RMM's


Enumeration & Presence/Absence:

Description and validation of a rapid (1 h) flow cytometry test for enumerating thermophilic bacteria in milk powders. Flint, S., K. Walker, et al. (2007). Journal of Applied Microbiology 102(4): 909-915.

Aims: The aim of this study was to develop a rapid assay for enumerating thermophilic bacteria in milk powder.
Methods and Results: The BactiFlowTM flow cytometer was used to count bacteria based on esterase activity in viable bacterial cells. A protocol for total viable bacteria was modified by heat-treating the sample to selectively label thermophilic bacteria. Samples of milk powder dissolved in 0·1% peptone were treated with 0·8% ethylenediaminetetraacetic acid to reduce background interference because of denatured milk proteins. Either thermophilic bacteria were added to the dissolved milk powder or milk powder solutions were incubated at 55°C for 2–3 h to enrich the natural thermophile population for testing. Results from the BactiFlow were compared with traditional plate count results.
Conclusions: Thermophilic bacteria in milk powder can be enumerated within 1 h using the BactiFlow flow cytometer.
Significance and Impact of the Study: Microbiological test results obtained within 1 h can potentially be used to monitor manufacturing processes, effectively trace problems and provide confidence in the manufacture of product
http://www.ncbi.nlm.nih.gov/pubmed/17381733

Determination of indicator bacteria in pharmaceutical samples by multiplex PCR. Faranjnia, S., M. Hassan, et al. (2009). Journal of Rapid Methods & Automation in Microbiology 17(3): 328-338.

Rapid and sensitive detection techniques for indicator pathogens are important in pharmaceutical industry. However, common detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 5-6 days to complete. Thus, the aim of this study was to develop a multiplex polymerase chain reaction (mPCR) assay for simultaneous detection and identification of four indicator pathogenic bacteria in a single reaction. Specific primers for indicator bacteria, namely Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella, were applied to allow simultaneous detection of them, and the sensitivity and specificity of each primer pairs were determined. In the mPCR with mixed DNA samples, specific bands for corresponding bacteria were simultaneously detected. Agarose gel electrophoresis of PCR products revealed 100% specificity of mPCR with single bands in the expected sizes. Low levels of microbial contamination less than 10 cfu per milliliter or gram of product were detected using mPCR assay. The detection of all four indicator pathogenic bacteria were completed in less than 8 h with this novel mPCR method, whereas the conventional United States Pharmacopeia methods and uniplex PCR required 5-6 days and 27 h for completion, respectively. Using mPCR assay, the microbial quality control of non-sterile pharmaceutical products can be performed in a cost-effective and timely manner in pharmaceutical industry. Detection of pathogenic indicators of Escherichia coli, Staphylococcus aureus, Salmonella and Pseudomonas aeruginosa is one of the mandatory tests in microbial quality of non-sterile pharmaceutical products; therefore, rapid and sensitive detection of the contaminations is of great importance for product release. According to the results of the present study, simultaneous detection of low levels of four major potential pathogenic bacteria in pharmaceutical finished products can be performed using mPCR in a cost-effective and timely manner, and upon these properties of the mPCR assay it could have potential applications in pharmaceutical industry.
http://onlinelibrary.wiley.com/doi/10.1111/j.1745-4581.2009.00154.x/abstract

Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products. Parveen S, Kaur S, Wilson-David SA, Kenney JL, McCormick WM, Gupta RK (2011). Vaccine, Issue 29, p. 8012-23

Most biological products, including vaccines, administered by the parenteral route are required to be tested for sterility at the final container and also at various stages during manufacture. The sterility testing method described in the Code of Federal Regulations (21 CFR 610.12) and the United States Pharmacopoeia (USP, Chapter <71 >) is based on the observation of turbidity in liquid culture media due to growth of potential contaminants. We evaluated rapid microbiological methods (RMM) based on detection of growth 1) by adenosine triphosphate (ATP) bioluminescence technology (Rapid Milliflex® Detection System [RMDS]), and 2) by CO2 monitoring technologies (BacT/Alert and the BACTEC systems), as alternate sterility methods. Microorganisms representing Gram negative, Gram positive, aerobic, anaerobic, spore forming, slow growing bacteria, yeast, and fungi were prepared in aliquots of Fluid A or a biological matrix (including inactivated influenza vaccines) to contain approximately 0.1, 1, 10 and 100 colony forming units (CFU) in an inoculum of 10 ml. These preparations were inoculated to the specific media required for the various methods: 1) fluid thioglycollate medium (FTM) and tryptic soy broth (TSB) of the compendial sterility method (both membrane filtration and direct inoculation); 2) tryptic soy agar (TSA), Sabouraud dextrose agar (SDA) and Schaedler blood agar (SBA) of the RMDS; 3) iASTand iNST media of the BacT/Alert system and 4) Standard 10 Aerobic/F and Standard Anaerobic/F media of the BACTEC system. RMDS was significantly more sensitive in detecting various microorganisms at 0.1 CFU than the compendial methods (p < 0.05), whereas the compendial membrane filtration method was significantly more sensitive than the BACTEC and BacT/Alert methods (p < 0.05). RMDS detected all microorganisms significantly faster than the compendial method (p < 0.05). BacT/Alert and BACTEC methods detected most microorganisms significantly faster than the compendial method (p < 0.05), but took almost the same time to detect the slow growing microorganism P. acnes, compared to the compendial method. RMDS using SBA detected all test microorganisms in the presence of a matrix containing preservative 0.01% thimerosal, whereas the BacT/Alert and BACTEC systems did not consistently detect all the test microorganisms in the presence of 0.01% thimerosal. RMDS was compatible with inactivated influenza vaccines and aluminum phosphate or aluminum hydroxide adjuvants at up to 8 mg/ml without any interference in bioluminescence. RMDS was shown to be acceptable as an alternate sterility method taking 5 days as compared to the 14 days required of the compendial method. Isolation of microorganisms from the RMDS was accomplished by re-incubation of membranes with fresh SBA medium and microbial identification was confirmed using the MicroSEQ Identification System. BacT/Alert and BACTEC systems may be applicable as alternate methods to the compendial direct inoculation sterility method for products that do not contain preservatives or anti-microbial agents.
http://cat.inist.fr/?aModele=afficheN&cpsidt=24627047

Flow cytometric analysis of bacteria- and virus-like particles in lake sediments. Duhamel, S. and Stéphan Jacquet (2006). Journal of Microbiological Methods 64: 316-332 

Flow cytometry (FCM) was successfully used to analyze freshwater bacteria and viruses in lake sediments after relatively simple sample treatment and optimization of dilution/fixation/staining procedures. Biological particles from Lakes Geneva and Bourget were first separated from the sediments by using both Sodium Pyrophosphate (0.01 M final concentration) and Polyoxyethylene-Sorbitan Monooleate (10% final concentration) and sonicating for 3 min in a water bath. The best results (based on FCM signature and the highest virus and bacterial yields from the sediments) were obtained by formaldehyde fixation carried out within less than one hour (2% final concentration, vs. no fixation or using glutaraldehyde at different concentrations), SYBR-Green II staining (×1 / 20,000 stock solution concentration, vs. use of SYBR-Gold and SYBR-Green I dyes at different concentrations). There was a considerable loss of particles after only a few days of storage at either 4 or - 22 °C. For FCM analysis, the samples were diluted in Tris–EDTA buffer (pH 8) and heated for 10 min at 75 °C after incubating for 5 min in the dark. The bacterial and viral counts paralleled those obtained using epifluorescence microscopy (EFM), but EFM always gave lower counts than FCM. Analysis of the distribution of the viruses in the water column and in the sediments of Lakes Bourget revealed a marked gradient, with larger quantities in the top layer of the sediment than in the water above it. These results are discussed, as well as the possible novel application of flow cytometry in the study of aquatic viral ecology.
http://www.sciencedirect.com/science/article/pii/S016770120500151X

Raman spectroscopy and chemical imaging for quantification of filtered waterborne bacteria. Escoriza, M. F., J. M. van Briesen, et al. (2006). Journal of Microbiological Methods 66: 63-72

Rapid and reliable assessment of pathogenic microbial contamination in water is critically important. In the present work we evaluated the suitability of Raman Spectroscopy and Chemical Imaging as enumeration techniques for waterborne pathogens. The prominent C–H stretching band observed between 2800–3000 cm-1 of the spectrum is used for quantification purposes. This band provides the highest intensity of the bacterial-spectrum bands facilitating the detection of low number of microorganisms. The intensity of the Raman response correlates with number of cells present in drops of sample water on aluminum-coated slides. However, concentration of pathogens in drinking and recreational water is low, requiring a concentration step, i.e., filtering. Subsequent evaluation of filtering approaches for water sampling for Raman detection showed significant background signal from alumina and silver membranes that reduces method sensitivity. Samples concentrated by filtration show good correlation between Raman spectroscopy and other quantification methods including turbidity (R2=0.92), plate counts (R2=0.87) and dry weight (R2=0.97). Background interferences did not allow for evaluation of this relationship at low cell concentrations.
http://www.sciencedirect.com/science/article/pii/S0167701205003325

Rapid and Simple Quantification of Bacterial Cells by Using a Microfluidic Device. Chieko Sakamoto, Nobuyasu Yamaguchi, and Masao Nasu (2005). Applied and Environmental Microbiology, p. 1117–1121 Vol. 71, No. 2

This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.
http://aem.asm.org/content/71/2/1117.full

Validation of the BacT/ALERT 3D System for Rapid Sterility Testing of Biopharmaceutical Samples. L. Jimenez, N. Rana, J. Amalraj, K. Walker, and K. Travers (2012). PDA J Pharm Sci and Tech, 66 38-54

ABSTRACT: The BacT/ALERT 3D system was validated to determine the sterility of different types of biopharmaceutical samples such as water for injection, unprocessed bulk, and finished bulk. The installation, operation, and performance qualification were completed and verified under good manufacturing practices. During the installation and operation validation stages, the functionality and security of the system and software were completed and verified.
For the performance qualification, 11 microorganisms were evaluated, six compendial (Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Candida albicans, Aspergillus niger, Clostridium sporogenes), one representing the number one microbial species in sterile product recalls (Burkholderia cepacia), and four environmental isolates (Kocuria rhizophila, Staphylococcus haemolyticus, Methylobacterium radiotolerans, and Penicillium spp.). Nine of the microorganisms were spiked into three different types of biopharmaceutical samples by three different analysts on different days to ascertain the equivalence, ruggedness, sensitivity, time of detection, and repeatability. In all samples, the BacT/ALERT exhibited equivalent or better detection than the standard test. With the exception of M. radiotolerans, all 11 microorganisms were detected within 2.5 days using the BacT/ALERT system and the standard test. The detection times for M. radiotolerans in the three sample types averaged 5.77 days. The minimum detectable level of cells for all the microorganisms tested was found to be within 1 to 2 CFU. The system optimized sterility testing by the simultaneous on-line, non-destructive incubation and detection of microbial growth.
http://journal.pda.org/content/66/1/38.abstract


Identification of Microorganisms

Improvement of the cDNA-AFLP method using fluorescent primers for transcription analysis in bacteria. Decorosi, F., C. Viti, et al. (2005). Journal of Microbiological Methods 63(2): 211-215

Here the cDNA-fluorescent amplified fragment length polymorphism (cDNA-FAFLP) technique, an improvement of cDNA-AFLP method, was used in order to analyse expression profiling in bacteria. The obtained results were validated by real-time PCR. This is the first report that validates the foreseen transcription pattern by cDNA-FAFLP with the application of real-time PCR in bacteria. This new protocol offers the possibility of quick and reliable analysis of transcription profiles, also avoids the problem linked with the use of radioisotopes, and allows a quick identification of genes differentially expressed in bacteria.
http://www.ncbi.nlm.nih.gov/pubmed/15939496



Gas Consumption

Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products. Parveen S, Kaur S, Wilson-David SA, Kenney JL, McCormick WM, Gupta RK (2011). Vaccine, Issue 29, p. 8012-23

Most biological products, including vaccines, administered by the parenteral route are required to be tested for sterility at the final container and also at various stages during manufacture. The sterility testing method described in the Code of Federal Regulations (21 CFR 610.12) and the United States Pharmacopoeia (USP, Chapter <71 >) is based on the observation of turbidity in liquid culture media due to growth of potential contaminants. We evaluated rapid microbiological methods (RMM) based on detection of growth 1) by adenosine triphosphate (ATP) bioluminescence technology (Rapid Milliflex® Detection System [RMDS]), and 2) by CO2 monitoring technologies (BacT/Alert and the BACTEC systems), as alternate sterility methods. Microorganisms representing Gram negative, Gram positive, aerobic, anaerobic, spore forming, slow growing bacteria, yeast, and fungi were prepared in aliquots of Fluid A or a biological matrix (including inactivated influenza vaccines) to contain approximately 0.1, 1, 10 and 100 colony forming units (CFU) in an inoculum of 10 ml. These preparations were inoculated to the specific media required for the various methods: 1) fluid thioglycollate medium (FTM) and tryptic soy broth (TSB) of the compendial sterility method (both membrane filtration and direct inoculation); 2) tryptic soy agar (TSA), Sabouraud dextrose agar (SDA) and Schaedler blood agar (SBA) of the RMDS; 3) iASTand iNST media of the BacT/Alert system and 4) Standard 10 Aerobic/F and Standard Anaerobic/F media of the BACTEC system. RMDS was significantly more sensitive in detecting various microorganisms at 0.1 CFU than the compendial methods (p < 0.05), whereas the compendial membrane filtration method was significantly more sensitive than the BACTEC and BacT/Alert methods (p < 0.05). RMDS detected all microorganisms significantly faster than the compendial method (p < 0.05). BacT/Alert and BACTEC methods detected most microorganisms significantly faster than the compendial method (p < 0.05), but took almost the same time to detect the slow growing microorganism P. acnes, compared to the compendial method. RMDS using SBA detected all test microorganisms in the presence of a matrix containing preservative 0.01% thimerosal, whereas the BacT/Alert and BACTEC systems did not consistently detect all the test microorganisms in the presence of 0.01% thimerosal. RMDS was compatible with inactivated influenza vaccines and aluminum phosphate or aluminum hydroxide adjuvants at up to 8 mg/ml without any interference in bioluminescence. RMDS was shown to be acceptable as an alternate sterility method taking 5 days as compared to the 14 days required of the compendial method. Isolation of microorganisms from the RMDS was accomplished by re-incubation of membranes with fresh SBA medium and microbial identification was confirmed using the MicroSEQ Identification System. BacT/Alert and BACTEC systems may be applicable as alternate methods to the compendial direct inoculation sterility method for products that do not contain preservatives or anti-microbial agents.
http://cat.inist.fr/?aModele=afficheN&cpsidt=24627047

Validation of the BacT/ALERT 3D System for Rapid Sterility Testing of Biopharmaceutical Samples. L. Jimenez, N. Rana, J. Amalraj, K. Walker, and K. Travers (2012). PDA J Pharm Sci and Tech, 66 38-54

ABSTRACT: The BacT/ALERT 3D system was validated to determine the sterility of different types of biopharmaceutical samples such as water for injection, unprocessed bulk, and finished bulk. The installation, operation, and performance qualification were completed and verified under good manufacturing practices. During the installation and operation validation stages, the functionality and security of the system and software were


Bioluminescence:

Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products. Parveen S, Kaur S, Wilson-David SA, Kenney JL, McCormick WM, Gupta RK (2011). Vaccine, Issue 29, p. 8012-23

Most biological products, including vaccines, administered by the parenteral route are required to be tested for sterility at the final container and also at various stages during manufacture. The sterility testing method described in the Code of Federal Regulations (21 CFR 610.12) and the United States Pharmacopoeia (USP, Chapter <71 >) is based on the observation of turbidity in liquid culture media due to growth of potential contaminants. We evaluated rapid microbiological methods (RMM) based on detection of growth 1) by adenosine triphosphate (ATP) bioluminescence technology (Rapid Milliflex® Detection System [RMDS]), and 2) by CO2 monitoring technologies (BacT/Alert and the BACTEC systems), as alternate sterility methods. Microorganisms representing Gram negative, Gram positive, aerobic, anaerobic, spore forming, slow growing bacteria, yeast, and fungi were prepared in aliquots of Fluid A or a biological matrix (including inactivated influenza vaccines) to contain approximately 0.1, 1, 10 and 100 colony forming units (CFU) in an inoculum of 10 ml. These preparations were inoculated to the specific media required for the various methods: 1) fluid thioglycollate medium (FTM) and tryptic soy broth (TSB) of the compendial sterility method (both membrane filtration and direct inoculation); 2) tryptic soy agar (TSA), Sabouraud dextrose agar (SDA) and Schaedler blood agar (SBA) of the RMDS; 3) iASTand iNST media of the BacT/Alert system and 4) Standard 10 Aerobic/F and Standard Anaerobic/F media of the BACTEC system. RMDS was significantly more sensitive in detecting various microorganisms at 0.1 CFU than the compendial methods (p < 0.05), whereas the compendial membrane filtration method was significantly more sensitive than the BACTEC and BacT/Alert methods (p < 0.05). RMDS detected all microorganisms significantly faster than the compendial method (p < 0.05). BacT/Alert and BACTEC methods detected most microorganisms significantly faster than the compendial method (p < 0.05), but took almost the same time to detect the slow growing microorganism P. acnes, compared to the compendial method. RMDS using SBA detected all test microorganisms in the presence of a matrix containing preservative 0.01% thimerosal, whereas the BacT/Alert and BACTEC systems did not consistently detect all the test microorganisms in the presence of 0.01% thimerosal. RMDS was compatible with inactivated influenza vaccines and aluminum phosphate or aluminum hydroxide adjuvants at up to 8 mg/ml without any interference in bioluminescence. RMDS was shown to be acceptable as an alternate sterility method taking 5 days as compared to the 14 days required of the compendial method. Isolation of microorganisms from the RMDS was accomplished by re-incubation of membranes with fresh SBA medium and microbial identification was confirmed using the MicroSEQ Identification System. BacT/Alert and BACTEC systems may be applicable as alternate methods to the compendial direct inoculation sterility method for products that do not contain preservatives or anti-microbial agents.
http://cat.inist.fr/?aModele=afficheN&cpsidt=24627047

Validation of the BacT/ALERT 3D System for Rapid Sterility Testing of Biopharmaceutical Samples. L. Jimenez, N. Rana, J. Amalraj, K. Walker, and K. Travers (2012). PDA J Pharm Sci and Tech, 66 38-54

ABSTRACT: The BacT/ALERT 3D system was validated to determine the sterility of different types of biopharmaceutical samples such as water for injection, unprocessed bulk, and finished bulk. The installation, operation, and performance qualification were completed and verified under good manufacturing practices. During the installation and operation validation stages, the functionality and security of the system and software were completed and verified.
For the performance qualification, 11 microorganisms were evaluated, six compendial (Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Candida albicans, Aspergillus niger, Clostridium sporogenes), one representing the number one microbial species in sterile product recalls (Burkholderia cepacia), and four environmental isolates (Kocuria rhizophila, Staphylococcus haemolyticus, Methylobacterium radiotolerans, and Penicillium spp.). Nine of the microorganisms were spiked into three different types of biopharmaceutical samples by three different analysts on different days to ascertain the equivalence, ruggedness, sensitivity, time of detection, and repeatability. In all samples, the BacT/ALERT exhibited equivalent or better detection than the standard test. With the exception of M. radiotolerans, all 11 microorganisms were detected within 2.5 days using the BacT/ALERT system and the standard test. The detection times for M. radiotolerans in the three sample types averaged 5.77 days. The minimum detectable level of cells for all the microorganisms tested was found to be within 1 to 2 CFU. The system optimized sterility testing by the simultaneous on-line, non-destructive incubation and detection of microbial growth.
http://journal.pda.org/content/66/1/38.abstract


Flow Cytometry

Description and validation of a rapid (1 h) flow cytometry test for enumerating thermophilic bacteria in milk powders. Flint, S., K. Walker, et al. (2007). Journal of Applied Microbiology 102(4): 909-915.

Aims: The aim of this study was to develop a rapid assay for enumerating thermophilic bacteria in milk powder.
Methods and Results: The BactiFlowTM flow cytometer was used to count bacteria based on esterase activity in viable bacterial cells. A protocol for total viable bacteria was modified by heat-treating the sample to selectively label thermophilic bacteria. Samples of milk powder dissolved in 0·1% peptone were treated with 0·8% ethylenediaminetetraacetic acid to reduce background interference because of denatured milk proteins. Either thermophilic bacteria were added to the dissolved milk powder or milk powder solutions were incubated at 55°C for 2–3 h to enrich the natural thermophile population for testing. Results from the BactiFlow were compared with traditional plate count results.
Conclusions: Thermophilic bacteria in milk powder can be enumerated within 1 h using the BactiFlow flow cytometer.
Significance and Impact of the Study: Microbiological test results obtained within 1 h can potentially be used to monitor manufacturing processes, effectively trace problems and provide confidence in the manufacture of product
http://www.ncbi.nlm.nih.gov/pubmed/17381733 

Flow cytometric analysis of bacteria- and virus-like particles in lake sediments. Duhamel, S. and Stéphan Jacquet (2006). Journal of Microbiological Methods 64: 316-332

Flow cytometry (FCM) was successfully used to analyze freshwater bacteria and viruses in lake sediments after relatively simple sample treatment and optimization of dilution/fixation/staining procedures. Biological particles from Lakes Geneva and Bourget were first separated from the sediments by using both Sodium Pyrophosphate (0.01 M final concentration) and Polyoxyethylene-Sorbitan Monooleate (10% final concentration) and sonicating for 3 min in a water bath. The best results (based on FCM signature and the highest virus and bacterial yields from the sediments) were obtained by formaldehyde fixation carried out within less than one hour (2% final concentration, vs. no fixation or using glutaraldehyde at different concentrations), SYBR-Green II staining (×1 / 20,000 stock solution concentration, vs. use of SYBR-Gold and SYBR-Green I dyes at different concentrations). There was a considerable loss of particles after only a few days of storage at either 4 or - 22 °C. For FCM analysis, the samples were diluted in Tris–EDTA buffer (pH 8) and heated for 10 min at 75 °C after incubating for 5 min in the dark. The bacterial and viral counts paralleled those obtained using epifluorescence microscopy (EFM), but EFM always gave lower counts than FCM. Analysis of the distribution of the viruses in the water column and in the sediments of Lakes Bourget revealed a marked gradient, with larger quantities in the top layer of the sediment than in the water above it. These results are discussed, as well as the possible novel application of flow cytometry in the study of aquatic viral ecology.
http://www.sciencedirect.com/science/article/pii/S016770120500151X

Rapid and Simple Quantification of Bacterial Cells by Using a Microfluidic Device. Chieko Sakamoto, Nobuyasu Yamaguchi, and Masao Nasu (2005). Applied and Environmental Microbiology, 2005, p. 1117–1121 Vol. 71, No. 2

This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.
http://aem.asm.org/content/71/2/1117.full


Spectroscopy

Raman spectroscopy and chemical imaging for quantification of filtered waterborne bacteria. Escoriza, M. F., J. M. VanBriesen, et al. (2006). Journal of Microbiological Methods 66: 63-72

Rapid and reliable assessment of pathogenic microbial contamination in water is critically important. In the present work we evaluated the suitability of Raman Spectroscopy and Chemical Imaging as enumeration techniques for waterborne pathogens. The prominent C–H stretching band observed between 2800–3000 cm-1 of the spectrum is used for quantification purposes. This band provides the highest intensity of the bacterial-spectrum bands facilitating the detection of low number of microorganisms. The intensity of the Raman response correlates with number of cells present in drops of sample water on aluminum-coated slides. However, concentration of pathogens in drinking and recreational water is low, requiring a concentration step, i.e., filtering. Subsequent evaluation of filtering approaches for water sampling for Raman detection showed significant background signal from alumina and silver membranes that reduces method sensitivity. Samples concentrated by filtration show good correlation between Raman spectroscopy and other quantification methods including turbidity (R2=0.92), plate counts (R2=0.87) and dry weight (R2=0.97). Background interferences did not allow for evaluation of this relationship at low cell concentrations.
http://www.sciencedirect.com/science/article/pii/S0167701205003325


Nucleic Acid Amplification techniques (NAT)

Determination of indicator bacteria in pharmaceutical samples by multiplex PCR. Faranjnia, S., M. Hassan, et al. (2009). Journal of Rapid Methods & Automation in Microbiology 17(3): 328-338.

Rapid and sensitive detection techniques for indicator pathogens are important in pharmaceutical industry. However, common detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 5-6 days to complete. Thus, the aim of this study was to develop a multiplex polymerase chain reaction (mPCR) assay for simultaneous detection and identification of four indicator pathogenic bacteria in a single reaction. Specific primers for indicator bacteria, namely Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella, were applied to allow simultaneous detection of them, and the sensitivity and specificity of each primer pairs were determined. In the mPCR with mixed DNA samples, specific bands for corresponding bacteria were simultaneously detected. Agarose gel electrophoresis of PCR products revealed 100% specificity of mPCR with single bands in the expected sizes. Low levels of microbial contamination less than 10 cfu per milliliter or gram of product were detected using mPCR assay. The detection of all four indicator pathogenic bacteria were completed in less than 8 h with this novel mPCR method, whereas the conventional United States Pharmacopeia methods and uniplex PCR required 5-6 days and 27 h for completion, respectively. Using mPCR assay, the microbial quality control of non-sterile pharmaceutical products can be performed in a cost-effective and timely manner in pharmaceutical industry. Detection of pathogenic indicators of Escherichia coli, Staphylococcus aureus, Salmonella and Pseudomonas aeruginosa is one of the mandatory tests in microbial quality of non-sterile pharmaceutical products; therefore, rapid and sensitive detection of the contaminations is of great importance for product release. According to the results of the present study, simultaneous detection of low levels of four major potential pathogenic bacteria in pharmaceutical finished products can be performed using mPCR in a cost-effective and timely manner, and upon these properties of the mPCR assay it could have potential applications in pharmaceutical industry.
http://onlinelibrary.wiley.com/doi/10.1111/j.1745-4581.2009.00154.x/abstract


Genetic Fingerprinting

Improvement of the cDNA-AFLP method using fluorescent primers for transcription analysis in bacteria. Decorosi, F., C. Viti, et al. (2005). Journal of Microbiological Methods 63(2): 211-215

Here the cDNA-fluorescent amplified fragment length polymorphism (cDNA-FAFLP) technique, an improvement of cDNA-AFLP method, was used in order to analyse expression profiling in bacteria. The obtained results were validated by real-time PCR. This is the first report that validates the foreseen transcription pattern by cDNA-FAFLP with the application of real-time PCR in bacteria. This new protocol offers the possibility of quick and reliable analysis of transcription profiles, also avoids the problem linked with the use of radioisotopes, and allows a quick identification of genes differentially expressed in bacteria.
http://www.ncbi.nlm.nih.gov/pubmed/15939496