Violet Red Bile Research Articles

High-Sensitivity Dry Rehydratable Film Method for Enumeration of Coliforms in Dairy Products: Collaborative Study

A dry-film coliform count plate that is inoculated with 5 mL sample was compared with the Violet Red Bile Agar plate method in a collaborative study by 18 laboratories. Products analyzed were 2% milk, chocolate milk, cream, vanilla ice cream, cottage cheese, and cheese. Collaborators tested blind duplicate uninoculated samples and samples inoculated at low, medium, and high level. Significantly (P < 0.05) higher numbers of coliforms were recovered by the dry-film method from 2% milk samples at the 3 inoculum levels, the chocolate milk at the low- and high-inoculum levels, and the cream at the high-inoculum level. Significantly higher counts were obtained by the agar method for cottage cheese samples at the low-inoculum level. The repeatability standard deviation for the dry-film method was significantly higher for the high-inoculum level chocolate milk sample and the medium-inoculum level cottage cheese. The same statistic was significantly higher for the agar method at all 3 inoculum levels in the 2% milk and the medium-inoculum level cream. The high-sensitivity dry rehydratable film method for enumeration of coliforms in dairy products has been adopted first action by AOAC INTERNATIONAL.

Bacterial populations associated with poultry processing in a South African abattoir

Bacteria were isolated from yeast extract supplemented tryptone soya agar (TSAYE) plates (357 isolates) and violet red bile glucose agar (VRBGA) plates (311 isolates) of micro- biological surveys in a South African Grade B poultry abattoir. Bacterial populations from TSAYE plates of carcasses (neck skin) were dominated by Acinetobacterspp., Escherichia coli, Micrococcusspp. and Flavobacteriumspp., which collectively comprised 75.4% of isolates, while isolates from VRBGA plates showed dominance of Escherichia coli(80.0%). Environmental isolates dominant on TSAYE plates were Micrococcusspp. from equipment surfaces and air samples of the defeathering area of the abattoir (48.5 and 68.7%, respectively) and Bacillusspp. from air samples of the packaging area (37.4%). Isolates from TSAYE plates of scald tank water were dominated by E. coli(47.8%) and Micrococcusspp. (39.1%), while isolates from immersion chiller water were predominantly E. coli(54.1%). Environmental isolates dominant on VRBGA plates were E. coliin water samples and air of the defeathering and packaging areas (from 93.7 to 100%). E. coli, Enterobacterspp. and Serratiaspp. were dominant on VRBGA plates of equipment surfaces, collectively comprising 77.8% of isolates.

The enumeration of coliforms and E. Coli on naturally contaminated beef: A comparison of the petrifilm ™ method with the Australian Standard

Petrifilm ™ PEC was compared to Australian Standard (AS) methods for the enumeration of coliforms and Escherichia coli on 50 naturally contaminated beef samples from three meat works. The standard methods consist of a 3-tube most probable number test or a direct plate count on tryptone bile agar for E. coli, and violet red bile agar and the most probable number test for conliorms (AS 1766.2.12.1984 and AS 1766.2.3.1992). No significant difference was found between the methods, except that the count of E. coli on the direct plate method with a resuscitation step was significantly higher than the count on Petrifilm ™, most probable number or direct plate count without resuscitation.

High-Pressure Transient Sensitization of Escherichia coli to Lysozyme and Nisin by Disruption of Outer-Membrane Permeability

Escherichia coli MG1655 suspensions in 10mM phosphate buffer (pH 7.0) were subjected to high pressures in the range of 180 to 320 MPa for 15 min. Cell death was evident at 220 MPa and increased exponentially with pressure. Surviving populations were sublethally injured, as demonstrated by their reduced ability to form colonies on violet red bile glucose agar, a selective growth medium containing crystal violet and bile salts. During exposure to high pressure (> 180 MPa), cells were sensitive to lysozyme, nisin, and ethylenediaminetetraacetic acid (EDTA), as was apparent from an increased lethality of pressure in the presence of these agents. Sublethal injury in the surviving population was lower in the presence of nisin and lysozyme, but higher in the presence of EDTA. Combinations of EDTA with nisin or lysozyme present during pressure treatment increased lethality in an additive manner. However, the addition of lysozyme, nisin and/or EDTA to pressurized cell suspensions immediately after pressure treatment did not cause any viable count reduction. Finally, we observed leakage of the periplasmic enzyme β-lactamase from an ampicillin-resistant recombinant E. coli MG1655 under high pressure. These results suggest that high pressure transiently disrupts the permeability of the E. coli outer membrane for water-soluble proteins.

Enumeration and Identification of Bacteria in Chicken Semen

Three experiments were conducted to determine the bacteriological quality of chicken semen. Semen was collected from donor males, diluted, and surface inoculated onto seven different bacteriological media, from which randomly selected colonies were identified. Bacterial counts in semen averaged 5.14 log10 cfu/mL. Tryptic soy agar (TSA) was the best medium for the isolation of Gram-positive bacteria, whereas TSA + .3% bile salts (TSABS) and violet red bile agar + 1% glucose (VRBAG) were the best media for the isolation of Gram-negative and enteric bacteria. The genera of bacteria that were isolated depended on the medium that was used for isolation. The most frequently isolated genera included Escherichia, Staphylococcus, Micrococcus, Enterococcus, and Salmonella. Most of the bacteria that were isolated were endemic to poultry and were common environmental bacteria. This indicates that the environment and feed are important sources of bacterial contamination in broilers.

Coliforms in processed mango: Significance and control

The aims of this investigation were to enumerate coliforms in fresh mangoes, puree, cheeks, and cheeks-in-puree in order to determine the source of these organisms in the processed products, to determine methods for their control, and to identify coliforms isolated from cheeks-in-puree to determine whether they have any public health significance. Product from four processors was tested on two occasions. The retail packs of cheeks-in-puree having the highest coliform counts were those in which raw puree was added to the cheeks. Coliform counts in these samples ranged between 1.4 × 10 3 and 5.4 × 10 4 cfu g . Pasteurisation reduced the coliform count of raw puree to < 5 cfu g . Forty-seven percent of the 73 colonies, isolated as coliforms on the basis of their colony morphology on violet red bile agar, were identified as Klebsiella pneumoniae using the ATB 32E Identification System. Klebsiella strains were tested for growth at 10 °C, faecal coliform response, and fermentation of d-melizitose, to differentiate the three phenotypically similar strains, K. pneumoniae, K. terrigena and K planticola. Results indicated that 41% of K. pneumoniae isolates gave reactions typical of K. pneumoniae. A further 44% of strains gave an atypical reaction pattern for these tests and were designated ‘psychrotrophic’ K. pneumoniae. Klebsiella pneumoniae counts of between 2.1 × 10 3 and 4.9 × 10 4 cfu g were predicted to occur in the retail packs of mango cheeks-in-puree produced by the processors who constituted this product with raw puree. In view of the opportunistic pathogenic nature of K. pneumoniae, its presence in these products is considered undesirable and steps, such as pasteurisation of puree, should be taken in order to inactivate it.

Coliform Bacteria and Staphylococcus aureus in Retail Natural Cheeses

Fifty commercial retail samples of natural cheeses (American and non-American-type) were examined for coliform bacteria, Escherichia coli, E. coli O157:H7, and thermonuclease-positive S. aureus. Nine cheese samples had coliform group bacteria ranging from 10 to 1.1 × 103 CFU/ml by the violet red bile agar procedure. Twenty-four or 48% of the cheeses were positive for coliform group bacteria when a 25-g sample was used in an enrichment broth. One sample had a confirmed E. coli by both the violet red bile agar and enrichment methods. No E. coli O157:H7 were found in the retail natural cheeses examined. Also, no thermonuclease-positive S. aureus were isolated. The levels of potentially pathogenic microorganisms in cheeses were lower in this study than reported in a 1974 to 1976 Canadian study.

The Effect of GDL-induced pH Decrease on the Formation of Biogenic Amines in Meat

The effect of pH on the formation of biogenic amines has mainly been studied in broths in which pH has been fixed before incubation. However, in the fermentation of dry sausage, pH quite rapidly decreases from the initial value to a certain level. In this study glucono-delta-lactone (GDL) was used to decrease pH in meat, Six minced meat samples were each divided into three portions (A–C): 0% (A), 0.5% (B), or 1.0% (C) of GDL was added and the samples were incubated at 20–22°C for 7 d. The amounts of biogenic amines (histamine, tyramine, putrescine, cadaverine, phenylethylamine, tryptamine, spermine, and spermidine) as well as pH, water activity, and the bacterial counts of lactic acid bacteria, fecal streptococci, coliforms, and total plate count were measured. Addition of GDL resulted in a significant decrease in pH and in the levels of histamine and putrescine as well as in the levels of fecal streptococci, coliforms, and total plate counts. Of 87 fecal streptococci, seven Enterococcus faecalis strains produced tyramine. All the coliforms and related strains isolated from violet red bile agar produced tyramine, putrescine, and cadaverine on agar plates. However, the proportion of histamine-positive strains of these strains, especially Hafnia alvei, increased from 0 to 57% during the incubation. The rate and level of pH decrease clearly affected amine formation in meat, indicating that the levels of, e.g., histamine produced could be decreased by optimizing the pH decrease during fermentation. Addition of GDL facilitates study of the effect of pH decrease without interactions between the starter culture and contaminant flora.

Bacterial populations associated with Vienna sausage packaging

Bacteria were isolated from plate counts on Standard One Nutrient agar (total aerobic plate count), Violet Red Bile Glucose agar (Gram-negative count) and modified MRS agar (lactic acid bacteria count) taken during an environmental survey of a Vienna sausage manufacturing and packaging plant. Isolates were purified and characterized to determine the composition of the bacterial population. On Standard One Nutrient agar, the genera Bacillus, Micrococcus and Staphylococcus comprised 71·4% of the 259 isolates, followed by Pseudomonas, Proteus and several other Gram-negative bacteria in smaller proportions. On Violet Red Bile Glucose agar, Pseudomonas (47·3%), Serratia, Vibrio, Acinetobacter, Enterobacter and Proteus comprised 83·9% of the 175 isolates. A wide spectrum of lactic acid bacteria (181 isolates) comprising homo- and heterofermentative lactobacilli, leuconostocs, pediococci, streptococci/enterococci and thermobacteria were characterized, with no clear predominance of any particular group. Of all the environmental bacterial genera isolated, it was only the homofermentative lactobacilii and leuconostocs which were able to dominate the spoilage populations of the vacuum-packaged Vienna sausages manufactured in the plant.

Sanitary Evaluation of Target Flowmeter Used in a Dairy Processing Plant

A target flowmeter, used to measure raw milk flow, was examined for sanitary conditions in a university dairy plant 10 times over a period of eight weeks. The flowmeter connection was swabbed at four different locations along the dairy plant connection at four different times during the work day: i) after chlorine sanitization, before product; ii) after product, before cleaning in place (CIP); iii) after CIP, before acid sanitization; and iv) after acid sanitization, at end of day. Samples were plated in duplicate on standard plate count agar and on violet red bile agar. After routine CIP cleaning and sanitization procedures, bacterial counts were low. Additionally, no finished product contamination problems were detected over the 7 months of flowmeter use as shown by routine quality control tests on pasteurized milk which had flowed past the in-line meter as raw milk. These results indicate that normal cleaning and sanitization procedures were adequate for the in-line flowmeter.

Evaluation of a rapid fluorogenic method for the detection ofEscherichia coliin dairy products

A rapid fluorogenic medium was evaluated for the detection of Escherichia coli in dairy products. The medium was capable of detecting Esch. coli after 7.5 h incubation at 41.5 degrees C. Samples of pasteurized milk (136), raw milk (63), soft cheese (60) and pasteurized cream (39) were examined with media based on 4-methylumbelliferyl-beta-D-glucuronide (MUG-7) and Violet red bile agar and there were no significant differences between the numbers of Esch. coli detected on the two media. MUG-7 medium had a specificity of 98.6% and the small number of organisms giving a false positive reaction were identified as Klebsiella pneumoniae. The incidence of false negative results was approximately 2%. MUG-7 medium was suitable for pour plate, spread plate and membrane filtration methods. Possible applications of the method are discussed.

Temperature in agar plates and its influence on the results of quantitative microbiological food analyses

The numbers of colony forming units (cfu) of some strains of Enterobacteriaceae growing in Violet Red bile agar at 44°C can vary considerably depending on incubation conditions and location of an individual agar plate in a stack. The reason is that the heating-up rates of the agar in plates incubated at elevated temperatures are slower if the incubators do not have an air circulator and/or if the plates are located in the centre of a stack. Variability in agar temperatures among different plates after 24 h of incubation at 44°C was higher if plates were placed in an incubator with an air circulator than in an incubator without one. This effect could be avoided if the plates were incubated enclosed in a plastic bag.

A Model for Statistical Evaluation of Precision Parameters of Microbiological Methods: Application to Dry Rehydratable Film Methods and IDF Reference Methods for Enumeration of Total Aerobic Mesophilic Flora and Coliforms in Raw Milk

Abstract A new statistical approach for collaborative study data of microbiological methods Is proposed. This Includes a confirmatory test to the Polsson distribution of the number of colonies. In addition, 2 new statistical parameters are used to express precision as a percent of the original unit: the geometric relative standard deviation (GRSD) and the critical relative difference between 2 measurements (RD95). This statistical approach was applied to an Interlaboratory study to assess and compare the precision of both dry rehydratable film (PetrlfUrn® SM and Petrifllm® VRB) methods and International Dairy Federation (IDF) reference methods [total aerobic mesophilic plate count (TAMPC) and violet red bile lactose agar (VRBL) methods] for estimation of total bacteria and collform, respectively, in raw milk. Each of the 14 laboratories In the study analyzed 40 laboratory samples (20 different materials In blind duplicates) for total bacteria and collform counts by both the Petrifllm and standard methods. Repeatability standard deviations (In log10 unit) of TAMPC, Petrifllm SM, VRBL, and Petrifllm VRB were 0.106, 0.089, 0.219, and 0.171, respectively; their reproducibility standard deviations were 0.170,0.167,0.348, and 0.199, respectively.

Acriflavine violet red bile agar for the isolation and enumeration of Klebsiella pneumoniae

Acriflavine violet red bile agar, developed by Fung and Niemeic (1977, Health Lab. Sci. 14, 273–278), was retested and refined. The results indicated that 0·01% acriflavine in VRB agar is most suitable for enumeration of Klebsiella pneumoniae from cultures as well as from food and livestock beddings (straw, sand and sawdust). Other organisms are either inhibited or can be distinguished easily from the typical K. pneumoniae colonies, which are large, mucous, and golden- or dull-yellow. With pure cultures, 45·2–98·9% recovery of K. pneumoniae was observed. From artificially contaminated meat, the recovery rate averaged 81%. Accuracy in identifying suspected K. pneumoniae colonies on AVRB plates, as confirmed by API-20E biochemical tests, was 85%. Of three types of food samples (raw milk, mastitis milk and raw hamburger meat) examined, none were positive for K. pneumoniae. The most important advantage of this selective medium over other, proposed, selective Klebsiella media is simplicity.

Effect of selective media on loss of congo red binding inShigella flexneri

The effect of growth ofShigella flexneri on various selective media on retention of congo red (CR) binding ability was determined to evaluate the effectiveness of isolation techniques regarding maintenance of the virulence plasmid. WhenS. flexneri was surface-plated onto selective agars and the resulting colonies replica plated onto CR plates, no white colonies indicative of loss of virulence were found despite repeated trials. However, whenS. flexneri was grown in liquid media (agar was removed from agar-containing media by centrifugation), white colonies were found upon plating onto CR plates. Most common selective media for shigellae produced fewer than 5–10 white colonies/1000 red colonies. However, growth in broth prepared from violet red bile agar, desoxycholate citrate agar, and SS agar gave more than 100 white colonies/1000 red colonies. Loss of CR binding was demonstrated whenS. flexneri was grown in broth containing tergitol 7, sodium dodecyl sulfate, bile salts #3, crystal violet, eosin y or methylen blue. However, concentrations of selective agents that led to loss of CR binding were much higher than those used in selective media. Results indicate that under usual conditions of isolation ofS. flexneri from food and clinical specimens, CR binding appears to be a relatively stable character with most selective media; however, use of violet red bile agar, desoxycholate citrate agar, and SS agar may lead to substantial loss of congo red binding indicating that the isolates may not be virulent.

Capability of Selective Media to Detect Heat-Injured Shigella flexneri

Heat-injured cells of Shigella flexneri 5348 were detected by using a two-media plating system: tryptic soy broth containing 2% agar plus 1% sodium pyruvate (TSBA + P) on which both injured and non-injured cells produce colonies and semisynthetic agar medium plus 0.15% bile salts #3 (SSBA) on which only non-injured cells grew. Subtracting the count on SSBA from that on TSBA + P gave an estimation of injury. Heating washed cells of S. flexneri in phosphate buffer gave about 3 log units of injury after 40 min at 52°C. A number of broths and agars specific for shigellae and gram-negative bacteria (2% agar was added to selective broths) were tested for their ability to supported colony formation of heat-injured S. flexneri. Only one medium, shigella broth containing novobiocin, gave recovery of heat-injured cells similar to that on TSBA + P. Lauryl tryptose broth, Levine EMB agar, and EMB agar were the least inhibitory to repair of injured cells (as compared to SSBA). Media that were more inhibitory to repair of heat-injured cells (but not as inhibitory as was SSBA) included tergitol 7 agar, desoxycholate lactose agar, brilliant green 2% bile broth, GN broth, Mac-Conkey agar, and Endo agar. Desoxycholate agar, violet red bile agar, SS agar, and desoxycholate citrate agar were more inhibitory than SSBA to repair. The addition of 1% sodium pyruvate to MacConkey agar, desoxycholate lactose agar, lauryl tryptose broth, tergitol 7 agar, EMB agar, and SSBA resulted in increased recoveries of heat-injured S. flexneri but did not lead to complete recovery (i.e., the counts on the pyruvate containing selective media did not give counts equal to TSBA + P). These results indicate that most media used for isolation of shigellae do not permit recovery of heat-injured cells, and special techniques must be incorporated into the isolation procedures to recover injured cells.

Influence of wilting on chemical and microbial parameters of grass relevant to ensiling.

Plots of Lolium perenne were subjected to the normal farm routine for making wilted silage. The grass was sampled periodically during (generally moist) wilting and analysed for chemical and microbial factors. The daily increase of the ammonium-N content was 5.8 g/kg N; the CF content also tended to increase during wilting (6 +or- 4.3 g/kg DM), though the correlation was poor. The sugar content of the grass and the in vitro digestibility decreased during wilting (9 +or- 6.1 g/kg DM per day and 1.1 +or- 0.11 percentage units/d, resp.). Counts on rogosa agar increased on av. by 0.24 +or- 0.16 logarithmic units (LU)/g DM per day and on MRS agar by 0.13 +or- 0.17 LU/g DM per day. Enterobacteria enumerated on violet red bile glucose agar increased by 0.23 +or- 0.18 LU/g DM per day. Inoculation by spraying the standing grass as an alternative to application on the forage harvester was investigated. In 4 separate trials the standing grass was sprayed with approximately 6.6 LU/g DM of a silage inoculant containing Lactobacillus plantarum. During wilting the counts on rogosa agar declined by 0.72 +or- 0.33 LU/g DM per day and on MRS agar by 0.65 +or- 0.35 LU/g DM per day whereas the counts on the uninoculated control field increased by 0.17 +or- 0.19 and 0.17 +or- 0.17 LU/g DM per day for the rogosa and MRS media, resp. It was concluded that inoculation of the standing grass was probably only useful in combination with a short wilting period. (Abstract retrieved from CAB Abstracts by CABI’s permission)

Use of Petrifilm™ to Evaluate the Microflora of Frozen Dessert Mixes

Ninety vanilla frozen dessert mix samples were analyzed to determine total microbial populations and coliforms present in samples inoculated with a coliform isolated from raw milk. Standard methods agar (SMA, PCA, Difco) and violet red bile agar (VRBA, Difco) as well as Petrifilm™ (PSM, PVRB) were used for plating of samples. Standard VRBA 1:10 method produced significantly higher counts of colony-forming units (CFU) than PVRB 2:3 and VRBA 2:3 methods. VRBA 2:3 colony-forming unit counts were also significantly higher than those on PVRB 2:3, but both methods showed a moderately strong linear relationship. Repeatabilities of all three coliform plating methods (VRBA 1:10, VRBA 2:3, and PVRB 2:3) were acceptably low. Less than 10% of samples plated on SMA and PSM resulted in total aerobic colony-forming units in the countable range, making evaluation of data difficult and resulting in a lack of strong linear relationship between PSM and SMA. An additional 70 local retail store samples containing naturally occurring coliforms were evaluated using PVRB 2:3 and VRBA 1:10 methodology, confirmed in brilliant green lactose bile broth (BGLB, Difco) and compared to standard VRBA 1:10 previously analyzed. All methods were equivalent for mean log coliforms, i.e., 1.38,1.33, and 1.31 for PVRB 2:3, VRBA 2:3, and VRBA 1:10, respectively. Petrifilm™ methods were comparable to standard methods for enumerating coliforms in frozen dairy products, and would be a valid alternative to standard coliform and total plate count methods.

Temperature-Independent Pectin Gel Method for Coliform Determination in Dairy Products: Collaborative Study

Ten laboratories participated in a collaborative study to compare a pectin-based violet red bile (VRB) method with the VRB agar-based standard method for estimation of coliform bacterial counts in 7 different dairy food groups: cream, cheddar cheese, cottage cheese, homogenized milk, raw milk, sour cream, and yogurt. Each laboratory analyzed 8 samples of each food group as sample pairs prepared at high, medium, and low inoculum levels, and an uninoculated control pair. Overall mean log counts were higher for the pectin gel method in 18 of 21 cases (7 samples at 3 inoculum levels); 12 results were significantly higher (P less than 0.01) than those for the VRB agar method. Of the 3 higher VRB agar method means, 2 were not significant at P less than 0.10, and one was of borderline significance (0.05 less than P less than 0.06). Repeatability variation (sr) favored the pectin gel method in 14 of 21 cases; 7 were highly significant differences (P less than 0.01). None of the 7 results favoring the VRB agar method was statistically significant. Reproducibility variation (sR) favored the pectin gel method in 14 of 21 results. These data indicate that the pectin gel method gives higher recovery of coliforms with better precision than does the VRB agar method, and strongly support the suitability of the pectin gel method as an alternative to the agar-based VRB method for coliform counts in dairy products. The pectin gel method has been adopted official first action.

Optimal Pyruvate Concentration for the Recovery of Conforms from Food and Water

Recoveries of coliforms in water and food samples were increased by adding sodium pyruvate to the Tryptic Soy Agar (TSA) base layer, and Violet Red Bile (VRBA) overlay, of the Modified VRBA procedure described in Standard Methods for the Examination of Dairy Products. Six pyruvate levels (0, 0.005, 0.01, 0.05, 0.1, and 1.0%) were tested. Counts were significantly lower (P≤0.05) on media containing 0% and 1.0% pyruvate than on the other media. Although 0.05% yielded the highest counts overall, there were no significant differences (P≤0.05) among 0.005, 0.01, 0.05, and 0.1% pyruvate. Analysis by using a general linear model procedure revealed that 0.02% pyruvate was the statistically predicted optimal level to use in the Modified VRBA procedure.