Comparison of Atmospheric Conditions for Culture of Clinical Specimens of Neisseria gonorrhoeae

We cultured 55 clinical specimens of Neisseria gonorrhoeae in the following atmospheric conditions: (i) 10% carbon dioxide in a CO(2) incubator; (ii) a candle extinction jar; (iii) an air convection incubator; and (iv) an anaerobic jar without added CO(2). The number and size of colonies growing on modified Thayer-Martin medium were evaluated after incubation of cultures for 24 and 48 h at 36 C. After 24 h, the specimens from the candle extinction jar had the greatest number and size of colonies, but after 48 h growth was approximately equal for specimens from the candle jar and the CO(2) incubator. Only 19 of 55 specimens grew in the air convention incubator. None of 55 clinical specimens or of 10 laboratory strains grew anaerobically. Development of colonial morphology for colony types 1, 2, 3, and 4 was studied at 24 h on a base medium that contained no hemoglobin. The relative numbers of the four colony types in specimens were comparable after 24 h of incubation in any of the three atmospheric conditions under which growth occurred, but the different types were distinguished most readily when grown in the candle extinction jar.     

Practicability of Hygienic Wrapping of Touchscreen Operated Mobile Devices in a Clinical Setting

Background

To prove effectiveness of wrapping tablet computers in order to reduce microbiological contamination and to evaluate whether a plastic bag-covered tablet leads to impaired user satisfaction or touchscreen functionality.

Materials and Methods

Within a period of 11 days 115 patients were provided with a tablet computer while waiting for their magnetic resonance imaging examination. Every day the contamination of the surface of the tablet was determined before the first and after the final use. Before the device was handed over to a patient, it was enclosed in a customized single-use plastic bag, which was analyzed for bacterial contamination after each use. A questionnaire was applied to determine whether the plastic bag impairs the user satisfaction and the functionality of the touchscreen.

Results

Following the use by patients the outside of the plastic bags was found to be contaminated with various bacteria (657.5 ± 368.5 colony forming units/day); some of them were potentially pathogenic. In contrast, the plastic bag covered surface of the tablet was significantly less contaminated (1.7 ± 1.9 colony forming units/day). Likewise, unused plastic bags did not show any contamination. 11% of the patients reported problems with the functionality of the touchscreen. These patients admitted that they had never used a tablet or a smartphone before.

Conclusions

Tablets get severely contaminated during usage in a clinical setting. Wrapping with a customized single-use plastic bag significantly reduces microbiological contamination of the device, protects patients from the acquisition of potentially pathogenic bacteria and hardly impairs the user satisfaction and the functionality of the touchscreen.     

Evaluation of the Gonosticon Dri Dot Test in Females with a Low Incidence of Gonorrhea

A group of 765 females attending a Planned Parenthood Clinic was screened for gonorrhea by inoculating Thayer-Martin plates and Transgrow bottles with specimens from the cervix. Blood was obtained at the same time and tested for anti-gonococcal antibody by using the Gonosticon Dri Dot test. In this low-incidence group, 18 positive cultures were detected by culture on Thayer-Martin plates, whereas Transgrow detected only 15 positive cultures. Of the 18 patients with gonorrhea, 11 exhibited reactive serum (agglutination of the latex particles). In the total population, 64% of the patients had nonreactive serum (no agglutination) and negative cultures; 25% had reactive serum and negative cultures. When this latter group was subdivided on the basis of race, blacks and Latin Americans were found to have a higher incidence of reactive serum with a corresponding negative culture than was found in whites. Patients who were originally culture positive and nonreactive in the Gonosticon Test were retested; three out of four patients retested within 6 to 11 days after the initial screening had converted to a positive Gonosticon test.     

Enzyme immunoassay (EIA) in the rapid diagnosis of gonorrhoea

The diagnostic value of a new, modified enzyme immunoassay (EIA) (Gonozyme; Abbott Laboratories, North Chicago III) was evaluated for the rapid antigenic detection of Neisseria gonorrhoeae in endocervical and urethral specimens. EIA results were compared with those of Gram stain (GS) and conventional culture tests. EIA sensitivity and specificity for male patients attending dermatovenerological clinic were 100% and 96.8% respectively in comparison to 86.7% and 96.8% obtained by Gram staining. For female Obstetrics-Gynaecology patients EIA sensitivity of 100% was highly significant compared to 50% sensitivity by the Gram stain. In culture, 30 strains of N. gonorrhoeae were isolated from 125 male specimens and 2 from 105 specimens from females; this suggests a prevalence of N. gonorrhoeae of 24% in males and 1.9% in females. In vitro antibiotic sensitivity testing indicated 55% resistance to penicillin and 43% to ampicillin in these isolated strains; all were sensitive to erythromycin/tetracycline. 12% of the strains were beta-lactamase producers.     

Rapid large-scale growth of Helicobacter pylori in flasks and fermentors.

We developed procedures for large-scale cultivation of Helicobacter pylori in flasks and fermentors. Flasks incubated closed under a microaerophilic gas phase with a cotton plug covered by a plastic bag, followed by removal of the bag after 8 h, gave excellent growth. Growth in a 10-liter fermentor led to excessive foaming if the medium was sparged with gas; silicone- or polyglycol-based antifoaming agents were severely inhibitory. Use of fermentor surface gassing, first with a microaerophilic 6% oxygen gas mixture, then with air, and then with 95% oxygen, allowed the culture to grow to an A600 of 2.5 in < 24 h. This method was modified for scale-up to a 100-liter fermentor.     

Coagglutination as a test for Neisseria gonorrhoeae

After growth on Thayer-Martin medium, 196 strains of freshly isolated Neisseria gonorrhoeae were subjected to a coagglutination reaction. The sensitivity of the test was 94% and did not vary much in the hands of four consecutive technicians. In a group of 99 strains tested by one of the technicians non-interpretable results were obtained with 17% of the strains when the test was performed with cells taken from the first or primary plate, against 9% when cells from the secondary (subcultured) plate were used. The lowest number of non-interpretable results was found with a modified Thayer-Martin medium, which also showed the lowest number of false negatives (2%).No non-interpretable results were obtained when the bacterial suspension was first heated to 100°C for 3 min. In a group of 14 recently isolated strains of non-gonococcal species there was only one, preventable, false-positive strain and there were none in a group of 12 meningococci (all of them laboratory strains).In comparison with the fermentation test with Lingelsheim's sugars, the coagglutination test with cells taken from the primary plate with Thayer-Martin medium yielded a conclusive result more often. The test is simple and rapid and does not require special technical equipment. It seems to deserve a place as a confirmative test in the search for gonococci in samples from the urogenital-anal area.     

红酵母固态发酵类胡萝卜素产物稳定性的研究

探讨了红酵母固态发酵类胡萝卜素产物的干燥及储存方法;比较了真空干燥和鼓风干燥2种方法对固态发酵产物稳定性的影响;考察了光照、空气、储藏温度对固态发酵产物稳定性的影响。结果表明:在相同温度下,真空干燥(5.67±0.58)h比鼓风干燥所需时间短(9.33±0.58)h、类胡萝卜素损失小,效果也较好;光照和空气对其储藏过程中色素稳定性影响很大,30 d后类胡萝卜素损失了53%;培养物烘干后用透明塑料袋真空避光储藏效果显著,6个月后类胡萝卜素只损失19%,不抽真空损失了46%;温度对培养产物中色素稳定性影响不显著;红酵母菌株固态发酵培养产物应选择真空避光室温储藏。     

Comparison of culture media and chairside assays for enumerating mutans streptococci

AIM: This study compared several traditional culture-based media and chairside cultural assays for ability to recover mutans streptococci (MS) from pure cultures and from saliva samples. METHODS AND RESULTS: When pure cultures were used with traditional culture-based media, mitis-salivarius bacitracin (MSB) agar demonstrated less support for bacterial recovery than trypticase-yeast extract-cysteine sucrose-bacitracin (TYCSB) agar and the modified medium of Ritz (HLR-S). One species of MS, Streptococcus ferus (c), was not recovered on MSB medium. Chairside cultural tests displayed considerable disparity between tests in recovering bacteria from pure cultures. On the glass adherence assay (Mucount), S. ferus was not detected and Streptococcus criceti was not detected on the dipslide assay (Cariescreen SM) or on the plastic adherence assay (Dentocult SM Strip mutans). The frequency of isolation of pure strains of bacteria other than MS was common. From saliva samples, the frequency of isolation of MS on HLR-S and TYCSB media and the glass adherence assay was 91-97%. The frequency of isolation on MSB medium and on the dip-slide and plastic adherence assays was significantly decreased (37, 47 and 69%, respectively). Recovery scores varied considerably among the culture methods studied and tended to be highest on the HLR-S medium and on the glass adherence assay. CONCLUSIONS: Growth and recovery profiles of pure bacterial cultures and of saliva samples for the MS varied according to different media. SIGNIFICANCE AND IMPACT OF THE STUDY: Caution should be exercised in comparing results between studies that employ different cultural methods for MS enumeration.     

Glucocorticoid inhibition of vascular smooth muscle cell proliferation: influence of homologous extracellular matrix and serum mitogens

We examined the influence of glucocorticoid hormones on the proliferation of cultured adult bovine aortic smooth muscle cells (BASM) using both primary mass cultures and a cloned strain. Cloned BASM cells maintained on plastic culture dishes were inhibited by approximately 40% by dexamethasone treatment but showed no inhibition when grown of homologous extracellular matrix (ECM) coated dishes. Dexamethasone inhibited growth of primary cultures by 73% on plastic and by 45% on ECM. The inhibitory effect was specific for the glucocorticoids, dexamethasone, corticosterone, and cortisol and was not observed with progesterone, aldosterone, estradiol or 17-alpha OH progesterone. In cloned cells, the abolition of glucocorticoid inhibition by ECM was independent of seeding density and serum concentration. The inhibition on plastic was dependent on serum concentrations greater than 1% and resulted in both a slow rate of proliferation and a lower saturation density. A specific subset of peptides detected on two-dimensional gels was induced by glucocorticoids under growth inhibitory conditions but was not induced when the cells were grown on ECM. Primary cultures grown on ECM and exposed to Dulbecco's modified Eagle's Medium (DME) containing high density lipoprotein and transferrin grew at 40% of the rate observed for cultures exposed to DME with 10% serum. Both conditions showed growth inhibition of 70% in the presence of dexamethasone. The addition of epidermal and platelet-derived growth factors in DME containing high density lipoprotein and transferrin to cells grown on ECM resulted in growth rates comparable to that observed with cultures exposed to 10% serum and were inhibited 45% by dexamethasone. These results suggest that glucocorticoids inhibit smooth muscle proliferation by decreasing the sensitivity of the cells to mitogenic stimulation by high density lipoprotein when the cells are maintained on a homologous substrate.     

HRM confirmation of Neisseria gonorrhoeae in clinical specimens by G→A (position 857) mutation detection in the 16S rRNA gene before sequencing and after porA confirmation

A total of 2273 specimens submitted to the Austin Hospital Pathology Service for Neisseria gonorrhoeae screening between September 1, 2009 and May 11, 2011 were used in this study. Specimens were simultaneously screened and confirmed with a previously published real time PCR assay for the opa gene (extra primers were included to increase sensitivity) and the porA gene respectively. The opa gene screen and initial porA gene confirmation yielded an N. gonorrhoeae positivity rate of 0.88% (20/2273) and 0.49% (11/2191) for specimens and patients respectively. A 16S rDNA High Resolution Melt confirmatory PCR was developed subsequently; this reduced the N. gonorrhoeae positivity rate to 0.35% (8/2273) and 0.27% (6/2191) for specimens and patients respectively (not altered by 16S sequencing). The higher rate of secondary confirmation (16S HRM) in patients compared with samples was due to the detection of species other than N. gonorrhoeae detected by the initial screening and confirmation test. This underlines the importance of performing the secondary confirmatory test that has been developed in this study.     

A comparison of a new campylobacter selective medium (CAT) with membrane filtration for the isolation of thermophilic campylobacters including Campylobacter upsaliensis

The newly developed CAT campylobacter selective medium employing the blood-free charcoal-based agar containing cefoperazone (8 mg I⁻¹), amphotericin (10 mg I⁻¹) and teicoplanin (4 mg I⁻¹) was compared with the membrane filtration culture technique for isolation of Campylobacter spp. including Camp. upsaliensis. Nine hundred and fifty human, 275 dog and 65 cat faeces (in which modified CCDA medium was also compared) were tested. In addition, the recovery of Camp. upsaliensis from pure cultures and from spiked human faeces was examined after membrane filtration. A 50-fold reduction in recovery after filtration using the 0·65 μm filters and a 150-fold reduction using the 0·45 μm filters was found. Recovery of Camp. upsaliensis from spiked faeces was considerably improved using the CAT medium compared with filtration, especially with the lower concentration of organisms (approx. 10⁴ cfu ml⁻¹). Campylobacter upsaliensis was recovered from 91 specimens of animal faeces, with CCDA recovering 26 isolates (29%), CAT recovering 76 isolates (84%) and membrane filtration (0·65 μm) recovering 82 isolates (90%). CAT selective agar was found to be a suitable medium for the isolation of thermophilic campylobacters including Camp. upsaliensis from faecal samples.     

An Unclassified Gram-Negative Rod Isolated From the Pharynx on Thayer-Martin Medium (Selective Agar)

An oxidase-positive, small gram-negative rod was isolated on Thayer-Martin medium (TM) inoculated with pharyngeal swabs obtained during surveys to detect Neisseria carriers. In one survey, this organism was isolated from 48% of the subjects, and 50 or more colonies were present on the majority of the primary isolation plates. Other characteristics of the organism, which has been given the provisional designation "TM-1," include: delayed production (2 to 10 days) of acid from glucose, formation of gas during nitrate reduction, and the frequent formation of "pits" in the agar surface. On TM, nonpitting colonies of TM-1 are morphologically similar to colonies of Neisseria gonorrhoeae and N. lactamica. Comparison of the characteristics of TM-1 strains with other similar fastidious gram-negative organisms encountered in clinical laboratories indicates that TM-1 is a distinct species. Further studies are required before proper taxonomic placement can be made.     

Automatic surface inoculation of agar trays

A machine is described which automatically inoculates a plastic tray containing agar media with a culture by use of either a conventional inoculating loop or a cotton swab. Isolated colonies were obtained with an inoculating loop when a heavy inoculum (10⁹ cells/ml) was used or with a cotton swab when a light inoculum (ca. 10⁴ cells/ml) was used. Trays containing combinations of differential or selective media were used to (i) separate mixtures of gram-positive and gram-negative bacteria, (ii) facilitate isolation of organisms from clinical specimens, and (iii) compare colony growth characteristics of pure cultures. The design of the machine is simple, it is easy to use, and it relieves the operator from the manual task of streaking cultures.     

Evaluation of different culture systems with low oxygen tension on the development, quality and oxidative stress-related genes of bovine embryos produced in vitro

The present study was conducted to assess the development, quality and gene expression profile of oxidative stress-related genes of bovine embryos cultured in different culture systems with low oxygen tension (5% CO2, 5% O2 and 90% N2). The systems assessed included: (1) an incubator chamber; (2) a plastic bag; and (3) a foil bag. The choice of culture system had no effect on cleavage rate at 72 h. However, significant differences (P < 0.01) were observed in the rate of blastocysts registered at day 7 (29.8, 20.2 and 12.7% for incubator chamber, plastic bag and foil bag, respectively). Total number of cells did not differ between systems, although the proportion of ICM:total cells was affected particularly in the plastic bag (19.5%), compared with the incubator chamber (31.4%). In addition, significant differences were found in the apoptotic:total cell ratio (3.3, 6.5 and 8.8% for the incubator chamber, plastic bag and foil bag, respectively), with apoptotic nuclei localised mainly in the ICM compartment of the embryo. The amount of reactive oxygen species was also different between culture systems and this effect was correlated with a higher expression of SOD2, GSS and GPX1 genes in embryos cultured in the gassed bags as compared with embryos cultured in the incubator chamber. In conclusion, these results give evidence that, under low oxygen tension, the incubator chamber is more efficient and generates higher number of, and better quality, embryos than gassed bag systems evaluated here and this effect was probably due to an increased level of reactive oxygen species in the gassed bags, which upregulates the expression of some antioxidant enzymes to compensate for hyperoxia conditions.     

Auxotypes of Neisseria gonorrhoeae isolated from localized and disseminated infections in Montreal.

A survey recently made in the United States on the regional distribution of auxotypes of Neisseria gonorrhoeae suggested that isolates from different geographic areas often differ in auxotype. A subsequent auxotyping study in Montreal of 901 isolates of N. gonorrhoeae, 15 from patients with disseminated gonococcal infection, proved interesting in many regards. Gonococcal genetic medium, modified by the addition of other amino acids, was used. Most (93%) of the strains isolated from patients with localized infection belonged to one of the following three phenotypes: arginine-, hypoxanthine- and uracil-dependent (44%); prototrophic (33%); and proline-dependent (16%). Of the 15 strains responsible for disseminated infection 14 required arginine, hypoxanthine and uracil for growth.     

Studies on Crystalline Growth of O2-susceptible Proteins in Space

In order to meet the requirement for crystalline growth of O2-susceptible proteins in space, crystallization conditions on the earth was optimized for the proteins using a simple and suitable device for anaerobic addition of the protein samples. Nitrogenase is susceptible to O2. ΔnifZ MoFe protein from a nifZ deleted strain and MnFe protein from mutant strain UW3 grown on a medium containing Mn were crystallized at the first time in the world using an anaerobic device equipped with plastic bags or using a small simplified box, as a replacement for the cumbersome dry box. And the proteins could be also crystallized far from laboratory by sitting-drop method using a much lighter device. It was equipped with a smaller plastic food bag and a first-aid bag filled with Ar, as a substitute for the cumbersome dry box and the Ar cylinder, respectively. The results showed that the device could meet the requirement for studies on crystal growth of the above anaerobic proteins in space.     

Factors involved in the control of proliferation of bovine corneal endothelial cells maintained in serum-free medium

Experimental conditions have been defined that allow bovine corneal endothelial (BCE) cells to grow in the complete absence of serum. Low density BCE cell cultures maintained on extracellular matrix (ECM)-coated dishes and plated in the total absence of serum proliferate actively when exposed to a synthetic medium supplemented with high density lipoprotein (HDL 500 μg protein/ml), transferrin (10 μg/ml), insulin (5 μg/ml), and fibroblast (FGP) or epidermal growth factor (EGF) added at concentrations of 100 or 50 ng/ml, respectively. Omission of any of these components results in a lower growth rate and/or final cell density of the cultures. BCE cell cultures plated on plastic dishes and exposed to the same synthetic medium grow very poorly. The longevity of BCE cultures maintained on plastic versus ECM and exposed to serum-free versus serum-containing medium has been studied. The use of ECM-coated dishes extended the life span of BCE cultures maintained in serum-supplemented medium to over 120 generations, as compared to less than 20 generations for cultures maintained on plastic. Likewise, BCE cells maintained on ECM and exposed to a synthetic medium supplemented with optimal concentrations of HDL, transferrin, insulin, and FGF underwent 85 generations, whereas control cultures maintained on plastic could not be passaged. The enhancing effect of ECM on BCE cell growth and culture longevity clearly illustrates the importance of the cell substrate in the control of proliferation of these cells.     

Reconstituted basement-membrane matrix modulates fibroblast activities in vitro

Cellular growth and collagen biosynthesis were compared in dermal calf fibroblasts cultured on plastic or on a reconstituted basement membrane gel, termed matrigel. This matrix, extracted from Engelbreth-Holm-Swarm tumors, consists mainly of laminin, entactin, type IV collagen, and heparan sulfate proteoglycan. The multiplication rate of fibroblasts grown on matrigel was stimulated compared to that of monolayered cells cultured on plastic, and these cells formed multilayers after 4 days. Protein and collagen biosynthesis was reduced in fibroblasts cultured on matrigel. A higher proportion of the newly synthesized collagen (40%) was incorporated to the extracellular matrix in cultures grown on matrigel than in those grown on plastic (14%). Type III collagen was the preferential collagen type deposited on matrigel, and the ratio of type III:type I collagens secreted in the medium was also slightly higher in cultures grown on matrigel. Partially processed collagen was more abundant in fibroblasts grown on matrigel than in cells cultured on plastic. Finally, cells grown on matrigel exhibited a higher catabolic activity than cells grown on plastic. In this experimental model, the reconstituted basement-membrane matrix seems to influence the activities of fibroblasts significantly.     

Induced changes in the surface of Neisseria gonorrhoeae

Growth of Neisseria gonorrhoeae strain F62 on medium containing pyruvate and a high ratio of cysteine to cystine resulted in functional and structural changes that are consistent with phenotypic changes in lipopolysaccharide. Both transparent (O-) and moderately opaque (O+) variants became more sensitive to killing by normal human serum and resistant to killing by pyocin G, a bacteriocin from Pseudomonas aeruginosa. Electrophoresis of outer membranes in the presence of sodium dodecyl sulfate demonstrated differences also dependent upon the growth medium. When gels were treated with periodic acid and stained with silver, lanes containing outer membranes obtained after growth in the modified medium demonstrated two bands in addition to those independent of the growth medium. The enhancement of these additional bands by periodate treatment indicated that they represent material containing carbohydrate. The mechanism by which the changes in the growth medium affected the surface of N. gonorrhoeae is not known; however, the changes demonstrated by electrophoresis were dependent upon either the high concentration of cysteine or the high ratio of cysteine to cystine.     

Selective isolation of mycobacteria from soil: a statistical analysis approach

We compared four decontamination methods for the isolation of mycobacteria from soil specimens. Different media were used: L?wenstein-Jensen, Ogawa and various modified Ogawa media. Statistical analysis demonstrated that the best results (low contamination and high positivity rates) were obtained when the specimens were incubated in trypticase soy broth, treated with solutions containing malachite green and cycloheximide, then decontaminated with sodium hydroxide and inoculated onto Ogawa media. The lowest contamination rates were obtained with Ogawa medium containing 500 micrograms cycloheximide ml-1. The use of these techniques is proposed for the isolation of mycobacteria from heavily contaminated clinical specimens as well as from soil.