Culture Incubation Time Research Articles

Incubation Time of Mycobacterial Cultures: How Long Is Long Enough To Issue a Final Negative Report to the Clinician?

Traditionally, solid culture media for mycobacteria are kept for up to 8 weeks before a negative result is reported to the physician. Even though it is well documented that liquid media detect mycobacteria much earlier, most incubation protocols still require a maximum of 6 weeks ([2][1]). In

In vitro induction of the acrosome reaction in spermatozoa from endangered Spanish bulls: Effect of breed, culture media and incubation time

Current conservation programs for endangered species have been widely benefited from the use of modern assisted reproductive techniques. However, at the present time, there is little information available regarding the use of locally adapted cattle breeds on IVF programs, and even less about their sperm behavior. The aims of this study were to (1) evaluate standard acrosome reaction (AR) protocols in cryopreserved semen doses of Andalusian endangered cattle breeds, and (2) investigate the effect of breed, culture medium supplementation and incubation time on AR test results. To this end, 80 frozen semen doses from 16 bulls (5 semen samples per bull) of five different cattle breeds (3 Berrendo en Colorado, 6 Berrendo en Negro, 2 Cardena Andaluza, 1 Pajuna and 4 Retinta) were in vitro incubated in three test media (Heparin, BSA or their combination), using Sp-TALP as a control, to induce in vitro AR. The percentage of acrosome-reacted spermatozoa was evaluated using fluorescence techniques (FITC-PNA and Propidium Iodide) at four time-points (0, 30, 60 or 120min.). Throughout the whole experiment, spermatozoa incubated with a combination of both supplements (Heparin and BSA) showed significantly (P<0.05) higher results than those processed with Heparin or BSA alone. In contrast, the control media (without supplementation) showed the lowest results. We also found a significant (P<0.05) influence of cattle breed on the dynamics of the in vitro induced AR using different protocols. Based on our results, we can conclude that the response pattern of in vitro induced AR depends not only on culture media and incubation time but also on the cattle breed. Therefore, all these factors must be taken in consideration to assess the rate of AR sperm in bulls.

Response surface methodology to determine optimal measles-specific cytokine responses in human peripheral blood mononuclear cells

Limitations of assay variability, labor costs, and availability of cells can affect the conduct of large population-based studies. The ability to determine optimal conditions for laboratory assessment of immune outcomes, including measurement of cytokines, can reduce the number of peripheral blood mononuclear cells (PBMCs) needed, reduce the labor costs involved, and the variability in secreted cytokine response by pooling cytokines from the same cell culture supernatant. Previously, we used response surface methodology to predict optimal conditions for vaccinia virus-stimulated cytokine responses in recipients of smallpox vaccine. Here, we apply the same approach for a measles vaccine study.PBMCs were collected from vaccinated subjects, and seven cytokines (IFN-γ, IL-2, TNF-α, IL-10, IFN-α, IFN-λ1, and IL-6) involved in measles virus-specific cytokine immune responses were examined. PBMCs were stimulated with differing multiplicity of infection (MOI) and days in culture (incubation time). Response surface methodology was used to select the optimal MOI and incubation time for each secreted cytokine.Our results demonstrate that each cytokine's optimal conditions (MOI and incubation time) differ for each virus (measles vs. vaccinia) and each cytokine's optimal conditions for each virus can be predicted using response surface methodology. These conditions allow for cytokines with overlapping optimal conditions to be pooled from the same supernatant in culture to reduce the number of PBMCs used, the costs involved, and assay variability. Therefore, response surface methodology is an effective technique that can be used to optimize antigen-specific secreted cytokines prior to population-based studies.

Chitosan-chondroitin sulphate nanoparticles for controlled delivery of platelet lysates in bone regenerative medicine

In this study, a new formulation of nanoparticles (NPs) based on the electrostatic interaction between chitosan and chondroitin sulphate (CH-CS NPs) is proposed for the controlled release of proteins and growth factors (GFs), specifically platelet lysates (PLs). These nanoparticulate carriers are particularly promising for protein entrapment because the interactions between the polysaccharides and the entrapped proteins mimic the interactions between chondroitin sulphate and proteins in the native extracellular matrix (ECM). Spherical non-cytotoxic NPs were successfully produced, exhibiting high encapsulation efficiency for physiological levels of GFs and a controlled protein release profile for > 1 month. Moreover, it was also observed that these NPs can be uptaken by human adipose-derived stem cells (hASCs), depending on the concentration of NPs in the culture medium and incubation time. This shows the versatility of the developed NPs, which, besides acting as a protein delivery system, can also be used in the future as intracellular carriers for bioactive agents, such as nucleotides. When the PL-loaded NPs were used as a replacement of bovine serum for in vitro hASCs culture, the viability and proliferation of hASCs was not compromised. The release of PLs from CH-CS NPs also proved to be effective for the enhancement of in vitro osteogenic differentiation of hASCs, as shown by the increased levels of mineralization, suggesting not only the effective role of the delivery system but also the role of PLs as an osteogenic supplement for bone tissue engineering and regenerative medicine applications.

PRODUCTION AND CHARACTERIZATION OF PARTIALLY PURIFIED EXTRACELLULAR THERMOSTABLE α-AMYLASE BY Bacillus subtilis IN SUBMERGED FERMENTATION (SmF)

A Bacillus strain was isolated from soil samples from the campus area of Dicle University. Based on 16S ribosomal RNA sequencing, the microorganism was closely related to Bacillus subtilis. Effects of different culture medium, incubation time, carbon and nitrogen sources, and various starches, flours, and chemicals on α-amylase production were examined. Maximum enzyme production (7516 U/mL) was obtained in a basal medium A containing 0.05% Tween 40 in 24 h. Partially purified enzyme showed maximum activity at 60°C with an optimum pH of 6.0. The effects of 0.2% detergents (sodium dodecyl sulfate [SDS], CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], and commercial detergent Omo Matic) on partially purified enzyme activity over a period of time (15–150 min) were examined and the order of inhibition effect from the most to the least was found as SDS > Omo Matic > CHAPS. Different metal ions inhibited α-amylase activity at low concentrations (1.5 mM). Co2+ was a mild inhibitor and Hg2+ and Cd2+ were potent inhibitors, whereas Ca2+ and Mg2+ increased the enzyme activity. At 20 mM, Ca2+ enhanced enzyme activity, and different Ca2+ concentrations (10–300 mM) were studied.

Incubation of fungal cultures: how long is long enough?

Fungal cultures are traditionally incubated for 4 weeks or longer to maximise the recovery of slowly growing fungi. However, the data in support of this are scarce. The objectives of this study were to determine the optimum incubation time for specimens in which moulds or yeast are suspected and to review the literature. A total of 3036 fungal cultures of 2216 dermatological and 820 non-dermatological specimens were analysed. The day on which fungal growth was first noted, was recorded. Eleven of 820 non-dermatological specimens were positive after day 14; in 10 cases, the fungus was considered clinically non-relevant and in one case, the cerebrospinal fluid of a patient receiving therapy for cryptococcosis was positive with Cryptococcus neoformans. Fourteen and three of 2216 dermatological samples showed the growth of a dermatophyte in the third week and fourth week respectively. The results indicate that for specimens sent for the detection of yeast or moulds (except dermatophytes and systemic dimorphic fungi), an incubation period of 2 weeks is sufficient, whereas for dermatophytes, a 4-week incubation period is necessary. Based on these results and previous literature, an algorithm for the incubation time of fungal cultures is proposed.

Longer Incubation Time of Frozen Cord Blood CFU-GM Cultures Allows Cells to Recover and Show Similar Growth Compared to CFU-GM Assays Performed on Fresh Cells

Longer Incubation Time of Frozen Cord Blood CFU-GM Cultures Allows Cells to Recover and Show Similar Growth Compared to CFU-GM Assays Performed on Fresh Cells

Time to positivity of neonatal blood cultures: fast and furious?

The aim of this study was to determine the time to positivity (TTP) of neonatal blood cultures, to investigate differences between early onset versus late-onset sepsis, and non-proven versus proven sepsis, and to examine differences in TTP by organism type using a retrospective observational study at the Neonatal Intensive Care Unit, Antwerp University Hospital, Belgium. The subjects were 1828 neonates with suspected sepsis who were treated with antimicrobials for at least 3 days. The TTP was recorded for all episodes of suspected sepsis in an approximately 6.5 year period. A total of 2916 blood cultures were collected, of which 437 (15%) became positive. The overall TTP was 21.33 h (Q1-Q3 13.17-32.46). The difference between the median TTP in early onset versus late-onset sepsis was 0.83 h (22.00 versus 21.17 h, P=0.75). The median TTP for Gram-negative organisms was 11.17 h (Q1-Q3 8.84-15.67), whereas the median TTP for Gram-positive organisms was 23.59 h (Q1-Q3 15.29-34.58, P<0.001). In Gram-positive isolates, the median TTP for coagulase-negative staphylococci (CNS) was 26.67 h (Q1-Q3 19.00-38.17), whereas the median TTP for non-CNS was 12.83 h (Q1-Q3 10.50-18.17, P<0.001). The median TTP in proven sepsis was 20.17 h (Q1-Q3 13.00-30.37), whereas it was 29.67 h (Q1-Q3 21.17-50.63, P<0.001) in non-proven sepsis. TTP of neonatal blood cultures was significantly shorter for Gram-negative organisms. We suggest shortening the total incubation time of neonatal blood cultures to a maximum of 3 days. However, blood cultures collected in infants<72 h of age might require a longer incubation time. According to our results, it may be safe to narrow the antimicrobial spectrum to solely target Gram-positive bacteria when the culture is still negative after 48 h, and to cease antimicrobial therapy when the culture is still negative after 72 h in clinically well infants.

Secretory expression and purification of a soluble NADH cytochrome b5 reductase enzyme from Mucor racemosus in Pichia pastoris based on codon usage adaptation

The genome of Mucor racemosus was analyzed to determine the relative levels of codon usage. The codon bias differed from that of Escherichia coli. The active, soluble isoform of NADH cytochrome b5 reductase containing 228 amino acids was successfully overexpressed and secreted using alpha factor in Pichia pastoris under the control of the alcohol oxidase promoter and finally purified. The culture medium and incubation time were optimized, and the maximum expression level observed was about 23 U/ml using X-33 recombinant yeast grown for 120 h with 0.5% (v/v) methanol in complex media.

ATP-Content and Kinetics of Acrosome Reaction in Human Spermatozoa: Influence of Various Culture Media and Incubation Time

The ATP content and the kinetics of acrosome reaction of isolated human spermatozoa were investigated during an in vitro culture period of 3 hours in 3 serum-enriched media commonly adopted in IVF and GIFT procedures (Earle solution, Ham F10 and Menezo B2). The ATP concentration in spermatozoa did not change between 1 and 3 hr of incubation and no differences were seen using the three media under investigation. The percentage of acrosome reactions significantly increased during culture, to a similar extent in the three media. The results suggest that each of the three media is equally well suitable for human sperm culture in preparation of IVF and GIFT procedures.

A One-Day, Dispense-Only IP-One HTRF Assay for High-Throughput Screening of Gαq Protein-Coupled Receptors: Towards Cells as Reagents

Abstract: Compared to biochemical high-throughput screening (HTS) assays, cell-based functional assays are generally thought to be more time consuming and complex because of additional efforts for running continuous cell cultures as well as the numerous assay steps when transferring media and compounds. A common strategy to compensate the anticipated reduction in overall throughput is to implement highly automated cell culture and screening systems. However, such systems require substantial investments in sophisticated hardware and highly specialized personnel. In trying to set up alternatives to increasing throughput in functional cell-based screening, we combined several approaches. By using (1) cryopreserved cell aliquots instead of continuous cell culture, (2) cells in suspension instead of adherent cells, and (3) "ready-to-screen" assay plates with nanoliter aliquots of test compounds, an assay procedure was developed that very much resembles a standard biochemical, enzymatic assay comprising only a few dispense steps. Chinese hamster ovary cells stably overexpressing a Galphaq-coupled receptor were used as a model system to measure receptor activation by detection of intracellular D-myo-inositol 1-phosphate with the help of homogeneous time-resolved fluorescence (HTRF, CISbio International, Bagnols-sur-Cèze, France). Initially established in 384-well adherent cell format, the assay was successfully transferred to 1,536-well format. The assay quality was sufficient to run HTS campaigns in both formats with good Z'-factors and excellent reproducibility of antagonists. Subsequently, the assay procedure was optimized for usage of suspension cells. The influences of cell culture media, plate type, cell number, and incubation time were assessed. Finally, the suspension cell assay was applied to pharmacological characterization of a small molecule antagonist by Schild plot analysis. Our data demonstrate not only the application of the IP-One HTRF assay (CISbio International) for HTS in a high-density format, but furthermore the successful use of cryopreserved and suspension cells in a one-day functional cell-based assay.

Comparison of microbiological culture methods in screening allograft tissue. Swab versus nutrient broth

In order to reduce the risk of transmission of infectious diseases through transplantation of tissue allografts, one should examine tissues for the presence of microorganisms. However, there are no detailed tissue banking guidelines describing the culture method or incubation time to be used. Therefore, we compared two culture methods – blood agar plate versus Wilkins Chalgren broth– and three incubation times – 2, 7 or 14 days for their performance. The ultimate aim is to use the optimal setting as standard operating procedure (SOP) for tissue allograft cultures. From 70 consecutive donors, 919 tissue samples were taken. All 919 tissue samples were incubated on blood agar as well as in Wilkins Chalgren broth for 7 days. 567 of these 919 tissue samples were left to incubate up to 14 days. Wilkins Chalgren broth yielded 24.5% (139/567) positive cultures after 14 days of incubation. This was slightly more than the growth on blood agar after 14 days (22.9% — 130/567) ( p = n.s.) and significantly more than the growth in Wilkins Chalgren broth after 7 days of incubation (21.9% — 124/567) (p < 0.05). Based on these results, Wilkins Chalgren broth has been implemented as the SOP. Since the yield of positive cultures increased from 2 to 7 days of incubation in broth (1.8 times) and the variability of species cultured from 7 to 14 days of incubation shifted towards mostly microorganisms known to be common contaminants, we established the cut-off at 7 days of incubation in Wilkins Chalgren broth.

Relative Quantitation of Protein–Protein Interaction Strength Within the Yeast Two-Hybrid System via Fluorescenceβ-Galactosidase Activity Detection in a High-Throughput and Low-Cost Manner

The yeast two-hybrid (Y2H) method is capable of delivering vast amounts of interacting positive yeast colonies from a single library screen, particularly if a multifunctional protein is used as bait. However, the selection of definitive colonies for further molecular analysis is limited by both technical practicality and high costs. Here we demonstrate a cost-effective and simple method for the rapid selection and ranking of those Y2H-positive interaction clones that are suitable for further analysis. We performed a Y2H screen for the identification of human transforming growth factor beta2- interacting proteins in a human skin keratinocyte library. The identified clones were ranked by the amount of beta-galactosidase enzyme produced, as well as by the interaction strength of the positive colonies. The combination of high-throughput microplate fluorescence readers and specific fluorescence assays can be utilized for relative quantitation of protein-protein interaction strength of Y2H-positive colonies in crude yeast-cell lysates. We demonstrate here that the high sensitivity of the fluorescence approach can bypass cumbersome conventional methods of cell lysis used in beta-galactosidase assays, and still deliver accurate values for analysis of protein interaction data. Finally, we also achieved a better understanding of general aspects of beta-galactosidase measurements in the Y2H system, such as protein normalization, the influence of yeast culture incubation time on optimal beta-galactosidase detection, and the linearity of beta-galactosidase detection in crude cell lysates.

Rapid Optimization of Antibotulinum Toxin Antibody Fragment Production by an Integral Approach Utilizing RC-SELDI Mass Spectrometry and Statistical Design

A process for the rapid development and optimization of the fermentation process for an antibotulinum neurotoxin antibody fragment (bt-Fab) production expressed in Escherichia coli was achieved via a high-throughput process proteomics and statistical experimental design. This process, using retentate chromatography-surface enhanced laser desorption/ionization mass spectrometry (RC-SELDI MS), was employed for identifying and quantifying bt-Fab antibody in complex biological samples for the optimization of microbial fermentation conditions. Five variables (type of culture media, glycerol concentration, post-induction temperature, IPTG concentration, and incubation time after induction) were statistically combined using an experimental 2(5)(-1) fractional factorial design and tested for their effects on maximal bt-Fab antibody production. When the effects of individual variables and their interactions were assessed, type of media and post-induction temperature showed statistically significant increase in yield of the fermentation process for the maximal bt-Fab antibody production. This study establishes an integral approach as a valuable tool for the rapid development of manufacturing processes for producing various biological materials. To verify the RC-SELDI MS method, a Fab-specific immuno-affinity HPLC assay developed here was also employed for the quantification of the bt-Fab antibody in crude lysate samples obtained during the fermentation optimization process. Similar results were obtained.

The impact of incubating the throat culture for 72 h on the diagnosis of group A beta-hemolytic streptococci

Objective The accurate detection and treatment of group A beta-hemolytic streptococcal pharyngitis is important to prevent the potential sequels. Throat culture remains the gold standard for the diagnosis of streptococcal pharyngitis. Many authors recommended 48 h incubation time for the isolation of group A Streptococcus (GAS). In this study the diagnostic efficacy of prolonging the incubation time of throat cultures to 72 h for isolation of GAS was researched. Methods Throat swab specimens were inoculated to 5% sheep blood agar plate and incubated at 37 °C, and evaluated at the 24th hour. GAS negative plates were incubated again for another 24 h and read at the 48th hour. Negative plates at 48 h were incubated again, read at the 72nd hour, and positive results of all measurements were evaluated. Results A total of 367 cultures were examined. At the 24th hour evaluation 34 (9.3%) cultures were positive for GAS. Evaluation of 333 cultures that were found to be negative in the first 24th hour evaluation revealed 44 (13.6%) positive cultures at 48th hour evaluation. Out of 289 cultures that were negative during the 48th hour evaluation, an additional 13 (4.7%) cultures were found to be positive during the 72nd hour evaluation. The agreement between 24th–48th and 24th and 72nd were both moderate (kappa 0.54 and 0. 46, respectively), and between 48th and 72nd hour was very good (kappa: 0.90). Conclusion Throat cultures that are negative for GAS at 24 h should be incubated for second day. We conclude that, to confirm the throat cultures GAS negative, results of throat cultures need to be incubated for 72 h, since 72nd hour evaluations revealed additional 5% positive results.

Production of Xyloglucanolytic Enzymes by Trichoderma viride, Paecilomyces farinosus, Wardomyces inflatus, and Pleurotus ostreatus

Different conditions of culture medium, incubation time, concentration and surfactant were tested to determine xyloglucanase activity. Trichoderma viride, Paecilomyces farinosus, Wardomyces inflatus and Pleurotus ostreatus showed increased xyloglucanase activities when the fungi grown on microcrystalline cellulose as the sole carbon source. Endoxyloglucanase activity increased with the growth of the fungi and reached a peak on day 14 of incubation, practically 95% of the activity was associated with the extracellular fraction. Precipitation with ammonium sulfate was the best concentration method for detection of endoxyloglucanase activity of the fungi. Endoxyloglucanase activity of the fungi was increased by 4 fold with the use of the non-ionic surfactant Tween 20. Six and three bands of xyloglucanase activities were observed in T. viride and P. ostreatus, respectively, whereas both P. farinosus and W. inflatus presented only one xyloglucanase activity band. These results indicate the presence of several xyloglucanases in the saprophytic fungi examined.

Production of xyloglucanolytic enzymes by Trichoderma viride, Paecilomyces farinosus, Wardomyces inflatus, and Pleurotus ostreatus

Different conditions of culture medium, incubation time, concentration and surfactant were tested to determine xyloglucanase activity. Trichoderma viride, Paecilomyces farinosus, Wardomyces inflatus and Pleurotus ostreatus showed increased xyloglucanase activities when the fungi grown on microcrystalline cellulose as the sole carbon source. Endoxyloglucanase activity increased with the growth of the fungi and reached a peak on day 14 of incubation, practically 95% of the activity was associated with the extracellular fraction. Precipitation with ammonium sulfate was the best concentration method for detection of endoxyloglucanase activity of the fungi. Endoxyloglucanase activity of the fungi was increased by 4 fold with the use of the non-ionic surfactant Tween 20. Six and three bands of xyloglucanase activities were observed in T. viride and P. ostreatus, respectively, whereas both P. farinosus and W. inflatus presented only one xyloglucanase activity band. These results indicate the presence of several xyloglucanases in the saprophytic fungi examined.

Modelling and laboratory investigation of microbial enhanced oil recovery

A one-dimensional model was developed to simulate the process of enhanced oil recovery by microorganisms. The model involves five components (oil, water, bacteria, nutrient and metabolites), with adsorption, diffusion, chemotaxis, growth and decay of bacteria, nutrient consumption, permeability damage and porosity reduction effects. Experiments were conducted to identify the parameters affecting the transport and growth of three bacterial strains: Streptococcus, Staphylococcus and Bacillus in porous media. Several correlations were developed from the experimental laboratory data and were used in the simulator. Comparison between the experimental and simulated results emphasized the validity of the developed simulator and determined its degree of accuracy (average absolute relative error = 8.323%). The simulator was used to investigate the effects of indigenous bacteria, slug size, incubation time, residual oil saturation, absolute permeability, and injection flow rate on oil recovery. Results show that more oil can be recovered by using Streptococcus with molasses as a medium. Oil recovery is sensitive to variation in concentration of injected indigenous bacteria, size of bacterial culture slug, incubation time and residual oil saturation. The change of absolute permeability, or injection flow rate, has no effect on oil recovery efficiency by bacteria.

Factors affecting the electroporation of Bacillus subtilis

Bacillus subtilis 168 trp- was found to be transformable with the tetracycline resistance plasmid pAB124 by electroporation of whole cells, inconsistently and at very low frequencies. Supplementation of the growth medium with glycine, or particularly DL-threonine, produced cells that could be electrotransformed much more efficiently at frequencies up to 2.5 x 10(3) transformants per microgram plasmid DNA. Transformation was optimal with cells grown in medium containing a racemic mixture of the D- and L-isomers of threonine, and no transformants were obtained when pure forms of the D- and L-threonine isomers were used. The cell walls of B. subtilis grown in the presence or absence of D-, L- and DL-threonine had a similar amino acid composition which did not include threonine. A more complex biochemical explanation of the enhancement of electroporation by growth in DL-threonine is likely, and this is discussed. Lysozyme treatments to weaken the cell wall and possibly mimic the effect of DL-threonine did not yield any transformants. The effects of buffer composition and culture incubation time were also determined and the electroporation protocol optimized accordingly. The response of a range of other B. subtilis strains to electroporation by the method produced was found to be variable. In all cases, transformation was verified by recovery of the plasmid DNA from putative transformants.

Major histocompatibility complex class I-restricted cytotoxic T-lymphocyte responses in horses infected with equine herpesvirus 1

An experimental system that permits sensitive and reproducible detection of equine herpesvirus 1 (EHV-1)-specific cytotoxic T-lymphocyte (CTL) activity in the horse was developed. Peripheral blood mononuclear cells (PBMC) collected from immune horses were restimulated in vitro by culture with live EHV-1. Cytotoxic activity against virus-infected, pokeweed mitogen-stimulated lymphoblast targets was assessed in a 4-h 51Cr release assay. The optimal conditions for in vitro stimulation of equine memory CTLs and for preparation of EHV-1-infected target cells expressing viral antigens were systematically identified by individually testing the effects of variations in responder cell concentration, culture medium composition, serum type, incubation time, antigen form, and exogenous mediator content. By using this optimized system for generation and assay of equine CTLs, the development of EHV-1-specific cytotoxic responses in 12 horses was evaluated after experimental viral infection. CTLs with the capacity for killing EHV-1-infected target cells were detected in equine PBMC as early as 1 week postinfection, reached maximal levels by 2 to 3 weeks, and remained detectable for a year after infection. Equine effector cells mediating lysis of EHV-1-infected targets were predominantly CD8+ T lymphocytes, and the cytotoxicity was specific for virus and restricted by major histocompatibility complex class I molecules. The results define a reliable and convenient experimental system for generation and assay of EHV-1 CTLs which can now be used for more-detailed characterization of the equine CTL response to infection by this herpesvirus pathogen.