The icaADBC gene locus of Staphylococcus aureus and its polysaccharide intercellular adhesin (PIA/PNSG) were recently identified, but biofilm formation has rarely been detected in vitro. In this study we evaluated a tissue culture plate (TCP) assay and a tube test, as well as Congo red agar, using the two basic media trypticase soy broth (TSB) and brain heart infusion (BHI) broth with different sugar supplements for detection of biofilm formation in 128 ica-positive S. aureus isolates. Of the S. aureus strains, 57.1% displayed a biofilm-positive phenotype under optimized conditions in the TCP test. The tube test correlated well with the TCP test for strongly biofilm-producing strains, whereas weak producers were not safely discriminated from biofilm-negative strains. Screening on Congo red agar displayed a strong correlation with the TCP and the tube test for only 3.8%, and is therefore not recommended for investigation of biofilm formation in S. aureus.

A serum-free medium (HMRI-2) has been developed for the outgrowth and subculture of epithelial cells from normal adult human ureter and bladder. Medium HMRI-2 consists of Ham's MCDB 152 with double the amounts of the essential amino acids in Stock 1, low Ca2+(0.06 mM) and is supplemented with epithelial growth factor, 5 ng/ml; transferrin, 5 micrograms/ml; insulin, 5 micrograms/ml; ethanolamine and phosphoethanolamine, 0.1 mM each; hydrocortisone, 2.8 X 10(-6) M; and bovine pituitary extract, 126 micrograms protein/ml. The cultured cells showed ultrastructural markers of epithelial cells (prekeratin fibers, tonofilaments, surface microvilli with glycocalyx), exhibited ABO antigens, and had a normal human diploid karyotype. Primary cultures could be subcultured and also cryopreserved in HMRI-2 in liquid nitrogen. Cells in mass cultures showed a population doubling time of 40.5 +/- 4.5 h and had a maximum in vitro life span of 20 to 25 population doublings. It was observed that primary outgrowths, secondary cultures, and even cryopreserved cells all retained the capacity to respond to high Ca2+ and serum by differentiation and desquamation. This study has resulted in the availability of easily obtainable serum-free epithelial cultures from normal adult human ureter and bladder. The useful in vitro life span of these cultures may be important in future studies of carcinogenesis.

Osmotic stress constitutes a major bacterial stress factor in the soil and during industrial fermentation. In this paper, we quantified the metabolic response, in terms of metabolic flux redistribution, of a lysine-overproducing strain of Corynebacterium glutamicum grown under continuous culture, to gradually increasing osmolality. Oxygen and carbon dioxide evolution rates, and the changes in concentration of extracellular, as well as intracellular, metabolites were measured throughout the osmotic gradient. The metabolic fluxes were estimated from these measurements and from the mass balance constraints at each metabolite-node of the assumed metabolic reaction network. Our results show that formation rates of compatible solutes--trehalose first and proline at a later stage of the gradient--increased with osmotic stress to equilibrate the external osmotic pressure. Estimated flux distributions indicate that the observed increase in the glucose specific uptake rate with osmotic stress is channeled through the main energy generating pathways-- glycolysis and the tricarboxylic acid cycle--while the flux through the pentose phosphate pathway remains constant throughout the gradient. This results in a significant increase in the net specific ATP production rate, which may possibly be used to support the higher energy requirements required for cellular maintenance at high osmolalities. Finally, nodal analysis confirmed that the PEP/pyruvate node is essentially rigid and that the glucose-6-phosphate, oxaloacetate and alpha-ketoglutarate nodes are flexible and therefore adaptable to changes in osmotic pressure in C. glutamicum.

The purpose of this study was to determine the ability of long shelf-life milk to serve as a temporary storage medium for the maintenance of periodontal ligament (PDL) cell viability on avulsed teeth. PDL cells were plated onto 24-well culture plates and allowed to attach for 24 h. Minimal Essential Medium was replaced with regular pasteurized milk (refrigerated milk), long shelf-life milk (Parmalat), or Save-A-Tooth. Tap water served as the negative control, and Minimal Essential Medium served as the positive control. The tissue culture plates were incubated at 37 degrees C for 1, 2, 4, or 8 h. Cell viability was determined using a cell proliferation assay (CellTiter 96 AQ Assay) and absorbance read at 490 nm. ANOVA indicated that all media performed significantly better than tap water at all time periods. At 8 h, PDL cell viability in regular pasteurized milk and long shelf-life milk were significantly greater than in Save-A-Tooth (p < or = 0.001). There was no significant difference between regular pasteurized milk and long shelf-life milk at any time period. These results suggest that long shelf-life milk, which has the advantage of not requiring refrigeration, is as effective a storage medium for avulsed teeth as regular pasteurized milk and more effective than Save-A-Tooth.

Parallel testing for culture recovery of Clostridium difficile was performed using three selective media in each of four anaerobic incubation environmental systems. Testing was completed on 67 stool samples from 60 hospitalized patients in whom C difficile-associated diarrhea was suspected. Three different media were evaluated: CCFA (modified cycloserine-cefoxitin-fructose agar), CCFA-PRAS (CCFA, prereduced-anaerobically-sterilized) and CMBA (modified cycloserine-mannitol-blood agar). The incubation systems compared were an anaerobic chamber (Model 800 Anaerobic Environmental System, Anaerobe Systems, San Jose, CA), the anaerobic jar (BBL, Cockeysville, MD), the anaerobic Bio-Bag (BBL), and the anaerobic pouch (BBL). C difficile was found in 16 samples collected from 15 patients. Comparing recovery on the various types of media in all four anaerobic atmospheres, C difficile was recovered on all (64) CCFA plates, 56 of 64 CCFA-PRAS plates, and 43 of 64 CMBA plates (P <.03 comparing CCFA and CMBA). Of the 48 plates in each incubation system that were inoculated with specimens positive for C difficile, the organism was recovered from 43 plates in the anaerobe chamber, 41 incubated in an anaerobe jar, 40 in the Bio-Bag, and 39 incubated in the GasPak pouch, all providing similar recovery of C difficile (P =.08). The CCFA-PRAS and CMBA media demonstrated less inhibition of normal stool flora compared to the CCFA. Overall CCFA that was anaerobically reduced at least 4 hours before use, and contained the original concentration of antimicrobial agents described by George and colleagues, was superior to CMBA for recovery of C. difficile.

BACKGROUND Old, stationary cultures of Mycobacterium tuberculosis contain a majority of bacteria that can grow in broth cultures but cannot grow on solid medium plates. These may be in a non-replicating, dormant growth phase. We hypothesised that a similar population might be present in chronic, murine tuberculosis. METHODS Estimates of the numbers of viable M. tuberculosis, strain H37Rv, in the spleens and lungs of mice in a 7-day acute infection and in a 10-month chronic infection were made by conventional plate counts and, as broth counts, by noting presence or absence of growth in serial replicate dilutions in liquid medium. RESULTS Plate and broth counts in 6 mice gave similar mean values in the acute infection, 7 days after infection. However, the broth counts were much higher in 36 mice with a chronic infection at 10 months. Broth counts averaged 5.290 log10 cfu /organ from spleens and 5.523 log10 cfu/organ from lungs, while plate counts were 3.858 log10 cfu/organ from spleens and 3.662 log10 cfu/organ from lungs, indicating that the total bacterial population contained only 3.7% bacilli in spleens and 1.4% bacilli in lungs, capable of growth on plates. CONCLUSION The proportion growing on plates might be a measure of the "dormancy" of the bacilli equally applicable to cultural and animal models.

OBJECTIVE: Our purpose was to compare the yield of positive group B Streptococcus cultures with standard medium for transport of culture swabs compared with use of selective medium during transport. STUDY DESIGN: Cultures of introitus, perineum, and rectum were obtained on prenatal patients; one was placed in standard transport medium, and the other directly in selective growth medium. Swabs in standard transport medium were plated for routine culture and then transferred to selective growth medium, Todd-Hewitt broth, in the laboratory. RESULTS: A total of 307 of 1222 (25.1%) patients had a positive result by any method. With direct inoculation into selective growth medium at the time of sampling, 4.6% of positive cultures were missed. With delayed inoculation into selective growth medium, 16.3% were missed (p < 0.001). Without use of selective media (routine culture), 31.9% were missed (p < 0.001). CONCLUSIONS: Use of standard transport medium with subsequent transfer into selective growth medium results in a significantly decreased yield of positive group B Streptococcus cultures.

OBJECTIVE To analyze critically the reasons justifying the choice of two-step protocols requiring two media for the culture of human preimplantation embryos from the zygote to the blastocyst. DESIGN Literature review. RESULT(S) Two types of protocol are used for the culture of human preimplantation embryos from the zygote to the blastocyst, using either one medium (one-step protocol) or two media of different composition (two-step protocol). Two-step protocols are the most widely used, largely because all but one of the commercially available protocols are of this type. The reasons for the adoption of two-step protocols are described and critically analyzed. They are based on considerations of the functions of glucose, ethylenediaminetetraacetic acid (EDTA), glutamine, and amino acids that are included in the media. A reappraisal of the reasons for selecting two-step protocols is important because recent animal experiments and clinical observations have raised doubts as to whether the more complex, two-step protocols have any advantage over one-step protocols. The analyses show that all of conclusions reached should be considered equivocal. CONCLUSION(S) Clinical embryologists should evaluate the justification for selecting two-step protocols for the culture of human preimplantation embryos from the zygote to the blastocyst.

A major limitation of the National Committee for Clinical Laboratory Standards M27-A methodology is reliable detection of amphotericin B (AMB) resistance. The results obtained by using Iso-Sensitest, a synthetic medium, to detect AMB resistance were analyzed and compared with those obtained with RPMI and antibiotic medium 3 (AM3). The ability to detect AMB resistance with RPMI is not enhanced by using a higher inoculum, glucose supplementation at a final concentration of 20 g/liter, spectrophotometric reading, or 24 h of incubation time. Testing using AM3 and an inoculum of 10(3) CFU/ml detects resistance. Identification of resistant isolates is not improved by glucose supplementation, changes in reading method, or changes in incubation time. However, the use of AM3 as assay medium and an inoculum of 10(5) CFU/ml did not allow detection of AMB resistance. Testing using Iso-Sensitest medium appears to be similar to AM3 in detecting resistance. The most pronounced discrimination is achieved by testing in Iso-Sensitest supplemented with glucose and spectrophotometric reading after 24 h of incubation. The reproducibility of MIC testing was greatest for Iso-Sensitest-based procedures. Use of Iso-Sensitest produces both highly reproducible MICs and reliable identification of AMB-resistant Candida isolates.