The Limulus Lysate Endotoxin Assay as a Test of Microbial Quality of Ground Beef

The Limulus lysate test (LLT) for endotoxin assay has been found to be an excellent, simple and rapid test of microbial quality of refrigerated ground beef. In fresh ground beef held at 5°C for 7–12 d, LLT titres increased from 10²–10⁵ and correlated very highly with extract-release volume (ERV) data and total viable Gram negative counts at both 5° and 30°C. The LLT was negative for fresh beef containing low numbers of bacteria and on aged beef in the absence of increasing numbers of Gram negative bacteria. Of 14 Gram negative meat isolates, all gave a positive LLT while none of eight miscellaneous Gram positive bacteria did. The use of this test provides objective information on the microbial quality of fresh refrigerated ground meats in 1 h. Based upon this study, it is suggested that a 0·1 ml inoculum from a 10³ dilution of good quality ground beef should produce a negative lysate test and thus serve as an additional rapid screening test of meat microbial quality.     

Comparison of microbial counts on beef carcasses by using the moist-swab contact method and secondary tissue removal technique.

When a tissue removal rinse technique was compared to the moist-swab contact method, significantly greater numbers of bacteria were recovered from beef carcasses, especially when the flora exceeded log10 4.5/6.45 cm2. Secondary treatment of the removed surface tissue by blending resulted in a significantly greater number of bacteria being recovered than when the same sample was swabbed and/or rinsed. Data indicate that blending of the carcass surface tissue provides a more representative value of the true microbial flora.     

Comparison of microbial counts on beef carcasses by using the moist-swab contact method and secondary tissue removal technique.

When a tissue removal rinse technique was compared to the moist-swab contact method, significantly greater numbers of bacteria were recovered from beef carcasses, especially when the flora exceeded log10 4.5/6.45 cm2. Secondary treatment of the removed surface tissue by blending resulted in a significantly greater number of bacteria being recovered than when the same sample was swabbed and/or rinsed. Data indicate that blending of the carcass surface tissue provides a more representative value of the true microbial flora.     

发酵中含氮类原料对保加利亚乳杆菌活菌数的影响

目的研究生产发酵过程中培养基含氮原料与发酵的相关性。方法实验通过对不同生产厂家三种原料进行理化指标检测,并通过正交试验,比较不同原料发酵后保加利亚乳杆菌发酵液活菌数。结果最佳条件为酪蛋白胨氨基氮含量高,酵母浸粉的炽灼残渣低,牛肉粉总氮含量高。结论经过相同原料不同生产厂家的配方发酵后的发酵液活菌数存在差异,其理化指标对发酵有一定影响。     

Quantification and Qualification of Bacteria Trapped in Chewed Gum

Chewing of gum contributes to the maintenance of oral health. Many oral diseases, including caries and periodontal disease, are caused by bacteria. However, it is unknown whether chewing of gum can remove bacteria from the oral cavity. Here, we hypothesize that chewing of gum can trap bacteria and remove them from the oral cavity. To test this hypothesis, we developed two methods to quantify numbers of bacteria trapped in chewed gum. In the first method, known numbers of bacteria were finger-chewed into gum and chewed gums were molded to standard dimensions, sonicated and plated to determine numbers of colony-forming-units incorporated, yielding calibration curves of colony-forming-units retrieved versus finger-chewed in. In a second method, calibration curves were created by finger-chewing known numbers of bacteria into gum and subsequently dissolving the gum in a mixture of chloroform and tris-ethylenediaminetetraacetic-acid (TE)-buffer. The TE-buffer was analyzed using quantitative Polymerase-Chain-Reaction (qPCR), yielding calibration curves of total numbers of bacteria versus finger-chewed in. Next, five volunteers were requested to chew gum up to 10 min after which numbers of colony-forming-units and total numbers of bacteria trapped in chewed gum were determined using the above methods. The qPCR method, involving both dead and live bacteria yielded higher numbers of retrieved bacteria than plating, involving only viable bacteria. Numbers of trapped bacteria were maximal during initial chewing after which a slow decrease over time up to 10 min was observed. Around 10⁸ bacteria were detected per gum piece depending on the method and gum considered. The number of species trapped in chewed gum increased with chewing time. Trapped bacteria were clearly visualized in chewed gum using scanning-electron-microscopy. Summarizing, using novel methods to quantify and qualify oral bacteria trapped in chewed gum, the hypothesis is confirmed that chewing of gum can trap and remove bacteria from the oral cavity.     

8种不同方法保藏病原菌效果的对比观察

采用８种不同的菌种保藏方法,对１８种常见病原菌的保藏时间及生物学特性的影响进行了对比观察。结果表明：冷冻干燥法保藏菌种时间最长（本实验室已保存１５年）;肉浸汤琼脂平板法保藏菌种时间最短（２～３月）。保藏时间由长到短依次为：冷冻干燥法＞半固体冷冻法≥半固体斜面加液体石蜡覆盖法＞半固体斜面法＞肉浸汤加液体石蜡覆盖法＞血琼脂平板法＞肉浸汤法＞肉浸汤琼脂平板法。且相同方法对不同菌种保藏时间不同。保藏时间在１年以内的菌种,其生物学特性无明显变异;而经冷冻干燥法保藏时间较长的白喉棒状杆菌、金黄色葡萄球菌、甲型副伤寒沙     

Rapid detection of meat spoilage by measuring volatile organic compounds by using proton transfer reaction mass spectrometry

The evolution of the microbial spoilage population for air- and vacuum-packaged meat (beef and pork) stored at 4 degrees C was investigated over 11 days. We monitored the viable counts (mesophilic total aerobic bacteria, Pseudomonas spp., Enterobacteriaceae, lactic acid bacteria, and Enterococcus spp.) by the microbiological standard technique and by measuring the emission of volatile organic compounds (VOCs) with the recently developed proton transfer reaction mass spectrometry system. Storage time, packaging type, and meat type had statistically significant (P < 0.05) effects on the development of the bacterial numbers. The concentrations of many of the measured VOCs, e.g., sulfur compounds, largely increased over the storage time. We also observed a large difference in the emissions between vacuum- and air-packaged meat. We found statistically significant strong correlations (up to 99%) between some of the VOCs and the bacterial contamination. The concentrations of these VOCs increased linearly with the bacterial numbers. This study is a first step toward replacing the time-consuming plate counting by fast headspace air measurements, where the bacterial spoilage can be determined within minutes instead of days.     

气调包装生鲜冷却牛肉贮藏中微生物多样性分析

【目的】分析普通包装和气调包装(65%O2和35%CO2)生鲜冷却牛肉在贮藏(4°C)过程中的微生物多样性。【方法】通过16S rDNA V3区PCR-DGGE(变性梯度凝胶电泳)方法和16S rDNA克隆分析法研究生鲜冷却牛肉中微生物菌落结构及菌相变化规律。【结果】初始菌相主要有嗜冷杆菌属(Psychrobacter)和假单胞菌属(Pseudomonas)。贮藏过程中,普通包装和气调包装生鲜冷却牛肉中优势菌均为Brochothrix和Pseudomonas。气调包装冷却牛肉中细菌种类较少,两种包装生鲜牛肉在贮藏前期菌相变化明显。【结论】不同包装冷却牛肉中微生物菌落结构有较大差异,气调包装中CO2对Pseudomonas等细菌起到了一定的抑制作用。     

Sensitive detection of Escherichia coli O157:H7 in food and water by immunomagnetic separation and solid-phase laser cytometry

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r2 = 0. 95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r2 = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.     

市售冷却牛肉中主要细菌的常规分离与鉴定

利用常用纯培养的方法, 根据细菌的菌落形态、菌落颜色、革兰氏染色等常见特征, 从市售冷却牛肉中, 选取菌落形态差别比较明显的菌株共32株, 其中保鲜膜包装冷却牛肉样品共12株, 未包装冷却牛肉样品共20株; 同时选取两样品中的优势菌株各4株进行进一步的研究(8株细菌编号为：S01~S08, 其中S01~S04为未包装冷却牛肉样品; S05~S08为保鲜膜包装冷却牛肉样品), 通过ARDRA(Amplified ribosomal DNA restriction analysis) 以及16S rDNA 序列等进行分类研究, 确定该细菌的分类地位, 并结合形态、常规生理生化特性进行鉴定, 确定各细菌所属种。实验表明：S01为假单胞菌属中的恶臭假单胞菌(Pseudomonas putida), S02为希瓦氏菌属下的(Shewanella cincia sp.), 而S03和S05为希瓦氏菌属下的腐败希瓦氏菌(Shewanella putrefaciens), S04为窄食单胞菌属中的嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia), S06为嗜冷杆菌(Psychrobacter sp.), S07为葡萄球菌属中的松鼠葡萄球菌(Staphylococcu sciuri), S08为微杆菌属中的产左聚糖微杆菌(Microbacterium laevaniformans)。证明两样品的共有优势菌为希瓦氏菌属。通过对样品可培养微生物情况进行初步的调查分析, 为冷却肉加工工艺提供一定的理论基础。     

A Survey of the Hygienic Quality of Beef and Pork Carcasses in Norway

The bacteriological quality of beef and pig carcasses was assessed at 9 Norwegian abattoirs by sampling 10 carcasses at multiple sites on each of several visits. On beef carcasses the following sites consistently carried higher numbers of bacteria: the Brisket, the Fore-ribs, the Flank groin, and the Round medial. There was no evidence that beef slaughter and dressing in the hanging position was superior to methods where the carcass was lying until the hide puller. On pork carcasses the Cheek, and the Abdomen lateral surface (belly) were most heavily contaminated. The hygienic quality of pork carcasses in abattoirs where singeing was a separate step tended to be better than where a combined singeing and dehairing machine was used. This survey suggests that bacteriological monitoring of slaughter at this level of sampling and visiting is able to detect consistently poor hygienic practices. Where direct comparisons with data from other countries could be made, the present investigation indicates that the bacterial counts on Norwegian beef and pork carcasses are of the same order or better.     

Genotypic analysis of Escherichia coli recovered from product and equipment at a beef-packing plant

AIMS: To identify sources of Escherichia coli on beef by characterizing strains of the organism on animals, equipment and product at beef-packing plant. METHODS AND RESULTS: Generic E. coli were recovered from hides, carcasses, beef trimmings, conveyers and ground beef during the summer of 2001 (750 isolates) and winter of 2002 (500 isolates). The isolates were characterized by Random Amplification of Polymorphic DNA (RAPD). The numbers of E. coli recovered from dressed carcasses were less than the numbers recovered from hides. The numbers recovered from chilled carcasses were too few for meaningful analysis of the strains present on them but the numbers recovered from trimmings and ground beef were larger. The RAPD patterns showed that the majority of isolates from hides, carcasses, beef trimmings, conveyers and ground beef were of similar RAPD types, but a few unique RAPD types were recovered from only one of those sources. The E. coli populations present on the hides of incoming animals and in the beef-processing environment were highly diverse. Randomly selected E. coli isolates from each of the five sources were further characterized by pulsed-field gel electrophoresis (PFGE). Most genotypes of E. coli defined by PFGE corresponded to the E. coli types defined by RAPD. CONCLUSIONS: The hides of the incoming animals appeared to be only one of the sources of the E. coli on trimmings and in ground beef, as additional sources were apparently present in equipment used for carcass breaking. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that hazardous microbiological contamination of meat may occur after the dressing of carcasses at commercial beef-packing plants, which suggests that attention should be given to the control of the contamination of meat during carcass breaking as well as during the dressing of carcasses.     

Rapid Detection of Meat Spoilage by Measuring Volatile Organic Compounds by Using Proton Transfer Reaction Mass Spectrometry

The evolution of the microbial spoilage population for air- and vacuum-packaged meat (beef and pork) stored at 4°C was investigated over 11 days. We monitored the viable counts (mesophilic total aerobic bacteria, Pseudomonas spp., Enterobacteriaceae, lactic acid bacteria, and Enterococcus spp.) by the microbiological standard technique and by measuring the emission of volatile organic compounds (VOCs) with the recently developed proton transfer reaction mass spectrometry system. Storage time, packaging type, and meat type had statistically significant (P < 0.05) effects on the development of the bacterial numbers. The concentrations of many of the measured VOCs, e.g., sulfur compounds, largely increased over the storage time. We also observed a large difference in the emissions between vacuum- and air-packaged meat. We found statistically significant strong correlations (up to 99%) between some of the VOCs and the bacterial contamination. The concentrations of these VOCs increased linearly with the bacterial numbers. This study is a first step toward replacing the time-consuming plate counting by fast headspace air measurements, where the bacterial spoilage can be determined within minutes instead of days.     

Animal viruses, coliphages, and bacteria in aerosols and wastewater at a spray irrigation site.

Aerosol samples collected at the Muskegon County Wastewater Management System Number 1 spray irrigation site in Michigan by using the Army prototype XM2 Biological Sampler/Collector were examined for the presence of animal viruses, coliphages, and bacteria. Air samples, collected in Earle lactalbumen hydrolysate, and wastewater samples were filtered through a 0.45- and 1.2-micron membrane filter sandwich, pretreated with 10% beef extract (pH 7.0), and assayed for animal viruses by the plaque method on Buffalo green monkey kidney cells. Untreated air and wastewater samples were assayed for coliphages by the soft agar overlay method with three Escherichia coli hosts (ATCC 13706, 15597, and 11303) and for bacteria by the heterotrophic plate count method. Filtered air samples were assayed for coliphages by the most-probable-number method with the same three hosts. Although no animal viruses were detected in the aerosol samples, coliphages and bacteria were recovered. E. coli ATCC 13706 coliphage were recovered more often and in greater numbers than either of the other two types of coliphages. Concentrations of animal viruses, coliphages, and bacteria detected in the raw influent decreased as the wastewater was aerated and stored in the lagoons. No animal viruses were detected in the wastewater at the pump station just before distribution to the spray irrigation rigs. The most-probable-number method was more sensitive and consistent than the overlay procedure in detecting low levels of coliphages in air samples.     

Estimation of abundance dynamics of gram-negative bacteria in soil

Bacterial succession in soil was studied for two variants of initiation (moistening and moistening with addition of glucose). To determine the numbers of viable gram-negative bacteria, the modified nalidixic acid method was applied. The numbers of gram-negative bacteria revealed by this method were 2 to 3.5 times higher than those determined by the traditional method. In a developing community, the highest total bacterial numbers were observed on day 7; afterwards their numbers decreased and stabilized at a level exceeding four-to fivefold the initial one. In both experimental variants, the highest numbers of viable gram-negative bacteria were revealed on day 15 (75–85% of the total bacterial numbers). Morphology of these bacteria suggests their classification as cytophagas (chitinophagas) utilizing chitin from the dead fungal mycelium.     

Animal viruses, coliphages, and bacteria in aerosols and wastewater at a spray irrigation site

Aerosol samples collected at the Muskegon County Wastewater Management System Number 1 spray irrigation site in Michigan by using the Army prototype XM2 Biological Sampler/Collector were examined for the presence of animal viruses, coliphages, and bacteria. Air samples, collected in Earle lactalbumen hydrolysate, and wastewater samples were filtered through a 0.45- and 1.2-micron membrane filter sandwich, pretreated with 10% beef extract (pH 7.0), and assayed for animal viruses by the plaque method on Buffalo green monkey kidney cells. Untreated air and wastewater samples were assayed for coliphages by the soft agar overlay method with three Escherichia coli hosts (ATCC 13706, 15597, and 11303) and for bacteria by the heterotrophic plate count method. Filtered air samples were assayed for coliphages by the most-probable-number method with the same three hosts. Although no animal viruses were detected in the aerosol samples, coliphages and bacteria were recovered. E. coli ATCC 13706 coliphage were recovered more often and in greater numbers than either of the other two types of coliphages. Concentrations of animal viruses, coliphages, and bacteria detected in the raw influent decreased as the wastewater was aerated and stored in the lagoons. No animal viruses were detected in the wastewater at the pump station just before distribution to the spray irrigation rigs. The most-probable-number method was more sensitive and consistent than the overlay procedure in detecting low levels of coliphages in air samples.     

Use of a Titrimetric Method to Assess the Bacterial Spoilage of Fresh Beef

A new method of determining bacterial spoilage in fresh beef is presented. The technique is based upon the fact that as beef undergoes refrigerator spoilage, there is a gradual increase in the production of alkaline substances by the spoilage flora. The level of these substances was measured by titrating meat homogenates to a pH 5.00 end point, employing 0.02 n HCl and an autotitrator. When 23 samples of ground beef from retail stores were tested, an average of 1.32 ml of acid was required for titration of 1 g of fresh beef to pH 5.00, whereas 2.58 ml was required for the same meat at the onset of spoilage. Preliminary data indicate that beef which requires more than 2 ml of 0.02 n HCl/g to lower its pH to 5.00 under the conditions of the test is in some state of incipient spoilage. The statistical correlation between titration values, log bacterial numbers, and extract-release volume was high (P < 0.001). The technique is simple to execute and is highly reproducible, and duplicate samples can be run within 15 min.     

Comparative Study of Two Methods for Detection of Clostridium perfringens in Ground Beef

The tryptose-sulfite-cycloserine agar pour plate method was superior to selective enrichment in liquid sulfite medium for isolation of small numbers of Clostridium perfringens from frozen ground beef.     

Sensitive Detection of Escherichia coli O157:H7 in Food and Water by Immunomagnetic Separation and Solid-Phase Laser Cytometry

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r² = 0.95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r² = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.     

Effect of sample transport systems on survival of bacteria in ground beef.

The effects of two transport systems and cryoprotective agents on the survival of bacteria in ground beef samples were evaluated. Survival of Clostridium perfringens in ground beef samples after simulated transport (72 h) was higher (about 99%) in Dry Ice than in Trans Temp shipping units (-3 degrees C). There were no significant differences between the two transport systems in survival of coliforms, Escherichia coli, Staphylococcus aureus, or aerobic bacteria. Mixing ground beef samples at a ratio of 1:1 (wt/vol) with 10, 20, or 30% buffered solutions of dimethyl sulfoxide or glycerol before freezing improved the survival of C. perfringens and coliforms in both transport systems. Recovery of E. coli was significantly higher with the addition of 10% dimethyl sulfoxide before Dry Ice transport. Addition of 10% dimethyl sulfoxide resulted in a 100% recovery of both S. aureus and aerobic bacteria from ground beef after simulated transport in Trans Temp shipping units. The use of cryoprotective agents can improve the survival of bacteria during transport of ground beef samples.