Lysozyme as a barrier to growth of Bacillus anthracis strain Sterne in liquid egg white, milk and beef

In this study, we investigated the role of lysozyme on the viability of Bacillus cereus, Bacillus subtilis, Bacillus pumilus and Bacillus anthracis (Sterne) in egg white (EW), ground beef and milk. At 35 °C in EW, growth rates (GR) for B. cereus, B. subtilis, B. pumilus and B. anthracis were 0.005, −0.018, −0.028 and −0.029 OD₆₀₀/h, respectively. Heat-treating EW at 55 and 60 °C reduced the inactivating effect of EW by 3.1 and 10.5-fold, respectively. Addition of lysozyme (2 mg/ml) to 60 °C-treated EW increased the inactivation rate 5.76-fold, indicating involvement of lysozyme in B. anthracis inactivation. B. anthracis inactivation was influenced by pH, as shown by a progressive increase in inactivation rate from 0.25 to −4.42 logs CFU/h over a pH range of 6.0-8.5. Adding 2 mg/ml lysozyme to milk and ground beef also suppressed the growth of B. anthracis 3.3 and 6.5-fold, respectively. These data indicate that lysozyme, as a natural component of EW or potential additive in other foods, could reduce biothreat risks presented by bioterror agents.     

Variation of lipid class composition in Nitzschia laevis as a response to growth temperature change

The lipid composition and the distribution of fatty acids in the lipid pool were determined in eicosapentaenoic acid (EPA)-producing microalga (Nitzschia laevis) grown under different temperatures. Both the relative amounts of lipid classes and the degree of fatty acid unsaturation in various lipid species were not greatly changed under tested growth conditions. Higher temperature up to 23 °C benefited the growth of N. laevis but only had a slight influence on EPA and lipid contents. Further increasing the culture temperature caused a serious inhibition of both the cell growth and fatty acid biosynthesis. Under all temperatures tested, triacylglycerol (TAG) was the predominant lipid constituent (64.5–69.1% of total lipid) and was highly saturated. Lower temperature favored the formation of polar lipids. The highest content of phosphatidylcholine (PC), the major phospholipids component, was reached at 15 °C (10.9% of total lipid). In sharp contrast to TAG, PC was highly unsaturated and contained a higher amount of EPA under lower temperature. The highest EPA content in polar lipid was achieved at 19 °C. The results from this investigation suggested that the low temperature could improve the distribution of polyunsaturated fatty acids in phospholipids, though it could not significantly influence their amount, especially in PC.     

Insect-derived compounds affect the behaviour of Liposcelis bostrychophila: Effects of combination and structure

A methanol fraction (MFr), prepared from 80% methanol extracts of whole Liposcelis bostrychophila, repels conspecifics when presented to groups of insects at 8000 ppm. To examine the reason for this repellent effect, bioassays tested a mixture of fatty acids and fatty acid methyl esters that are present in the MFr at concentrations ranging from 0.02 to 1.2 mM. This mixture of compounds contributes significantly to the repellence of the MFr. Therefore, an interaction among compounds can affect the settling behaviour of L. bostrychophila, even though the individual compounds do not have any effect. Other experiments further investigated the repellence of C₁₆ and C₁₈ fatty acids. In the majority of instances groups of insects consistently settled on the disc impregnated with either stearic acid or the compound with the most saturated bonds. The one exception was C_18:2 versus C_18:3 where more insects settled on C_18:3. This repellent effect of unsaturated fatty acids was also evident in five-way choice tests using a series of C₁₈ fatty acids, where stearic acid (10 mM) was consistently selected by groups of L. bostrychophila instead of oleic, linoleic and linolenic acid. The discrimination between and among fatty acids could be due to the differences in their physical properties which affect how the compounds are perceived by the insects. The fact that the most unsaturated fatty acids repel the insects presents the possibility that volatile insect-derived compounds could be utilised as repellents for this increasingly important pest.     

Effects of fatty acid supplements on milk yield and energy balance of lactating dairy cows

Saturated and unsaturated fatty acid supplements (FS) were evaluated for effects on yield of milk and milk components, concentration of milk components including milk fatty acid profile, and energy balance. Eight ruminally and duodenally cannulated cows and 8 noncannulated cows were used in a replicated 4 × 4 Latin square design experiment with 21-d periods. Treatments were control and a linear substitution of 2.5% fatty acids from saturated FS (SAT; prilled, hydrogenated free fatty acids) for partially unsaturated FS (UNS; calcium soaps of long-chain fatty acids). The SAT treatment did not change milk fat concentration, but UNS linearly decreased milk fat in cannulated cows and tended to decrease milk fat in noncannulated cows compared with control. Milk fat depression with UNS corresponded to increased concentrations of trans-10, cis-12 conjugated linoleic acid and trans C18:1 fatty acids in milk. Milk fat profile was similar for SAT and control, but UNS decreased concentration of short- and medium-chain FA. Digestible energy intake tended to decrease linearly with increasing unsaturated FS in cannulated and noncannulated cows. Increasing unsaturated FS linearly increased empty body weight and net energy gain in cannulated cows, whereas increasing saturated FS linearly increased plasma insulin. Efficiency of conversion of digestible energy to milk tended to decrease linearly with increasing unsaturated FS for cannulated cows only. Addition of SAT provided little benefit to production and energy balance, whereas UNS decreased energy intake and milk energy yield.     

Combined effect of lysozyme and nisin at different incubation temperature and mild heat treatment on the probability of time to growth of Bacillus cereus

Stochastic models can be useful to predict the risk of foodborne illness. The presence of Bacillus cereus in liquid egg can pose a serious hazard to the food industry, since a mild heat treatment cannot guarantee its complete inactivation. However, most of the information available in the scientific literature is deterministic, including growth of B. cereus. In this paper, a stochastic approach to evaluate growth of B. cereus cells influenced by different stresses (presence of nisin and lysozyme separately or in combination) was performed, using an individual-based approach of growth through OD measurements. Lag phase duration was derived from the growth curves obtained. From results obtained, histograms of the lag phase were generated and distributions were fitted. Normal and Weibull distributions were ranked as the bestfit distributions in experiments performed at 25 °C. At 16 °C, lag values (obtained in presence of combinations of both antimicrobials) were also fitted by a Gamma distribution. Predictions were compared with growth curves obtained in liquid egg exposed to mild heat, nisin and/or lysozyme to assess their validity.     

Survival of three Bifidobacterium animalis subsp. lactis strains is related to trans-vaccenic and α-linolenic acids contents in organic fermented milks

This study aims at relating the survival at 4 °C for 28 days in organic and conventional fermented milks of three strains of Bifidobacterium animalis subsp. lactis (BB12, B94 and BL04), in co-culture with Streptococcus thermophilus TA040 and Lactobacillus bulgaricus LB340, to milk fatty acids profile. Cultivability after 28 days of cold storage was improved in organic fermented milks as compared to conventional products, with slight differences among strains. In addition, the poly-unsaturated fatty acids fraction was higher in organic products, as well as the relative trans-vaccenic (TVA) and α-linolenic (ALA) fatty acids contents that were respectively 1.7 and 2.4 times higher in organic than in conventional fermented milks. From these results, it was concluded that elevated levels of TVA and ALA, together with a lower ratio between linoleic acid and α-linolenic acid, as found in organic products, improved the survival of the bifidobacteria during chilled storage.     

Fatty acid distribution in triacylglycerols and phospholipids of broad beans (Vicia faba)

Fatty acid distribution of triacylglycerols (TAG) and phospholipids (PL) obtained from the four cultivars of broad beans (Vicia faba) was investigated. Total lipids extracted from the beans were separated by thin-layer chromatography into eight fractions. The major lipid components were TAG (47.7–50.1%) and PL (47.5–50.5%), while hydrocarbons, steryl esters, free fatty acids, diacylglycerols (1,3-DAG and 1,2-DAG) and monoacylglycerols were present in minor proportions (1.8–2.4%). The major PL components were phosphatidylcholine (56.4–58.4%), phosphatidylethanolamine (20.3–21.7%) and phosphatidylinositol (16.6–18.6%). Phosphatidylinositol was unique in that it had the highest saturated fatty acid content among the three PL. No significant differences (P > 0.05) in fatty acid distribution existed when the different cultivars were compared. The fatty acid distributions in the TAG were evident among the four cultivars: unsaturated fatty acids were predominantly concentrated in the sn−2 position, and saturated fatty acids primarily occupied the sn−1 or sn−3 position in the oils. These results could be useful to both consumers and producers for manufacture of traditional foods in Japan and elsewhere.     

Synthesis of cyclopropane fatty acids in Lactobacillus helveticus and Lactobacillus sanfranciscensis and their cellular fatty acids changes following short term acid and cold stresses

An implemented GC method to separate and quantify the cell cyclopropane fatty acids lactobacillic (C19cyc11) and dehydrosterculic (C19cyc9) was used to study the adaptive response to sublethal acid and cold stresses in Lactobacillus helveticus and Lactobacillus sanfranciscensis. The comparison of the composition of cellular fatty acids of the two strains and their changes after 2 h of stress exposure under micro-aerobic and anaerobic conditions indicated that the aerobic biosynthetic pathway for unsaturated fatty acids is prevalent in L. sanfranciscensis, while the anaerobic pathway is prevalent in L. helveticus. Indeed in the latter strain, in the presence of a source of oleic acid and under micro-aerobic conditions, C18:1n11 and its post-synthetic derivative C19cyc11 accounted for overall proportion ranging from 52 to 28% of the total FAs. On the other hand L. sanfranciscensis synthesizes by aerobic pathway C18:1n9 and transforms it to C19cyc9. However in this species the cumulative level of these two FAs did not exceed 30%. The relevant proportion of dodecanoic acid in the latter species suggests that carbon chain shortening is the principal strategy of L. sanfranciscensis to modulate fluidity or chemico-physical properties of the membranes.     

Fatty acid composition of four potential aquatic weeds and their possible use as fish-feed neutraceuticals

A study was conducted to explore the fatty acid composition of four aquatic weeds, namely Salvinia cuculata, Trapa natans, Lemna minor and Ipomoea reptans collected from water bodies of Assam, India. It has been observed that C24:1 (Cis-15 Tetracosenoic acid) followed by C16:1 n-3 (9-Hexadecenoic acid) are the predominant fatty acids in these aquatic weeds and among them, Ipomoea reptans and Trapa natans contain higher proportions of C18:2 and C18:3 PUFA. The ratio of saturated fatty acid to unsaturated fatty acid is extremely low in the case of all four plants and they contain a predominant proportion of highly unsaturated fatty acids (HUFA), essential for the growth and proper performance of fish.     

Persistence strategies of Bacillus cereus spores isolated from dairy silo tanks

Survival of Bacillus cereus spores of dairy silo tank origin was investigated under conditions simulating those in operational dairy silos. Twenty-three strains were selected to represent all B. cereus isolates (n = 457) with genotypes (RAPD-PCR) that frequently colonised the silo tanks of at least two of the sampled eight dairies. The spores were studied for survival when immersed in liquids used for cleaning-in-place (1.0% sodium hydroxide at pH 13.1, 75 °C; 0.9% nitric acid at pH 0.8, 65 °C), for adhesion onto nonliving surfaces at 4 °C and for germination and biofilm formation in milk. Four groups with different strategies for survival were identified. First, high survival (log 15 min kill ≤1.5) in the hot-alkaline wash liquid. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterised by slow germination in rich medium and well preserved viability when exposed to heating at 90 °C. Fourth, spores capable of germinating at 8 °C and possessing the cspA gene. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot-alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus.     

Growth and predictive model of Bacillus cereus on blanched spinach with or without seasoning at various temperatures

The growth of Bacillus cereus on blanched spinach with or without seasoning at various temperatures (15, 20, 25, 30, and 35°C) was investigated. The number of B. cereus on blanched spinach stored at 35°C was significantly increased and resulted in maximum populations (7.8 log CFU/g) after 10 h. However, the growth rate of B. cereus on blanched spinach with seasoning stored at 35oC was lower than on blanched spinach without seasoning. The growth rate (GR) of B. cereus on blanched spinach stored at 15°C was lower than the other temperatures. The lag time (LT) of B. cereus on blanched spinach with seasoning was longer than blanched spinach without seasoning. Primary growth models of B. cereus on blanched spinach fit well to a modified Gompertz model (blanched spinach R² =0.98; blanched spinach with seasoning R² =0.96). These results might be used for developing safe storage guidelines for cooked vegetable at various temperatures.     

A combination of TiO2–UV photocatalysis and high hydrostatic pressure to inactivate Bacillus cereus in freshly squeezed Angelica keiskei juice

High hydrostatic pressure (HHP) is an effective nonthermal food processing method for microbial inactivation with minimal change to sensory and nutritional values. However, the resistance of endospores to high pressure hinders application of HHP to freshly squeezed vegetable juice, especially from leafy vegetables that are susceptible to contamination of soil bacteria, such as endospore-forming Bacillus cereus. A combination of TiO₂–UV photocatalysis and HHP was used in the processing of freshly squeezed Angelica keiskei juice, and inactivation of naturally occurring microbes, especially B. cereus, was investigated. Yeasts and molds, coliform bacteria, and Pseudomonas were inactivated by HHP to levels below the detection limit, but B. cereus survived HHP and grew during 8 days of subsequent refrigerated storage (4 °C). However, no colonies of yeasts and molds, coliform bacteria, Pseudomonas, or B. cereus were detected in A. keiskei juice after processing with a combination of TiO₂–UV photocatalysis and HHP. Although B. cereus in juice processed with the combination treatment recovered and grew to 2.02 log CFU/mL on day 6 of storage, this level was less than the population of B. cereus in unprocessed Angelica keiskei juice immediately after squeezing.     

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R² values of 0.9969 and 0.9958 respectively. Linear correlations between the log₁₀ input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10¹ CFU/mL to 1.65 × 10⁶ CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.     

Modelling the effect of temperature and water activity on the growth rate and growth/no growth interface of Byssochlamys fulva and Byssochlamys nivea

The purpose of the present study was to apply a modelling approach to define the growth rate and growth/no growth interface of Byssochlamys fulva and Byssochlamys nivea on a synthetic medium as a function of temperature and water activity. Both fungal species were grown on malt extract agar at different temperatures (10, 15, 20, 25, 30, 35, 40 and 45 °C) and a_w levels (0.88, 0.90, 0.92, 0.94, 0.96 and 0.99) for a period of 30 days. Growth responses were evaluated over time in terms of colony diameter changes. Growth data were fitted to the primary model of Baranyi and the resulting growth rates were further modeled as a function of temperature and water activity using the cardinal model with inflection (CMI) ( Rosso et al., 1993). A logistic regression quadratic polynomial model was also employed to predict the probability of growth over storage time. Estimated parameters for minimum, maximum and optimum temperatures for growth were 9.1 °C, 46.4 °C and 32.1 °C for B. fulva and 10.5 °C, 43.2 °C and 32.1 °C for B. nivea. The respective values for a_w were 0.893, 0.993 and 0.985 for B. fulva and 0.892, 0.992 and 0.984 for B. nivea. No growth was observed at 0.88 a_w regardless of temperature for both species, whereas B. nivea ascospores could not grow at 10 and 45 °C irrespective of a_w. Regarding growth boundaries, the degree of agreement between predictions and observations was >98% concordant for both species. The erroneously predicted growth cases were 1.4–4.2% false positive and 2.1–3.5% false negative for B. nivea and B. fulva, respectively. The developed logistic model was validated with two literature data sets as well as with data from independent experiments carried out on fruit juices. Validation results showed that agreement with literature data for growth was 25 out of 36 (69.4%) cases, whereas validation on fruit juice data failed in only 6 cases (5 false positives and 1 false negative) out of 128 cases.     

The effect of ethanol and specific growth rate on the lipid content and composition of Saccharomyces cerevisiae grown anaerobically in a chemostat

The effects of produced ethanol and specific growth rate on the lipid content and composition of Saccharomyces cerevisiae CBS 2806 were studied using anaerobic chemostat cultures. The cells adapted to increased concentrations of produced ethanol by increasing the proportion of ergosterol at the expense of lanosterol, by increasing the proportion of phosphatidylinositol at the expense of phosphatidylcholine, and by increasing the amount of C_18:0 fatty acids in total phospholipids at the expense of C_16:0 fatty acids. The produced ethanol had no effect on the phospholipid content nor on the proportion of unsaturated fatty acids in the phospholipids. The specific growth rate had no effect on the phospholipid content, the sterol composition, the phospholipid composition, the fatty acid composition of total phospholipids, or on the proportion of unsaturated fatty acids in the phospholipids of S. cerevisiae. It was not possible to separate the effects of produced ethanol and growth rate on the ergosterol content of the chemostat-grown S. cerevisiae cells.     

Heterotrophic growth and nutritional aspects of the diatom Cyclotella cryptica (Bacillariophyceae): Effect of some environmental factors

To investigate the nutritional value of the diatom Cyclotella cryptica (Reimann, Lewin, and Guillard) as an alternative feed for use in the aquaculture industry, the heterotrophic growth characteristics and resultant fatty acid profile of the microalga were studied when cultivated under a variety of controlled salinity and temperature conditions. In addition, the effects of pH on the growth characteristics were also studied. The maximum specific growth rate was affected by initial pH and cultivation temperature, but not by salinity. The optimal pH and temperature ranges for growth were 7.2 to 8.1 and 22.5 to 25.0 °C, respectively. Lipid accumulation and the fatty acid composition were also affected by cultivation temperature and salinity. The optimal temperature range and salinity level for lipid accumulation were 18.0 to 25.0 °C and 11.2 psu, respectively. In all cases the fatty acid distribution was similar, with the most abundant fatty acids being palmitic acid (16:0), palmitoleic acid (16:1 n-7), stearidonic acid (18:4 n-3, SDA), eicosapentaenoic acid (20:5 n-3, EPA), and decosahexaenoic acid (22:6 n-3, DHA).     

Amino acids improve acid tolerance and internal pH maintenance in Bacillus cereus ATCC14579 strain

This study investigated the involvement of glutamate-, arginine- and lysine-dependent systems in the Acid Tolerance Response (ATR) of Bacillus cereus ATCC14579 strain. Cells were grown in a chemostat at external pH (pH_e) 7.0 and 5.5. Population reduction after acid shock at pH 4.0 was strongly limited in cells grown at pH 5.5 (acid-adapted) compared with cells grown at pH 7.0 (unadapted), indicating that B. cereus cells grown at low pH_e were able to induce a marked ATR. Glutamate, arginine and lysine enhanced the resistance of unadapted cells to pH 4.0 acid shock of 1-log or 2-log populations, respectively. Amino acids had no detectable effect on acid resistance in acid-adapted cells. An acid shock at pH 4.0 resulted in a marked drop in internal pH (pH_i) in unadapted cells compared with acid-adapted cells. When acid shock was achieved in the presence of glutamate, arginine or lysine, pH_i was maintained at higher values (6.31, 6.69 or 6.99, respectively) compared with pH_i in the absence of amino acids (4.88). Acid-adapted cells maintained their pH_i at around 6.4 whatever the condition. Agmatine (a competitive inhibitor of arginine decarboxylase) had a negative effect on the ability of B. cereus cells to survive and maintain their pH_i during acid shock. Our data demonstrate that B. cereus is able to induce an ATR during growth at low pH. This adaptation depends on pH_i homeostasis and is enhanced in the presence of glutamate, arginine and lysine. Hence evaluations of the pathogenicity of B. cereus must take into account its ability to adapt to acid stress.     

Analysis and formation of trans fatty acids in hydrogenated soybean oil during heating

Hydrogenated oil has been widely used for production of shortenings or margarine, however, the presence of trans fatty acids may be detrimental to human health. The objectives of this study were to develop an improved method for analysis of trans fatty acids and evaluate their formation in both unhydrogenated and hydrogenated soybean oil during heating at 160, 180 and 200 °C for varied length of time. Results showed that among the four columns tested, an Agilent HP-88 column (100 × 0.25 mm I.D., 0.2-μm film thickness) could resolve eight trans fatty acids and nine cis fatty acids simultaneously within 31 min with injector temperature 240 °C, detector temperature 250 °C, and column temperature 170 °C in the beginning, maintained for 24 min, increased to 220 °C at 7.5 °C/min, 230 °C at 10 °C/min, and maintained for 5 min. The contents of both cis and trans fatty acids showed a decreased trend for the increase of heating time or temperature. No trans fatty acid formation was observed even after extensive heating of unhydrogenated and hydrogenated soybean oil for 24 h. This phenomenon demonstrated that trans fatty acids can only be formed under severe conditions.     

Attachment strength and biofilm forming ability of Bacillus cereus on green-leafy vegetables: cabbage and lettuce

The present study was designed to investigate the ability of six Bacillus cereus strains to attach and form biofilm on cabbage and lettuce surfaces. These six strains were; a reference strain DSMZ 345 and five biofilm-producing strains (aquatic strains; TUB8, TUB30, TUB31, TUB32 and TUB33) isolated from drinking-water distribution network. Hydrophobicity, biofilm formation ability, attachment strength (S_R) of spores and vegetative cells of the six B. cereus strains were also determined. Due to their high hydrophobicity, spores of all strains had high ability to attach polystyrene and did not affect by dilution of tryptone soy broth (TSB, 1:20 v/v) in the in vitro experiment. Significant (p < 0.05) enhancement in vitro biofilm formation by vegetative cells of B. cereus was recorded in the diluted TSB. The highest biofilm formation on cabbage and lettuce surfaces was obtained by spores and vegetative cells of all tested strains on the 4^th hour of the incubation period. These populations were significantly (p < 0.05) increased by elongating incubation time from 4 h to 24 h except DSMZ 345 and TUB8. Biofilm formation behavior obtained by B. cereus spores and vegetative cells on the polystyrene surface was different compared with that recorded on produce surface. The S_R of both spores and vegetative cells of the studied strains to the lettuce surface was higher than that of the cabbage surface. The hydrophobicity, biofilm formation and S_R of spores and vegetative cells of the biofilm-producing strains were higher than that of the reference strain DSMZ 345. Scanning electron microscopy (SEM) exposed random distribution of cells either on the surface or cut edge, without clear obvious affinity for the surface structures. Increasing in the presence of large clusters of cells on leaf surfaces was demonstrated after 4 and 24 h.In conclusion, use of aquatic environmental isolates is more useful for studying biofilm formation than the reference strain. Lettuce surface supported the attachment of B. cereus spores and vegetative cells compared with the cabbage surface. Further investigations are required to improve our knowledge of biofilm formation mechanisms by the human pathogenic microorganisms, especially by using the environmental and clinical isolates. To ensure safety level of green-leafy vegetables, biofilm formation after harvest should be considered as critical control point during handling of these vegetables.     

Low temperatures and fermentative metabolism limit peptidoglycan digestion of Bacillus cereus. Impact on colony forming unit counts

The impact of fermentative metabolism at low temperature on cell division of Bacillus cereus was studied. Fermentation at 37 °C had no influence on the division of bacteria. Aerobic cultures at 15 °C produced larger cells than at 37 °C, but cell division was normal. In fermentative cultures at 15 °C, no increase in CFU ml⁻¹ was observed. However, A₆₀₀ increased, due to formation of long filaments. Transmission electronic microscopy and light microscopy with fluorescent staining showed several nucleic acid entities separated by a hydrophobic membrane, indicating that each filament contained several individual cells attached by peptidoglycan. When left in air at room temperature, one filament gave several daughter cells, this means that one CFU formed by one filament may represent a greater contamination potential than one CFU formed by a single cell. Division was observed in cultures at 15 °C with anaerobic respiration in the presence of nitrates. Possible filamentous growth must thus be taken into account to avoid underestimating B. cereus growth in vacuum or modified atmosphere packaged foods stored at low temperature.