Determining endotoxin content of ground beef by the Limulus amoebocyte lysate test as a rapid indicator of microbial quality.

Eighty-four samples of ground beef were placed into five half-log cycle groups based upon aerobic plate count (APC) results. Endotoxins were determined by the Limulus amoebocyte lysate test (LAL), and gram-negative viable counts were determined by a violet red bile agar overlay method. Ten samples with a log of APC of less than 5.50 had an APC mean of less than 5.24 and mean endotoxin content by the LAL of 51 ng/g. The 15 samples with APCs between a log of 5.50 and 5.99 had an APC mean of 5.79/g and an endotoxin mean of 103.8 ng/g. Twenty-eight samples had APCs between a log of 6.00 and 6.49 with a mean of 5.28/g and an endotoxin mean of 1106.4 ng/g. The 20 samples with APCs between a log of 6.50 and 7.00 had a mean of 6.77/g and an endotoxin mean of 5067.6 ng/g, while 11 samples had a log of APCs of greater than 7.00 with a mean of 7.53 and an endotoxin mean of 7,472 ng/g. Correlation of half-log cycle mean APC and violet red bile agar counts with mean endotoxin content were both highly significant, indicating that LAL-determined endotoxin content can be used to make a rapid approximation of viable plate counts. Because results can be obtained by LAL in 1 h, the finding of low levels of endotoxins can be taken to indicate low-count meat. The use of additional tests of microbial quality may be necessary when high endotoxin levels are found because the LAL detects both viable and nonviable cells.     

Comparison of homogenizing, shaking, and blending on the recovery of microorganisms and endotoxins from fresh and frozen ground beef as assessed by plate counts and the Limulus amoebocyte lysate test.

Of three methods studied, brisk shaking of samples in dilution blanks by hand and homogenization by a stomacher were compared relative to their capacity to recover the endotoxins and viable bacteria; blending with a Waring blender was compared with these two methods only on the recovery of viable cells. Aerobic plate counts were essentially the same by the three methods for fresh meats, with the stomacher producing slightly higher aerobic plate counts and significantly higher gram-negative counts determined by violet red bile agar. The stomacher produced significantly higher aerobic plate counts and violet red bile agar results on frozen meats than did shaking. Endotoxins were determined by the Limulus amoebocyte lysate test; results by shaking and stomacher on 15 single samples of frozen meat were identical. Of Limulus amoebocyte lysate-negative beef which was spiked with known endotoxin, a higher percentage of recovery was obtained with the stomacher. Although both aerobic plate counts and violet red bile agar counts were found by shaking and stomacher to decrease significantly in frozen meats, endotoxin content was not significantly affected. The stomacher was found to be the better method overall, especially when meats are to be examined for their content of viable gram-negative bacteria, endotoxins, or both.     

Comparison of homogenizing, shaking, and blending on the recovery of microorganisms and endotoxins from fresh and frozen ground beef as assessed by plate counts and the Limulus amoebocyte lysate test.

Of three methods studied, brisk shaking of samples in dilution blanks by hand and homogenization by a stomacher were compared relative to their capacity to recover the endotoxins and viable bacteria; blending with a Waring blender was compared with these two methods only on the recovery of viable cells. Aerobic plate counts were essentially the same by the three methods for fresh meats, with the stomacher producing slightly higher aerobic plate counts and significantly higher gram-negative counts determined by violet red bile agar. The stomacher produced significantly higher aerobic plate counts and violet red bile agar results on frozen meats than did shaking. Endotoxins were determined by the Limulus amoebocyte lysate test; results by shaking and stomacher on 15 single samples of frozen meat were identical. Of Limulus amoebocyte lysate-negative beef which was spiked with known endotoxin, a higher percentage of recovery was obtained with the stomacher. Although both aerobic plate counts and violet red bile agar counts were found by shaking and stomacher to decrease significantly in frozen meats, endotoxin content was not significantly affected. The stomacher was found to be the better method overall, especially when meats are to be examined for their content of viable gram-negative bacteria, endotoxins, or both.     

Culture medium for selective isolation and enumeration of Gram-negative bacteria from ground meats.

We developed a new medium, designated peptone bile amphotericin cycloheximide (PBAC) agar, which contains (per liter) 10 g of peptone, 300 mg of bile salts, 1 mg of amphotericin B, 1 g of cycloheximide, and 15 g of agar. When 21 samples of fresh ground beef were studied and plate count agar counts were used as references, we obtained a mean recovery of 28% of total counts with violet red bile agar overlay, whereas we obtained 48% recovery with PBAC agar. With 12 samples of frozen ground beef, recovery on violet red bile agar overlay was 29% of the recovery on plate count agar, whereas the corresponding value on PBAC agar was 45%. PBAC agar allowed the enumeration of 1.4 times as many gram-negative bacteria as violet red bile agar overlay. None of eight strains of gram-positive bacteria and none of eight strains of yeasts grew on PBAC agar. Of 158 colonies randomly selected from pour plates of eight fresh ground meat samples, 95% stained gram negative. In comparison, only 70% of 151 colonies selected from corresponding plate count agar plates were gram negative. The lack of background color, turbidity, and ease of use make PBAC agar easier to handle than other media used for gram-negative bacteria, such as violet red bile agar, violet red bile agar overlay, and crystal violet tetrazolium agar. In the preparation PBAC agar, all ingredients are autoclaved together except amphotericin B, which is filter sterilized and added before the plates are poured.     

Violet Red Bile 2 Agar for Stressed Coliforms

Counts on a new, autoclave-sterilizable violet red bile (VRB-2) agar were compared with counts on freshly boiled VRB agar. Yields on VRB-2 agar averaged 217, 180, 130, and 112% of counts obtained on the control medium for samples of water, cottage cheese, frozen vegetables, and raw milk, respectively. The general principle used for the development of VRB-2 agar could be applied to many other kinds of selective plating media.     

人用狂犬病疫苗浓缩前后内毒素含量变化的研究

本文采用显色鲎试验法测定人用狂犬病疫苗浓缩前后半成品中内毒素含量。经鲎试验抑制增强实验确定人用狂犬病疫苗半成品对鲎试验法测定内毒素无干扰作用。样品经系列稀释,测定光吸收值（５４５ｎｍ）,然后将其对数回归直线与标准品的反应直线比较,结果表明,两者间有平行关系,经对四个生物制品研究所的样品进行测定,结果显示,人用狂犬病疫苗经超滤浓缩后内毒素含量有所增加,但在被检３１批样品中,除一所二批浓苗外,其余２９批均低于以往测得的最低家兔致热剂量（３．３２ＥＵ／ｍｌ／Ｋｇ）。     

Distribution and sources of microbial contamination on beef carcasses

Three beef dressing lines of different capacity (160, 440 and 800 head d(-1)) were investigated with respect to contamination associated with carcass/hide and carcass/faeces contacts, the distribution of microbial contamination on carcasses and the antimicrobial efficacy of cold water carcass washes. Swab samples were taken from up to 17 sites for determination of Aerobic Plate Counts at 37 degrees C (APC 37 degrees C) and Escherichia coli enumeration using the Petrifilm procedure. The three beef dressing systems produced virtually identical patterns of microbial contamination. High contamination was found at those sites associated with opening cuts and/or subject to hide contact during hide removal. Where contamination is intermittent, the use of mean microbial data tended to obscure evidence of faecal or hide contact. Consequently, worst-case results, as represented by the 95th percentile value, were used to identify probable instances and sources of contact contamination. Sites not subject to faecal contamination or hide contact typically had swab sample APC (37 degrees C) values of less than log 2.00 cfu cm(-2) accompanied by the occasional detection of E. coli at levels below log 1.00 cfu cm(-2). Sites contacted by 'clean' hide typically had APC (37 degrees C) counts of log 3.00 cfu cm(-2) or greater accompanied by occasional E. coli counts not exceeding log 2.00 cfu cm(-2). Sites contaminated by direct faecal contact or contact with faecally contaminated hides typically had APC (37 degrees C) counts equal to, or greater than, log 4.00 cfu cm(-2) accompanied by E. coli counts exceeding log 2.00 cfu cm(-2). Cold water carcass washing was ineffective in removing microbial contamination and tended to bring about a posterior to anterior redistribution, resulting in increased counts at forequarter sites.     

Fermentation characteristics of exopolysaccharide-producing lactic acid bacteria from sourdough and assessment of the isolates for industrial potential

Lactic acid bacteria (LAB) with antimicrobial activity and high exopolysaccharide (EPS) production ability isolated from sourdough were studied for their fermentation characteristics as potential new starter cultures. The values of pH, titratable acidity, and viable cell counts were 4.06+/-0.009-4.50+/- 0.015, 0.787+/-0.020%-1.172+/-0.018%, and 8.78+/-0.08-8.98+/- 0.06 log CFU/ml, respectively. In order to select probiotics with a high survival rate in the gut, isolates were tested to assess resistance against the artificial gastric acid and bile juice. Viable LAB counts were significantly (p<0.05) affected by the acidity. At pH 2.0, the total declines in the initial bacterial counts were 4.52+/-0.07 log for S. thermophilus St-Body-1, >7.98+/-0.03 log for E. flavescens DU-10, >7.95+/-0.05 log for E. faecium DU-12, and 3.15+/- 0.06 log for L. amylovorus DU-21. Among the strains, L. amylovorus DU-21 was the only strain that had bile tolerance under simulated gastrointestinal conditions. In order to improve EPS production by L. amylovorus DU- 21, the influence of carbon source was studied. When glucose was used as a carbon source, EPS production dramatically increased to 17.19+/-0.28 g/l (p<0.05). The maximum cell growth (10.012+/-0.012 log CFU/ml) and EPS production (18.71+/-0.19 g/l) were achieved when 15 g/ l of glucose was employed as the carbon source.     

Survival of cold-stressed Campylobacter jejuni on ground chicken and chicken skin during frozen storage

Campylobacter jejuni is prevalent in poultry, but the effect of combined refrigerated and frozen storage on its survival, conditions relevant to poultry processing and storage, has not been evaluated. Therefore, the effects of refrigeration at 4 degrees C, freezing at -20 degrees C, and a combination of refrigeration and freezing on the survival of C. jejuni in ground chicken and on chicken skin were examined. Samples were enumerated using tryptic soy agar containing sheep's blood and modified cefoperazone charcoal deoxycholate agar. Refrigerated storage alone for 3 to 7 days produced a reduction in cell counts of 0.34 to 0.81 log10 CFU/g in ground chicken and a reduction in cell counts of 0.31 to 0.63 log10 CFU/g on chicken skin. Declines were comparable for each sample type using either plating medium. Frozen storage, alone and with prerefrigeration, produced a reduction in cell counts of 0.56 to 1.57 log10 CFU/g in ground chicken and a reduction in cell counts of 1.38 to 3.39 log10 CFU/g on chicken skin over a 2-week period. The recovery of C. jejuni following freezing was similar on both plating media. The survival following frozen storage was greater in ground chicken than on chicken skin with or without prerefrigeration. Cell counts after freezing were lower on chicken skin samples that had been prerefrigerated for 7 days than in those that had been prerefrigerated for 0, 1, or 3 days. This was not observed for ground chicken samples, possibly due to their composition. C. jejuni survived storage at 4 and -20 degrees C with either sample type. This study indicates that, individually or in combination, refrigeration and freezing are not a substitute for safe handling and proper cooking of poultry.     

ADVISORY MICROBIOLOGICAL STANDARDS AND TOLERANCES FOR RAW MILK

SUMMARY: To study the value of recent modifications of microbiological tests used for advisory purposes, samples of tuberculin tested milk, taken at weekly intervals over a 5 year period from the Trawscoed Experimental Husbandry Farm and selected at random during a 12 months period from farms in Wales, were examined by a temperature compensated keeping quality test at 22°, colony count on Yeastrel milk agar in 3 days at 30° and coli-aerogenes colony count on violet red bile agar in 20–24 hr at 30°.
The results show that milk produced and handled under hygienic conditions can be expected to have colony counts of less than 2 × 10⁴/ml and coli-aerogenes colony counts less than 10/ml when examined within 18 hr of milking.     

Escherichiacoli endotoxin absorption from the ewe's uterus and peritoneal cavity

An experiment was conducted to assess the effects of estrogen or parturition on absorption of endotoxin from the ovine uterus. Twelve cycling ewes were assigned to one of four treatment groups (three ewes/group): Group I, no estrogen (NE) + intrauterine infusion of sterile saline (IUS); Group II, NE + intrauterine infusion of 100 mg endotoxin - Lipopolysaccharide W./$\text{E.}$$\text{coli}$ 0127:B8, Difco Laboratories, Detroit, MI (IUE); Group III, 3 days pretreatment with estradiol-17β (50 μg/da, E) + IUS; and Group IV, E + IUE. In addition, the uteri of three early postpartum ewes were infused with 100 mg endotoxin (Group V). Rectal temperatures (RT) and jugular blood samples were obtained at ?40, ?20, 0 (infusion), 20, 40, 60, 80, 100, 120, 180, and 240 minutes. The blood samples were analyzed for total white blood cell counts (WBC) and Limulus Amebocyte Lysate Assays (LAL). There were no alterations in RT, WBC, or LAL observed in Groups I–V. These results indicated that neither prior treatment with estradiol in cycling ewes nor parturition affected absorption of $\text{E.}$$\text{coli}$ endotoxin from the ovine uterus.A second study was conducted to characterize the changes in RT, WBC, and LAL during endotoxemia in cycling ewes. Three ewes received intraperitoneal infusions of 100 mg endotoxin and three ewes received intraperitoneal infusions of sterile saline. Evidence that endotoxin was absorbed from the peritoneal cavity was a decrease (P<0.10) in WBC and positive LAL in endotoxin-infused ewes. WBC and LAL did not change in saline-infused ewes. No changes in RT were observed in either group.     

A novel endotoxin-specific assay by turbidimetry with Limulus amoebocyte lysate containing beta-glucan

The gelation of standard Limulus amoebocyte lysate (LAL) is triggered by the addition of a small amount of beta-glucan (1-1000 ng/ml plasma), but in the presence of an excessive amount of beta-glucan (1 mg/ml plasma), the gelation becomes insensitive to beta-glucan. Utilizing this property, a method to determine quantitatively the amount of endotoxin circulating in humans was developed. When a modified LAL, or LAL-ES, which contains an excessive amount of CM-curdlan as beta-glucan, was used for the assay, a linear relation in the logarithmic scales was obtained between the gelation time measured by the turbidimetry (min) and the concentration of endotoxin. This relation was not affected by a considerable amount of beta-glucan (100 ng/ml). The sensitivity of the endotoxin assay was estimated to be as low as 3 pg/ml. The following aspects of the method were found by clinical application to normal and febrile subjects. (1) Using both LAL and LAL-ES, it was possible to distinguish the effect of endotoxin from that of beta-glucan in plasma, i.e., bacterial sepsis from fungal sepsis. (2) The amount of circulating endotoxin determined by the present method showed good correlation to those obtained by chromogenic assay using modified LAL devoid of Factor G which could be activated by beta-glucan.     

The enumeration of thermotrophic types amongst the Enterobacteriaceae colonizing perishable foods

A total of 41 pure cultures of Enterobacteriaceae, comprising 32 thermotrophic and nine psychrotrophic strains, pathogens or marker organisms, were examined for numbers of colony forming units obtained at 37 degrees and 42.5 degrees C (thermotrophs) and 30 degrees C (psychrotrophs), when surface-plated on a rich infusion agar and violet red bile agar. In addition 42 food and water samples, collected in a rural area of the Philippines, were examined by surface inoculating violet red bile AIPC (agar immersion plating and contact; 'dip') slides and incubating at 37 degrees and 42.5 degrees C. At 42.5 degrees C there was almost total recovery of the thermotrophic Enterobacteriaceae, whereas the psychrotrophic strains were completely suppressed. At 37 degrees C the psychrotrophs were only slightly inhibited. The Philippine foods, predominantly cooked meals, milk and drinking water, appeared to be significantly colonized by thermotrophic Enterobacteriaceae. It is concluded that incubation at 42.5 degrees C satisfactorily selects enteropathogenic and other enteric Enterobacteriaceae while suppressing the psychrotrophic types which are mainly of vegetable origin. It is emphasized that, regardless of the temperature used, a resuscitation procedure for Enterobacteriaceae populations that have incurred sublethal injury in food has to precede counts on or in the usual selective media.     

Quantitative measurement of endotoxin in rainbow trout (Salmo gairdneri) serum by the chromogenic substrate method

The amount of endotoxin in serum collected from normal rainbow trout ( Salmo gairdneri) and trout inoculated with viable Vibrio anguillarum or lipopolysaccharide (LPS) extracted from bacteria was determined by the chromogenic substrate method. The mean values of endotoxin in four different groups of normal rainbow trout sera ranged from 31.9 to 65.3 pg/ml. When fish were inoculated with viable bacteria (1 × 10⁸), they became septicaemic and a large amount of endotoxin (> 14 ng/ml) was detected in the sera. In fish inoculated with a smaller number of bacteria the amount of endotoxin was several times higher than that of normal fish in spite of failure of bacterial isolation. Although the endotoxin level in serum increased rapidly (> 100 ng/ml) after intraperitoneal inoculation with purified V. anguillarum LPS (540 μg), no fish died during the experiment. The high level of endotoxin in normal rainbow trout and the resistance of trout to endotoxin are in striking contrast to those of mammalian and avian species.     

Use of magnesium to increase sensitivity of Limulus amoebocyte lysate for detection of endotoxin.

Increased sensitivity of the Limulus amoebocyte lysate (LAL) assay for the detection of endotoxin was attained by the reconstitution of commercially available LAL reagent with a magnesium-containing solution. As little as 2 to 6 pg (0.002 to 0.006 ng) of Escherichia coli O127:B8 endotoxin per ml was detected, an increase in sensitivity of 10 to 30 times. The optimum magnesium concentration range for the LAL reagent used and the optimum pH range were approximately 50 to 65 mM and pH 6.0 to 8.0, respectively. Reconstitution of five commercially available brands of LAL with a solution containing magnesium resulted in greater assay sensitivity than the identical LAL reconstituted with pyrogen-free water. Use of LAL reconstituted with a solution containing magnesium is crucial for the assay of some parenteral products, wherein increased sensitivity is essential to meet the requirement for the maximum valid dilution criteria. The mode of action of magnesium for enhanced sensitivity of LAL has been postulated.     

The enumeration of thermotrophic types amongst the Enterobacteriaceae colonizing perishable foods

A total of 41 pure cultures of Enterobacteriaceae, comprising 32 thermotrophic and nine psychrotrophic strains, pathogens or marker organisms, were examined for numbers of colony forming units obtained at 37° and 42°5°C (thermotrophs) and 30°C (psychrotrophs), when surface-plated on a rich infusion agar and violet red bile agar. In addition 42 food and water samples, collected in a rural area of the Philippines, were examined by surface inoculating violet red bile AIPC (agar immersion plating and contact; 'dip') slides and incubating at 37° and 42°5°C. At 42°5°C there was almost total recovery of the thermotrophic Enterobacteriaceae, whereas the psychrotrophic strains were completely suppressed. At 37°C the psychrotrophs were only slightly inhibited. The Philippine foods, predominantly cooked meals, milk and drinking water, appeared to be significantly colonized by thermotrophic Enterobacteriaceae. It is concluded that incubation at 42°5°C satisfactorily selects enteropathogenic and other enteric Enterobacteriaceae while suppressing the psychrotrophic types which are mainly of vegetable origin. It is emphasized that, regardless of the temperature used, a resuscitation procedure for Enterobacteriaceae populations that have incurred sublethal injury in food has to precede counts on or in the usual selective media.     

Comparison of the Effects of Liquid Medium Repair and the Incorporation of Catalase in MacConkey Type Media on the Recovery of Enterobacteriaceae Sublethally Stressed by Freezing

Nine pure cultures of species of Enterobacteriaceae were stressed by rapid freezing in tryptone soya broth (TSB) to — 22°C and subsequent storage at that temperature for 7 d. About one to two log cycles kill and at least one additional log cycle sublethal impairment was achieved. Numbers of colonies of these cultures in poured plates of violet red bile glucose (VRBG) agar, with 67 u/ml of catalase added at 47°C, were only slightly higher than those in plain VRBG, both incubated overnight at 30°C. Two hours incubation of TSB suspensions at 17–25° C resulted in almost complete restoration of the ability of cells to develop colonies in VRBG, without, however, leading to any significant multiplication.
Similar experiments with 32 samples of frozen minced meat, 27 samples of frozen surface water, 18 of frozen chicken liver and 14 of fresh sausage substantiated the results obtained in the studies on pure cultures.
In the experiments with the nine pure cultures the influence of the nutrient composition of the solid enumeration media: 'minimal' agar, TSB agar (TSBA) and Mueller-Hinton agar with Polyvitex nutrient supplement (MHA), on the recovery of Enterobacteriaceae stressed by freezing was also studied. Colony numbers in TSBA and MHA were virtually identical. The glucose mineral salts medium led to lower recovery, indicating that so-called 'minimal medium recovery' of stressed bacterial populations is not a common phenomenon.     

Correlation of Campylobacter bacteriophage with reduced presence of hosts in broiler chicken ceca

Campylobacter jejuni and Campylobacter-specific bacteriophage were enumerated from broiler chicken ceca selected from 90 United Kingdom flocks (n = 205). C. jejuni counts in the presence of bacteriophage (mean log(10) 5.1 CFU/g) were associated with a significant (P < 0.001) reduction compared to samples with Campylobacter alone (mean log(10) 6.9 CFU/g).     

Estimation of microbial contamination of food from prevalence and concentration data: application to Listeria monocytogenes in fresh vegetables

A normal distribution and a mixture model of two normal distributions in a Bayesian approach using prevalence and concentration data were used to establish the distribution of contamination of the food-borne pathogenic bacteria Listeria monocytogenes in unprocessed and minimally processed fresh vegetables. A total of 165 prevalence studies, including 15 studies with concentration data, were taken from the scientific literature and from technical reports and used for statistical analysis. The predicted mean of the normal distribution of the logarithms of viable L. monocytogenes per gram of fresh vegetables was -2.63 log viable L. monocytogenes organisms/g, and its standard deviation was 1.48 log viable L. monocytogenes organisms/g. These values were determined by considering one contaminated sample in prevalence studies in which samples are in fact negative. This deliberate overestimation is necessary to complete calculations. With the mixture model, the predicted mean of the distribution of the logarithm of viable L. monocytogenes per gram of fresh vegetables was -3.38 log viable L. monocytogenes organisms/g and its standard deviation was 1.46 log viable L. monocytogenes organisms/g. The probabilities of fresh unprocessed and minimally processed vegetables being contaminated with concentrations higher than 1, 2, and 3 log viable L. monocytogenes organisms/g were 1.44, 0.63, and 0.17%, respectively. Introducing a sensitivity rate of 80 or 95% in the mixture model had a small effect on the estimation of the contamination. In contrast, introducing a low sensitivity rate (40%) resulted in marked differences, especially for high percentiles. There was a significantly lower estimation of contamination in the papers and reports of 2000 to 2005 than in those of 1988 to 1999 and a lower estimation of contamination of leafy salads than that of sprouts and other vegetables. The interest of the mixture model for the estimation of microbial contamination is discussed.     

Enhanced recovery of injured Escherichia coli by compounds that degrade hydrogen peroxide or block its formation.

Escherichia coli LSUFS was injured either by freezing at -10 degrees C or by heating at 57 degrees C for 12 min. Surviving cells were recovered on nonselective tryptone-glucose extract agar and selective violet red bile agar supplemented with compounds that degrade hydrogen peroxide or block its formation. Various concentrations of the following compounds were tested: sodium pyruvate, 3,3'-thiodipropionic acid, catalase, ascorbic acid, potassium permanganate, sodium thioglycolate, dimethylsulfoxide, ethoxyquin, n-propyl gallate, alpha-tocopherol sodium metabisulfite, and ferrous sulfate. Sodium pyruvate and 3,3'-thiodipropionic acid, when added to either medium, significantly (P greater than 0.01) increased recovery of injured cells. More than 90% of the heat-injured cells and 40 to 90% of the freeze-injured cells failed to grow on unsupplemented tryptone-glucose extract agar. Supplementation of violet red bile agar increased recovery, but the counts remained considerably lower than the tryptone-glucose extract agar counts. The repair detection procedure of Speck et al. (M. Speck, B. Ray, R. Read, Jr., Appl. Microbiol. 29:549-550, 1975) was greatly improved by the addition of pyruvate or 3,3'-thiodipropionic acid. However, when this improved repair detection procedure was applied to foods, pyruvate-supplemented media showed some false-positives. We therefore recommend that 3,3'-thiodipropionic acid be used to supplement media in the repair detection procedure.