desiccated microdroplet into rehydration in MALF. Thirty-one of these genes are non-essential under conventional laboratory conditions and seven have unknown functions. Thirteen of the 35 genes were additionally required for Mtb to survive in macrophage-like cells cultured at the air-liquid interface with pulmonary epithelial cells. This study nominates additional members of the transmission survival genome of Mtb, illustrates that different genes may contribute to the survival of Mtb at different stages of transmission, and suggests that modeled transmission can shed light on the functions of Mtb genes whose contributions have been unknown.

Journal Article↗
PubMed1975

Determination of the glycogen content in single neutrophil leukocytes using a micromodel of leukocyte glycogen.

The kinetics of the periodic acid oxidation as part of the periodic acid-Schiff reaction was studied by combined microinterferometry and microspectrophotometry in micromodel systems of liver glycogen and leukocyte glycogen as well as in neutrophil leukocytes. The initial formation of Schiff-positive chromogens was more rapid in neutrophil leukocytes than in liver or leukocyte glycogen. The chromogen formation was, however, practically complete within 60 min in both neutrophil leukocytes and leukocyte glycogen, but this did not appear to be the case in liver glycogen. Differences in the rate of chromogen formation may depend on various factors such as differences in the source and treatment of the glycogen. The complete periodic acid-Schiff reaction appears to be a measure of the glycogen amount in neutrophil leukocytes and the microdroplet system of leukocyte glycogen is considered to be an appropriate model for the estimation of the glycogen amount in single neutrophil leukocytes. A mean value of 13.3 10-12 g glycogen per normal human neutrophil was found.

Glycogen↗
PubMed1977

HLA antigens in osteitis condensans ilii and ankylosing spondylitis.

Using a standard microdroplet lymphocyte cytotoxicity test for tissue typing, the distribution of the HLA antigens was determined in 37 female patients; 25 with osteitis condensans ilii (OCI) and 12 with ankylosing spondylitis (AS). Although low back pain was a common feature of OCI, none of these patients exhibited the limitation of spinal involvement, radiological evidence of spondylitis, or progressive clinical course seen in the AS group. Four of the 25 patients with OCI (16 per cent) were B27 positive vs 11 of the 12 patients with AS (92 per cent). These results suggest that OCI is not a variant of AS in women.

Cytotoxicity Tests, Immunologic↗
PubMed1978

Refractoriness to migration inhibitory factor of macrophages of LPS nonresponder mouse strains.

The responsiveness to macrophage migration inhibitory factor (MIF) of peritoneal exudate cells (PEC) from the LPS unresponsive C3H/HeJ and C57BL/10ScCR mice was assessed by the indirect agarose microdroplet macrophage migration inhibition assay. No migration inhibition with PEC from C3H/HeJ nor C57BL/10ScCR mice was detected, whereas PEC from both C3H/HeN and C57BL/10Sn mice were significantly inhibited by even a 1/32 dilution of MIF-containing supernatants. Responsiveness to MIF of C3H/HeJ PEC could, however, be induced. In vivo inoculations of Mycobacterium bovis, strain BCG, 7 days before in vitro assay rendered C3H/HeJ PEC responsive to MIF. The lack of responsiveness to MIF by C3H/HeJ PEC appeared related to some form of suppression, since a mixture of PEC from C3H/HeN mice with 10 to 15% PEC from C3H/HeJ mice resulted in undetectable migration inhibition at any MIF dilution. In contrast to the usual lack of responsiveness of their macrophage to MIF, C3H/HeJ mice were able to produce MIK in response to PPD as well as their counterpart C3H/HeN mice after BCG sensitization. These results demonstrate that macrophages from C3H/HeJ and C57BL/10ScCR mice are unable to be inhibited in their in vitro migration of MIF (possibly being directly or indirectly influenced by a suppressor cell), whereas lymphoid cells from at least one of these strains, the C3H/HeJ mice, can produce MIF in response to antigenic stimulation.

Animals↗
PubMed1979

Studies of assay conditions for macrophage migration from an agarose droplet.

The agarose microdroplet method is a relatively simple and economic technique to determine migration inhibition of leukocytes or macrophages in vitro. In the present study, further cultural and technical requirements of this method for the determination of macrophage migration inhibition have been defined: influence of macrophage handling before the assay, kinetics of migration and dependence on the pH of the medium. Considering defined conditions, the agarose microdoplet assay gives highly reliable and reproducible results. In comparative experiments, it proved to be as sensitive and valid as the capillary tube technique.

Animals↗