Evaluation of a rapid microbial detection method via phage lytic amplification assay coupled with Live/Dead fluorochromic stains

AIMS: To develop a method for rapid detection of bacteria via bacteriophage amplification coupled with exogenous fluorochromic stains. METHODS AND RESULTS: A method for the rapid detection of bacteria was developed which consisted of exposing the sample suspected to contain target cells to host-specific phage. After at least one infection cycle, bacteria known to be infected by the phage (helper cells) were added and the number of nascent phage particles was estimated using the Live/Dead BacLight Bacterial Viability kit. Using Pseudomonas aeruginosa, it was shown that the dead helper cell population following phage infection was proportional to the initial number of target cells present in the original sample. Approximately 1 x 10(1) CFU per ml of P. aeruginosa could be detected within 4 h without the need for enrichment. CONCLUSIONS: The phage lytic amplification assay coupled with exogenous fluorochromic stains was able to detect approx. 1 x 10(1) CFU per ml of the target bacterium within 4 h. SIGNIFICANCE AND IMPACT OF THE STUDY: A method to detect low number of bacterial cells in a sample within 4 h without the need for enrichment was developed.     

Some Morphological Types of Bacteriophages in Bovine Rumen Contents

Six morphological types of bacteriophage were found in bovine rumen contents. Minimal total phage count was 5 x 10(7) per ml of rumen fluid.     

Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms

To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n = 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 x 10(8) purified phage per ml for 24 h at 37 degrees C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10(10) and 10(12) phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.     

Factors Affecting Infection of Protoplasts with Deoxyribonucleic Acid of Actinophage PK-66

To establish a method for transmission of genetic materials in the genus Streptomyces, the conditions of infection of protoplasts of S. kanamyceticus by actinophage PK-66 deoxyribonucleic acid (DNA) were studied. The protoplasts of Streptomyces were prepared by treatments with lysozyme and trypsin. The infectivity of the phage DNA was enhanced by the presence of NaCl in the medium. The optimal concentration of the protoplasts for infection with DNA was 7 x 10(7) to 4 x 10(8)/ml. A proportional relationship was found between the infectivity and the DNA concentration within a certain range. The maximal production of mature phage was achieved after 19 hr of incubation. The number of phage propagated in the infection mixture reached 10(4) plaque-forming units per ml under the appropriate conditions. The phage DNA infected not only protoplasts prepared from S. kanamyceticus but also those prepared from S. violaceoniger and S. acidomyceticus, which were resistant to intact phage PK-66.     

Transfection of Escherichia coli Spheroplasts I. General Facilitation of Double-Stranded Deoxyribonucleic Acid Infectivity by Protamine Sulfate

The addition of 25 mug of protamine sulfate per ml to lysozyme-ethylenediamine-tetraacetic acid spheroplasts of Escherichia coli stimulates transfection not only for T1 phage deoxyribonucleic acid (DNA; Hotz and Mauser, 1969) but also for the following phage DNA species: lambda, 10,000-fold to an efficiency of 10(-3) infective centers per DNA molecule; phiX174 replicative form, 300-fold to an efficiency of 5 x 10(-2); fd replicative form, 300-fold to 10(-6); T7, 300-fold to 3 x 10(-7). Three native phage DNA species were not infective at all in the absence of protamine sulfate but were infective in the presence of protamine sulfate with the following efficiencies: T4, 10(-5); T5, 3 x 10(-6); and P22, 3 x 10(-9). The effect of protamine sulfate is specific for double-stranded DNA. The application of infectivity assays to the study of phage DNA replication, recombination, prophage integration, prophage excision, and interspecies transfection are discussed.     

Effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes

This study was carried out to investigate the effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes. The concentrations of frozen-thawed sperm were 0.2 x 10(7), 2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml, respectively. Culture media were NCSU-23, HEPES-buffered (25 mM) NCSU-23, PZM-3 and PZM-4, respectively. Increasing the sperm concentration from 0.2 x 10(7) to 2 x 10(7)/ml, significantly increased the penetration rate. Also, increasing the sperm concentration from 20 x 10(7) to 200 x 10(7)/ml increased the penetration rate from 62.1% to 69.9%, respectively, with no differences between these two concentrations. A similar pattern was observed for polyspermic penetration and male pronucleus formation. The mean number of sperm per oocyte significantly increased in the 20 x 10(7)/ml and again in the 200 x 10(7)/ml sperm concentrations. The percentage of blastocysts from cleaved oocytes at the 2 x 10(7)/ml sperm concentration was significantly higher than that at the 0.2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml sperm concentrations. The percentage of blastocysts from cleaved oocytes and the cell numbers per blastocyst were significantly higher in the HEPES-buffered NCSU-23 culture medium than in the NCSU-23, PZM-3 and PZM-4 culture media under a gas atmosphere of 5% CO2 in air.     

Detection of Vibrio parahaemolyticus in shellfish by use of multiplexed real-time PCR with TaqMan fluorescent probes

We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 10(4) CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3x10(9) CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.     

Detection and precipitation of hepatitis B core antigen using a fusion bacteriophage

The nucleocapsids of hepatitis B virus (HBV) are made of 180 or 240 subunits of core proteins or known as core antigens (HBcAg). A fusion bacteriophage bearing the WSFFSNI sequence that interacts tightly to HBcAg was employed as a diagnostic reagent for the detection of the antigen using the phage-enzyme-linked immunosorbent (phage-ELISA), dot blot and immunoprecipitation assays. The results from phage-ELISA and dot blot assay showed that as low as 10 ng of HBcAg can be detected optimally by 1.0x10(12) pfu/ml fusion M13 bacteriophage. The sensitivity of the dot blot assay corresponds with that of the phage-ELISA. HBcAg in HBV positive serum samples can also be detected using the fusion phage via the phage-ELISA and phage-dot blot assay. The phage cross-linked to cyanogen bromide (CNBr) activated agarose can also be used to precipitate HBcAg in bacterial lysate. The optimum amount of phage needed for cross-linking to 1 g of agarose is about 7.0x10(6) pfu/ml which could also precipitate purified and unpurified HBcAg in bacterial lysate. This study demonstrates the potential of fusion bacteriophage bearing the sequence WSFFSNI as a diagnostic reagent and a ligand for the detection and purification of HBcAg respectively.     

Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents

AIM: To incorporate into the lambda phage genome, a luxI-based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux-based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. METHODS AND RESULTS: The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage-based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL-specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target-specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 x 10(8) to 1 CFU ml(-1) within 1.5-10.3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 x 10(8) to 130 CFU ml(-1) was achieved within 2.6-22.4 h, respectively. CONCLUSIONS: The initial feasibility of binary phage-based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. SIGNIFICANCE AND IMPACT OF STUDY: With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.     

Effects of heparin and sperm concentration on cleavage and blastocyst development rates of bovine oocytes inseminated with flow cytometrically-sorted sperm

The objective was to determine the optimal concentration of heparin for sperm capacitation, as well as the optimal sperm concentration for in vitro fertilization using flow cytometrically-sorted sperm from individual bulls. A total of 5327 bovine oocytes and sperm from four bulls were examined. Oocytes from slaughterhouse ovaries were matured in TCM199 for 22-24 h. Flow cytometrically-sorted sperm as well as unsorted control sperm from the same bulls were cryopreserved. For sperm from each of the four bulls, oocytes were inseminated in a three-by-three factorial design plus one control group (three heparin concentrations: 0, 2, and 10 microg/ml and three sperm concentrations: 0.5 x 10(6), 1.5 x 10(6), and 4.5 x 10(6) ml(-1); 10 microg/ml of heparin and 1.5 x 10(6) ml(-1) of sperm were used for the unsorted control). Presumptive zygotes were cultured in chemically defined media, CDM-1 and CDM-2 for 52-54 h and 96 h, respectively. Samples of about 10 oocytes from each of the 10 treatment groups per replicate were fixed at 18-20 h after insemination to determine sperm pronuclei formation and polyspermy. Increased polyspermy resulted as heparin and sperm concentrations increased (P < 0.05). A higher rate of polyspermy was found in oocytes inseminated with unsorted control sperm compared with sorted sperm (P < 0.05). Sperm of one of four bulls tested required no heparin and lower concentration (0.5 x 10(6) ml(-1)) to obtain optimal cleavage and blastocyst rates while optimal parameters for another bull were higher heparin (10 microg/ml) and sperm concentrations (4.5 x 10(6) ml(-1)). Optimal parameters for the other two were intermediate levels of heparin and sperm. Sperm appeared to be partially capacitated during the flow cytometric-sorting process used for sex pre-determination. When heparin and sperm concentrations were optimized for individual bulls, blastocyst production per oocyte was similar for sorted and unsorted sperm for three of the four bulls studied.     

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in milk as assessed by FASTPlaqueTB phage assay and culture

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in Middlebrook 7H9 broth and whole and semiskim milk was investigated using a laboratory-scale UV machine that incorporated static mixers within UV-penetrable pipes. UV treatment proved to be less effective in killing M. avium subsp. paratuberculosis suspended in milk (0.5- to 1.0-log(10) reduction per 1,000 mJ/ml) than that suspended in Middlebrook 7H9 broth (2.5- to 3.3-log(10) reduction per 1,000 mJ/ml). The FASTPlaqueTB phage assay provided more rapid enumeration of surviving M. avium subsp. paratuberculosis (within 24 h) than culture on Herrold's egg yolk medium (6 to 8 weeks). Despite the fact that plaque counts were consistently 1 to 2 log(10) lower than colony counts throughout the study, UV inactivation rates for M. avium subsp. paratuberculosis derived using the phage assay and culture results were not significantly different (P = 0.077).     

Production of Bacteriophage by Temperature-Sensitive Sporulation Mutants of Bacillus cereus T

Five temperature-sensitive sporulation mutants of Bacillus cereus T have been isolated. These mutants are blocked at stage 0 of sporulation at the restrictive temperature (37 C) but are able to sporulate at nearly normal frequencies at the permissive temperature (26 C). A bacteriophage that forms a stable lysogen in the parent strain is induced at increased frequencies in the mutants. This induction is accompanied, in some of the mutants, by a reduction in immunity to the phage. Revertants, selected for their ability to sporulate normally at both temperatures, lose their ability to produce high titers of the phage. In addition to this lytic phage, an apparently defective phage has been found in lysates of the mutants. Strains cured of the plaque-forming phage still carry the defective phage. Comparisons of physical and biological properties of the plaque-forming phage with those of the two Bacillus cereus phages most similar to it have shown that this phage is not identical to either of them. The maximal titer of phage produced in cultures of the parent strain is about 10(3) plaque-forming units (PFU) per ml at both temperatures. The maximal titers of phage produced by the mutant are 4 x 10(9) PFU/ml at 37 C and 7 x 10(8) PFU/ml at 26 C. Both mutant and parent strains release over 90% of the phage they produce after the onset of stationary phase.     

Estimation of ruminal bacteriophage numbers by pulsed-field gel electrophoresis and laser densitometry.

To investigate phage activity in the rumen, a method for quantifying phage has been developed. By differential centrifugation and ultrafiltration, phage particles were separated and concentrated from ruminal fluid. Linear double-stranded DNA from this fraction containing predominantly tailed phage was isolated and separated by size, using pulsed-field gel electrophoresis (PFGE). Laser densitometry of gel photographs allowed the numbers of phages with DNA in each size region to be calculated and, therefore, the total numbers per milliliter of ruminal fluid to be estimated. Phage numbers were estimated to be between 3 x 10(9) and 1.6 x 10(10) particles ml of ruminal fluid-1. The phage population, as gauged by the appearance of DNA on PFGE gels, had two major components. A broad region of DNA between 30 and 200 kb was always present on PFGE gels. It appears this region comprises DNA from a great many different phages and would include most of the temperate phages. In addition, discrete DNA bands ranging in size from 10 to 850 kb were frequently observed. DNA from one such band, of 12 kb in size, was shown to consist primarily of a single DNA type, suggesting that it originated from a specific phage. It is postulated that the discrete bands are due to epidemics or blooms of phage activity from specific, probably lytic, phages. The method that has been developed will greatly enhance future investigations into the interactions between the ruminal phage population, the ruminal bacterial population, and animal nutrition and growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phage immobilized magnetoelastic sensor for the detection of Salmonella typhimurium

In this article, a phage-based magnetoelastic sensor for the detection of Salmonella typhimurium is reported. Filamentous bacteriophage specific to S. typhimurium was used as a biorecognition element in order to ensure specific and selective binding of bacteria onto the sensor surface. Phage was immobilized onto the surface of the sensors by physical adsorption. The phage immobilized magnetoelastic sensors were exposed to S. typhimurium cultures with different concentrations ranging from 5x10(1) to 5x10(8) cfu/ml, and the corresponding changes in resonance frequency response of the sensor were studied. It was experimentally established that the sensitivity of the magnetoelastic sensors was higher for sensors with smaller physical dimensions. An increase in sensitivity from 159 Hz/decade for a 2 mm sensor to 770 Hz/decade for a 1 mm sensor was observed. Scanning electron microscopy (SEM) analysis of previously assayed biosensors provided visual verification of frequency changes that were caused by S. typhimurium binding to phage immobilized on the sensor surface. The detection limit on the order of 10(3) cfu/ml was obtained for a sensor with dimensions 1x0.2x0.015 mm.     

Enumeration of Bacteriophages and Host Bacteria in Sewage and the Activated-Sludge Treatment Process

Bacteriophage populations in an activated-sludge sewage treatment plant were enumerated. A newly developed assay for quantitation of total phages, employing direct electron microscopic counts, was used in conjunction with the plaque assay. The total concentration of phages was significantly higher in reactor mixed liquor and effluent than in influent sewage, indicating a net production of phages within the reactor. Maximum total phage concentrations in the fluid phase of sewage, activated-sludge mixed liquor, and reactor effluent were 2.2 × 10⁷, 9.5 × 10⁷, and 8.4 × 10⁷/ml, respectively. Conditions were optimized for isolation of predominant heterotrophic aerobic bacteria from sewage and mixed liquor. Blending at ice water temperatures was superior to ultrasound or enzyme treatments for maximum release of viable bacteria from microbial floc. A solidified extract of mixed liquor was superior to standard media for cultivating maximum numbers of heterotrophic bacteria. The highest culture counts for sewage and mixed liquor were 1.4 × 10⁷ and 1.3 × 10⁹/ml, respectively, which represented only 3 and 6.8% of the total microscopic cell counts. Only 3 out of 48 dominant bacterial isolates from either mixed liquor or sewage were hosts for phages present in the system. The sum of phage populations infecting these three hosts accounted for, at best, 3.8% (sewage) and 0.2% (mixed liquor) of the total number of phages present. Generally, specific phage titers were lower in mixed liquor than in sewage, indicating that these hosts were not responsible for the net production of phages in the reactor. This study emphasizes the limitations of the plaque assay for ecological studies of phages, and it suggests that bacteria responsible for phage production in activated-sludge mixed liquor are either minor components of the heterotrophic population, floc-producing strains, or members of other physiological groups.     

Significance of Bacteriophages for Controlling Bacterioplankton Growth in a Mesotrophic Lake

Bacterium-specific viruses have attracted much interest in aquatic microbial ecology because they have been shown to be about 10 times more abundant than planktonic bacteria. So far most of the studies of interactions of planktonic bacteria and viruses have been done in marine environments, and very little is known about these interactions in lakes. Therefore, we studied phage proliferation in Lake Constance, a large mesotrophic lake in Germany. We enumerated bacteria and quantified the fraction of bacteria with mature intracellular phage particles and the number of free viruses by transmission electron microscopy. Between the end of March and early August 1992, peaks of bacterial abundance were followed in 1 to 2 weeks by peaks in the fraction of bacteria containing visible phage particles (0 to 1.7%) and in the number of free viruses (1 x 10(sup7) to 4 x 10(sup7) ml(sup-1)). We estimated that 1 to 17% +/- 12% of all bacteria were phage infected, implying that phage-induced mortality was <34% +/- 24% of total mortality. A direct comparison between phage-induced mortality, the net decrease of bacterial numbers, and bacterial growth rates indicated that phage-induced mortality accounted for <11% of total bacterial mortality during the phytoplankton spring bloom and 18 to 21% following the bloom. Estimated burst sizes ranged from 21 to 121 phages. Phage production rates of 0.5 x 10(sup6) to 2.5 x 10(sup6) ml(sup-1) day(sup-1) accounted for 70 to 380% of the observed net increase rates of free phages, implying high rates of simultaneous phage decay. The cyclic dynamics between bacteria and phages and the varying size structure of the intracellular mature phage particles suggested that phage infection was important in structuring the bacterial host assemblage during the study period.     

Characteristics of φT, the Temperate Bacteriophage Carried by Bacillus megaterium 899a

Phage T was the only phage observed in lysates of Bacillus megaterium 899a induced with mitomycin C, 0.35 mug/ml. The phage adsorbed slowly to its host in nutrient agar, giving rise to plaques of varying sizes and turbidity. Only clear plaques were observed when the phage and host cells were preincubated in an adsorption buffer and plated under optimum conditions. Plaque turbidity was caused by either the addition of 0.5 x 10(-2) to 1.0 x 10(-2) M CaCl(2) to the phage assay medium, or by raising the incubation temperature to 34 C. Phage T purified on a CsCl gradient had a density of 1.48 g/ml in CsCl and the extracted phage DNA had a buoyant density in CsCl of 1.6975 g/ml, equivalent to 38.2% guanine plus cytosine. The phage was rapidly inactivated at 75 C and was unstable in the presence of chloroform at 4 C, but it was stable in buffer stored in ice. When stage I sporulating cells were induced with mitomycin C, phage were carried into spores which when germinated lyse with the release of phi T. The burst size on induction of early-log vegetative cells was 52, whereas the burst size of induced T(0) sporulating cells, diluted in fresh medium, was 47 for a sporulating strain and 140 for an asporogenous mutant. A typical phage T had a long, noncontracting tail 240 nm long, 9 to 11 nm wide, with a repeating disk unit along the tail, 4 nm in size center to center. The tail ended in a small disk (15 nm wide) which is presumably for attachment to the host. The hexagonal head measures 68 by 57 nm and is composed of donut-shaped units 9 nm in diameter.     

Transfection of Lysostaphin-treated Cells of Staphylococcus aureus

After treatment with 1 unit of lysostaphin per ml for 3 min, two strains of Staphylococcus aureus, 233 and PS 44A HJD, were transfected with phenol-extracted deoxyribonucleic acid (DNA) from the staphylococcal bacteriophages, 53 and 44A HJD, respectively. The number of transfected cells was low in both systems, approximately two in 10(7) enzyme-treated cells. There was a saturation effect at high concentrations of DNA; optimal results were obtained at concentrations between 10 to 25 mug/ml. Growth curves and fluctuation tests indicated that cells of strain 44A HJD infected with phage, then converted to protoplasts by a 10-min treatment with lysostaphin, produce only one phage particle and lose their ability to lyse spontaneously in hypertonic media.     

Affinity-selected filamentous bacteriophage as a probe for acoustic wave biodetectors of Salmonella typhimurium

Proof-in-concept biosensors were prepared for the rapid detection of Salmonella typhimurium in solution, based on affinity-selected filamentous phage prepared as probes physically adsorbed to piezoelectric transducers. Quantitative deposition studies indicated that approximately 3 x 10(10)phage particles/cm(2) could be irreversibly adsorbed for 1 h at room temperature to prepare working biosensors. The quality of phage deposition was monitored by fluorescent microscopy. Specific-bacterial binding resulted in resonance frequency changes of prepared sensors, which were evaluated using linear regression analysis. Sensors possessed a rapid response time of <180 s, had a low-detection limit of 10(2)cells/ml and were linear over a range of 10(1)-10(7)cells/ml with a sensitivity of 10.9 Hz per order of magnitude of S. typhimurium concentration. Viscosity effects due to increasing bacterial concentration and non-specific binding were not significant to the piezoelectric platform as confirmed by dose-response analysis. Phage-bacterial binding was confirmed by fluorescence and scanning electron microscopy. Overall, phage may constitute effective bioreceptors for use with analytical platforms for detecting and monitoring bacterial agents, including use in food products and possibly biological warfare applications.     

Photodynamic Action of Proflavine on Coliphage T3 I. Kinetics of Inactivation

The photodynamic inactivation of coliphage T3 was studied over a wide range of concentrations of the dye proflavine. With 2 x 10(7) phage/ml, two modes of inactivation were observed. Between 0.25 and 12 to 13 mug/ml, inactivation was biphasic. There was an initial first-order inactivation (Rx1) which became temporally associated with an apparently multiorder process (Rx2) at higher light doses. Dye concentrations above 12 to 13 mug/ml showed only two-target inactivation curves (Rx3), except at high dye concentrations where processes kinetically identical to Rx1 and Rx2 reappeared. Rx2 showed a normal rectangular hyperbolic saturation curve but Rx1 and Rx3 appeared to saturate prematurely. The saturation behavior of Rx1 and Rx2 was independent of phage concentration, but Rx3 was lost at phage titers above 2 x 10(7)/ml. No dark inactivation was seen with Rx1 and Rx2 subsequent to a period of illumination. With Rx3, an exponential dark inactivation was seen for at least 1 hr after a period of illumination. The dye-phage system equilibrated immediately, at any temperature, at proflavine concentrations where Rx1 and Rx2 occurred. With Rx3, prolonged equilibration times were necessary. Moreover, there was a temperature effect. The rate of inactivation at equilibrium was temperature-dependent, whereas the initial rate at which equilibrium was approached was essentially temperature-independent.