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PubMed1996

Microbial contamination of antiseptics and disinfectants.

BACKGROUND: There have been a number of reports on microbial contamination of antiseptics and disinfectants. At present, however, the necessity of measures to prevent contamination do not seem to be fully appreciated. We investigated microbial contamination of antiseptics and disinfectants that are used in our hospital. METHODS: Fifty-one samples of benzalkonium chloride and chlorhexidine gluconate that were being used in the hospital were examined. Viability of the contaminants detected in these samples was also tested in the agents. Then we examined measures to prevent contamination of these agents. RESULTS: Microbial contamination was detected at 10(2) to 10(7) CFU/ml in the following samples: 6 of 23 samples of cotton balls soaked in 0.02% benzalkonium chloride kept in a canister for antisepsis and disinfection (26.1%); 7 of 13 samples of 0.02%, benzalkonium chloride or 0.02% chlorhexidine gluconate in an irrigation apparatus kept at 37 degrees C for vaginal douching (53.8%); and 9 of 15 samples of 0.02% benzalkonium chloride or 0.05% chlorhexidine gluconate for storage of suction catheters in a plastic bottle (60%). The major contaminants were Burkholderia cepacia, Pseudomonas aeruginosa, Xanthomonas maltophilia, and Pseudomonas fluorescens. The first two organisms examined grew in the agents. After improvements in the handling of the antiseptics and disinfectants, no microbial contamination was observed. CONCLUSIONS: It is necessary to check microbial contamination of diluted benzalkonium chloride and diluted chlorhexidine gluconate that are in use. Such products are not recommended as antiseptics.

Anti-Infective Agents, Local↗

PubMed2003

Evaluation of screening test for detection of antimicrobial residues in ewe milk.

The effects of preservatives (potassium dichromate and sodium azide), heat treatment (untreated and 82 degrees C/10 min), and lactation stage upon the response of the microbial tests (BRT AiM and Delvotest) utilized for the detection of residues of antimicrobial substances in ewe milk were examined. Milk samples were collected from the morning milking of 50 Manchega ewes every 2 wk, from 15 d postpartum until the end of lactation. A total of 2322 samples were analyzed by BRT AiM with prediffusion and Delvotest microbial tests. The specificity of preservative-free milk samples without heat treatment was high (96.3% for BRT and 97.7% for Delvotest), with results improving for those samples thermally treated at 82 degrees C/10 min (99.0% for BRT and 98.7% for Delvotest). Potassium dichromate produced a total inhibition of growth of Bacillus stearothermophilus with both methods. When acidiol was utilized, the specificity of the samples not treated thermally was lower compared with preservative-free milk samples for the BRT AiM (90.2%) and Delvotest (91.0%) methods, improving when the samples were thermally treated, both for BRT AiM (94.8%) and Delvotest (95.3%), given that the presence of the preservative increased the frequency of doubtful results. The lactation stage significantly affected the results of the methods, with a greater frequency of false-positive and doubtful cases toward the end of the cycle, especially in those samples preserved with acidiol. The greater selectivity in both methods was therefore obtained for preservative-free ewe milk samples with prior heat treatment taken at the beginning or in midlactation period.

Animals↗

PubMed1998

The automation of sediment urinalysis using a new urine flow cytometer (UF-100)

The UF-100 analyser is a fully automated instrument that stains the DNA and the membranes of the formed elements in native urine. The sample then passes as a laminar flow through a laser beam and light scattering, fluorescence and impedance are measured. The main purpose of the present work was to assess the analytical performance and the accuracy of the measurements of the UF-100 analyser. No carryover was observed, while the linearity was higher then the upper limit (40000 total particles microl(-1)) suggested by the manufacturer. The within-run imprecision was low, ranging from 17.7 % to 2.4% and was up to threefold better than manual microscopy. Comparison of results obtained by sediment microscopy (performed according to National Committee for Clinical Laboratory Standards (NCCLS) recommendations) and by the UF-100 analyser showed a linear correlation with r = 0.833 for erythrocytes, r = 0.934 for leukocytes, r = 0.880 for epithelial cells and r = 0.40 for casts. To evaluate the reliability of the UF-100 analyser in detecting bacteria we compared the results with the microbial culture (n = 608). Using a cut-off value of bacterial count above 1800 degrees l(-1) and at leukocyte count above 45 microl(-1), the analyser detected positive cultures with a sensitivity of 87 % and a specificity of 80 %. In conclusion, the UF-100 analyser can improve the work flow, increasing the output of urinalysis by reducing the number of specimens submitted for microscopy. Also the method provides reliable information for the identification of urinary tract inflammation and bacterial infection.

Bacteria↗

PubMed2002

Emerging waterborne infections: contributing factors, agents, and detection tools.

Because microorganisms are easily dispersed, display physiological diversity, and tolerate extreme conditions, they are ubiquitous and may contaminate and grow in water. The presence of waterborne enteric pathogens (bacteria, viruses, and protozoa) in domestic water supplies represents a potentially significant human health risk. Even though major outbreaks of waterborne disease are comparatively rare, there is substantial evidence that human enteric pathogens that are frequently present in domestic water supplies are responsible for low-level incidence of waterborne microbial disease. Although these diseases are rarely debilitating to healthy adults for more than a few hours to a few days, enteric pathogens can cause severe illness, even death, for young children, the elderly, or those with compromised immune systems. As the epidemiology of waterborne diseases is changing, there is a growing global public health concern about new and reemerging infectious diseases that are occurring through a complex interaction of social, economic, evolutionary, and ecological factors. New microbial pathogens have emerged, and some have spread worldwide. Alternative testing strategies for waterborne diseases should significantly improve the ability to detect and control the causative pathogenic agents. In this article, we provide an overview of the current state of knowledge of waterborne microbial pathogens, their detection, and the future of new methods in controlling these infectious agents.

Adaptation, Physiological↗

PubMed2003

Automated enumeration of groups of marine picoplankton after fluorescence in situ hybridization.

We describe here an automated system for the counting of multiple samples of double-stained microbial cells on sections of membrane filters. The application integrates an epifluorescence microscope equipped with motorized z-axis drive, shutters, and filter wheels with a scanning stage, a digital camera, and image analysis software. The relative abundances of specific microbial taxa are quantified in samples of marine picoplankton, as detected by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition. Pairs of microscopic images are automatically acquired from numerous positions at two wavelengths, and microbial cells with both general DNA and FISH staining are counted after object edge detection and signal-to-background ratio thresholding. Microscopic fields that are inappropriate for cell counting are automatically excluded prior to measurements. Two nested walk paths guide the device across a series of triangular preparations until a user-defined number of total cells has been analyzed per sample. A backup autofocusing routine at incident light allows automated refocusing between individual samples and can reestablish the focal plane after fatal focusing errors at epifluorescence illumination. The system was calibrated to produce relative abundances of FISH-stained cells in North Sea samples that were comparable to results obtained by manual evaluation. Up to 28 preparations could be analyzed within 4 h without operator interference. The device was subsequently applied for the counting of different microbial populations in incubation series of North Sea waters. Automated digital microscopy greatly facilitates the processing of numerous FISH-stained samples and might thus open new perspectives for bacterioplankton population ecology.

Automation↗

PubMed1988

DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community.

We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridizations. Using single-stranded, P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect Bradyrhizobium japonicum at densities as low as 4.3 x 10 cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a 1-mug DNA sample.

Journal Article↗

PubMed1998

Microbiological profile of raw Nile water.

With the increase of population on the Nile banks, a remarkable increase in industrial, agricultural, human activities, and recreational activities have also occurred. The effluents of such activities are discharged directly into the Nile or through some agricultural drains which finally discharge their wastes into the Nile. Thus, the microbiological monitoring is one of the main objectives to protect the river Nile. This study was conducted to determine the microbiological profile of river Nile at Cairo segment. Eight sites were chosen along the distance (60 Km) from El-Shobak to El-Kanater and tested for two successive years (summer 1994-spring 1996). To achieve this goal, microbiological parameters (total bacterial counts at both 22 degrees C and 37 degrees C, total coliforms, faecal coliforms, faecal streptococci, total yeasts, Candida albicans, Aeromonas hydrophila, salmonellae, total staphylococci, total vibrios and Listeria group) were evaluated. The results showed that the count of the previous parameters ranged between 1.0x10(3)-7.8x10(5) CFU/ml, 5.0x10(2)-6.4x10(5) CFU/ml, 4.9x10-1.6x10(4) MPN/100 ml, 2.0-5.2x0(3) MPN/100 ml, 2.0x10-1.4x10(3) MPN/100 ml, 1.7x10(3)-2.6x10(5) CFU/100 ml, 1.5x10(2)-1.7x10(5) CFU/100 ml, 1.0x10(3)-2.4x10(5) CFU/100 ml, 1.3x10(2)-5.1x10(3) CFU/100 ml, 1.0x10(2)-3.3x10(3) CFU/100 ml, 1.0x10(2)-3.8x10(4) CFU/100 ml and 1.0x10(2)-1.4x10(5) CFU/100 ml, respectively. The total bacterial counts and bacterial indicators (total coliforms, faecal coliforms and faecal streptococci) were detected in all samples during the period of study, and the count increased in samples collected during summer than other seasons. Also total yeasts, A. hydrophila and total staphylococci were detected in all samples with differences in count between the sites. In contrast, other microbial parameters, Candida albicans, salmonellae, total vibrios and Listeria group were not detected in some samples at some sites. According to our results it can be concluded that the river Nile is categorized as an intermediately polluted river.

Aeromonas hydrophila↗

PubMed2005

Increased precision of microbial RNA quantification using NASBA with an internal control.

Detection and quantification of low abundance target RNA has wide utility in the fields of clinical diagnostics, environmental monitoring, gene expression analysis, and biodefense. Nucleic acid based sequence amplification (NASBA) is an isothermal amplification method that provides the sensitivity needed for these applications. However, the requirement for three separate enzymes in NASBA often results in a greater variability between replicate samples than that seen in PCR-based assays. To overcome this problem, we have adapted the bioMérieux Nuclisens Basic Kit and Nuclisens EasyQ Analyzer along with the introduction of a synthetic internal control RNA (IC-RNA) for quantification of potentially any RNA sequence. Using the rbcL gene from the Florida red tide organism Karenia brevis as our target, we describe a simple method to accurately quantify the native target by computing the ratio of the time to positivity (TTP) values for both the wild-type and IC-RNA, and plotting this ratio against the starting number of target molecules or cells. By utilizing this simple method, we have significantly increased our accuracy and precision of prediction over the standard TTP calculations.

Animals↗

PubMed1978

Testing the environment for dispersed mutagens: use of plant bioconcentrators coupled with microbial mutagen assays.

Mutagens dispersed in ecosystems are usually in low concentration and episodic in occurrence. The possibility of detecting such dispersed mutagens by utilizing indigenous bioconcentrator organisms coupled with a microbial mutagen assay offer a useful screening protocol. There are numerous examples of plant and animal species which concentrate toxic substances from the environment. Body extracts of these bioconcentrators can be suitably fractioned and tested for mutagens with various microbial mutagen assays. The fractions may be tested with a broad range of microbial assays covering numerous genetic end points as well as both with and without mammalian microsomal activation. This kind of environmental screening has an advantage over physicochemical techniques, in that sampling techniques are simpler and a wider chemical spectrum can be screened. There are problems inherent with testing a complex biological extract, however. If a reversion assay is used, the metabolite necessary for growth may be present. Toxins may be introduced, either concentrated from the environment in the same way as the mutagen, or produced by the concentrator itself. Finally, the concentrator may also produce an endogenous mutagen which will give spuriously active extracts. Methods for minimizing some of these difficulties are discussed.

Bacteria↗

PubMed1993

Binding of streptavidin to bacteria or fungi and its applications in detecting these microbes.

We have investigated the characteristics and utilities of streptavidin-binding to gram-negative and gram-positive bacteria and Candida spp. The pre-treatment of these microbes with chemical reagents such as CHCl3, NaOH, and Tween 20 have allowed colorimetric visualization under light microscopy or quantitation on nitrocellulose membranes, using streptavidin/biotinylated alkaline phosphatase conjugates. Analysis of this binding was confirmed by western blot. These binding reactions were due to the specific interaction of streptavidin with biotinylated proteins present in the microbes. Competition assays with free biotin or inhibition by an antibiotin antibody confirmed binding to these proteins. With knowledge of these strongly specific interactions, we attempted to reveal the biotinylated proteins within these microbes using clinical specimens. Using phagocyte-smears from blood, urine, and ascites, these intracellular microbes were easily detected by light microscopy. One of the septic blood samples stained by our technique revealed semi-digested microbial signals despite the absence of a signal with routine staining. This detection system, which combines streptavidin as a probe and biotinylated proteins as a microbial marker, is useful in staining for intracellular bacteria or fungi (e.g., microbial infections in phagocyte-smears).

Alkaline Phosphatase↗

PubMed1996

The detection of Mycoplasma pneumoniae in nasal polyps.

The aetiology and microbial flora of nasal polyps is not well understood. No study in the literature has reported an association between the sub-bacterium Mycoplasma pneumoniae and nasal polyps. We have developed an assay method using the Polymerase Chain Reaction (PCR) to amplify a specific region of the M. pneumoniae DNA in extracts of clinical samples using species-specific primers designed to a region of the 16S rRNA. The presence of M. pneumoniae was detected in 13/14 (93%) nasal polyps, in 4/5 (80%) rhinosinusitis mucosal samples but only in 1/7 (14%) of control samples (obstructive turbinates). An epidemic of infections due to M. pneumoniae is expected to occur in 1995. We believe this assay could form the basis of a rapid technique for M. pneumoniae detection. We also propose that the presence of M. pneumoniae may be of importance in the aetiology of nasal polyps.

Blotting, Southern↗

PubMed1999

Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy.

Deep microbial biofilms are a major problem in many industrial, environmental, and medical settings. Novel approaches are needed to understand the structure and metabolism of these biofilms. Two-photon excitation microscopy (TPE) and conventional confocal laser scanning microscopy (CLSM) were compared quantitatively for the ability to visualize bacteria within deep in vitro biofilms. pH gradients within these biofilms were determined by fluorescence lifetime imaging, together with TPE. A constant-depth film fermentor (CDFF) was inoculated for 8 h at 50 ml. h(-1) with a defined mixed culture of 10 species of bacteria grown in continuous culture. Biofilms of fixed depths were developed in the CDFF for 10 or 11 days. The microbial compositions of the biofilms were determined by using viable counts on selective and nonselective agar media; diverse mixed-culture biofilms developed, including aerobic, facultative, and anaerobic species. TPE was able to record images four times deeper than CLSM. Importantly, in contrast to CLSM images, TPE images recorded deep within the biofilm showed no loss of contrast. The pH within the biofilms was measured directly by means of fluorescence lifetime imaging; the fluorescence decay of carboxyfluorescein was correlated with biofilm pH and was used to construct a calibration curve. pH gradients were detectable, in both the lateral and axial directions, in steady-state biofilms. When biofilms were overlaid with 14 mM sucrose for 1 h, distinct pH gradients developed. Microcolonies with pH values of below pH 3.0 were visible, in some cases adjacent to areas with a much higher pH (>5.0). TPE allowed resolution of images at significantly greater depths (as deep as 140 microm) than were possible with CLSM. Fluorescence lifetime imaging allowed the in situ, real-time imaging of pH and the detection of sharp gradients of pH within microbial biofilms.

Biofilms↗

PubMed1998

Detection of intimins alpha, beta, gamma, and delta, four intimin derivatives expressed by attaching and effacing microbial pathogens.

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), and Citrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated alpha, beta, delta, and gamma, respectively.

Adhesins, Bacterial↗

PubMed2001

A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems.

BACKGROUND: Discrimination among viable, active, and inactive cells in aquatic ecosystems is of great importance to understand which species participate in microbial processes. In this study, a new approach combining flow cytometry (FCM), cell sorting, and molecular analyses was developed to compare the diversity of viable cells determined by different methods with the diversity of total cells and active cells. METHODS: Total bacteria were determined by SYBR-II staining. Viable bacteria were determined in water samples from different sites by plate count techniques and by the direct viable count (DVC) method. Substrate-responsive cells (i.e., DVC(+) cells) were distinguished from nonresponsive cells (i.e., DVC(-) cells) by FCM and sorted. The genetic diversity of the sorted cell fraction was compared with the diversity of the total microbial community and with that of the culturable cell fraction by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. The same approach was applied to a seawater sample enriched with nutrients. In this case, actively respiring cells (CTC+) were also enumerated by FCM, sorted, and analyzed by DGGE. RESULTS: The diversity of viable cells varied depending on the methods (traditional culture or DVC) used for viability assessment. Some phylotypes detected in the fraction of viable cells were not detectable at the community level (from total DNA). Similar results were found for actively respiring cells. Inversely, some phylotypes found at the community level were not found in viable and active cell-sorted fractions. It suggests that diversity determined at the community level includes nonactive and nonviable cells. CONCLUSION: This new approach allows investigation of the genetic diversity of viable and active cells in aquatic ecosystems. The diversity determined from sorted cells provides relevant ecological information and uncultured organisms can also be detected. New investigations in the field of microbial ecology such as the identification of species able to maintain cellular activity under environmental changes or in the presence of toxic compounds are now possible.

Bacteria↗

PubMed1985

Variation in microbial survival and growth in intravenous fluids.

The survival and growth of aerobic and anaerobic bacteria, as well as a yeast, was studied in intravenous fluids over a 3-day period. Solutions were experimentally contaminated with pathogenic strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacteroides fragilis and Candida albicans. Samples of these solutions were tested on each day up to 3 days for determining viable numbers of the organisms by a membrane filtration method. Each organism showed a different survival/growth pattern in various infusion fluids. In 5% dextrose, C. albicans multiplied but only 2-3% of the initial viable cells of E. coli, P. aeruginosa, and S. aureus were detected after 3 days. In 0.9% sodium chloride, C. albicans and S. aureus showed growth and E. coli remained viable, but P. aeruginosa was not detected after 72 h. In lactated Ringer's solution, P. aeruginosa and C. albicans grew but E. coli and S. aureus were not detected after 72 h. In the amino acid solution, C. albicans, S. aureus and E. coli grew but P. aeruginosa and B. fragilis were not detected. Results indicate that the pattern of microbial survival and growth may be substantially different in various intravenous fluids.

Bacteria↗

PubMed2026

Ruminosignatures associated with methane emissions and feed efficiency across geographies and cattle breeds.

The cattle rumen microbiota represents a complex and dynamic ecosystem whose organization and relationship to host phenotypes are important for food security and environmental sustainability. We analyzed rumen microbiota profiles from 2496 cattle representing five breeds and production systems across five countries, identifying microbial co-abundance groups termed Ruminosignatures. We detected 14 distinct Ruminosignatures, including 2 observed across all populations dominated by Prevotella and UBA2810. Additional Ruminosignatures showed breed- and diet-specific patterns and collectively explained 96%-99% of variance in rumen microbial composition. Integrative cross-country analysis confirmed 10 out of 14 Ruminosignatures identified in cohort-specific analyses. Several Ruminosignatures were associated with methane emissions and feed efficiency traits and were partially under host genetic control, with heritability estimates ranging from 0.09 to 0.58. Structural equation modeling revealed consistent negative genetic and phenotypic correlations between the UBA2810-dominated Ruminosignature (RS_UBA2) and methane emissions across cohorts (rg = -0.40 to -0.65), with structural coefficients concordant in sign across all populations, supporting the expected direction of phenotypic response to selection on RS_UBA2. Meta-analysis confirmed positive associations of RS_UBA2 with average daily gain and negative associations with methane-related traits and feed conversion ratio. Functional genome-based predictions suggested RS_UBA2 may reduce methanogenesis through alternative hydrogen utilization pathways competing with methanogenic archaea. Production system type influenced both Ruminosignature occurrence and relationships with host phenotypes, emphasizing the relevance of context-specific strategies for microbiome modulation. Our findings highlight the potential of the Ruminosignatures framework for microbiome-informed breeding programs aimed at improving feed efficiency while reducing the environmental impact of cattle production.

Animals↗

PubMed2004

PCR method based on the ogdH gene for the detection of Salmonella spp. from chicken meat samples.

In a previous paper, the ogdH gene that encodes 2-oxoglutarate dehydrogenase was isolated from Salmonella typhimurium. The catalytic N-terminal region in the enzyme was found to be very specific for the Salmonella species. Therefore, the aim of the present study was to detect S. typhimurium in food sources using primers designed for OGDH-1 and OGDH-2 which were based on the salmonella-specific region of the ogdH gene. A simple polymerase chain reaction (PCR) detection method was developed to detect low numbers of S. typhimurium in a chicken meat microbial consortium. Using the ogdH-specific primers under stringent amplification conditions and for gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the PCR assay was run on S. typhimurium-contaminated meat contents, only the positive meat samples containing as few as 200 CFUs reacted to the assay. The method employed for sample processing is simple and it was determined to provide a sensitive means of detecting trace amounts of S. typhimurium-specific sequences in the presence of mixed meat microbial populations. When compared with six representative intestinal gram-negative bacterial strains in foods, including Vibrio parahaemolyticus, V. vulnificus, Enterobacter cloacae, E. coli O157:H7, Pseudomonas aeruginosa, and Proteus sp., S. typhimurium had a unique and distinct PCR product (796 bp). In conclusion, the two OGDH primers were found to be rapid and sensitive detectors of Salmonella spp for the PCR method.

Animals↗

PubMed2000

Use of PCR analysis for detecting low levels of bacteria and mold contamination in pharmaceutical samples.

PCR assays were developed and compared to standard methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples were artificially contaminated with less than 10 CFU of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger. Bacterial DNA was extracted from each enrichment broth by mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling samples in Tris-EDTA-SDS buffer for 1 h. A 10-microl aliquot of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50-microl aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. Standard methods required 6-8 days while PCR detection of all microorganisms was completed within 27 h. Low levels of microbial contamination were detected in all raw materials and products using PCR assays. Rapid quality evaluation of pharmaceutical samples resulted in optimization of product manufacturing, quality control, and release of finished products.

Aspergillus niger↗