Use of the Chromogenic Substrate 5-Bromo-4-Chloro-3-Indolyl-B-D-Glucuronide (X-GLUC) for Enumerating Escherichia coli in 24 H from Ground Beef

1990 ◽  
Vol 53 (6) ◽  
pp. 508-510 ◽  
Author(s):  
LAWRENCE RESTAINO ◽  
ELON W. FRAMPTON ◽  
RICHARD H. LYON
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Most Probable Number ◽  
Chromogenic Substrate ◽  
Probable Number ◽  
E Coli ◽  
Glucuronidase Activity ◽  
Significant Difference ◽  
Plating Method ◽  
Positive Linear Correlation

A 24-h direct plating method for Escherichia coli using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-B-D-glucuronide (X-GLUC) incorporated into a Peptone-tergitol agar base (PTX) was compared with the standard 3-tube Most Probable Number (MPN) method on 50 naturally contaminated ground beef samples. A paired-comparisons t-test showed no significant difference between the two methods. A positive linear correlation between the two methods was observed over the entire range of values. Ninety-seven percent of the positive colonies (blue colonies) on PTX agar were indentified as E. coli, whereas no atypical colonies (nonblue) were characterized as such. Thus, a simple and reliable enumeration of E. coli can be made within 24 h using the X-GLUC substrate in a selective agar as an indicator of B-glucuronidase activity.

Comparison of β-Glucuronidase and Indole-Based Direct Plating Methods for Enumerating Escherichia coli in Artificially Inoculated Ground Meats

1990 ◽  
Vol 53 (11) ◽  
pp. 933-935 ◽  
Author(s):  
ELON W. FRAMPTON ◽  
LAWRENCE RESTAINO ◽  
NANCY BLASZKO
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Plate Count ◽  
Most Probable Number ◽  
Probable Number ◽  
E Coli ◽  
Glucuronidase Activity ◽  
Plate Count Agar ◽  
Significant Difference ◽  
Indole Production

Peptone tergitol glucuronide (PTG) agar containing 4-methylumbelliferyl-β-D glucuronide (MUG) (for β-glucuronidase activity), the Holbrook, Anderson, Baird-Parker (HABP) method (for detecting indole production), and the standard 3-tube most probable number (MPN) method were compared with plate count agar (PCA) for enumerating three strains of unstressed Escherichia coli artificially inoculated into ground beef and chicken at 1–6 × 106 cells/g. No significant difference (P>0.05) was determined between PTG agar and PCA in the recovery of E. coli. The MPN method enumerated a significantly greater (P<0.05) number of E. coli cells than PCA. Compared with PCA, the HABP method recovered a significantly lower (P<0.05) number of E. coli cells from chicken, whereas no significant difference (P>0.05) was obtained with ground beef. When combining all data from chicken and beef, the recovery of E. coli cells by the HABP method was also significantly lower (P<0.05). Overall, based on the enumeration of E. coli on PCA, the HABP method, PTG agar, and MPN method recovered 57, 102, and 144%, respectively.

scholarly journals Lessons from a large outbreak of Escherichia coli O157[ratio ]H7 infections: insights into the infectious dose and method of widespread contamination of hamburger patties

1999 ◽  
Vol 122 (2) ◽  
pp. 185-192 ◽  
Author(s):  
J. TUTTLE ◽  
T. GOMEZ ◽  
M. P. DOYLE ◽  
J. G. WELLS ◽  
T. ZHAO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Most Probable Number ◽  
Strong Argument ◽  
Probable Number ◽  
Infectious Dose ◽  
E Coli ◽  
Beef Patties ◽  
Large Outbreak ◽  
Microbiological Testing

Between November 1992 and February 1993, a large outbreak of Escherichia coli O157[ratio ]H7 infections occurred in the western USA and was associated with eating ground beef patties at restaurants of one fast-food chain. Restaurants that were epidemiologically linked with cases served patties produced on two consecutive dates; cultures of recalled ground beef patties produced on those dates yielded E. coli O157[ratio ]H7 strains indistinguishable from those isolated from patients, confirming the vehicle of illness. Seventy-six ground beef patty samples were cultured quantitatively for E. coli O157[ratio ]H7. The median most probable number of organisms was 1·5 per gram (range, <0·3–15) or 67·5 organisms per patty (range, <13·5–675). Correlation of the presence of E. coli O157[ratio ]H7 with other bacterial indicators yielded a significant association between coliform count and the presence of E. coli O157[ratio ]H7 (P=0·04). A meat traceback to investigate possible sources of contamination revealed cattle were probably initially colonized with E. coli O157[ratio ]H7, and that their slaughter caused surface contamination of meat, which once combined with meat from other sources, resulted in a large number of contaminated ground beef patties. Microbiological testing of meat from lots consumed by persons who became ill was suggestive of an infectious dose for E. coli O157[ratio ]H7 of fewer than 700 organisms. These findings present a strong argument for enforcing zero tolerance for this organism in processed food and for markedly decreasing contamination of raw ground beef. Process controls that incorporate microbiological testing of meat may assist these efforts.

Rapid Fluorogenic Enumeration of Escherichia coli in Selected, Naturally Contaminated High Moisture Foods

1987 ◽  
Vol 70 (6) ◽  
pp. 991-993
Author(s):  
Paul L Poelma ◽  
Clyde R Wilson ◽  
Wallace H Andrews
Keyword(s):  
Escherichia Coli ◽  
Ultraviolet Light ◽  
Ground Beef ◽  
Most Probable Number ◽  
High Moisture ◽  
Probable Number ◽  
Pork Sausage ◽  
E Coli

Abstract An assay for the enzyme glucuronidase was used to determine the presence of Escherichia coli in selected, naturally contaminated high moisture foods. Raw pork sausage, ground turkey, and ground beef were inoculated into tubes containing the substrate 4-methylumbelliferyl beta-D-glucuronide (MUG) in lauryl tryptose (LT) medium. After incubation at 35°C for 24 h, the inoculated LT-MUG tubes were examined under longwave ultraviolet light for the presence of a fluorogenic glucuronidase end product. A fluorescing tube indicated the presumptive presence of E. coli. The 10 day most probable number method of the AOAC and the LT-MUG procedure gave comparable recoveries of E. coli.

Recovery of Escherichia coli Biotype I and Enterococcus spp. during Refrigerated Storage of Beef Carcasses Inoculated with a Fecal Slurry

1999 ◽  
Vol 62 (8) ◽  
pp. 944-947 ◽  
Author(s):  
M. CALICIOGLU ◽  
D. R. BUEGE ◽  
S. C. INGHAM ◽  
J. B. LUCHANSKY
Keyword(s):  
Escherichia Coli ◽  
Storage Period ◽  
Most Probable Number ◽  
Probable Number ◽  
E Coli ◽  
Viable Cells ◽  
Beef Carcasses ◽  
Significant Difference ◽  
Enterococcus Spp ◽  
Appreciable Reduction

Three beef front quarters/carcasses were inoculated with a slurry of cattle manure. During storage at 4°C, two sponge samples from each of three sites (i.e., 100 cm2 from each of two fat surfaces and 100 cm2 from a lean surface) were taken from each of the three carcasses on days 0, 1, 3, 7, and 10 after inoculation. The initial numbers of Escherichia coli averaged 2.0 log10 CFU/cm2 (1.21 to 2.47 log10 CFU/cm2) using the Petrifilm method and 2.09 log10 most probable number (MPN)/cm2 (0.88 to 2.96 log10 MPN/cm2) using the MPN method. The initial numbers of enterococci averaged 3.34 log10 CFU/cm2 (3.07 to 3.79 log10 CFU/cm2) using kanamycin esculin azide agar. In general, an appreciable reduction in the numbers of E. coli occurred during the first 24 h of storage; for the Petrifilm method an average reduction of 1.37 log10 CFU/cm2 (0.69 to 1.71 log10 CFU/cm2) was observed, and for the MPN method an average reduction of 1.52 log10 MPN/cm2 (0.47 to 2.08 log10 MPN/cm2) was observed. E. coli were not detected (&lt;−0.12 log10 CFU/cm2) using Petrifilm on day 7 of the storage period on two (initial counts of 1.21 and 2.29 log10 CFU/cm2) of the three carcasses. However, viable E. coli cells were recovered from these two carcasses after a 24-h enrichment at 37°C in EC broth. Viable E. coli cells were detected at levels of −0.10 log10 CFU/cm2 on the third carcass (initial count of 2.47 log10 CFU/cm2) after 7 days at 4°C. No significant difference in recovery of viable cells was observed between the MPN and Petrifilm methods on days 0, 1, and 3 (P &gt; 0.05). However, viable E. coli cells were recovered from all three carcasses by the MPN method on day 7 at an average of −0.29 log10 MPN/cm2 (−0.6 to −0.1 log10 MPN/cm2). On day 10, viable cells were recovered by the MPN method from two of the three carcasses at −0.63 and −0.48 log10 MPN/cm2 but were not recovered from the remaining carcass (&lt;−0.8 log10 MPN/cm2). Similar to E. coli, the greatest reduction (average of 1.26 log10 CFU/cm2, range = 1.06 to 1.45 log10 CFU/cm2) in the numbers of enterococci occurred during the first 24 h of storage. Because of higher initial numbers and a slightly slower rate of decrease, the numbers of Enterococcus spp. were significantly higher (P &lt; 0.017) than the numbers of E. coli Biotype I after 3, 7, and 10 days of storage. These results suggest that enterococci may be useful as an indicator of fecal contamination of beef carcasses.

Evaluation of Colilert-18® as an alternative method for monitoring total coliforms and Escherichia coli in some faecally polluted river waters

2014 ◽  
Vol 4 (4) ◽  
pp. 604-611
Author(s):  
Amanda S. Brand ◽  
Jo M. Barnes
Keyword(s):  
Escherichia Coli ◽  
Rank Correlation ◽  
Water Source ◽  
Polluted Water ◽  
Most Probable Number ◽  
Probable Number ◽  
Total Coliforms ◽  
E Coli ◽  
True Value ◽  
Significant Difference

The increase in numbers and contamination levels of faecally polluted water has resulted in shifts worldwide towards methods which enumerate faecal indicator bacteria faster. Rapid methods enable more timely remedial and preventative actions which protect the health of water users. However, especially in the developing world, straightforward methods are also preferred as they reduce the requirement for highly qualified analysts. This study investigates the feasibility of using the rapid, semi-automated enzyme substrate test Colilert-18® instead of multiple-tube fermentation (MTF) in total coliform and Escherichia coli enumeration for South African river water, as one example of a surface water source carrying considerable faecal pollution, which needs monitoring. Spearman rank correlation coefficients (ρ) of 0.83 and 0.86 were obtained for total coliforms and E. coli respectively, indicating Colilert-18® performed acceptably in the pollution ranges encountered. A Bland–Altman plot further revealed that Colilert-18® showed no significant difference (p &gt; 0.05) from MTF values below 100,000 E. coli most probable number/100 mL (estimated true value). Above this level Colilert-18® was found to progressively underestimate E. coli. This inadequacy of Colilert-18® was considered acceptable from a health risk assessment viewpoint as such high counts should have sounded the alarm for preventative and corrective action irrespective of method inaccuracy.

Comparison of culturable fecal coliforms and Escherichia coli enumeration in freshwaters

2007 ◽  
Vol 53 (6) ◽  
pp. 798-801 ◽  
Author(s):  
Tamara Garcia-Armisen ◽  
Josué Prats ◽  
Pierre Servais
Keyword(s):  
Escherichia Coli ◽  
Microbiological Quality ◽  
Fecal Coliforms ◽  
Quality Data ◽  
Plate Count ◽  
Most Probable Number ◽  
Tetrazolium Chloride ◽  
Probable Number ◽  
E Coli ◽  
Glucuronidase Activity

Fecal coliforms (FC) counts were compared with Escherichia coli counts in differently contaminated freshwater samples (n = 166). FC were enumerated by plate count on triphenyl 2,3,5-tetrazolium chloride Tergitol medium. Escherichia coli were enumerated by the most probable number microplate method based on the detection of glucuronidase activity. FC and E. coli counts were highly correlated; an average E. coli/FC ratio equal to 0.77 was found, meaning that on average, 77% of FC were E. coli. Knowing the E. coli/FC ratio allows us to convert the historical microbiological quality data expressed in FC counts into E. coli abundance and thus to compare with present and future monitoring data that are (or will be) based on E. coli enumeration.

Comparison of the Traditional Three-Tube Most Probable Number Method with the Petrifilm, SimPlate, BioSys Optical, and Bactometer Conductance Methods for Enumerating Escherichia coli from Chicken Carcasses and Ground Beef

2000 ◽  
Vol 63 (9) ◽  
pp. 1179-1183 ◽  
Author(s):  
SCOTT M. RUSSELL
Keyword(s):  
Escherichia Coli ◽  
Correlation Coefficients ◽  
Ground Beef ◽  
Foodborne Illness ◽  
Most Probable Number ◽  
Probable Number ◽  
Rapid Methods ◽  
E Coli ◽  
Microbiological Methods ◽  
The Cost

A study was conducted to compare commonly used methods, such as Petrifilm and SimPlate, and the rapid microbiological methods BioSys optical and Bactometer conductance to the standard most probable number (MPN) procedure for enumerating Escherichia coli from poultry carcasses and ground beef. Broiler carcasses and ground beef were evaluated in each of three replicate trials. Five groups of carcasses or ground beef were sampled and analyzed using Petrifilm, SimPlate, BioSys optical, and Bactometer conductance measurements after temperature abuse at 37°C for 0 (Petrifilm and SimPlate only), 2, 4, 6, or 8 h. The correlation coefficients for the regression lines comparing the standard E. coli MPN procedure to Petrifilm and SimPlate for chicken and ground beef, respectively, were as follows: 0.95, 0.94, 0.93, and 0.91. The correlation coefficients for the regression lines comparing the standard E. coli MPN procedure to BioSys optical and Bactometer conductance measurements for chicken and ground beef, respectively, were −0.91, −0.90, −0.93, and −0.96. Although Petrifilm and SimPlate performed well, E. coli could not be enumerated from 16.7 and 10% of samples, respectively, using these methods. The BioSys optical and Bactometer conductance methods performed very well when compared with Petrifilm and SimPlate. Using rapid methods (BioSys optical and Bactometer conductance), results were obtained in 1 to 11 h rather than the 48 h required to conduct Petrifilm or SimPlate or the 5 days required to conduct the MPN procedure. These methods may allow processors to test products and obtain results before shipping, avoiding the cost and loss of reputation associated with a recall or foodborne illness outbreak.

scholarly journals Dry Rehydratable Film Method for Enumerating Confirmed Escherichia coli in Poultry, Meats, and Seafood: Collaborative Study

1999 ◽  
Vol 82 (1) ◽  
pp. 73-78 ◽  
Author(s):  
Vidhya Gangar ◽  
Michael S Curiale ◽  
Kathryn Lindberg ◽  
Sonya Gambrel-Lenarz ◽  
E Adamson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Collaborative Study ◽  
Most Probable Number ◽  
Probable Number ◽  
E Coli ◽  
Fish Samples ◽  
Plating Method ◽  
Medium Level ◽  
Repeatability And Reproducibility ◽  
Film Method

Abstract A rehydratable dry-film plating method for Escherichia coli, the Petrifilm E. coli/Coliform (EC) Count Plate in foods, has been compared with the AOAC INTERNATIONAL most probable number (MPN) method. Eleven laboratories participated in the collaborative study. Three E. coli levels in 8 samples each of frozen raw ground turkey, frozen raw ground beef, and frozen cooked fish were tested in duplicate. Mean log counts for the Petri film plate procedure were not significantly different from those for the MPN procedure for cooked fish samples inoculated with low or high inocula levels, for samples of raw turkey inoculated at medium level, and for beef inoculated at low, medium, and high levels. Repeatability and reproducibility vari ances of the Petrifilm EC Plate method recorded at 24 h were as good as or better than those of the MPN method. The dry rehydratable film method for enumerating confirmed E. coli in poultry, meats, and seafood has been adopted first action by AOAC INTERNATIONAL.

Enumeration of Escherichia coli O157:H7 in Outbreak-Associated Beef Patties

2016 ◽  
Vol 79 (7) ◽  
pp. 1266-1268 ◽  
Author(s):  
ALEXANDER GILL ◽  
GEORGE HUSZCZYNSKI
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Most Probable Number ◽  
Escherichia Coli O157 ◽  
Cross Contamination ◽  
Probable Number ◽  
E Coli ◽  
Beef Patties ◽  
Selective Agar ◽  
Agar Media

ABSTRACT An outbreak of five cases of Escherichia coli O157 infection that occurred in Canada in 2012 was linked to frozen beef patties seasoned with garlic and peppercorn. Unopened retail packs of beef patties from the implicated production lot were recovered and analyzed to enumerate E. coli O157, other E. coli strains, and total coliforms. E. coli O157 was not recovered by direct enumeration on selective agar media. E. coli O157 in the samples was estimated at 3.1 most probable number per 140 g of beef patty, other E. coli was 11 CFU/g, and coliforms were 120 CFU/g. These results indicate that the presence of E. coli O157 in ground beef at levels below 0.1 CFU/g may cause outbreaks. However, the roles of temperature abuse, undercooking, and cross-contamination in amplifying the risk are unknown.

Efficacy of Petrifilm™ VRB for Enumerating Coliforms and Escherichia coli from Frozen Raw Beef

1987 ◽  
Vol 50 (12) ◽  
pp. 1017-1022 ◽  
Author(s):  
LAWRENCE RESTAINO ◽  
RICHARD H. LYON
Keyword(s):  
Escherichia Coli ◽  
Incubation Temperature ◽  
Ground Beef ◽  
Gas Bubbles ◽  
Most Probable Number ◽  
Type I ◽  
Type Ii ◽  
Probable Number ◽  
E Coli ◽  
Colony Diameter

Petrifilm™ violet red bile (PVRB) compared favorably to the most probable number method (MPN) and violet red bile agar (VRBA) methods for enumerating coliforms from frozen raw ground beef. When comparing PVRB and VRBA incubated at 35°C, coliform enumeration displayed a linear relationship (correlation coefficient of 0.932). However, by analyzing 64 ground beef samples, PVRB enumerated 41% more coliforms/g than did VRBA. Two distinct colony types were observed on PVRB: (a) type I (butterfly in appearence) with a colony diameter equal to or greater than 1 mm and gas bubbles 2–4 mm in diameter touching the associated colony; and (b) type II with a colony diameter less than 1 mm in diameter and gas bubbles of the associated colony not necessarily touching the colony but within a colony diameter. The disparity between PVRB and VRBA for enumerating coliforms was attributed to non-coliforms representing approximately 50% of the type II coliform colonies. At 35°C, 83.7% of the type I colonies were Escherichia coli, whereas only 10.9%, of the type II colonies were E. coli. By elevating the incubation temperature from 35°C to 44.5°C, over 90% of the colonies in the counting dilution were type I of which 99.2% were E. coli. At 44.5°C, 39.4% of the type II colonies were E. coli; however, this colony type represented only 9.5% of the total colonies on PVRB. Therefore, a reliable method for enumerating E. coli from raw meat was developed by counting only the type I colonies on PVRB incubated at 44.5°C.

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