Viability Of Phage Research Articles

Efficacy of Different Encapsulation Techniques on the Viability and Stability of Diverse Phage under Simulated Gastric Conditions.

Salmonella causes a range of diseases in humans and livestock of considerable public health and economic importance. Widespread antimicrobial use, particularly in intensively produced livestock (e.g., poultry and pigs) may contribute to the rise of multidrug-resistant Salmonella strains. Alternative treatments such as bacteriophages have shown promise when used to reduce the intestinal carriage of Salmonella in livestock. However, the digestive enzymes and low pH encountered in the monogastric GI tract can significantly reduce phage viability and impact therapeutic outcomes. This study deployed alginate-carrageenan microcapsules with and without CaCO3 to protect a genomically diverse set of five Salmonella bacteriophages from simulated gastrointestinal conditions. None of the unprotected phage could be recovered following exposure to pH < 3 for 10 min. Alginate-carrageenan encapsulation improved phage viability at pH 2-2.5 after exposure for 10 min, but not at pH 2 after 1 h. Including 1% (w/v) CaCO3 in the formulation further reduced phage loss to <0.5 log10 PFU/mL, even after 1 h at pH 2. In all cases, phage were efficiently released from the microcapsules following a shift to a neutral pH (7.5), simulating passage to the duodenum. In summary, alginate-carrageenan-CaCO3 encapsulation is a promising approach for targeted intestinal delivery of genomically diverse Salmonella bacteriophages.

Fabrication of gelatin/silk fibroin/phage nanofiber scaffold effective against multidrug resistant Pseudomonas aeruginosa

Objective The alarming rise of multi-drug resistant (MDR) Pseudomonas aeruginosa has prompted the World Health Organization to consider it a serious threat to human health. Although phage (Phg), an effective antibacterial treatment option, can maintain long-term infectivity via lyophilized storage, freeze-drying can be expensive and time-consuming. Thus, we propose electrospun gelatin/fibroin (G/F) nanofibrous formulation for dehydrating and storing phage against MDR P. aeruginosa. Significance The formulation of phage within the nanofibrous structure of the electrospun G/F scaffold would result in antimicrobial activity against MDR P. aeruginosa leading to enhanced wound healing. Methods Phg effective against MDR P. aeruginosa was isolated, characterized and loaded within G/F nanofibers by electrospinning. Morphology, crystallinity and thermal stability as well as the antimicrobial activity and the biocompatibility of the developed G/F/Phg nanofibers were determined. Results Phg-loaded G/F nanofibers revealed an amorphous structure with good thermal stability at temperatures below 300 °C and exhibited effective antibacterial activity against MDR P. aeruginosa with ∼2 log reduction in the bacterial count which increased to ∼4 log reduction in bacterial count after 16 h as compared to both the G/F nanofibers and the negative control. Lack of cytotoxic effects on cultured fibroblasts supported the biocompatibility of G/F/Phg nanofibers. Conclusion The developed G/F/Phg nanofibers are able to maintain the viability of phage and represent a promising antimicrobial dressing for wounds infected with MDR P. aeruginosa.

Construction and identification of the recombinant M13-IN5 phage and its effect on Chlamydia trachomatis

Objective To construct active phages against Chlamydia trachomatis, and to evaluate its effect on Chlamydia trachomatis. Methods The M13 phage was recombined with the IN5 sequences encoding the capsid protein VP1 of chlamydiophage phiCPG1, and then the recombinant M13-IN5 phage was obtained. PCR amplification, enzyme digestion and sequencing were performed to verify whether the target fragment was inserted into the phage successfully. The viability of the phage was evaluated by plaque formation assay. Cell counting kit-8 (CCK8) assay was conducted to evaluate the effect of M13 phage and recombinant M13-IN5 phage at the titer of 1011 plaque-forming units (PFU) /ml on the proliferation of Hela cells, and Hela cells uninfected with chlamydia served as the blank control group. Western blot analysis was performed to determine the expression of the IN5 loop protein in the recombinant M13-IN5 phage, M13 phage and Escherichia coli ER2738 at exponential growth phase. Cultured standard Chlamydia trachomatis serovar E strain was treated with M13 phage and recombinant M13-IN5 phage at the titer of 1011 PFU/ml separately, and chlamydia control group without the treatment with phages was set up. After 36-hour infection, confocal microscopy was performed to detect the location of the M13 phage and the recombinant M13-IN5 phage. Moreover, iodine staining was conducted to count inclusion bodies at 36, 48, 60 and 72 hours separately after infection. Statistical analysis was carried out by a two-sample t-test for comparisons between two groups, one-way analysis of variance (ANOVA) for intergroup comparison, and Bonferroni test for multiple comparisons. Results The bioactive recombinant M13 phage containing the IN5 loop gene was constructed successfully, and Western blot analysis confirmed that the recombinant phage expressed IN5 loop/pⅢ fusion protein with a high titer of 3.05 × 1011 PFU/ml. As CCK8 assay showed, there was no significant difference in proliferation of Hela cells among the blank control group, M13 phage group and recombinant M13-IN5 phage group (A450 values: 3.63 ± 0.01, 3.55 ± 0.02, 3.70 ± 0.01, respectively, F = 12.0, P > 0.05) . Confocal microscopy showed overlap between the phage fluorescence and chlamydial inclusion body fluorescence. The M13-IN5 phage group and M13 phage group both showed significantly decreased number of inclusion bodies compared with the control group (both P 0.05) . After 48, and 60 hours of chlamydial infection, the number of inclusion bodies did not differ among the M13 phage group, M13-IN5 phage group and control group (both P > 0.05) . Conclusions The recombinant M13-IN5 phage was bioactive and could successfully express the IN5 loop protein. In the in vitro experiments, the recombinant phage could enter into chlamydia inclusion bodies, and markedly inhibited the infection of Chlamydia trachomatis. Key words: Chlamydia trachomatis; Bacteriophage M13; DNA, recombinant; Inclusion bodies; IN5 loop

Manufacturing and ambient stability of shelf freeze dried bacteriophage powder formulations

The severity of multidrug resistance to antibiotics has urged development of alternative treatment approaches, including bacteriophage therapy. Given the complexity of the bacteriophage structure, formulation and stability are primary concerns. Our present work optimized process and formulations of phage powder manufacturing and investigated the stability of lyophilized bacteriophage powders under ambient storage. The model phage M13 was formulated with trehalose, mannitol, sucrose and PEG6000 and lyophilized in different conditions. Bacteriophage viability was examined by titering and was considered as the assessment of phage stability. Less titer loss of trehalose and sucrose formulations were observed compared to mannitol and PEG groups both immediately after lyophilization and upon long term storage. When evaluating lyophilization conditions, an additional 1 log titer was preserved by reduction of product drying stress. Trehalose was stabilized in the amorphous state whereas mannitol stayed in crystalline state in lyophilized powders. Increased moisture content was demonstrated to have a positive impact on viability of phage after lyophilization and upon storage. Overall, 2% trehalose or sucrose (w/v) can sufficiently stabilize phage during lyophilization process and storage in ambient conditions. There is a positive correlation between residual water and stability of phage. These collective findings highlight the potential of long-term, ambient storage of bacteriophage towards their successful use in diverse healthcare settings.

Nebulization effects on structural stability of bacteriophage PEV 44

Reduced infectivity of phage due to the nebulization process has been reported previously, but no visual evidence on structural change upon nebulization has been established, or whether these structural changes can be attributed to the titer reduction. In this study, transmission electron microscopy (TEM) was employed to assess the impact of three different types of nebulizers, air-jet, vibrating-mesh and static-mesh nebulizers, on the structural stability of a Myoviridae phage, PEV44, active against Pseudomonas aeruginosa. The morphology of the phage in the nebulized samples was categorized into two groups: “whole” (the capsid and tail held together) and “broken” (the capsid separated from the tail) phages. The “whole” phage group was further divided into three sub-groups: (1) intact, (2) contracted tail and (3) empty capsid. The starting stock suspension was found to contain considerable portions of “broken” phages (35.5 ± 6.7%), “whole” phages with contracted tails (9.9 ± 5.4%) and empty capsids (19.3 ± 8.9%). The fraction of “broken” phages was significantly increased after nebulization, with the air-jet nebulizer (83%) being more pronounced than the mesh type nebulizers (50–60%). While the amount of intact phages (2–10%) and whole phages with contracted tails (0–3%) were significantly decreased, the proportion of phages with empty capsids (15–36%) were not significantly different. Phages with broken features obtained by TEM quantification were 92.9 ± 3.2%, 74.8 ± 10.4% and 71.2 ± 11.0% for the jet, vibrating-mesh and static-mesh nebulizers, respectively. These results were found to be comparable with the titer loss obtained by the conventional plaque assay results. The in vitro aerosol performance and viable phage delivery of the three nebulizers was also assessed. The Omron nebulizer achieved a significantly higher viable respirable fraction (VRF) than the SideStream and Aeroneb Go (15.1 ± 5.8%, 2.4 ± 2.0%, 4.1 ± 2.7% respectively). In conclusion, this study identified various changes in the phage structure and viability of phage from different types of nebulizers. Understanding these effects and the phage tolerance to nebulization stresses can potentially improve our choice of the delivery method for inhaled phage therapy.

Encapsulation of specific Salmonella Enteritidis phage f3αSE on alginate-spheres as a method for protection and dosification

Background: Bacteriophages have been proposed as an alternative to control pathogenic bacteria resistant to antibiotics. However, they are not extensively used due to different factors such as vulnerability under environmental conditions and the lack of efficient administration methods. A potential solution is the encapsulation of bacteriophages in hydrogel polymers to increase their viability and as a controlled release method. This work describes the use of alginate-Ca+2 matrixes as mechanisms for protection and dosification of the phage f3αSE which has been successfully used to prevent infections produced by Salmonella Enteritidis. Results: The viability of the pure phage is reduced in near 100% after 1-h incubation at pH 2 or 3. However, the encapsulated phage remains active in 80, 6% at pH 3, while no differences were observed at pH 2, 4 or 7. Exposition of f3αSE to different T° showed that the viability of this phage decreased with increased T° to near 15% at 60°C, while the encapsulated phage remains with 50% viability at same temperature. Finally, the encapsulation of phages showed to extend their presence for 100 h in the medium compared to non-encapsulated phages in a water flow system, which simulate automatic birdbath used in poultry industry, maintaining the phage concentration between 102 and 104 PFU/mL during 250 h. Conclusions: Encapsulation in alginate-Ca+2 spheres can be a good alternative to extend viability of phages and can be used as a phage method dosification method in water flow systems.

The viability of lytic bacteriophage ΦD5 in potato-associated environments and its effect on Dickeya solani in potato (Solanum tuberosum L.) plants.

Dickeya solani is one of the most important pectinolytic phytopathogens responsible for high losses in potato, especially in seed potato production in Europe. Lytic bacteriophages can affect the structure of the host population and may influence spread, survival and virulence of the pathogen and in consequence, infection of the plant. In this study, we aimed to acquire information on the viability of the broad host lytic bacteriophage ΦD5 on potato, as well as to apprehend the specific effect of this bacteriophage on its host D. solani type-strain in different settings, as a preliminary step to target co-adaptation of phages and host bacteria in plant environment. Viability of the ΦD5 phage in tuber extract, on tuber surface, in potting compost, in rainwater and on the leaf surface, as well as the effect of copper sulfate, were examined under laboratory conditions. Also, the interaction of ΦD5 with the target host D. solani in vitro and in compost-grown potato plants was evaluated. ΦD5 remained infectious in potato tuber extract and rain water for up to 72 h but was inactivated in solutions containing 50 mM of copper. The phage population was stable for up to 28 days on potato tuber surface and in potting compost. In both, tissue culture and compost-grown potato plants, ΦD5 reduced infection by D. solani by more than 50%. The implications of these findings are discussed.

Aislamiento y caracterización del bacteriófago Va1 específico a Vibrio alginolyticus

Vibrio alginolyticus está asociado a enfermedades en acuicultura. El uso indiscriminado de antibióticos ha conllevado a la búsqueda de alternativas en el tratamiento de enfermedades bacterianas, entre ellas la aplicación de bacteriófagos, los cuales infectan y destruyen selectivamente bacterias. En ese sentido, en este trabajo se aisló un bacteriófago altamente lítico a V. alginolyticus el cual fue denominado Va1, con el objetivo de evaluar los parámetros físicos químicos en los cuales es viable. Para esto, se evaluó al bacteriófago Va1 en diferentes condiciones de pH, temperatura, cloroformo. El fago Va1 presenta mayores títulos a 20 y 30 °C y pH de 5 a 8 disminuyendo su viabilidad a partir de 40 °C y en unidades de pH menores de 5. La exposición al cloroformo redujo la viabilidad del fago Va1 en un 25%. A partir de la curva de un paso se determinó que el periodo de latencia y el tamaño de la explosión fueron de 20 minutos y 192 UFP/centro infectivo respectivamente. Al microscopio electrónico de transmisión el fago Va1 evidencio una cabeza icosaedrica y una cola no contráctil, características propias de la familia Podoviridae. En conclusión, el fago Va1 presenta características potenciales para su uso en fagoterapia.

Poly-γ-Glutamic Acid: Biodegradable Polymer for Potential Protection of Beneficial Viruses.

Poly-γ-glutamic acid (γ-PGA) is a naturally occurring polymer, which due to its biodegradable, non-toxic and non-immunogenic properties has been used successfully in the food, medical and wastewater industries. A major hurdle in bacteriophage application is the inability of phage to persist for extended periods in the environment due to their susceptibility to environmental factors such as temperature, sunlight, desiccation and irradiation. Thus, the aim of this study was to protect useful phage from the harmful effect of these environmental factors using the γ-PGA biodegradable polymer. In addition, the association between γ-PGA and phage was investigated. Formulated phage (with 1% γ-PGA) and non-formulated phage were exposed to 50 °C. A clear difference was noticed as viability of non-formulated phage was reduced to 21% at log10 1.3 PFU/mL, while phage formulated with γ-PGA was 84% at log10 5.2 PFU/mL after 24 h of exposure. In addition, formulated phage remained viable at log10 2.5 PFU/mL even after 24 h of exposure at pH 3 solution. In contrast, non-formulated phages were totally inactivated after the same time of exposure. In addition, non-formulated phages when exposed to UV irradiation died within 10 min. In contrast also phages formulated with 1% γ-PGA had a viability of log10 4.1 PFU/mL at the same exposure time. Microscopy showed a clear interaction between γ-PGA and phages. In conclusion, the results suggest that γ-PGA has an unique protective effect on phage particles.

Preparation and characterization of dry powder bacteriophage K for intestinal delivery through oral administration

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the prevalent pathogens, causing various diseases in humans and farm animals. Phage therapy is a potential mean to control the MRSA as an alternative to antibiotics. The objective of this paper was to develop a dry powder phage K preparation for oral delivery using alginate-whey protein microspheres. A number of protective agents were tested for enhancing the viability of phage during dehydration. The viabilities of free and encapsulated phage K were evaluated under simulated gastrointestinal conditions. Among the protective agents tested, maltose provided the best protection to encapsulated phage K during dehydration. Phage K is very sensitive to stomach acids. Encapsulation in the alginate-whey protein microspheres remarkably improved the gastric resistance of phage K. The viability of encapsulated phage was fully maintained in simulated gastric fluid at pH 2.5 after 2.0 h incubation. The microsphere size and polymer concentrations in the encapsulation matrix were important factors determining the degree of protection against stomach acids. Encapsulated phage was completely released in simulated intestinal juice within 2 h. In conclusion, the dried alginate-whey protein microspheres could be an effective delivery vehicle for oral administration of phage K to control Staphylococcus aureus.

Whey protein improves survival and release characteristics of bacteriophage Felix O1 encapsulated in alginate microspheres

Bacterial contamination is a major cause of food poisoning in humans worldwide. Phage therapy is a potential alternative to antibiotics in food animal production. Gastric acidity has been shown to compromise the viability of phage in vitro, hence, may affect the effectiveness of the phage treatment. Microencapsulation of bacteriophage in alginate microspheres protected phage against gastric acids; however, the release rate of phage was too slow when applied in chicks. The current study investigated the in vitro protection and release characteristics of alginate–whey protein microspheres (AWM) containing phage Felix O1 prepared by an extrusion method. Free phage was completely inactivated within minutes in simulated gastric fluid (SGF) at pH 2.0 and pH 2.5, while phage encapsulated in AWM mostly retained their viability after 2.0h incubation. Encapsulated phage was completely released from the AWM in simulated intestinal fluid (SIF) within 3h. Moreover, addition of maltodextrin to the encapsulation formula improved phage stability during air drying and storage. Our results show that addition of whey to alginate microsphere improved phage protection against acidic conditions and accelerated the release rate in SIF. AWM could be more suitable for oral phage therapy in chickens.

Comparison of the efficiency and the specificity of DNA-bound and free cationic porphyrin in photodynamic virus inactivation

The risk of transmitting infections by blood transfusion has been substantially reduced. However, alternative methods for inactivation of pathogens in blood and its components are needed. Application of photoactivated cationic porphyrins can offer an approach to remove non-enveloped viruses from aqueous media. Here we tested the virus inactivation capability of meso-Tetrakis(4- N-methylpyridyl)porphyrin (TMPyP) and meso-Tri-(4- N-methylpyridyl)monophenylporphyrin (TMPyMPP) in the dark and upon irradiation. T7 bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. TMPyP and TMPyMPP reduce the viability of T7 phage already in the dark, which can be explained by their selective binding to nucleic acid. Both compounds proved to be efficient photosensitizers of virus inactivation. The binding of porphyrin to phage DNA was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by DNA bound porphyrin. As optical melting studies and agarose gel electrophoresis of T7 nucleoprotein revealed, photoreactions of TMPyP and TMPyMPP affect the structural integrity of DNA and also of viral proteins, despite their selective DNA binding.

Lethal effects of 32P decay on transfecting activity of Bacillus subtilis phage φe DNA

Disintegration of 32P present in the DNA of Bacillus subtilis phage φe (a phage containing double-strand DNA) results in the loss of viability of intact phage as well as transfecting activity of isolated DNA. Only 1 12 of the 32P disintegrations per phage DNA equivalent inactivates the intact phage while nearly every disintegration inactivates the transfecting DNA. This result provides evidence for a single-strand intermediate in the transfection of B. subtilis by φe DNA.

Ultraviolet reactivation of lambda phage: Assay of infectivity of DNA molecules by spheroplast transfection

Prior irradiation of non-lysogenic bacteria by ultraviolet light leads to an increase in the viability of infecting irradiated λ phage (ultraviolet reactivation). Similarly, u.v. irradiation of wild type or uvrD bacteria lysogenic for λcIind − increased the fraction of closed circular duplex phage DNA molecules formed after infection with u.v.-irradiated λ phage. The closed circular molecules isolated from the irradiated lysogens were shown to be free from u.v. damage by a spheroplast transfection assay. The increase of closed circular molecules is sufficient to explain the ultraviolet reactivation observed by the increase of viability of irradiated phage. In ultraviolet reactivation, damage must be erased on irradiated DNA molecules and the repair is independent of total replication of phage genomes, exchange of sister chromatids or recombination between phage genomes. Protein synthesis is necessary to increase the level of closed circular molecules of irradiated λ phage after irradiation of bacteria.

Characterization of a nocardiophage for Nocardia restrictus.

Summary A nocardiophage, called Ri, was isolated from soil samples. It lysed Nocardia restrictus and a few other species of Nocardia, but none of the eight streptomycetes tested. On N. restrictus phage Ri produced plaques very irregular in shape, size and turbidity; replacement of NaCl by Ca(NO3)2 in the medium increased the uniformity of plaque morphology and gave higher counts. Electron microscope examination of the phage particles revealed a hexagonal head, 75 nm. in diameter, and a long, flexible, non-contractile tail, 330 × 10 nm., bearing a round terminal plate; a collar was sometimes visible in particles with empty heads. The phage nucleic acid is most probably double-stranded DNA. Maximum viability of phage was at pH 7; the survival was less than 0·001 % for an exposure of 30 min. at pH 5; about 20% survived at pH 6, and 10% at pH 8. When exposed to 55° or to ultraviolet irradiation, phage Ri showed similar survival curves, and was very sensitive to both treatments. Adsorption of phage to its host was rapid and efficient (80% in 5 min.); the latent period was 28 min., the rise period occurred between 28 and 37 min., and the burst size was 61. Mutation of N. restrictus to resistance to phage Ri was very rare; the apparent mutation rate was 3·4 × 10−11, which can be partly attributed to the lower growth rate of the resistant mutants. In chromium-shadowed preparations, N. restrictus appeared as short rods, 1·3 × 0·5 μ., dividing by transverse fission, showing no lateral branching characteristic of the genus Nocardia, and very few chains (never longer than 2 or 3 cells); thin sections of infected cells showed a few intracellular phage particles.

Some factors affecting the viability of freeze-thawed T4 bacteriophage. II. The influence of certain electrolytes on the degree of inactivation.

SUMMARYThe effects of various salts on the viabifity of T4 and T4 Bo phages during freezing and thawing have been studied. The effectiveness of different salts in causing inactivation or protection of phage during freeze—thawing was of the same order as has been observed for their effects on the conformational stability of macromolecules. There were two important contributory factors causing inactivation of phage during freeze—thawing: (i) concentration of solutes during freezing, and (ii) lowering the temperature, without change in salt concentration.The viability of phage following storage at subzero temperatures was dependent on the storage temperature, the species of ions in the suspending medium and the initial salt concentration. Viability was greatest when phage had been initially suspended in dilute solutions.Survival of T4 Bo phage, following cooling to subzero temperatures and rewarming, was greater in D2O solutions than in H2O solutions.It was found that 20% glycerol completely protected against inactivation of T4 Bo phage, suspended in 3·5 molal NaBr, which were cooled to subzero temperatures and rewarmed in the absence of ice. Without added glycerol the viability of T4 Bo phage suspended in 3·5 molal NaBr, which were cooled to −20° C and rewarmed was less than 1%. This protective effect of glycerol is in contradiction to the accepted views of its mode of action in prevention of freezing injury.This work has been supported in part by grants from the Medical Research Council and the Office of Naval Research under contract F 61052–68-C-0041.

The effect of radiophosphorus decay on cistron function in bacteriophage T4: I. Long-range and short-range hits

The effect of disintegration of incorporated 32P atoms on the functional survival of different viral cistrons was investigated by infecting non-permissive host bacteria jointly with a non-radioactive rII or am T4 mutant and with highly 32P-labeled T4 am+ wild-type phage, and then following upon radioactive decay the loss of ability of the wild type to complement growth of the conditionally lethal mutant phage in the same cell. These experiments showed that the function of each of eleven different viral cistrons is abolished by 32P decay at about half the rate of loss of viability of the whole phage. The relative 32P decay sensitivity of one-half cannot represent the functional inactivation of about half of the viral genome by each lethal 32P disintegration, since the ability of the wild-type phage to complement a variety of am double mutants is lost only slightly faster than, and never at twice, the rate of loss of complementation of single am mutants. Nevertheless, the sensitivity of multiple cistron function does increase as more and more am mutant cistrons are brought into play, until in the case of complementation of a sextuple am mutant, joint functional survival of six cistrons is lost at 0.8 the rate of loss of viability of the whole am+ phage. These observations can be interpreted by postulating two types of lethal 32P disintegrations, long-range hits that cause functional inactivation of the whole, and short-range hits that inactivate only part of the viral genome. Analysis of the results from this point of view shows that about half of the lethal 32P hits are of the short-range type if decay proceeds either at −196°C or at + 4°C. The frequency of short-range hits is sharply reduced, however, if the free radical trap AET‡ is present during decay at 4°C, suggesting that short-range hits represent radiochemical lesions, in the production of which free radicals play an important part. Calculation of the fraction of all 32P disintegrations that inactivates a given cistron by short-range hits shows that this fraction is equal to about half the total share of the whole genome represented by that cistron. This coincidence leads to the notion that half the 32P disintegrations occurring within the confines of a viral cistron produce sub-lethal damage preventing its functional expression, unless the damage had first been erased by a repair process which can proceed in the infected cell. Short-range hits would then correspond to lesions in the viral DNA that render non-erasable, and thus permanently fix, all sublethal cistron damages that have already occurred, or may yet occur, upon further decay.

The Effect of Buffered Glycerol Saline and Formalized Buffered Glycerol Saline on the Viability of Enteric Bacteria and Enteric Phage

of the specimens. The author noted that it was more difficult to isolate the dysentery bacilli after the acute stages of the disease had passed. Carter (2), from specimens that contained blood and mucus, reported SO* /^ positive isolations only, while Topley and Wilson (3) obtained 60.2*^. Several investigators?Teague and Clurman (4); Wu and Sia (5); Holmes (6)?have attempted to develop a preservative for fecal specimens which cannot be plated immediately. Most of these preservatives contained glycerine. Sachs (7) pointed out that the acidity of the glycerated solutions is incompatible with the survival of shigella organisms. He suggested titrating these solutions to pH 8.0 with a saturated aqueous solution of phenol sulphonphthalein in 5^o sodium phosphate. Such a solution was evaluated by Hirschberg (8) who found it to be of value in preserving shigella and salmonella organisms. Friedmann (9) suggested that the presence of shigella bacteriophage in stools of dysenteric patients is the cause of poor isolation of shigella organisms. Feemster (10) recommended the isolation of these viruses as a diagnostic criterion for the presence of dysentery