Hydroxyapatite is a calcium phosphate bioceramic that has been shown by many authors to be biocompatible with bioactive properties. It is widely accepted as the best synthetic material available for surgical use as a bone graft substitute. HA granules produced by AMREC-SIRIM from local materials underwent 5 types of sterilisation techniques with different ageing periods. Samples were tested for chemical and phase composition and microbial contamination before and after being sterilised. From the microbiological tests done, none of the unsterilised positive control yielded a positive culture. Results from X-Ray diffraction studies found that all the sterilisation techniques did not chemically degrade or structurally change the HA granules significantly.
Bacteriological Techniques ; Bone Substitutes/*analysis ; Calcium Phosphates/*analysis ; Materials Testing/*methods ; Sterilization/*methods ; X-Ray Diffraction

A magnetic separator was used to separate magnetic bacteria based on their magnetotactic characteristics. Acidithiobacillus ferrooxidans, a bacterium that could synthesize intra-cellular nanometer magnetic particles, was investigated as an example. Strong magnetic and weak magnetic cells were separated and collected. On average, the number of the magnetic particles present in the strong magnetic cells is more than that of the weak magnetic cells. Moreover, semisolid-plate magnetophoresis showed that the magnetotaxis of strong magnetic cells was stronger than the weak magnetic cells. These results suggest that the magnetic separator can be used to isolate the magnetic bacteria, which will facilitate the research of magnetic bacteria.
Acidithiobacillus ; isolation & purification ; metabolism ; Bacteria ; isolation & purification ; metabolism ; Bacterial Physiological Phenomena ; Bacteriological Techniques ; methods ; Magnetics

In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth (Luria-Bertani agar plates for 12-18 h) and lysis conditions (4 h incubation with 500 µg/mL lysozyme) produced sharp bands on the gel. Restriction enzyme NotI was chosen as the most suitable. Twenty-two isolates were analyzed by NotI digestion, using three electrophoretic parameters (EPs). The EP-a was optimal for distinguishing between isolates. The optimized protocol could be completed within 40 h which is a significant improvement over the previous methods.
Bacillus cereus ; genetics ; isolation & purification ; Bacteriological Techniques ; DNA, Bacterial ; chemistry ; genetics ; Electrophoresis, Gel, Pulsed-Field ; methods

Clostridium cellulolyticum, as one of obligate anaerobic bacteria capable of secreting cellulosome, has not been efficiently cultured due to its strict requirement of growing conditions. In this study, culture conditions of C. cellulolyticum were optimized using response surface methodology. Plackett-Burman design was first used to screen the dominant impact factors for the growth of C. cellulolyticum, which were determined as yeast extract concentration, cellobiose concentration and culture temperature. The steepest ascent path design was then applied to gain the suitable range close to the optimal culture conditions for obtaining high cell density. The central composite design and the response surface analysis were finally used to determine the optimal levels of the influential factors, which were 3 g/L for yeast extract concentration, 7 g/L cellobiose concentration and 34 degrees C for culture temperature. The optimized medium was used for flask culture, and OD600 of C. cellulolyticum was increased from 0.303 to 0.586. With a pH-controlled fermentor at batch mode, OD600 reached 3.432, which was 2.8 times higher than elsewhere reported. These results support further study on the high-density culture of C. cellulolyticum and its application.
Bacteriological Techniques ; methods ; Clostridium cellulolyticum ; growth & development ; Culture Media ; Population Density

This study was aimed to analyze the results of false positive reaction in bacterial detection of blood samples with BacT/ALERT 3D system, to evaluate the specificity of this system, and to decrease the false positive reaction. Each reaction flasks in past five years were processed for bacteria isolation and identification. When the initial cultures were positive, the remaining samples and the corresponding units were recultured if still available. 11395 blood samples were detected. It is worthy of note that the incubator temperature should be stabilized, avoiding fluctuation; when the cultures were alarmed, the reaction flasks showed be kept some hours for further incubation so as to trace a sharply increasing signal to support the judgement of true bacterial growth. The results indicated that 122 samples (1.07%) wee positive at initial culture, out of them 107 samples (88.7%) were found bacterial, and 15 samples (12.3%) were found nothing. The detection curves of positive samples resulted from bacterial growth showed ascent. In conclusion, maintenance of temperature stability and avoidance of temperature fluctuation in incubator could decrease the occurrence of false-positive reaction in detection process. The reaction flasks with positive results at initial culture should be recultured, and whether existence of a sharply ascending logarilhimic growth phase in bacterial growth curve should be further detected, which are helpful to distinguish false-positive reactions from true positive, and thus increase the specificity of the BacT/ALERT system.
Automation, Laboratory ; Bacteria ; isolation & purification ; Bacteriological Techniques ; methods ; Blood ; microbiology ; Culture Media ; False Positive Reactions ; Humans

To seek a practical and inexpensive method to preserve gonococcal cultures, a few methods were compared. The following methods kept the cultures alive for only a short period of time: skim milk at -20 degrees C; tryptic soy broth with 15% glycerol at -20 degrees C; cystine tryptic agar at 35 degrees C. Most of the test cultures survived for more than 4 weeks in the following media: one half strength dextrose starch agar; one half strength dextrose starch agar with ferric nitrate; one half strength dextrose starch agar with antimicrobic CNV. Dextrose starch agar could be substituted by GC medium base with a slight modification. It is concluded that preservation of gonococci in one half strength dextrose starch agar with CNV, which produces less frequent contamination, is a practical method to maintain cultures for teaching and quality control.
Agar ; Bacteriological Techniques ; Culture Media* ; Glucose ; Neisseria gonorrhoeae* ; Preservation, Biological/methods* ; Starch

OBJECTIVETo establish a highly efficient culture method for detecting Yersinia enterocolitica in stool samples from diarrheic patients.

METHODSStool samples collected from 200 diarrheic patients were detected with a modified and 3 conventional methods, and the positivity rates of the bacterium were compared statistically.

RESULTSWith the modified method, 18 positive samples for Yersinia enterocolitica were detected from 200 stool samples, while with the 3 conventional methods, only 3, 5, and 8 positive samples were identified, respectively. Statistical analysis with McNemer test suggested significant difference in the positive detection rate between the modified and the 3 conventional methods.

CONCLUSIONThe modified method for Yersinia enterocolitica detection has much higher sensitivity than the 3 conventional methods, and can be applied in clinical detection.

Bacteriological Techniques ; Culture Techniques ; methods ; Diarrhea ; microbiology ; Feces ; microbiology ; Humans ; Temperature ; Yersinia enterocolitica ; growth & development ; isolation & purification

Listeria monocytogenes is a pathogenic bacterium, therefore, it is essential for food safety monitoring to establish a rapid and specific detecting method. In this study, immunomagnetic beads and selective medium were combined to detect Listeria monocytogenes at different concentrations (10(1)-10(5) CFU/mL). Other three types of Listeria spp., Staphylococcus aureus and Vibrio parahaemolyticus were also detected to conduct the cross-reaction analysis. Meanwhile, contaminated milk samples were prepared to explore the limit of detection of immunomagnetic beads combining with selective medium. Results showed that Listeria monocytogenes with the concentration of 10(3) CFU/mL and above was successfully detected. Milk samples were detected within 6 hours, with a detection limit of 0.7 CFU/mL. The method developed is capable of detecting milk samples within 30 h, which is 38 h faster compared with national standard method with the same sensitivity.
Bacteriological Techniques ; methods ; Culture Media ; chemistry ; Immunomagnetic Separation ; methods ; Listeria monocytogenes ; growth & development ; isolation & purification ; Sensitivity and Specificity

BACKGROUND: Early laboratory detection of Mycobacterium tuberculosis is crucial for controlling tuberculosis. We developed a hydrogel mycobacterial culture method that retains the advantages of both solid and liquid methods in terms of speed, cost, and efficiency. METHODS: Mycobacterium bovis bacillus Calmette-Guerin (BCG) suspensions and 200 acid-fast bacilli (AFB)-positive clinical specimens were inoculated in Middlebrook 7H9 liquid media (Becton-Dickinson and Company, USA) and mixed with 75 microL of 9-fluorenylmethoxycarbonyl (Fmoc)-Phe-Phe-OH hydrogel stock solution in an Eppendorf tube just before culture incubation. The mixtures were cultured at 37degrees C for as long as 14 days to monitor culture status. RESULTS: The number of M. bovis BCG increased with time. For 200 AFB smear-positive specimens, 155 of 158 conventional culture-positive specimens and 4 culture-negative or contaminated specimens yielded positive cultures within 14 days. For 128 specimens positive with the liquid culture method, the time to positive culture using the hydrogel method (mean, 12.6 days; range, 7 to 14 days) was significantly shorter than that for conventional liquid culture (mean, 16.2 days; range, 6 to 31 days; P<0.0001). CONCLUSIONS: The hydrogel scaffold culture system is useful for timely, economical, and efficient detection of mycobacteria in clinical specimens.
Bacteriological Techniques/*methods ; Culture Media/chemistry ; Early Diagnosis ; Humans ; Hydrogel/*chemistry ; Mycobacterium tuberculosis/*isolation & purification ; Tuberculosis/diagnosis/*microbiology

OBJECTIVETo establish a set of procedure for recovery and species identification of Legionella from the surface environmental water.

METHODSForty-four water samples were collected in eight parks of Guangzhou city from August to November in 2006. The bacteriologic examination was performed by cultivation on BCYEalpha plate, and 108 presumptive Legionella colonies were picked and their homogeneous relationship was analyzed by using an amplified fragment length polymorphism (AFLP) method. Species identification was carried out by latex agglutination test, biochemical characterization, analysis of cellular fatty acids composition, 16 S rRNA gene and mip gene sequencing.

RESULTSLegionella was recovered among 27 (61.36%) samples of all eight parks, and 31 different strains were identified from those 108 presumptive Legionella isolates by AFLP method, including 20 strains of L. pneumophila, five strains of L. feeleii, four strains of L. longbeachae, one strain of L. oakridgensis and one strain of L. sainthelensi, and L. pneumophila could be easily differentiated by phenotypic and biochemical characteristics, latex agglutination test or analysis of the cellular fatty acids composition . However, uncertain factors were existing in those phenotypic identification methods as compared to the sequence analysis.

CONCLUSIONThe taxonomic analysis of the Legionellae family should be dependent on the 16 S rRNA gene or mip gene.

Bacteriological Techniques ; DNA, Bacterial ; genetics ; Environmental Monitoring ; methods ; Legionella ; genetics ; isolation & purification ; RNA, Bacterial ; RNA, Ribosomal, 16S ; Water Microbiology