Histamine and biogenic amine production by Morganella morganii isolated from temperature-abused albacore

Histamine-producing bacteria were isolated from albacore stored at 0, 25, 30, and 37 degrees C. They were screened using Niven's differential medium, and their histamine production was confirmed by high-pressure liquid chromatography analysis. The optimum temperature for growth of histamine-producing bacteria was 25 degrees C. The bacterium producing the highest level of histamine was isolated from fish abused at 25 degrees C. It was identified as Morganella morganii by morphological, cultural, biochemical, and antimicrobial characteristics and by the Vitek microbial identification system. The M. morganii isolate was inoculated into tuna fish infusion broth medium, and the effect of temperature was determined for microbial growth and formation of histamine and other biogenic amines. The isolate produced the highest level of histamine, 5,253 ppm, at 25 degrees C in the stationary phase. At 15 degrees C, histamine production was reduced to 2,769 ppm. Neither microbial growth nor histamine formation was detected at 4 degrees C. To determine whether the isolate can also produce other biogenic amines that can potentiate histamine toxicity, production of cadaverine, putrescine, serotonin, tryptamine, tyramine, phenylethylamine, spermidine, and spermine by the isolate was also monitored. Cadaverine, putrescine, and phenylethylamine were detected with microbial growth in the tuna fish infusion broth medium. The optimum temperature for cadaverine, putrescine, and phenylethylamine formation was found to be 25 degrees C, as it was for histamine.     

Effects of on-board and dockside handling on the formation of biogenic amines in mahimahi (Coryphaena hippurus), skipjack tuna (Katsuwonus pelamis), and yellowfin tuna (Thunnus albacares)

Consumer illnesses by scombroid poisonings have been a continuing problem for many years. The intoxications follow the ingestion of fish such as tuna and mahimahi that have undergone bacterial decomposition, leading to the formation of biogenic amines. Research studies have concluded that histamine is one of the indicators of scombrotoxic fish and that other amines, such as cadaverine, could be involved in the illnesses. Guidance for the handling of fish on board fishing vessels to prevent the production of scombrotoxic fish has been limited by a lack of data addressing changes that occur in fish from the water to delivery at dockside. In this study, the changes in selected biogenic amines were determined in mahimahi and tuna, which were captured and held in seawater at 25 to 35 degrees C for incubation times up to 18 h. The fillets from the treated fish were sectioned by transverse cuts and analyzed for histamine, cadaverine, and putrescine. Results showed that at 26 degrees C, more than 12 h of incubation were required before a histamine concentration of 50 ppm was reached in mahimahi. At 35 degrees C, 50 ppm histamine formed within 9 h. Similar results were found for skipjack and yellowfin tuna. Histamine concentrations exceeded 500 ppm within an additional 3 h of incubation in mahimahi. At both temperatures, an increase in the concentration of cadaverine preceded an increase in histamine levels. Changes in putrescine concentrations in the fish were less pronounced. The study also demonstrated that histidine decarboxylase activity was retained in some frozen samples of fish and could result in further increases in histamine on thawing.     

Histamine Formation and Bacterial Spoilage of Albacore Harvested off the U.S. Northwest Coast

Iced and previously frozen albacore were monitored for histamine formation and bacterial growth during storage at 0–37°C. The optimum temperature for histamine formation in albacore tuna (Thunnus alalunga) was 25°C, and whole fish were more susceptible to histamine formation than dressed fish at that temperature. Storage at 25°C resulted in the highest histamine level, 60.4 mg/100g in whole fish stored for 7 days. When albacore were frozen prior to storage, reduced amount of histamine was found at 7.14 mg/100 g after 7 day storage at 25°C, only after decomposition became obvious. No histamine was found in any of the albacore samples stored in ice for 18 days.     

Source and identification of histamine-producing bacteria from fresh and temperature-abused albacore.

Histamine-producing bacteria were isolated from fresh and temperature-abused albacore using two different isolation procedures. Typically, the bacterial isolates on Niven's or modified Niven's medium produced negligible or low levels of histamine (<300 ppm) in histamine enumeration broth. The most frequently found species using this approach was Hafnia alvei. By prescreening on selective media (eosin methylene blue [EMB] agar for enteric bacteria; deMan Rogosa Sharpe agar for lactic acid bacteria: KF streptococcus agar for streptococci; pseudomonas isolation [PI] agar for pseudomonads; and staphylococcus medium 110 agar for staphylococci) prior to plating on histidine decarboxylase differential media, detection rate of true histamine formers increased. Prolific histamine producers capable of forming >1,000 ppm histamine in culture broth were isolated when PI and EMB agars were used for prescreening. Among the selective media tested, EMB agar was most effective in selecting high histamine producers, as demonstrated by the highest rate of true positives based on histamine analysis. Histamine-producing isolates were mostly enteric bacteria, including Morganella morganii, H. alvei, Klebsiella spp., Citrobacter freundii, Enterobacter spp., and Serratia spp. M. morganii isolated on PI agar from temperature-abused albacore muscle was found to be the highest histamine former. This species was not isolated from fresh albacore. while other enteric bacteria were frequently detected on the gills. However, only a few species isolated from both fresh and temperature-abused muscles were identified as high histamine formers.     

Histamine content and histamine-forming bacteria in dried milkfish (Chanos chanos) products

Thirty-two dried milkfish products sold in five retail markets in southern Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. Except for histamine and cadaverine, the average content of various biogenic amines in tested samples was less than 8.5 mg/100 g. Most of the tested dried milkfish products (78.1%) had histamine levels greater than the FDA guideline of 5 mg/100 g for scombroid fish and/or product, while fourteen of them (43.7%) contained > 50 mg/100 g hazard action level. Thirty histamine-producing bacterial strains, capable of producing 5.4 ppm to 562 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine (TSBH), were identified as Enterobacter aerogenes (seven strains), Citrobacter sp. (one strain), Staphylococcusxylosus (ten strains), S. sciuri (one strain), Bacillus thuringiensis (two strains), Citrobacter freundii (five strains), Klebsiella pneumoniae (one strain) and E. cloacae (three strains), by 16S rDNA sequencing with PCR amplification.     

Histamine, Cadaverine and Putrescine Forming Bacteria from Ripened Spanish Semipreserved Anchovies

Histidine decarboxylase activity and production of putrescine and cadaverine were assessed in 200 bacterial isolates from ripened semipreserved Spanish anchovies. Highest levels of histidine decarboxylase activity were observed in Morganella morganii, with 2336.74 ± 356.32 ppm of histamine, produced in laboratory media, after 24 hr incubation at 37°C. Microorganisms producing histamine also produced detectable putrescine and cadaverine. The Niven medium was used to evaluate the 200 isolates for histidine decarboxylase activity, after incubation 24 hr at 37°C. An enzymic technique was used to distinguish false positives and to quantify bacterial histamine levels. The number of microorganisms was the most important factor in the accumulation of histamine.     

Antibacterial activities of blueberry and muscadine phenolic extracts

Phenolics are one category of phyto-antimicrobials that refer to the antimicrobial substances extracted from plant sources. This study was undertaken to determine the influence of blueberry and muscadine phenolic extracts on the growths of 2 important foodborne bacterial pathogens, Salmonella Enteritidis and Listeria monocytogenes. Cells of S. Enteritidis (n = 4) or L. monocytogenes (n = 4) strains were inoculated (3 log CFU/mL) into tryptic soy broth (TSB) supplemented with 46.25 ppm of muscadine phenolics and 24 ppm of blueberry phenolics, respectively. The inoculated and un-inoculated broth with or without the supplemented phenolics were incubated at 37 °C for 24 h. Samples were drawn periodically, and cell populations of Salmonella and Listeria were determined on tryptic soy agar (TSA). It was observed that Salmonella was relatively more susceptible than Listeria to the phenolic extracts used in the study. The growth of Salmonella was significantly inhibited in all samples at all sampling points except for the sample that was supplemented with muscadine water extract and drawn at the 24-h sampling point. Blueberry phenolics were relatively more effective than muscadine phenolic extracts in inhibiting the growth of Salmonella. One tested strain of Listeria was more susceptible to ethanol than water phenolic extracts. The study revealed the potentials and limitations of using blueberry and muscadine phenolics to control the growths of selected Salmonella and Listeria strains.     

Biogenic Amines Formation in Bigeye Tuna Steaks and Whole Skipjack Tuna

ABSTRACT: Putrescine, cadaverine and histamine formation was monitored in fresh and frozen/thawed Bigeye tuna steaks ( Thunnus obesus ) and whole Skipjack ( Katsuwonus pelamis ) stored at room temperature and in ice and to determine the potential use of putrescine and cadaverine as indicators of decomposition. While all biogenic amines were formed during both storage conditions, putrescine was formed at a much lower rate and concentration. For both storage conditions and species, cadaverine appeared prior to and/or accumulated at a faster rate than histamine. Skipjack tuna had a faster accumulation of cadaverine than Bigeye under the same storage conditions. The results indicated that cadaverine, either alone or with histamine, could be used as an index of decomposition for Skipjack and Bigeye tuna.     

Determination of histamine and histamine-forming bacteria in tuna dumpling implicated in a food-borne poisoning

An incident of food-borne poisoning causing illness in seven victims, due to ingestion of tuna dumpling, occurred in March 2006, in Chiayi Prefecture, southern Taiwan. The leftovers of the victims’ tuna dumpling and the five other tuna dumpling samples from five other retail stores were collected and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, aerobic plate count (APC), total volatile basic nitrogen (TVBN), total coliform (TC) and Escherichia coli in all samples ranged from 6.08 to 6.43, 0.46% to 0.81%, 5.90 to 8.95 log CFU/g, 6.38 to 21.29 mg/100 g, 750 to 8000 most probable number (MPN)/g, and <3 to 1000 MPN/g, respectively. The suspected tuna dumpling contained 160.8 mg/100 g of histamine greater than the hazard action level of 50 mg/100 g set by the US Food and Drug Administration (FDA) for tuna fish. Given the allergy-like symptoms of the victims and the high histamine content in the suspected tuna dumpling, this food-borne poisoning was strongly suspected to be due to histamine intoxication. In addition, although thirteen histamine-producing bacteria strains capable of producing 8.1–19.7 ppm of histamine in trypticase soy broth (TSB) supplemented with 1.0% l-histidine (TSBH), were identified as Enterobacter sp. (three strains), Pantoea agglomerans (two strains), Klebsiella variicola (four strains) and Serratia marcescens (four strains), by 16S rDNA sequencing with PCR amplification, they were not determined to be the main contributors to histamine accumulation in suspected tuna dumpling.     

Effect of prior growth conditions on the thermal inactivation of 13 strains of Listeria monocytogenes in two heating menstrua

The thermal tolerance of 13 Listeria monocytogenes strains was tested using a submerged heating coil apparatus. The strains were grown individually for 18 h at 37 degrees C in acidogenic tryptic soy broth (without dextrose) supplemented with 1% glucose and 1% glutamine (TSB+G) or nonacidogenic tryptic soy broth supplemented with 1% glutamine but containing no glucose (dextrose) (TSB-G). The former medium results in cells induced for pH-dependent, stationary-phase acid resistance, whereas the latter medium allows L. monocytogenes to grow to high numbers in the absence of glucose, yielding cells that are not induced for pH-dependent, stationary-phase acid resistance. The average final pH values of the 18-h TSB+G and the TSB-G cultures were 4.7 and 6.7, respectively. The cells grown in the acid resistance-inducing and non-acid resistance-inducing media were then tested in two heating menstrua that consisted of brain heart infusion broth adjusted to pH 3.0 and water activity (a(w)) of 0.987 or pH 7.0 and a(w) 0.970. In 14 of the 26 menstruum-strain combinations tested, the acid resistance-induced strains were more heat resistant then the equivalent noninduced cultures. No difference in the pattern of thermal resistance in response to induction of acid resistance was apparent among the different serovars tested. The results suggest that the ability of prior induction of acid resistanceto enhance thermal resistance can vary substantially among L. monocytogenes strains.     

Histamine and Other Biogenic Amines and Bacterial Isolation in Retail Canned Anchovies

ABSTRACT: Histamine and biogenic amine contents in retail canned anchovies were determined. Bacterial strains were isolated, and their histamine-producing capability was determined. The majority of canned anchovy products (80%) had histamine levels below the FDA guideline of 50 ppm. The sensory quality of products was relatively good. A few samples contained high levels of histamine (>1000 ppm). Overall, histamine contents in the products showed great lot-to-lot variations. Spermine and tyramine were commonly detected in all samples analyzed, regardless of their histamine contents. Bacterial counts in the products were mostly below the detection limit (10² CFU/g), and bacteria were frequently recovered with the enrichment of test samples in tryptic soy broth supplemented with 0.5% or 5% NaCl. Only Bacillus spp., the nonhistamine formers, were isolated from these test products. Prolific histamine-forming bacteria were not detected in these canned anchovies.     

Biogenic amine survey and organoleptic changes in fresh, stored, and temperature-abused bluefish (Pomatomus saltatrix).

Changes in histamine, putrescine, and cadaverine concentrations in bluefish filets (Pomatomus saltatrix) stored at 5, 10, and 15 degrees C were determined using high-performance liquid chromatography. An organoleptic assessment was conducted simultaneously with the biogenic amine analyses. The histamine levels found in fresh bluefish obtained from wholesale seafood distributors ranged between <1 ppm and 99 with an average of 39 ppm. Putrescine and cadaverine were not found in fresh bluefish. Fish fillets stored at each of the three temperatures developed histamine. The greatest accumulation of histamine was observed in fish stored at 15 degrees C, which developed histamine levels as high as 2,200 ppm. Putrescine levels increased at each temperature during storage. Cadaverine was present only in uninoculated bluefish stored at 15 degrees C. Histamine achieved higher levels in bluefish pieces inoculated with Morganella morganii, which demonstrates that bluefish support bacterial histamine formation. Histamine levels at each temperature exceeded the 50-ppm advisory level established by the Food and Drug Administration before 100% sensory rejection. Standard plate counts increased during storage of fish at all temperatures, but the correlation between histamine levels and standard plate count was not significant.     

Significant histamine formation in tuna (Thunnus albacares) at 2 degrees C--effect of vacuum- and modified atmosphere-packaging on psychrotolerant bacteria

Occurrence and importance of psychrotolerant histamine producing bacteria in chilled fresh tuna were demonstrated in the present study. The objective was to evaluate microbial formation of histamine and biogenic amines in chilled fresh tuna from the Indian Ocean and stored either vacuum-packed (VP) or modified atmosphere-packed (MAP). Firstly, biogenic amines and the dominating microbiota were determined in VP tuna involved in an outbreak of histamine fish poisoning in Denmark. Secondly, the microbiota of fresh MAP tuna was evaluated at the time of processing in Sri Lanka and chemical, microbial and sensory changes were evaluated during storage at 1-3 degrees C. To explain the results obtained with naturally contaminated tuna the effect of VP and MAP on biogenic amine formation by psychrotolerant bacteria was evaluated in challenge tests at 2 degrees C and 10 degrees C. The VP tuna that caused histamine fish poisoning had a histamine concentration of >7000 mg/kg and this high concentration was most likely produced by psychrotolerant Morganella morganii-like bacteria or by Photobacterium phosphoreum. Similar psychrotolerant M. morganii-like bacteria dominated the spoilage microbiota of fresh MAP tuna with 60% CO2/40% N2 and formed >5000 mg/kg of histamine after 24 days at 1.7 degrees C. These psychrotolerant bacteria were biochemically similar to M. morganii subsp. morganii and their 16S rDNA (1495 bp) showed >98% sequence similarity to the type strain of this species. Toxic concentrations of histamine were produced at 2.1 degrees C in inoculated VP tuna by both the psychrotolerant M. morganii-like bacteria (7400+/-1050 mg/kg) and P. phosphoreum (4250+/-2050 mg/kg). Interestingly, MAP with 40% CO2/60% O2, in challenge tests, had a strong inhibitory effect on growth and histamine formation by both the psychrotolerant M. morganii-like bacteria and P. phosphoreum. In agreement with this, no formation of histamine was found in naturally contaminated fresh MAP tuna with 40% CO2/60% O2 during 28 days of storage at 1.0 degrees C. To reduce current problems with histamine fish poisoning due to VP tuna it is suggested, for lean tuna loins, to replace vacuum packaging with MAP containing approximately 40% CO2 and approximately 60% O2.     

High-pressure resistance variation of Escherichia coli O157:H7 strains and Salmonella serovars in tryptic soy broth, distilled water, and fruit juice

The effect of high pressure on the log reduction of six strains of Escherichia coli O157:H7 and five serovars of Salmonella enterica was investigated in tryptic soy broth, sterile distilled water, and commercially sterile orange juice (for Salmonella) and apple cider (for E. coli). Samples were subjected to high-pressure processing treatment at 300 and 550 MPa for 2 min at 6 degrees C. Samples were plated onto tryptic soy agar directly after pressurization and after being held for 24 h at 4 degrees C. At 300 MPa, little effect was seen on E. coli O157:H7 strains, while Salmonella serovars varied in resistance, showing reductions between 0.26 and 3.95 log CFU/ml. At 550 MPa, E. coli O157:H7 strains exhibited a range of reductions (0.28 to 4.39 log CFU/ml), while most Salmonella populations decreased beyond the detection limit (> 5-log CFU/ml reduction). The most resistant strains tested were E. coli E009 and Salmonella Agona. Generally, bacterial populations in fruit juices showed larger decreases than did populations in tryptic soy broth and distilled water. E. coli O157:H7 cultures held for 24 h at 4 degrees C after treatment at 550 MPa showed a significant log decrease as compared with cultures directly after treatment (P < or = 0.05), while Salmonella serovars did not show this significant decrease (P > 0.05). All Salmonella serovars tested in orange juice treated at 550 MPa for 2 min at 6 degrees C and held for 24 h showed a > 5-log decrease, while E. coli O157:H7 strains require a higher pressure, higher temperature, longer pressurization, or a chemical additive to achieve a 5-log decrease.     

Biofilm formation, cellulose production, and curli biosynthesis by Salmonella originating from produce, animal, and clinical sources

The ability of 71 strains of Salmonella enterica originating from produce, meat, or clinical sources to form biofilms was investigated. A crystal violet binding assay demonstrated no significant differences in biofilm formation by isolates from any source when tested in any of the following three media: Luria-Bertani broth supplemented with 2% glucose, tryptic soy broth (TSB), or 1/20th-strength TSB. Incubation was overnight at 30 degrees C under static conditions. Curli production and cellulose production were monitored by assessing morphotypes on Luria-Bertani agar without salt containing Congo red and by assessing fluorescence on Luria-Bertani agar containing calcofluor, respectively. One hundred percent of the clinical isolates exhibited curli biosynthesis, and 73% demonstrated cellulose production. All meat-related isolates formed curli, and 84% produced cellulose. A total of 80% of produce-related isolates produced curli, but only 52% produced cellulose. Crystal violet binding was not statistically different between isolates representing the three morphotypes when grown in TSB; however, significant differences were observed when strains were cultured in the two other media tested. These data demonstrate that the ability to form biofilms is not dependent on the source of the test isolate and suggest a relationship between crystal violet binding and morphotype, with curli- and cellulose-deficient isolates being least effective in biofilm formation.     

Survival,growth, and inactivation of acid-stressed Shigella flexneri as affected by pH and temperature

A study was done to determine the survival, growth, and inactivation characteristics of unadapted, acid-adapted, and acid-shocked Shigella flexneri 2a cells as affected by pH and temperature. The pathogen was grown at 37 degrees C for 18 h in tryptic soy broth containing no glucose (TSBNG) (unadapted cells) and TSBNG supplemented with 1% glucose (TSBG) (acid-adapted cells). Cells grown in TSBNG were acid-shocked by adjusting 18-h cultures to pH 4.5+/-0.05 with lactic acid. All three cell types were separately inoculated into tryptic soy broth (6.6-7.0 log(10) cfu/ml) containing 0.25% glucose (TSB) acidified to pH 3.5-5.5 with lactic acid and incubated at 4, 12, 21, 30, and 48 degrees C for up to 144 h. Overall, inactivation of S. flexneri cells at low pH was enhanced with an increase in incubation temperature. All three types of cells survived for 144 h at 4 degrees C in TSB acidified to pH 3.5, compared to < 24 h at 30 degrees C and 2 h at 48 degrees C. The population of all three cell types increased significantly (alpha = 0.05) within 24 h when cells were incubated at 12, 21, or 30 degrees C in TSB at pH 5.0, 5.5, or 7.3. Prior exposure of the S. flexneri to an acidic environment (acid-adapted or acid-shocked cells) resulted in increased resistance to extreme acid and temperature conditions. Acid-adapted cells decreased by approximately 2.5 log(10) cfu/ml when incubated at 4 degrees C for 144 h, compared to a 6-log(10) reduction in control (unadapted) cells. When cells were exposed to low pH (3.5-4.5) and high temperature (48 degrees C), significantly higher (alpha = 0.05) populations were recovered on tryptic soy agar (TSA) than on TSA supplemented with 4% NaCl (TSAS), indicating that a portion of S. flexneri cells were injured. Results show that the ability of S. flexneri to survive and grow at a given pH is influenced by previous exposure to acidic environments and by incubation temperature.     

Effects of Frozen Storage on Survival of Staphylococcus aureus and Enterotoxin Production in Precooked Tuna Meat

This study investigated the survival of Staphylococcus aureus in precooked tuna meat for producing canned products during frozen storage (?20 ± 2 °C) as well as its growth and enterotoxin production at 35 to 37 °C after the storage. Samples (50 ± 5 g) of precooked albacore (loin, chunk, and flake) and skipjack (chunk and flake) tuna were inoculated with 5 enterotoxin‐producing strains of S. aureus at a level of approximately 3.5 log CFU/g and individually packed in a vacuum bag after 3 h incubation at 35 to 37 °C. Vacuum‐packed samples were stored in a freezer (?20 ± 2 °C) for 4 wk. The frozen samples were then thawed in 37 °C circulating water for 2 h and incubated at 35 to 37 °C for 22 h. Populations of S. aureus in all precooked tuna samples decreased slightly (<0.7 log CFU/g) after 4 wk of storage at ?20 ± 2 °C, but increased rapidly once the samples were thawed and held at 35 to 37 °C. Total S. aureus counts in albacore and skipjack samples increased by greater than 3 log CFU/g after 6 and 8 h of exposure to 35 to 37 °C, respectively. All samples became spoiled after 10 h of exposure to 35 to 37 °C, while no enterotoxin was detected in any samples. However, enterotoxins were detected in albacore loin and other samples after 12 and 24 h of incubation at 35 to 37 °C, respectively. Frozen precooked tuna meat should be used for producing canned tuna within 6 to 8 h of thawing to avoid product spoilage and potential enterotoxin production by S. aureus in contaminated precooked tuna meat.     

Detection and identification of histamine-producing bacteria associated with harvesting and processing mahimahi and yellowfin tuna

Histamine poisoning is one of the most common chemically induced seafoodborne illnesses reported in the United States today. The causative agents are biogenic amines, commonly produced by gram-negative bacteria. The purpose of this study was to detect and identify histamine-producing bacteria associated with standard industry practices during the harvesting, receiving, and processing of mahimahi and yellowfin tuna in North Carolina. Twenty-nine composite samples were obtained from 18 mahimahi and 11 yellowfin tuna and analyzed for their histamine content. No sample analyzed exceeded 2 ppm histamine, the lower detection limit. Composite fish muscle and environmental samples were screened (n = 386) for the presence of histamine-producing bacteria. Twenty-six percent (145) of 549 isolates selected on the basis of their morphological characteristics tested positive on Niven's media. Sixty-three Niven-positive isolates were gram negative, and 58 were gram positive. Of the 43 isolates tested further, 5 were confirmed as histamine producers, and all 5 produced at low levels (< 250 ppm in 48 h at > 15 degrees C). Three gram-negative and two gram-positive isolates were identified as Enterobacter cloacae and Staphylococcus kloosii, respectively. This study revealed that gram-negative bacteria might not be solely responsible for histamine production in at-risk fish. The confirmation of histamine-producing bacteria demonstrates the potential risk for histamine production. However, no detectable levels were found in the composite fish muscle samples analyzed even though 60% of the yellowfin tuna harvested did not meet the U.S. Food and Drug Administration's regulatory hazard analysis critical control point guidelines for temperature reduction. Therefore, no seafood safety risks were found under the standard industry practices observed in this study.     

Spore formation by Bacillus cereus in broth as affected by temperature, nutrient availability, and manganese

A study was done to determine the effect of interacting factors on sporulation of Bacillus cereus in broth. Vegetative cells (1.4 to 2.2 log CFU/ml) of B. cereus strain 038-2 (capable of growing at 12 degrees C) and strain F3812/84 (capable of growing at 8 degrees C) were inoculated into 30 ml of tryptic soy broth (TSB), TSB supplemented with manganese (50 microg/ml), diluted (10%) TSB (dTSB), and dTSB supplemented with manganese (50 microg/ml) and incubated at 8, 12, or 22 degrees C for up to 30, 30, or 10 days, respectively. Unheated and heated (80 degrees C for 10 min) cultures were plated on brain heart infusion agar to determine total cell counts (vegetative cells plus spores) and the number of spores produced, respectively. Both strains of B. cereus survived in TSB and dTSB for 30 days at 8 degrees C but did not sporulate. At 12 degrees C, cells grew in TSB to a population of 6.0 +/- 0.8 log CFU/ml, which was maintained for 30 days. Neither strain grew in dTSB at 12 degrees C and survived for at least 30 days. Spores were not produced in any of the test broths at 12 degrees C. At 22 degrees C, cells reached a stationary growth phase between 12 and 24 h in TSB, TSB supplemented with manganese, and dTSB supplemented with manganese, and approximately 1% of the CFU were spores. In dTSB, cell growth and spore formation were retarded at 22 degrees C and a significantly lower number of spores was produced compared with the number of spores produced in TSB, TSB supplemented with manganese, and dTSB supplemented with manganese. The addition of manganese to TSB did not affect cell growth or spore formation, but manganese did enhance sporulation in dTSB. This study provides useful information on spore formation by B. cereus as affected by conditions that may be imposed in liquid milieus on the surface of foods and on food contact surfaces in processing environments.     

Formation of biofilms by Listeria monocytogenes under various growth conditions

Eight strains of Listeria monocytogenes (7644, 19112, 15313, Scott A, LCDC, 10403S, SLCC, and 1370) produce biofilms when grown on polyvinyl chloride microtiter well plates. The growth medium (tryptic soy broth [TSB] or modified Welshimer's broth [MWB] at 32 degrees C) influenced the amount of biofilm formed; maximum biofilms were formed in MWB by six strains and in TSB by the remaining two strains. This result suggests that the growth medium is critical in development of L. monocytogenes biofilm. This organism also produced biofilms on stainless steel chips. Biofilm formation on these chips was observed following growth in TSB at 4, 20, and 37 degrees C. After 20 h of incubation at 20 or 37 degrees C, the cell density was approximately 10(6) CFU per chip, and after 4 days incubation at 4 degrees C, the cell density was 10(5) CFU per chip. L. monocytogenes strain Scott A biofilm formation on stainless steel chips was visualized using scanning electron microscopy, which revealed dense aggregates of cells held together by meshlike webbing.