Evidence that bacteria can form new cells in airborne particles.

Serratia marcescens incubated for 8 h at 31 degrees C in a chemically defined medium contained in shake flasks was aerosolized into rotating-drum aerosol chambers at 30 degrees C and saturated humidity. Cells furnished tryptone (Difco) and glycerol just before aerosolization increased (in viable numbers and countable cells) almost twofold within 1 to 2 h after becoming airborne, whereas cells not furnished additional tryptone decreased in viable numbers at a faster rate than the number of particles removed by gravitational settling. Limited tests with a Coulter Counter showed that cell volume changes occurred in growing cells that did not occur in the nongrowing population.     

Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures

Cells of Salmonella typhimurium LT2 were dehydrated on hydrophobic membranes (Millipore FGLP2500) placed in a controlled atmosphere chamber held at 57% equilibrium relative humidity (ERH) and 37 degrees C. Dehydration for 48 h under the above conditions increased the heat resistance of Salm. typhimurium LT2 when measured as the surviving fraction after a heat challenge of 135 degrees C for 30 min. Results also showed that little or no death occurred during heat challenges of 1 h at temperatures of up to 100 degrees C. The survival of Salm. typhimurium LT2 was measured as the ability to form colonies on solid media tryptone soy broth plus 1.2% agar (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of 0.2% into the recovery medium, did not enhance the recovery of heated Salm. typhimurium LT2. Dehydrated cells of S. typhimurium LT2 showed a triphasic death curve. Increasing the period of dehydration from 48 h to 34 d, reduced initial numbers due to die off but did not alter the shape of the subsequent survival curve.     

Effect of incubation media on the recovery of Escherichia coli K12 heated at 52 degrees C.

The exposure of exponentially grown Escherichia coli K12 to 52 degrees C for 30 min in Tris/Mg2+ buffer resulted in a considerable loss of viability when plated on tryptone agar. When such heated bacteria were held at 37 degrees C for 2 h in tryptone broth before plating on tryptone agar, there was a significant increase in viability. Thus, heat damage was repaired in tryptone broth but not on tryptone agar. Recovery was greater in tryptone broth than in synthetic medium. In tryptone broth, recA or polA mutants also recovered but a lex mutant did not. As a result of heating, the sensitivity of bacteria to ultraviolet radiation (u.v.), to mitomycin C and to plating on high salt medium was enhanced. After incubation for 2 h in tryptone broth at 37 degrees C, the bacteria regained their resistance to u.v. and mitomycin C and tolerance to high salt medium. Recovery of viability required RNA and protein synthesis, whereas recovery of u.v. resistance did not require protein synthesis. Heating for 30 min inhibited the release of acid-soluble material from DNA in all strains of E. coli used.     

Developmental induction of Myxococcus xanthus myxospores.

Myxospore differentiation during the developmental cycle of Myxococcus xanthus is characterized by several distinguishable morphological stages. Two experimentally useful criteria of myxospore induction are the conversion of vegetative rods to optically refractile short rods or ovoids and the development of resistance to sonic lysis. The use of optical refractility as the first morphological criterion of myxospore induction has facilitated an analysis of induction on developmental plates. The time-dependent changes in the cell population from vegetative rods to the final products of development, autolysed cells and myxospores, were determined in liquid suspension by interrupting cells from developmental plates before the first appearance of myxospores. The treatment of cells involved a two-step induction system. The cells were first aerated in buffer at 32 degrees C (preinduction) and then aerated in 1% tryptone (Difco) at 32 degrees C (induction). At early plate times (0 to 18 h) there was little or no response to these treatments. After 18 h, many of the cells undergoing development on plates responded to preinduction in buffer by subsequent induction to myxospores in tryptone medium (intermediate cells). After 32 h, cells induced to myxospores in tryptone medium and did not require preinduction (competent cells). After 36 h, cells begin to undergo differentiation to myxospores on plates. These results indicate that there was a sequence of physiological changes in developing cells that are defined by the differential response of cells to treatment in liquid suspension. The liquid induction system described here provides a means to analyze the regulation of developmental myxospore induction.     

Survival of Flavobacterium psychrophilum in rainbow trout (Oncorhynchus mykiss) serum in vitro

Virulent and non-virulent strains of Flavobacterium psychrophilum of different serotypes were examined for survival and growth in non-immune and immune rainbow trout serum, in vitro. A majority of the examined strains consumed complement of non-immune serum, but the complement cascade was not able to cause an immediate (after 3 h incubation) notable reduction in viability of the inoculated cells. After 24 h incubation a more pronounced reduction in the number of viable bacteria was observed in untreated serum as well as in serum heated at 45 degrees C. In serum heated at 56 degrees C this reduction in cell number was not observed, but an increase in cell number did not occur either. The serum survival of one of the examined strains was different from the others in showing cell multiplication after 24 h incubation in normal as well as heat-treated (45 and 56 degrees C) serum. In immune serum no immediate reduction in viability of inoculated cells, of all tested strains, was observed. The number of viable cells showed a slow decrease or remained almost unchanged for up to 72 h post-inoculation in untreated serum, at 5 degrees C as well as 15 degrees C. In heat-treated serum (45 degrees C) the number of viable cells decreased slowly at 5 degrees C and 15 degrees C for up to 72 h. The results suggest that the examined strains were unaffected by the alternative complement reaction present in fish serum as well as by antibodies against F. psychrophilum. However, some unknown component(s) in the fish sera, or lack of nutrients or essential growth factors, inhibited the growth of most of the examined strains in the tested fish sera.     

Fresh PBSC harvests, but not BM, show temperature-related loss of CD34 viability during storage and transport

BACKGROUND: The optimum conditions for storage and transport of freshly harvested HPC in the liquid state are uncertain. It is not specified in commonly applied standards for stem cell transplantation. We used a viable CD34 assay to determine the optimum temperature for maintaining progenitor cell viability in freshly harvested BM and PBSC. Our aim was to identify standardized conditions for storage and transport of marrow or peripheral blood products that would optimize CD34 recovery, leading to better transplant outcomes. METHODS: Samples were aseptically removed from 46 fresh HPC harvests (34 PBSC and 12 BM) and stored at refrigerated temperature (2-8 degrees C), room temperature (18-24 degrees C) and 37 degrees C for up to 72 h. Samples were analyzed for viable CD34+ cells/microL at 0, 24, 48 and 72 h. RESULTS: The mean viable CD34+ yield prior to storage was 7.7 x 10(6)/kg (range 0.7-30.3). The mean loss of viable CD34+ cells in HPC products at refrigerated temperature was 9.4%, 19.4% and 28% at 24, 48 and 72 h, respectively. In contrast, the mean loss of viable CD34+ cells at room temperature was 21.9%, 30.7% and 43.3% at 24, 48 and 72 h, respectively. No viable CD34+ cells remained after storage at 37 degrees C for 24 h. Only PBSC products and not BM showed temperature-related loss of CD34 viability. Greater loss of viable CD34+ cells was observed for allogeneic PBSC compared with autologous PBSC. DISCUSSION: These results demonstrate that the optimum temperature for maintaining the viability of CD34+ cells, during overnight storage and transport of freshly harvested HPC, is 2-8 degrees C. These findings will allow the development of standard guidelines for HPC storage and transport.     

Techniques for the optimum recovery of cold injured Campylobacter jejuni from milk or water

When broth was inoculated with cells of Campylobacter jejuni that had been injured by chilling there was a fall in the viable population of up to 90%. It was greater at 43 degrees than 37 degrees C and in the presence of certain antibiotics and in some cases resulted in a surviving population that was below the minimum inoculum for subsequent growth. A technique of pre-enrichment in non-selective culture broth at 37 degrees C for 2 h before the addition of antibiotics and incubation at 43 degrees C was found to significantly reduce the fall in numbers and to improve the detection of C. jejuni in samples of raw milk and water.     

Thermal inactivation of Listeria monocytogenes during a process simulating temperatures achieved during microwave heating

Conventional heating was used to expose cells of Listeria monocytogenes, either in broth or in situ on chicken skin, to the mean times and temperatures that are achieved during a 28 min period of microwave cooking of a whole chicken. Heating L. monocytogenes by this method in culture broth resulted in a reduction in viable cell numbers by a factor of greater than 10(6) upon reaching 70 degrees C. Simulated microwave cooking of L. monocytogenes in situ, on chicken skin, resulted in more variability in the numbers of survivors. Heating for the full cook time of 28 min, however, resulted in a mean measured temperature of 85 degrees C and no surviving listerias were detected. This indicated a reduction in viable numbers of greater than 10(6). To reduce temperature variation, cells were heated on skin in a submerged system in which exposure to 70 degrees C for 2 min resulted in a reduction in viable cell numbers of all strains of listerias tested of between 10(6) and 10(8). These results show that when a temperature of 70 degrees C is reached and maintained for at least 2 min throughout a food there is a substantial reduction in the numbers of L. monocytogenes. The survival of this organism during microwave heating when temperatures of over 70 degrees C are reported is probably due to uneven heating by microwave ovens resulting in the presence of cold spots in the product. The heat resistance of L. monocytogenes is comparable with that of many other non-sporing mesophilic bacteria.     

The detection of non-O157 E. coli in food by immunomagnetic separation

AIMS: To compare immunomagnetic separation (IMS) protocols (enrichment media and temperature) for the isolation of Escherichia coli serotypes O26 and O111 from four different foods. METHODS AND RESULTS: Foods (minced beef, cheese, apple juice and pepperoni) spiked with low numbers (<100 g(-1)) of stressed nalidixic mutant E. coli serotypes O26 and O111 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at temperatures of 37 and 42 degrees C to optimize the IMS technique. BPW enrichments gave increased recoveries of both serotypes compared with tryptone soya and EC broths. Elevated temperatures of incubation at 42 degrees C were superior to 37 degrees C. CONCLUSIONS: Positive detection of low numbers of stressed target pathogens in all replicate tests was only possible using BPW enrichments. The majority of tests from alternative enrichments resulted in zero or single colonies recovered post-IMS. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum IMS protocol would improve isolation rates of E. coli O26 and O111 from foods and lead to increased safety for the consumer. Sub-optimal IMS protocols could lead to foods being incorrectly labelled free from these pathogens.     

Fate of Enterobacter cloacae JP120 and Alcaligenes eutrophus AEO106(pRO101) in soil during water stress: effects on culturability and viability.

A sandy loam soil near field capacity moisture content (psi = -0.050 MPa) or air dried (psi = -300 MPa) was inoculated with about 3 x 10(7) CFU of Enterobacter cloacae JP120 and Alcaligenes eutrophus AEO106(pRO101) per g and incubated in 40-g portions at 17 degrees C in closed or open Erlenmeyer flasks. In the field-moist soil, selective plating, direct viable counts, and DNA hybridization showed only minor changes in the numbers of E. cloacae and A. eutrophus cells with time (14 days), and the results obtained with the three detection methods generally agreed. In the air-dried soil, the majority of both bacteria were found as intact DNA-carrying cells that were neither culturable nor viable by the methods employed in this study. The numbers of culturable E. cloacae and A. eutrophus cells dropped to 10(5) and 10(2) CFU/g, respectively, 2 h after inoculation. Direct viable counts showed that only about 1% of the cells detected by immunofluorescence microscopy were viable, but a fraction of viable nonculturable cells of both bacteria was present. A. eutrophus did not tolerate desiccation as well as E. cloacae. Only a minor fraction of the two test organisms regained their culturability or viability after rewetting of the air-dried soil; the number of total heterotrophic culturable bacteria, however, increased more than 10-fold and reached 73% of the level found in the field-moist soil at day 14.     

Escherichia coli O26 detection from foods using an enrichment procedure and an immunomagnetic separation method

We found effective enrichment procedures for detecting Escherichia coli O26 in foods using methods that are used for E. coli O157. Ground beef or radish sprouts inoculated with approximately 6 colony-forming units of E. coli O26 were homogenized in 225 ml of various broths. After static incubation at 37 degrees C or 42 degrees C for 6 h or 18 h, we isolated the inoculated bacterium by plating onto Rainbow Agar O157 with novobiocin. In combination with the immunomagnetic separation method, E. coli O26 was isolated from all samples by using enrichment in tryptone soy broth at 37 degrees C for 6 h and in modified E. coli broth with novobiocin (mEC + n) at 42 degrees C for 18 h in ground beef and radish sprouts, respectively. Enrichment in mEC + n at 42 degrees C for 18 h was effective for isolating both E. coli O26 and E. coli O157 from both ground beef and radish sprouts.     

Simple method to observe the adaptive response of Listeria monocytogenes in food

A simple, novel method for determining stress-adaptive response of Listeria monocytogenes in food systems is presented. The method involves plating samples on Listeria-selective agar (LSA) acidified to pH 5.25 with incubation at 36 degrees C for 60 h to detect acid adaptation and plating on LSA with 70 gl-1 NaCl and incubation at 7 degrees C for 7 d to detect cold-osmotic adaptation. Adapted cells produced larger colonies (> 1 mm) under these conditions than unadapted cells. Scot A (97%) and Brie-1 (100%) cells incubated in milk at pH 5 for 3 h manifested the acid-adapted colony type compared with 6% and 21% of viable cells in the unstressed control population. After a 5-d adaptation period at 4 degrees C in milk with 80 gl-1 salt, 29% of Scot A and 91% of Brie-1 viable cells exhibited the adapted colony type compared with < 1% of the unstressed control population. Stress-adapted L. monocytogenes were isolated from soft cheese held for 42 d at 10 C.     

Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures

Cells of Salmonella typhimurium LT2 were dehydrated on hydrophobic membranes (Millipore FGLP2500) placed in a controlled atmosphere chamber held at 57% equilibrium relative humidity (ERH) and 37°C. Dehydration for 48 h under the above conditions increased the heat resistance of Salm. typhimurium LT2 when measured as the surviving fraction after a heat challenge of 135°C for 30 min. Results also showed that little or no death occurred during heat challenges of 1 h at temperatures of up to 100°C. The survival of Salm. typhimurium LT2 was measured as the ability to form colonies on solid media tryptone soy broth plus 1.2% agar (TSBA) after 24 h at 37°C. Incorporation of sodium pyruvate, at a concentration of 0.2% into the recovery medium, did not enhance the recovery of heated Salm. typhimurium LT2. Dehydrated cells of S. typhimurium LT2 showed a triphasic death curve. Increasing the period of dehydration from 48 h to 34 d, reduced initial numbers due to die off but did not alter the shape of the subsequent survival curve. and accepted 22 June 1989     

Optimizing enrichment conditions for the isolation of Escherichia coli O157 in soils by immunomagnetic separation

AIMS: To compare a range of enrichment broths and enrichment temperatures for the isolation of Escherichia coli O157 by immunomagnetic separation (IMS) from sandy, loam and clay soils. METHODS AND RESULTS: Soils were spiked with cocktails of four atoxigenic strains of E. coli O157 and four strains of commensal E. coli. The organisms were stressed by subjecting soils to cycles of freeze/thawing, followed by drying at 20 degrees C for up to 4 days. Nine enrichment broths were trialled based on buffered peptone water, tryptone soya broths and EC broths supplemented with a range of selective additions. Enrichments were incubated for 6 h and assessed by target recovery after IMS on cefixime tellurite sorbitol MacConkey agar (CTSMAC) incubated at 37 degrees C for 24 h. A comparison of enrichment temperatures (37 and 42 degrees C) was also performed. Buffered peptone water (with or without vancomycin) and tryptone soya broth (with or without novobiocin) gave significant increases in recovery of E. coli O157 compared to others tested. In addition, broths incubated at 42 degrees C were superior to those at 37 degrees C for the recovery of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that sub-lethally damaged E. coli O157 surviving in soil can be sensitive to antimicrobial additions. The choice and concentration of these additions is vitally important to optimize target recovery. Some IMS protocols, established for the isolation of E. coli O157, may be unsuitable for the examination of soil samples.     

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.     

The effects of elevated temperatures and various time-temperature combinations on the development of Brachiola (Nosema) algerae N. Comb. in mammalian cell culture

Nosema algerae Vávra and Undeen 1970, a microsporidian known to cause infection in mosquitoes, develops in mammalian cell cultures at 24-35 degrees C and in the tails and footpads of athymic mice. More recently it has been reported to grow at 38 degrees C in human cell culture. The present study is a two-part temperature/development examination. The first part examines the development of N. algerae in rabbit kidney cell culture at 29 degrees C, which permits the formation of functional spores within 72 h, and compares the effect of elevated temperatures (36.0, 36.5, 37 degrees C) on parasite development. At these elevated temperatures, N. algerae infects but undergoes only one or two proliferative divisions, with no evidence of sporogony by 72 h post-inoculation. During this time, however, the host cells continue to divide resulting in fewer infected cells over time and giving the appearance of a diminished parasitemia. Additionally, at 37 degrees C some organisms degenerate/hibernate by 72 h while others remain viable/active. It is not until 96 h that the parasites appear in large clusters of proliferative stages in the few host cells that are infected. By 120 h post-inoculation, proliferative cells, sporoblasts, and early spores are observed. These results suggest that elevated temperatures impede proliferation rates and the onset of sporogony. The second part of this study evaluates developmental changes in N. algerae when incubation temperatures and times are varied during parasite growth, resulting in abnormal parasite morphology. These abnormalities were not present when parasites were grown at constant temperature (29-37 degrees C). This report demonstrates that N. algerae can successfully develop at high temperatures (37 degrees C), justifying its taxonomic relocation to the genus Brachiola.     

The cryobiology of rat and human dendritic cells: preservation and destruction of membrane integrity by freezing

Dendritic cells (DCs) are now regarded as specialized leucocytes with distinctive morphological and functional characteristics as accessory or stimulator cells for many lymphocyte responses. While knowledge of the response of other leucocytes (e.g., lymphocytes, macrophages, and granulocytes) to freezing and thawing has been established for some years, an understanding of the cryobiological properties of DCs has not, hitherto, been determined specifically. Such information is important both for establishing procedures for the long-term storage of these cells for use in immunological procedures and for defining freezing conditions that might selectively kill DCs in attempts to modulate the immunogenicity of transplantable tissues during cryopreservation. Preparations of rat and human spleen cells enriched for DCs were frozen to -60 degrees C at one of six cooling rates (0.3, 1.5, 10, 20, 70, or 150 degrees C/min) using a procedure that was established for pancreatic islets with 2 M dimethyl sulfoxide (Me2SO) as the cryoprotectant. Following storage at -196 degrees C the survival of thawed cells was assessed by evaluating both the numbers of cells recovered after the complete process and the membrane integrity of the recovered cells using a supravital fluorescent probe assay. Survival profiles for DCs showed a dependence upon cooling rate similar to other lymphoid cells but DCs were more sensitive to freezing injury than either lymphocytes or macrophages: Optimum survival (75% recovery of numbers and 57% membrane integrity) of rat DCs was achieved by slow cooling (0.3 degrees C/min). Optimal recovery of human DCs was significantly higher (83% recovery of numbers and 72% membrane integrity) after cooling at either 0.3 or 1.5 degrees C/min. The viable yield of DCs from both species declined abruptly as cooling rate was increased, with less than 10% survival after cooling at 20 degrees C/min and negligible survival after cooling at 70 degrees C/min or greater. Analysis of variance of the survival data showed that the response of DCs to freezing and thawing was significantly different (P less than 0.005) from that of either lymphocytes or macrophages, thus providing additional evidence that DCs are distinct from other leucocytes, especially macrophages. This study defines conditions that either will provide effective cryopreservation of DCs for immunological purposes or are most likely to bring about their inactivation in cryobiological approaches to modulating tissue immunogenicity.     

The Recovery of Sublethally Injured Escherichia coli from Frozen Meat

Sublethal injury to Escherichia coli , measured as the inability of surviving cells to grow on media containing bile salts, was monitored during frozen storage on meat at —5, —10 and —20° C. More rapid increases in injury occurred at the higher subzero temperatures and log phase cells were more susceptible than those in the stationary phase of growth. Repair of injury in non-selective liquid media took between 2 and 6 h at 25° and was often accompanied by an increase in total viable count. Incubation for a fixed period in broth was, therefore, unsuitable for the quantitative recovery of freeze-injured Esch. coli. Resuscitation on membrane filters avoided confusing repair of injury with multiplication of uninjured or repaired cells. The mean recovery of injured cells following incubation on membranes for 4 h at 35°C on tryptone soya agar, was 94%.     

Comparison of microbial numbers in soils by using various culture media and temperatures

The influence of different media and incubation temperatures on the quantification of microbial populations in sorghum, eucalyptus and forest soils was evaluated. Microbial growth was compared by using complex (tryptone soybean agar, TSA, casein-starch, CS, and Martin) and saline (Thorton, M3, Czapeck) media and incubation temperatures of 25 and 30 degrees C. Higher numbers of total bacterial and fungal colony-forming units (CFU) were observed in sorghum soils, and of spore-forming and Gram-negative bacteria in forest soils than other soils. Actinomycetes counts were highest in forest soil when using CS medium at 30 degrees C and in sorghum soil at 25 degrees C in M3 medium. Microorganism counts were dependent on the media and incubation temperatures. The counts at temperatures of 30 degrees C were significantly higher than at 25 degrees C. Microbial quantification was best when using TSA medium for total and spore-forming bacteria, Thorton for Gram-negative bacteria, M3 for actinomycetes, and Martin for fungi.     

A comparative study of the formation of extracellular proteins by Aeromonas salmonicida at two different temperatures

Aeromonas salmonicida was grown in a supplemented 3% (w/v) tryptone soya broth medium at 10 degrees C, a temperature at the lower end of the range over which furunculosis has been observed to occur in the field, and 25 degrees C, the optimum temperature for growth. Similar bacterial densities in the range 2.35 +/- 0.05 mg dry wt/ml were achieved in the two cultures at the beginning of the stationary phase of the growth cycle, after 125 h at 10 degrees C and 18 h at 25 degrees C. At this point, at the higher temperature 1.5 times more exoprotein was formed, 80 +/- 2.8 micrograms/ml compared with 54 +/- 1.7 micrograms/ml. Exoprotein contained the same proportion of haemolysin at both temperatures and twice as much protease at the higher temperature. The most marked difference was in an unidentified 100 kD protein which was formed in a 10-fold greater amount at 10 degrees C.