An in-use study of the relationship between bacterial contamination of food preparation surfaces and cleaning cloths

The relationship between bacterial contamination of food preparation surfaces and the cleaning cloths associated with these surfaces was investigated in a busy college kitchen. Cleaning cloths used in conjunction with detergent became heavily contaminated within hours of first use. Following 'cleaning' with these cloths with detergent, both surfaces and cloths were found to be more heavily contaminated and there was evidence of contamination transfer to and from cloths and surfaces. Following 'cleaning' with cloths impregnated with quaternary ammonium disinfectant, there was a significant reduction in contamination on both surfaces and cloths, together with a reduction in the numbers of enterobacteria and pseudomonads. The results suggest that significant improvements in kitchen hygiene could be achieved by ensuring that contaminated cloths are not used for food preparation activities.     

Role of EDTA in a simple method for determining toxicity using a bacterial indicator organism

A method to determine toxicity using a bacterium as the indicator organism previously developed (Botsford 1998) perceives most divalent cations as being toxic. Mercury is perceived as the most toxic, followed by cadmium, zinc and copper. It was found that adding 2.5 m EDTA to the reaction would relieve the toxicity of the 15 divalent cations tested. This effect does not appear to be simple chelation. One micromolar EDTA eliminated the toxicity of 1.6 m calcium or 0.006 m mercury. Thirty-six chemicals were tested for their toxicity in the presence and absence of 2.5 m EDTA and 25 ppm calcium. Twenty-one were less toxic and two of these, p-aminobenzoic acid and tetrachloroethylene would no longer appear to be toxic according to the assay when these additions were present. Six chemicals had the same toxicity with and without the additions. Nine chemicals were more toxic when the EDTA and calcium were present. This experiment was repeated with six chemicals and ten times the EDTA concentration and ten times the calcium concentration. The toxicity with 10× was compared with the toxicity with 1× the additions. The toxicity of 4 of the six chemicals changed with the higher concentration of EDTA and calcium when the absorbancy values observed in samples with the lower levels were compared with samples with the higher levels. Obviously before EDTA can be added to mitigate the toxicity of divalent cations, it must be determined how much EDTA is required to eliminate the toxicity by the ions present in the sample. Alternatively, if the nature of the contaminating organic chemical is known, it can be determined what the effect of EDTA and the divalent cation present is on the apparent toxicity of the compound.     

An artificial redox coenzyme based on a triazine dye template

A series of nicotinamide-containing compounds based on the structure of a triazine dye, C.I. Reactive Blue 2, which is known to interact at the coenzyme-binding sites of several NAD(P)(H)-dependent dehydrogenases,^1,2 were designed, synthesized, and characterized. The preparation of these compounds is described. Reduction of the coenzyme mimics with sodium borohydride led to an increase in absorption at 356 nm, analogous to the behavior of the natural coenzyme, NAD⁺.³ When incubated with horse liver alcohol dehydrogenase and ethanol at 25°C and pH 9.0, one of the mimics, Blue N-3, was converted into a new compound with an increased absorption at 356 nm and an R_f value on thin-layer chromatography identical to that of the reduced form produced by treatment with sodium borohydride. The oxidized and reduced forms of Blue N-3 could be separated by reverse-phase ion pair high-performance liquid chromatography. This technique could be used to measure the extent of Blue N-3 reduction: Approximately 90 turnovers were calculated for each enzyme active site over a 48-h period. Gas chromatography analysis suggested that ethanol was simultaneously converted to acetaldehyde. Blue N-3 represents the first example of a new generation of potentially inexpensive, stable, and active biomimetic redox coenzymes.     

In situ and laboratory studies of bacterial survival using a microporous membrane sandwich

A new device and procedure for the study of bacterial survival in an aquatic environment are described. The device uses two appressed presterilized microporous membranes to expose a bacterial cell suspension to the environment at a cell concentration that closely resembles those levels found in natural aquatic ecosystems. The device has been used under laboratory controlled conditions and in situ to study and compare bacterial survival times. In laboratory studies, Escherichia coli and Streptococcus faecalis survived the longest at 12 degrees C, pH 5, and in the presence of iron or calcium ions and cysteine. Cells in mid-stationary growth phase survived longer than those in mid- or late-logarithmic phase, whereas those maintained for a year or more as stock cultures survived for shorter period of time than did recent environmental isolates. In situ studies indicate that 5% of the starting number of E. coli and S. faecalis cells may survive longer than 96 h at 16 degrees C in potable lake water, whereas survival times in polluted lake water were approximately 12 h.     

An in situ method for cultivating microorganisms using a double encapsulation technique

The lack of cultured microorganisms represents a bottleneck for advancement in microbiology. The development of novel culturing techniques is, therefore, a crucial step in our understanding of microbial diversity in general, and the role of such diversity in the environment, in particular. This study presents an innovative method for cultivating microorganisms by encapsulating them within agar spheres, which are then encased in a polysulfonic polymeric membrane and incubated in a simulated or natural environment. This method stimulates growth of the entrapped microorganisms by allowing them access to essential nutrients and cues from the environment. It allows for the discovery of microorganisms from dilutions that are 10–100-fold greater than possible with conventional plating techniques. Analysis of microorganisms grown in such spheres incubated in and on a number of different substrates yielded numerous novel ribotypes. For example, spheres incubated on the mucus surface of a Fungiid coral yielded numerous ribotypes, with only 50% sharing similarity (85–96%) to previously identified microorganisms. This suggests that many of the species represent novel ribotypes. Hence, the technique reported here advances our ability to retrieve and successfully culture microorganisms and provides an innovative tool to access unknown microbial diversity.     

In situ and laboratory studies of bacterial survival using a microporous membrane sandwich.

A new device and procedure for the study of bacterial survival in an aquatic environment are described. The device uses two appressed presterilized microporous membranes to expose a bacterial cell suspension to the environment at a cell concentration that closely resembles those levels found in natural aquatic ecosystems. The device has been used under laboratory controlled conditions and in situ to study and compare bacterial survival times. In laboratory studies, Escherichia coli and Streptococcus faecalis survived the longest at 12 degrees C, pH 5, and in the presence of iron or calcium ions and cysteine. Cells in mid-stationary growth phase survived longer than those in mid- or late-logarithmic phase, whereas those maintained for a year or more as stock cultures survived for shorter period of time than did recent environmental isolates. In situ studies indicate that 5% of the starting number of E. coli and S. faecalis cells may survive longer than 96 h at 16 degrees C in potable lake water, whereas survival times in polluted lake water were approximately 12 h.     

Development of a rapid method for detecting bacterial cells in situ using 16S rRNA-targeted probes.

A rapid method for the identification of bacterial cells using 16S rRNA-directed, fluorescently tagged oligonucleotide probes has been developed. The parameters evaluated for their effect on labeling intensity included storage time, type of fixative, time of fixation, treatment time with methanol:formaldehyde and treatment time with borohydride. The results of tests using a variety of microorganisms, both Gram-positive and Gram-negative, are presented. Using this method, cells are spotted onto slides and stored desiccated until hybridized. This method may be especially applicable to environmental samples, which comprise diverse cell types and frequently require storage prior to examination.     

An automated method for determining buoyant density of nucleic acids using a preparative ultracentrifuge

We present a technique for analytical buoyant density sedimentation of nucleic acids which is performed in a preparative ultracentrifuge, in contrast to an analytical ultracentrifuge. Following centrifugation in a preparative rotor, small cylindrical quartz tubes are optically scanned; upon completion of the scan the data are processed immediately by a microcomputer and the buoyant density of the nucleic acid is calculated. Experimental data are presented employing several different deoxyribonucleic acids banded in neutral and alkaline cesium sulfate. Results are independent of rotor speed, location of bands within the gradient, and loading density of the cesium sulfate solution. Derived buoyant density values agree within 0.5% of previously published values.     

An improved boiling method for the preparation of bacterial plasmid and phage DNA

A streamlined, reproducible boiling method for preparing plasmid as well as phage replicative form DNA is described. Both quantity and quality of DNA purified are sufficient for restriction enzyme analysis and sequencing. A small loopful of bacteria will provide enough DNA for several experiments, and multiple samples can be processed and prepared for digestion or sequencing in under 20 minutes. DNA preparations are stable for at least 6 months at −20°C.     

Effect of food residues on norovirus survival on stainless steel surfaces

Background

In households and food processing plants, minute food residues left behind from improper cleaning may influence the survivability of human norovirus on surfaces. In this study, the survivability of norovirus on desiccated food residue-attached stainless steel coupons was investigated.

Methodology/Principal Findings

Using murine norovirus-1 (MNV-1) as a surrogate of human norovirus, the survivability of norovirus was investigated on lettuce, cabbage, or ground pork-attached stainless steel coupons. A 6.2 log MPN/ml of MNV-1 infectivity was completely lost at day 30 in residue-free coupons, whereas only a 1.4 log MPN/ml reduction was observed in coupons with residues. Moreover, the disinfective effect of sodium hypochlorite was reduced when residues were present on the coupons.

Conclusions/Significance

This study revealed that the food residues increased the survivability and the resistance to chemicals of norovirus, indicating the need of thorough cleaning in food processing plants and household settings.     

Fluorescence in situ hybridization: a new method for determining primary sex ratio in ants

The haplodiploid sex determining system in Hymenoptera, whereby males develop from haploid eggs and females from diploid eggs, allows females to control the primary sex ratio (the proportion of each sex at oviposition) in response to ecological and/or genetic conditions. Surprisingly, primary sex ratio adjustment by queens in eusocial Hymenoptera has been poorly studied, because of difficulties in sexing the eggs laid. Here, we show that fluorescence in situ hybridization (FISH) can be used to accurately determine the sex (haploid or diploid) of eggs, and hence the primary sex ratio, in ants. We first isolated the homologue coding sequences of the abdominal-A gene from 10 species of 8 subfamilies of Formicidae. Our data show that the nucleotide sequence of this gene is highly conserved among the different subfamilies. Second, we used a sequence of 4.5 kbp from this gene as a DNA probe for primary sex ratio determination by FISH. Our results show that this DNA probe hybridizes successfully with its complementary DNA sequence in all ant species tested, and allows reliable determination of the sex of eggs. Our proposed method should greatly facilitate empirical tests of primary sex ratio in ants.     

A rapid method for preparation of bacterial plasmids

A method for isolating plasmids from Escherichia coli which requires less than 8 h from cell pellet to purified plasmid essentially free of protein, RNA, and chromosomal DNA is presented. By this procedure, amplified plasmid pBR322 was isolated from E. coli strain RR1. The final product had no detectable protein or RNA, and plasmid comprised approximately 99% of the total DNA. The procedure includes lysozyme treatment in hypertonic solution followed by lysis with a mild detergent in the presence of high salt and an RNase inhibitor--conditions which prevent unfolding of the bacterial nucleoid. After centrifuging out the nucleoid and cell debris, the nucleic acids are selectively precipitated with a neutral solution of sodium trichloroacetate and ethanol. RNA is degraded with RNase and the degradation products and RNase are eliminated through a second trichloroacetate/ethanol precipitation. Finally, the plasmid is resuspended and passed through a nitrocellulose filter to remove aggregates and any residual protein and single-stranded DNA--giving a plasmid preparation suitable for electrophoretic fractionation or cleavage with restriction nucleases.     

A method for in situ embedding of cultured cells grown on plastic surfaces

Summary A method is described for the electron microscopic visualization of contact relationships between monolayer cells and the supporting surface of the plastic culture vessel.