Tryptone Medium Research Articles

Unraveling the microbiota of the fish parasite Pseudoterranova decipiens in codfish (Gadus morhua) reveals a fish-related bacterial community

Anisakidae, mainly represented by the species Anisakis simplex and Pseudoterranova decipiens, are one of the most commonly zoonotic nematodes present in marine fish species. Apart from public health risks directly linked to the parasite itself, little is known on the effects of the migrating nematodes on the hygienic quality of the fish fillet due to bacteria it carries. In the present study, the cultivated bacterial community on and in individual P. decipiens larvae deriving from codfish is reported. Four isolation media were included and evaluated to increase the bacterial diversity isolated, and identification of the bacterial growth was performed by a combination of Matrix-Assisted Laser Desorption Ionization Time-Of-Flight mass spectrometry and 16S rRNA gene sequencing. Results revealed that the microbiota of P. decipiens larvae comprises both potential spoilage bacteria and human opportunistic pathogens, and that a combined isolation on the general isolation medium tryptone soy agar and a medium supplemented with artificial seawater resulted in the highest bacterial recovery in terms of diversity and enumeration. Dissimilarity analysis also revealed similar, though unique, bacterial communities between nematodes originating from the same fish suggesting that anisakid microbiota compositions are reflections of the microbial assemblages in the fish host as an individual, and that the gut microbiome is diverse within gadoid fish species originating from the same geographical habitat. Future research should, based on the results in the present study, further elaborate on the comparison of the bacterial communities of both the larva and the codfish from which it was isolated, and, explore the extrapolation potential towards other fish and nematode species. Also, the actual degree of risk beyond the simple presence of the parasite due to carriage of opportunistic bacteria should be examined, as well as the nematode's true effect on spoilage.

Metabolic engineering of Bacillus subtilis with an endopolygalacturonase gene isolated from Pectobacterium. carotovorum; a plant pathogenic bacterial strain.

Pectinolytic enzymes or pectinases are synthesized naturally by numerous microbes and plants. These enzymes degrade various kinds of pectin which exist as the major component of the cell wall in plants. A pectinase gene encoding endo-polygalacturonase (endo-PGase) enzyme was isolated from Pectobacterium carotovorum a plant pathogenic strain of bacteria and successfully cloned into a secretion vector pHT43 having σA-dependent promoter for heterologous expression in Bacillus subtilis (WB800N).The desired PCR product was 1209bp which encoded an open reading frame of 402 amino acids. Recombinant proteins showed an estimated molecular weight of 48 kDa confirmed by sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. Transformed B. subtilis competent cells harbouring the engineered pHT43 vector with the foreign endo-PGase gene were cultured in 2X-yeast extract tryptone medium and subsequently screened for enzyme activity at various temperatures and pH ranges. Optimal activity of recombinant endo-PGase was found at 40°C and pH 5.0. To assay the catalytic effect of metal ions, the recombinant enzyme was incubated with 1 mM concentration of various metal ions. Potassium chloride increased the enzyme activity while EDTA, Zn++ and Ca++, strongly inhibited the activity. The chromatographic analysis of enzymatic hydrolysates of polygalacturonic acid (PGA) and pectin substrates using HPLC and TLC revealed tri and tetra-galacturonates as the end products of recombinant endo-PGase hydrolysis. Conclusively, endo-PGase gene from the plant pathogenic strain was successfully expressed in Bacillus subtilis for the first time using pHT43 expression vector and could be assessed for enzyme production using a very simple medium with IPTG induction. These findings proposed that the Bacillus expression system might be safer to escape endotoxins for commercial enzyme production as compared to yeast and fungi. Additionally, the hydrolysis products generated by the recombinant endo-PGase activity offer their useful applications in food and beverage industry for quality products.

Isolation And Characterization Of Biosurfactant Producing Bacteria From Different Environmental Soil Samples

Isolation And Characterization Of Biosurfactant Producing Bacteria From Different Environmental Soil Samples

Study on the reuse process of hydrolysate from γ-polyglutamic acid fermentation residues

In order to study the feasibility of using γ-polyglutamic acid (γ-PGA) waste biomass hydrolysate as nitrogen source of culture medium, waste biomass was treated with nitric acid to obtain hydrolysate. Through the orthogonal optimization experiment, the optimal conditions of biomass hydrolysis were obtained as follows: 2% nitric acid, solid-liquid ratio 4:10, 110 °C, and 24 h. Under the optimal conditions, the degree of hydrolysis reached 77.08 ± 0.48%. The fermentation medium was further optimized: biomass hydrolysate 60 mL, glucose 38 g/L, L-glutamate 25 g/L. In the optimal medium, γ-PGA yield was 30.69 ± 0.42 g/L, which was 4.49% higher than that in tryptone medium (29.37 ± 0.43 g/L). The results showed that the waste biomass hydrolysate could be used as tryptone for γ-PGA production. Moreover, the addition of waste biomass hydrolysate reduced the amount of glutamate in the medium. In addition, the waste biomass could be recycled for 2 times. This study provided a new idea for the cleaner production technology of γ-PGA.

Acetate and Potassium Modulate the Stationary-Phase Activation of lrgAB in Streptococcus mutans.

Fluctuating environments force bacteria to constantly adapt and optimize the uptake of substrates to maintain cellular and nutritional homeostasis. Our recent findings revealed that LrgAB functions as a pyruvate uptake system in Streptococcus mutans, and its activity is modulated in response to glucose and oxygen levels. Here, we show that the composition of the growth medium dramatically influences the magnitude and pattern of lrgAB activation. Specifically, tryptone (T) medium does not provide a preferred environment for stationary phase lrgAB activation, which is independent of external pyruvate concentration. The addition of pyruvate to T medium can elicit PlrgA activation during exponential growth, enabling the cell to utilize external pyruvate for improvement of cell growth. Through comparison of the medium composition and a series of GFP quantification assays for measurement of PlrgA activation, we found that acetate and potassium (K+) play important roles in eliciting PlrgA activation at stationary phase. Of note, supplementation of pooled human saliva to T medium induced lrgAB expression at stationary phase and in response to pyruvate, suggesting that LrgAB is likely functional in the oral cavity. High concentrations of acetate inhibit cell growth, while high concentrations of K+ negatively regulate lrgAB activation. qPCR analysis also revealed that growth in T medium (acetate/K+ limited) significantly affects the expression of genes related to the catabolic pathways of pyruvate, including the Pta/AckA pathway (acetate metabolism). Lastly, stationary phase lrgAB expression is not activated when S. mutans is cultured in T medium, even in a strain that overexpresses lytST. Taken together, these data suggest that lrgAB activation and pyruvate uptake in S. mutans are connected to acetate metabolism and potassium uptake systems, important for cellular and energy homeostasis. They also suggest that these factors need to be implemented when planning metabolic experiments and analyzing data in S. mutans studies that may be sensitive to stationary growth phase.

Characterization of the ammonification, the relevant protease production and activity in a high-efficiency ammonifier Bacillus amyloliquefaciens DT

High-efficiency ammonifiers are imperative for the treatment of wastewater with high organic nitrogen content. Ammonification characteristics and the corresponding protease production and activity of a Bacillus amyloliquefaciens was studied using simulated wastewater. High ammonification was observed with more than 500 mg/L NH4+ released from tryptone medium after 18-h cultivation and almost all the organic nitrogen was transformed to NH4+-N and biomass. Single factor experiments were performed to optimize the conditions. For ammonification, pH 5, 25–37 °C, and a C:N ratio <15 were optimal; for protease production, pH 7, 30 °C and addition of 2 mM Mn2+ were favorable, whereas pH 8, 50–60 °C and the presence of 1 mM Mn2+ showed the highest protease activity. Moreover, protease inhibition experiment indicated that Bacillus amyloliquefaciens DT simultaneously produces metalloprotease and serine protease. Correlation analysis indicated that protease production and ammonification were tightly coupled with cell growth during the logarithmic growth phase. However, temperature had different influence on the correlations between protease secretion and cell growth. These findings are expected to help in designing a high-efficiency reactor for the treatment of wastewater with high organic nitrogen and enhance our understanding of the production and activity of proteases during the ammonification process.

Anolyte in-situ functionalized carbon nanotubes electrons transport network as novel strategy for enhanced performance microbial fuel cells

Anolyte in-situ electrons transport network from functionalized multi-walled carbon nanotubes is introduced as a novel strategy to compile the electrons from the anode-away microorganisms. The strategy was investigated by a single-chamber air-cathode microbial fuel cell with Escherichia coli. Based on our best knowledge, for the utilized MFC configuration, unprecedented output power (1.1 W m−2) and current density (4.1 A m−2) were obtained when 6.06 mg ml−1 autoclave-treated MWCNTs (in tryptone medium at 121 °C) was used. Moreover, after 6 h working time, the observed current density was almost duplicated in case of using the best sample compared to the carbon nanotubes-free cell. FTIR and Bio-TEM analyses indicated that the proposed hydrothermal treatment leads to functionalize the carbon nanotubes by nitrogenous groups that strongly enhances the attachment with the bacterial cell wall and improves the biocompatibility. EIS measurements confirmed the good adhesion as a small charge-transfer resistance was observed; 33 Ω. Besides the treatment temperature of the carbon nanotubes, which should be 121 °C, the concentration in the anolyte should be optimized; 6.06 mg ml−1 reveals the best performance compared to 1.21, 3.63, 8.48 and 10.9 mg ml−1. On the other hand, due to formation of the carbonyl group, acid treatment converts the carbon nanotubes to have antibacterial activity toward the E. coli that decreases the cell performance drastically.

Hydrogen Production and Enzyme Activities in the Hyperthermophile Thermococcus paralvinellae Grown on Maltose, Tryptone, and Agricultural Waste.

Thermococcus may be an important alternative source of H2 in the hot subseafloor in otherwise low H2 environments such as some hydrothermal vents and oil reservoirs. It may also be useful in industry for rapid agricultural waste treatment and concomitant H2 production. Thermococcus paralvinellae grown at 82°C without sulfur produced up to 5 mmol of H2 L−1 at rates of 5–36 fmol H2 cell−1 h−1 on 0.5% (wt vol−1) maltose, 0.5% (wt vol−1) tryptone, and 0.5% maltose + 0.05% tryptone media. Two potentially inhibiting conditions, the presence of 10 mM acetate and low pH (pH 5) in maltose-only medium, did not significantly affect growth or H2 production. Growth rates, H2 production rates, and cell yields based on H2 production were the same as those for Pyrococcus furiosus grown at 95°C on the same media for comparison. Acetate, butyrate, succinate, isovalerate, and formate were also detected as end products. After 100 h, T. paralvinellae produced up to 5 mmol of H2 L−1 of medium when grown on up to 70% (vol vol−1) waste milk from cows undergoing treatment for mastitis with the bacterial antibiotic Ceftiofur and from untreated cows. The amount of H2 produced by T. paralvinellae increased with increasing waste concentrations, but decreased in P. furiosus cultures supplemented with waste milk above 1% concentration. All mesophilic bacteria from the waste milk that grew on Luria Bertani, Sheep's Blood (selective for Staphylococcus, the typical cause of mastitis), and MacConkey (selective for Gram-negative enteric bacteria) agar plates were killed by heat during incubation at 82°C. Ceftiofur, which is heat labile, was below the detection limit following incubation at 82°C. T. paralvinellae also produced up to 6 mmol of H2 L−1 of medium when grown on 0.1–10% (wt vol−1) spent brewery grain while P. furiosus produced < 1 mmol of H2 L−1. Twelve of 13 enzyme activities in T. paralvinellae showed significant (p < 0.05) differences across six different growth conditions; however, methyl viologen-dependent membrane hydrogenase activity remained constant across all media types. The results demonstrate the potential of at least some Thermococcus species to produce H2 if protein and α-glucosides are present as substrates.

Potential of natamycin in enhancing the antagonistic activity of Bacillus amyloliquefaciens BGP20 against post-harvest bacterial soft rot of green pepper

ABSTRACTBacillus amyloliquefaciens BGP20 is a promising antagonist in controlling post-harvest bacterial soft rot of vegetables caused by Erwinia carotovora subsp. carotovora (Ecc). The objective of this study was to screen a kind of natural and safe additive which could enhance the bio-control activity of BGP20 against post-harvest bacterial soft rot of green pepper. The results of this study indicated that the additive natamycin had stronger inhibition against the pathogen Ecc compared with bamboo vinegar and chitosan in the 2× Yeast extract and Tryptone (2YT) medium. However, natamycin had a slight negative effect on the growth of BGP20 in the 2YT medium. In preventative treatments, natamycin significantly improved the bio-efficacy of BGP20, and enhanced its competitive position against Ecc in the wounds of green pepper. Compared with the treatment with BGP20 alone, the viable count of BGP20 after 72 h of incubation increased by 115.8% in the wounds of green pepper treated with BGP20 and 0.1% natamycin, while that of Ecc decreased by 92.1%. In addition, natamycin remarkably promoted the flocculation of Ecc cells in the 2YT medium, while promoting the dispersion of BGP20. Natamycin had no negative effects on the spore germination of BGP20 and its shelf life. These results indicated that natamycin had perfect compatibility with the antagonist BGP20, and it had a great potential in enhancing the bio-control activity of BGP20 against post-harvest bacterial soft rot of green pepper in preventative treatments.

Eradication of Pseudomonas biofilm by disinfectants and some plants extracts

In the present study three isolates belonging to Pseudomonas sp and one reference strain of P. aeruginosa ATCC 27853 were tested for biofilm formation on two types of support (glass and polystyrene), using two cultures medium Tryptone Soy Broth (TSB) and Modified Biofilm Broth (MBB). The results showed that the quantity of biofilm formed depends on the nature of culture medium, where the rate of the adherent bacteria was more significant in TSB medium. Polystyrene was more favorable to bacteria for adherence compared to glass. We examined the effectiveness of three types of disinfectants, sodium hypochlorite, hydrogen peroxide and temperature on a biofim formed by the studied bacteria. Sodium hypochlorite reached good levels of biofilm eradication using all isolates adhered on the two types of support. Hydrogen peroxide exerted less significant effect compared to sodium hypochlorite, eliminating approximately 56% from the biofilm adhered on polystyrene at concentration of 3%. The elimination of biofilm temperature (80°C) was rather weak compared with the two chemical disinfectants. Our study included the testing of extracts of three plants: Allium sativum, Aloe vera and Lawsonia inermis on biofilm eradication formed by P. aeruginosa ATCC 27853. The effect of these plant extracts on planktonic cells was also studied. The results showed that Allium sativum and Lawsonia inermis inhibit both bacterial growth and biofilm formation and no activity was detected for Aloe vera extract.

Optimization of media and temperature for antimicrobial activity of Enterobacter sp. associated with entomopathogenic nematode Rhabditid sp.

An entomopathogenic bacterium isolated from the nematode, Rhabditis (Oscheius) sp. was found to produce secondary metabolites with antimicrobial activity. The bacterium isolated from the entomopathogenic nematode was identified as Enterobacter sp. by using biochemical and 16S rDNA sequence analysis. Media for the production of the bioactive metabolites were standardized with six carbon sources viz. glycerol, maltose, fructose, glucose, sucrose and lactose, and four nitrogen sources viz. tryptone, yeast extract, beef extract and peptone. Antimicrobial activity was found highest for culture filtrate solvent extract (CFSE) obtained from tryptone plus glycerol (T+G) combination. Addition of peptone to the media, irrespective of carbon sources, had the least antimicrobial activity. Fermentation with tryptone plus glycerol medium when carried out at temperature ranging from 25 to 40ºC, the highest antimicrobial activity was observed at 37ºC.

Efficacy of common disinfectants and terbinafine in inactivating the growth of Batrachochytrium dendrobatidis in culture

Use of disinfectants by biologists, veterinarians, and zoological facilities is a standard biosecurity procedure to prevent contamination and the spread of pathogens. We tested the efficacy of 5 disinfectants and 1 anti-fungal treatment, at 1 and 5 min contact durations, in inactivating Batrachochytrium dendrobatidis (Bd) grown on tryptone media. Our study focused on concentrations of disinfectants known to inactivate ranaviruses, which can be found at the same sites as Bd and can concurrently infect amphibians. Disinfectants tested were chlorhexidine gluconate (0.25, 0.75, and 2%), Pro-San (0.19, 0.35, and 0.47%), Virkon S (1%), household bleach (0.2, 1, and 3%), and Xtreme Mic (5%). The anti-fungal was terbinafine HCl at 0.005, 0.05, 0.1, and 1 mg ml-1. Inactivation of Bd was determined by microscopic evaluation of zoospore motility and growth of colony mass after 14 d. All disinfectants were effective at inactivating zoospore motility and colony growth of Bd at all concentrations and both contact times; however, terbinafine HCl inactivated Bd at only the highest concentration tested (1 mg ml-1) and 5 min duration. Thus, a minimum of 0.25% chlorhexidine gluconate, 0.19% Pro-San, 1% Virkon, 0.2% bleach, and 5% Xtreme Mic with 1 min contact was sufficient to inactivate Bd. Also, terbinafine HCl (1 mg ml-1) with a 5 min contact time might be effective in treating amphibians infected with Bd. Based on this study and previously published findings, 0.75% Nolvasan, 1% Virkon S, and 3% bleach with 1 min contact are sufficient to inactivate both Bd and ranaviruses.

Bacillussp. SXB andPantoeasp. IMH, aerobic As(V)-reducing bacteria isolated from arsenic-contaminated soil

To isolate highly effective aerobic As(V)-reducing bacteria from arsenic(As)-contaminated soils in Northwest China and to identify their dynamic As(V) reduction processes and genomic detoxification mechanisms. Enrichment cultures were performed aerobically in tryptone, yeast extract and glucose medium in the presence of As(V). Strain SXB isolated from soil in Shanxi Province, belonging to Bacillus genus, reduced As(V) more effectively under aerobic conditions than under anaerobic conditions. Strain IMH, a strictly aerobic isolate obtained from soil in Inner Mongolia, identified as Pantoea, is reported for the first time to reduce As(V). Both isolates could reduce over 90% As(V) in 36 h under aerobic conditions. Putative gene fragments for the ArsB efflux pump gene were obtained from both strains. The putative arsenate reductase gene was only amplified from strain SXB. A putative arsH gene was amplified from strain IMH. Strains SXB and IMH isolated from the As-contaminated soils reduce As(V) effectively under aerobic conditions via a detoxification mechanism regulated by ars operons. Pantoea genus is reported to reduce As(V) for the first time. This study provides a full understanding of the highly effective As(V)-reducing bacteria SXB and IMH, which could influence the As biogeochemical cycle in soils.

Isolation and characterization of a novel Micrococcus strain for bioremediation of strontium in radioactive residues

There has been increased interest in the isolation of bacteria with a potential role in bioremediation from extreme environments such as phosphate mines containing various metals and radionuclides. This paper describes the isolation and characterization of a novelMicrococcus species from a phosphate mining region in the South of Tunisia, designated as strain BRM7. Colonies of bacterial strain BRM7 produced on Tryptone-Glucose-Yeast Extract (TGY) agar plates were yellow, smooth, circular and 0.5–1.5 mm in diameter. Cells of strain BRM7 were Gram-positive cocci, with a diameter of 0.7–1.0 µm. The most abundant cellular fatty acids of strain BRM7 were found to be anteiso-C15: 0 (49.42%) and iso-C15: 0 (32.63%). On the basis of its morphological attributes, biochemical characteristics, and partial sequencing of 16S rRNA gene (rDNA), the strain BRM7 belongs to the genus Micrococcus with 99% identity to Micrococcus luteus. Strain BRM7 grew well in tryptone, glucose and yeast extract (TGY) medium, and tolerated (1) high salt concentrations (up to 20%), (2) a wide range of pH (5.0–12.0), and (3) high temperatures (up to 45°C). The bacterial isolate Micrococus sp. BRM7 showed a high tolerance to strontium (Sr, D10 (dose for 90% reduction in Colony Forming Units (CFUs)) = 350 mM) with a similar tolerance curve to Cupriavidus metallidurans CH34, best known for its high tolerance to a wide range of heavy metals. Interestingly, Micrococus sp. BRM7 has an ionizing-radiation (IR) resistance D10 (~800 Gy) four times higher than that of C. metallidurans. Immobilization into alginate beads indicated that Micrococus sp. BRM7 cells have the potential to adsorb 17 and 34% of Sr following an incubation time of 3 and 24 h, respectively. Overall, the results of this study suggest that BRM7 can be valorized to bioremediate Sr in radioactive residues like phosphogypsum (PG), an industrial concentrator of this toxic metal. Key words: Bioremediation, immobilization, ionizing radiation, Micrococcus, phosphate, strontium.

Isolation and characterization of Pseudomonas sp. DX7 capable of degrading sulfadoxine

Given that the intensive application of sulfonamides in aquaculture, animal husbandry and malaria treatment has lead to an increase in sulfonamide discharge into the environment, there is an increasing need to find a way to remediate sulfonamide-contaminated sites. The bacterial strain DX7 was isolated from a marine environment and is capable of degrading sulfadoxine. DX7 was identified as a Pseudomonas sp. based on 16S rRNA gene sequencing. Approximately 30% of sulfadoxine was degraded after Pseudomonas sp. DX7 was inoculated into mineral salt plus tryptone media containing 10mgl(-1) sulfadoxine for 2 days. The degradation efficiency under different environmental conditions was characterized using HPLC. The optimal temperature and pH for sulfadoxine biodegradation were around 30°C and 6.0, respectively. The optimal concentrations of sulfadoxine and tryptone for sulfadoxine biodegradation were determined to be approximately 30mgl(-1) and between 2.0 and 8.0gl(-1), respectively. Cytotoxicity analysis indicated that the metabolites of sulfadoxine generated by Pseudomonas sp. DX7 showed significantly reduced cytotoxicity to Hela cells. These results suggest that Pseudomonas sp. DX7 is a new bacterial resource for degrading sulfadoxine and indicate the potential of the isolated strain in the bioremediation of sulfadoxine-contaminated environments.

The occurrence of Escherichia coli O157:H7 in market and abattoir meat in Plateau State, Nigeria

Escherichia coli O157:H7 is a newly emerging pathogen frequently associated with the consumption of foods of bovine origin. The severity of the infections caused by this food borne pathogen in the young and the elderly has had a tremendous impact on human health and food industry. The present study evaluated the occurrence of E. coli O157:H7 in abattoir and market meat in seven selected sites in Plateau State, Nigeria. One hundred and forty samples of each of the various meat parts including muscle, liver, heart, kidney and intestine were collected to give a total of 700 meat samples. The samples were aseptically collected at monthly intervals from December 2008 to November 2009 after which they were transported to the laboratory for bacteriological analysis. Enrichment medium Tryptone Soya Broth (TSB) and selective agar media, namely; sorbitol MacConkey agar (SMAC)and cefixime tellurite-sorbitol MacConkey agar (CT-SMAC) were used for the isolation of E. coli O157:H7 from the various meat samples. The results of this study indicated that the intestinal samples had the highest percentage occurrence of the organism (3.39%), while muscle tissue had the lowest (0.43%). The organ meats had comparable percentage occurrence of the organism, with 1.0% for liver, 0.86% for kidney and 0.71% for heart samples. Significant difference (P 0.05) existed in the occurrence of the organism with respect to the sampling sites and the sources (abattoir and market). The presence of E. coli O157:H7 in beef should be regarded as being highly hazardous to consumers and handlers alike because of the severity of the infections associated with the organism. Thus, hygienic and good management of meats and meat environments are very essential.

Nutritional regulation of protease production by the feather-degrading bacterium Chryseobacterium sp. kr6

The effects of nutritional conditions on growth and protease production by the feather-degrading Chryseobacterium sp. kr6 were investigated. Higher growth was observed on feather-containing or tryptone (TR) medium when compared to casein (CA) or glucose-nitrogen (GN) base medium. Protease production occurred during growth on feather-containing and TR media, whereas no protease activity was detected on CA or GN medium, indicating that protease production is not constitutive, depending on the presence of specific complex nitrogen sources. Supplementation of whole feathers (WF) medium with glucose (WFG) or NH(4)Cl (WFN) did not result in major differences in growth and protease production, whereas soluble protein was lower in supplemented media. Glucose consumption and growth were higher on WFG than on GN medium, suggesting that the absence of a specific complex nitrogen source limited bacterial growth. On WF medium, this strain grew closely attached to the feather structures, initially on the barbules and subsequently on the feather rachis. It was observed, through zymogram analysis, that strain kr6 produced diverse proteolytic enzymes in response to different growth substrates. These results were confirmed by the differential behaviors of crude proteases towards protease inhibitors.

Optimisation of storage conditions for maintaining culturability of penicillin-susceptible and penicillin-resistant isolates of Streptococcus pneumoniae in transport medium

Methods employed by the World Health Organization (WHO) are used during this study to determine the optimum storage conditions for maintaining the culturability of Streptococcus pneumoniae in skimmed milk, tryptone, glucose and glycerin (STGG) transport medium. A comparison of S. pneumoniae strains sensitive and resistant to penicillin showed no significant difference in their survival ability in STGG medium. Furthermore, it is confirmed that storage at –70°C remains most effective for maintaining viability by culture of S. pneumoniae. Storage at –20°C would only be acceptable in the short-term, while storage at +4°C is not recommended. Of note, this study has shown STGG medium at room temperature to be an efficient growth medium for pneumococci in the short-term. This work will help to establish robust sampling protocols when performing community studies to ensure culturability of comparison between community and laboratory pneumococci survival.

Modelling the effect of sub(lethal) heat treatment of Bacillus subtilis spores on germination rate and outgrowth to exponentially growing vegetative cells

Spores of Bacillus subtilis were subjected to relatively mild heat treatments in distilled water and properties of these spores were studied. These spores had lost all or part of their dipicolinic acid (DPA) depending on the severity of the heat treatment. Even after relatively mild heat treatments these spore lost already a small but significant amount of DPA. When these spores were inoculated in nutrient medium—tryptone soy broth (TSA)—the non-lethally heated spores started to germinate. Results of classical optical density measurements showed that both phase darkening and subsequent outgrowth could be affected by sub-lethal heat. A study of single cells in TSB showed that lag times originating from exponentially growing cells followed a normal distribution, whereas lag times originating from spores followed a Weibull distribution. Besides classical optical density measurements were made to study the effect of previous heating on the kinetics of the first stages of germination. The germination kinetics could be described by the model as was proposed by Geeraerd et al. [Geeraerd, A.H., Herremans, C.H. and Van Impe, J.F., 2000. Structural model requirements to describe microbial inactivation during a mild heat treatment. International Journal of Food Microbiology 59, 185–209]. Two of the 4 parameters of the sigmoid model of Geeraerd were dependent on heating time and heating temperature, whereas the two other parameters were considered as independent of the heating conditions. Based on these observations, a secondary model could be developed that describes the combined effect of heating temperature and heating time on the kinetics of germination. To have more detailed information of the kinetics of germination samples incubated in TSB were tested at regular time intervals by flow cytometry. To that end the cells were stained with syto 9 to distinguish between the various germination stages. There was a qualitative agreement between the results of flow cytometry and those of optical density measurements, but there was a difference in quantitative terms. The results have shown that germination rate of spores is dependent on previous heating conditions both in the first stage when phase darkening occurs and also during the later stages of outgrowth when the phase dark spore develops to the vegetative cell.

Influence of Artificial Sweeteners on the Kinetic and Metabolic Behavior of Lactobacillus delbrueckii subsp. bulgaricus

The addition of artificial sweeteners to a LAPT (yeast extract, peptone, and tryptone) medium without supplemented sugar increased the growth rate and final biomass of Lactobacillus delbrueckii subsp. bulgaricus YOP 12 isolated from commercial yogurt. Saccharin and cyclamate were consumed during microorganism growth, while the uptake of aspartame began once the medium was glucose depleted. The pH of the media increased as a consequence of the ammonia released into the media supplemented with the sweeteners. The L. delbrueckii subsp. bulgaricus strain was able to grow in the presence of saccharin, cyclamate, or aspartame, and at low sweetener concentrations, the microorganism could utilize cyclamate and aspartame as an energy and carbon source.