The relationship between the detection of mRNA and cellular viability in Escherichia coli was investigated in cells killed by heat or ethanol. Reverse transcription-PCR (RT-PCR) methods were developed for detecting mRNA from rpoH,groEL, and tufA genes. mRNA from all three genes was detected immediately after the cells had been killed by heat or ethanol but gradually disappeared with time when dead cells were held at room temperature. In heat-killed cells, some mRNA targets became undetectable after 2 to 16 h, whereas after ethanol treatment, mRNA was still detected after 16 h. In contrast, 16S rRNA was detected by RT-PCR in all samples containing dead cells and did not disappear during a subsequent incubation of 16 h at room temperature. Of the different types of nucleic acid, mRNA is the most promising candidate for an indicator of viability in bacteria, but its persistence in dead cells depends on the inactivating treatment and subsequent holding conditions.

No abstract

Thermograms of whole cells of Escherichia coli obtained by differential Scanning calorimetry contained ten main peaks (denoted& l, m, , m2, m3, n,p, q, r and s) occurring at temperatures of approximately 25,54,61,71,76,81, 95, 105, 118 and 124 "C, respectively. After cooling to 5 "C and reheating, peaks denoted fr, m, and p r were observed at 23,73 and 94 "C, respectively. By examining thermograms of different cell fractions we have identified the following thermal denaturation events. During primary heating there is a broad endotherm ( f ) beginning below 20 "C and extending to just above 40 "C that is caused by melting of membrane lipids. Superimposed on'this is an exothermic process associated with a change of state of the peptidoglycan. The first irreversible denaturation event occurs just above 47 "C, associated with the onset of denaturation of the 30s ribosomal subunit and soluble cytoplasmic proteins. Ribosome melting is a complex process occurring between 47 and 85 "C and is characterized by peaks m, , m2 and n. Peak m3 at 75-76 "C is of unknown identity but may possibly represent melting of tRNA.Peak p at 95 "C results from melting of a portion of the cellular DNA combined with denaturation of a cell wall component. Peak q at 105 "C is multicomponent and may be caused by melting of a different region of DNA together with denaturation of another cell wall component. The complex events denoted r and s at 118 and 125 "C, respectively, are associated with denaturation of a component of the cell envelope, and possibly also of DNA.Following cooling and reheating there is a broad endotherm with a maximum at 23 "C caused by remelting of membrane lipid and a very broad endotherm extending between 40 and lo0 "C caused by the remelting of ribosomal RNA. Peak p , at 94 "C is caused by the melting of reannealed DNA. Additional features not appearing in whole cells were evident in some cell fractions. These observations should allow us to distinguish events that may lead to loss of viability from those that do not. IntroductionThe inactivation of micro-organisms by heat forms the basis of large sections of the food and pharmaceutical industries. Not surprisingly, the factors affecting microbial heat-resistance and the nature of heat damage are of continuing practical and scientific concern.The mechanisms of thermal inactivation of microorganism have commonly been investigated by using biochemical methods to examine the effect of heat on particular cell structures or processes. Membranes, nucleic acids and certain enzymes have all been identified as cellular sites of heat injury and, in some cases, information is available on the molecular nature of heat damage (Tomlins & Ordal, 1976 Gould, 1989). However, despite this large body of information, we still do not understand precisely how micro-organisms are killed by heat. 1984;Differential scanning calorimetry (DSC) enables denaturation and other thermal processes that occur when samples are heated to be detected and recorded as a time-temperature sequence. The techn...

The relationship between membrane damage and loss of viability following pressure treatment was examined in Escherichia coli strains C9490, H1071, and NCTC 8003. These strains showed high, medium, and low resistance to pressure, respectively, in stationary phase but similar resistance to pressure in exponential phase. Loss of membrane integrity was measured as loss of osmotic responsiveness or as increased uptake of the fluorescent dye propidium iodide. In exponential-phase cells, loss of viability was correlated with a permanent loss of membrane integrity in all strains, whereas in stationary-phase cells, a more complicated picture emerged in which cell membranes became leaky during pressure treatment but resealed to a greater or lesser extent following decompression. Strain H1071 displayed a very unusual pressure response in stationary phase in which survival decreased to a minimum at 300 MPa but then increased at 400 to 500 MPa before decreasing again. Membranes were unable to reseal after treatment at 300 MPa but could do so after treatment at higher pressures. Membrane damage in this strain was thus typical of exponential-phase cells under low-pressure conditions but of stationary-phase cells under higher-pressure conditions. Heat shock treatment of strain H1071 cells increased pressure resistance under low-pressure conditions and also allowed membrane damage to reseal. Growth in the presence of IPTG (isopropyl-␤-D-thiogalactopyranoside) increased resistance under highpressure conditions. The mechanisms of inactivation may thus differ at high and low pressures. These studies support the view that membrane damage is an important event in the inactivation of bacteria by high pressure, but the nature of membrane damage and its relation to cell death may differ between species and phases of growth.