A flow cytometry method for rapid detection and enumeration of total bacteria in milk

Application of flow cytometry (FCM) to microbial analysis of milk is hampered by the presence of milk proteins and lipid particles. Here we report on the development of a rapid (/= 0.98) between the FCM assay and the more conventional methods of plating and direct microscopic counting was achieved. Raw milk data showed a significant correlation (P < 0.01) and a good agreement (r = 0.91) between FCM and standard plate count methods. The detection limit of the FCM assay was 相似文献   

Suitability of a rapid gas chromatographic method for total mesophilic bacteria and coliform enumeration in hamburgers

An instrumental method for the rapid determination of total mesophilic bacteria and total coliforms in hamburgers is described. The two procedures are based on the gas chromatographic determination of metabolic CO₂ in equilibrium in the head space of sealed vials containing suitable media and inoculated with aliquots of the sample under examination. Both procedures proved to be suitable for quality control of meat products, being highly sensitive, reproducible, simple and rapid. Good correlation coefficients were obtained with plate counting for microbial concentrations ranging between 10¹ and 10⁸.     

荧光法快速检测活菌数的方法研究及应用

【背景】Calcein UltraGreen~(TM)AM是一种新型荧光染料,用于标记和监测活细胞。【目的】基于该荧光染料的荧光特性及其在活细胞内的稳定特性,建立一种荧光定量快速检测活细菌总数的方法,并在实际样品中应用校正。【方法】通过应用荧光染料对细菌进行染色,再进行荧光强度检测,同时以平板计数法作平行对照,建立荧光强度值-活菌数标准曲线。【结果】确定了染色细菌的最佳pH值为8.0。该检测方法仅需固定染色温度,染色时间在20-30min范围即可快速检测。建立了革兰氏阴性菌铜绿假单胞菌NY3、大肠杆菌和革兰氏阳性菌芽孢杆菌、红平红球菌FF、金黄色葡萄球菌和枯草芽孢杆菌的细菌总数与相对荧光强度值标准曲线。当菌悬液OD600值在0.01-0.30范围内时,上述6种细菌与荧光信号强度呈良好的线性关系(R20.99)。【结论】当样品菌悬液浓度范围控制在105-109CFU/mL时,建立的荧光检测方法快速便捷,精密度、重复性、稳定性、回收率和准确度均较好,可应用于微生物实验、固体菌剂发酵、食品卫生与安全、环境检测等领域的活细菌总数现场快速检测。     

Real-time PCR detection of Paenibacillus spp. in raw milk to predict shelf life performance of pasteurized fluid milk products

Psychrotolerant sporeformers, specifically Paenibacillus spp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. While Paenibacillus spp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number of Paenibacillus spp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detect Paenibacillus spp. in fluid milk and to discriminate between Paenibacillus and other closely related spore-forming bacteria. Specificity was confirmed using 16 Paenibacillus and 17 Bacillus isolates. All 16 Paenibacillus isolates were detected with a mean cycle threshold (C(T)) of 19.14 ± 0.54. While 14/17 Bacillus isolates showed no signal (C(T) > 40), 3 Bacillus isolates showed very weak positive signals (C(T) = 38.66 ± 0.65). The assay provided a detection limit of approximately 3.25 × 10(1) CFU/ml using total genomic DNA extracted from raw milk samples inoculated with Paenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection of Paenibacillus. Heat-treated milk samples where Paenibacillus (≥1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection of Paenibacillus that has the potential to identify raw milk with microbial spoilage potential as a pasteurized product.     

Improved membrane filtration method incorporating catalase and sodium pyruvate for detection of chlorine-stressed coliform bacteria

In vitro pure culture studies were conducted on three different strains of Escherichia coli (K-12, EPA 00244, and SWEI) to determine the effect of chlorination on catalase activity. In each case, stationary-phase cells exhibited significant (P less than 0.001) reductions in enzyme activity following exposure to chlorine. Mean differences in activity between control and chlorine-stressed cells ranged from 8.8 to 20.3 U/mg of protein for E. coli SWEI and EPA 00244, respectively. Following initial enzyme studies, resuscitation experiments utilizing the membrane filtration technique were conducted on chlorinated sewage effluent. Five different amendments, including catalase (1,000 U per plate), heat-inactivated catalase (1,000-U per plate), sodium pyruvate (0.05%), a catalase-sodium pyruvate combination (1,500 U/0.01%), and acetic acid (0.05%), were tested for the ability to enhance detection of chlorine-stressed cells on M-fecal coliform (M-FC), mT7, M-Endo, and tryptone-glucose-yeast extract (TGY) media. Significant (P less than 0.001) increases in recovery of fecal coliforms on M-FC, total coliforms on mT7 and M-Endo, and total heterotrophs on TGY were obtained on plates containing catalase, pyruvate, or the combination of these compounds. Supplementation with heat-inactivated catalase and acetic acid did not improve recovery of chlorine-stressed cells compared with recovery on nonamended media. Subsequent analysis of colonies from plates containing compounds which enhanced recovery indicated coliform verification percentages of greater than 80% on M-FC, greater than 90% on mT7, and greater than 94% on M-Endo media. These data suggest that the addition of peroxide-degrading compounds to various standard recovery media may improve detection of both coliform and heterotrophic bacteria in chlorinated waters.     

A method for the specific detection of resident bacteria in brine shrimp larvae

In this study, we describe an easy but efficient method to specifically target the intestinal resident microbiota in brine shrimp larvae during DGGE analysis, hereby excluding the interference of both transient (luminal) bacteria and body surface bacteria. This effective technique has several advantages over alternative methods, with respect of ease of use and rapidity.     

β-D-galactosidase activity of viable, non-culturable coliform bacteria in marine waters

The β-D-galactosidase activity of viable but non-culturable (vnc) Escherichia coli cells in seawater was investigated using a rapid fluorimetric enzyme assay. Results from microcosm studies showed that loss of culturability did not necessarily result in loss of the ability to produce the galactosidase enzyme. Even when no culturable cells were detected, a positive enzyme assay response was observed and the activity of the inducible enzyme over time more closely reflected the number of vnc cells present.     

Improved membrane filtration method incorporating catalase and sodium pyruvate for detection of chlorine-stressed coliform bacteria.

In vitro pure culture studies were conducted on three different strains of Escherichia coli (K-12, EPA 00244, and SWEI) to determine the effect of chlorination on catalase activity. In each case, stationary-phase cells exhibited significant (P less than 0.001) reductions in enzyme activity following exposure to chlorine. Mean differences in activity between control and chlorine-stressed cells ranged from 8.8 to 20.3 U/mg of protein for E. coli SWEI and EPA 00244, respectively. Following initial enzyme studies, resuscitation experiments utilizing the membrane filtration technique were conducted on chlorinated sewage effluent. Five different amendments, including catalase (1,000 U per plate), heat-inactivated catalase (1,000-U per plate), sodium pyruvate (0.05%), a catalase-sodium pyruvate combination (1,500 U/0.01%), and acetic acid (0.05%), were tested for the ability to enhance detection of chlorine-stressed cells on M-fecal coliform (M-FC), mT7, M-Endo, and tryptone-glucose-yeast extract (TGY) media. Significant (P less than 0.001) increases in recovery of fecal coliforms on M-FC, total coliforms on mT7 and M-Endo, and total heterotrophs on TGY were obtained on plates containing catalase, pyruvate, or the combination of these compounds. Supplementation with heat-inactivated catalase and acetic acid did not improve recovery of chlorine-stressed cells compared with recovery on nonamended media. Subsequent analysis of colonies from plates containing compounds which enhanced recovery indicated coliform verification percentages of greater than 80% on M-FC, greater than 90% on mT7, and greater than 94% on M-Endo media. These data suggest that the addition of peroxide-degrading compounds to various standard recovery media may improve detection of both coliform and heterotrophic bacteria in chlorinated waters.     

Flow cytometric detection of viable bacteria in compost

Abstract Flow cytometry employing several vital stains was used to study the colonisation of sterile compost by Bacillus subtilis 168 (pAB224). The dyes used included rhodamine 123 (Rh123), carboxyfluorescein diacetate (CFDA) and chemchrome B. The results demonstrated the ability of flow cytometry to detect and enumerate viable bacteria in filtered compost extracts. Flow cytometry was also used to detect and study the viability of an indigenous compost community. Although it was possible to detect a viable bacterial population, the numbers of viable bacteria estimated were significantly different to those estimated from cfu.     

Comparison of media for recovery of total coliform bacteria from chemically treated water

Five broth media and two solid media were compared for their ability to quantitatively recover total coliform bacteria from chemically treated water. M-Endo LES and mT7 media were used in the membrane filter technique. Lauryl tryptose broth, lactose broth, presence-absence broth, lactose broth with twice the amount of lactose, and lauryl tryptose broth with twice the amount of sodium lauryl sulfate were used in the fermentation tube procedure. The differences in recovery were not significant for the five broth media and M-Endo LES agar. The M-Endo LES and mT7 media were not significantly different; however, the five broth media did yield significantly higher counts than mT7.     

Spiral plate count method for the examination of raw and pasteurized milk.

The spiral plate count method (SPLPC) was compared with the standard plate count (SPC) method by examining 201 samples of raw and pasteurized milk. Although the means of the two methods differed significantly at alpha = 0.01,the difference was less than 10% and was not considered to be of any practical importance. The pooled replicate variances of both methods were less than 0.003, indicating good agreement between duplicate plates, with the variance of the SPLPC slightly less than that of the SPC. We believe this study indicates that the SPLPC could be substituted for the SPC in the bacteriological examination of milk.     

Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.     

Suitability of poor medium in counting total viable airborne bacteria

The purpose of this study was to test the effect of incubation temperature and culture medium on viable counts of airborne bacteria. The incubation temperature had different effect on indoor and outdoor air bacteria. Indoor air bacteria grew as well at 20°C as 37°C, but less at 10°C. Outdoor air bacteria grew equally well at 10°C and 20°C, but less at 37°C. Both indoor and outdoor air bacteria grew differently on poor and rich media. The counts of both indoor and outdoor air bacteria were higher on poor R2A medium (low nutrient concentration) than on rich TYG and blood media (high nutrient concentration). The results indicate that a poor medium incubated at 20°C is adequate for counting viable airborne bacteria.