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Cooked and raw duck eggs were held in saturated salt brine for periods up to four weeks. Fresh egg controls and brine pickled eggs were evaluated for flavor, texture and color by a panel consisting of about equal numbers of American and Thai ethnic origin. Albumen quality, microbial growth and pH were determined. American panel members preferred flavor, texture and color of control eggs and rejected or disliked flavor, texture and color of salt brined eggs. Thai panel members preferred flavor, texture and color of all salt brined eggs, especially those held in brine for four weeks at 21° C. Halophilic bacteria were enumerated in eggs after four weeks in salt brine, followed by four weeks at 3° C. out of brine. No halophilic organisms were detected in control eggs. Counts in uncooked eggs were lower than in those eggs cooked before brine immersion. After four weeks out of brine, counts in raw eggs reached 6.0 × 103, whereas those cooked before brine treatment reached 1.7 × 105.

Growth of 25 strains of red halophiles stopped below 15° and above 55°; heat resistance is less than two minutes at 98 °C. in 24 per cent saline. Range of relative humidity necessary for growth lies between 70 and 85 per cent. They have high resistance to ultra-violet light, surviving 120 minutes' exposure. Range of salt tolerance can be increased in both directions by gradual adaptation. Replacement of the Na ion by other cations inhibits growth.

In the previous paper1), the author reported on the invasion of the Takikawa's so-called pathogenic halophilic bacteria into the material of wooden fish container. It was also disclosed that the bacteria might easily migrate into inner part of such rough-grained wooden material as ceder or pine. In the present paper, studies have been undertaken to elucidate the fate of pathogenic halophilic bacteria being artificially polluted in the wooden test pieces for the period 60 to 90 days under various keeping conditions. The brief keeping conditions were as follows: Artificially polluted wooden pieces with the bacteria (pine and oak, 8×20×50mm; each piece has been dipped in the culture fluid of the test organisms at 30°C for 48 hours) were put in 6 large Petri dishes and each dish was let stand under following conditions. 1. Wet condition (moisture contents of the test pieces were higher than 57%). 1) Being kept in an refrigerator (0°C-5°C). 2) Being kept in a dark room during October to December at room temperature. 3) Being kept in an incubator at 30°C. For keeping wet condition, a small beaker filled with water was placed in each Petri dish, in addition, 2 to 3 drops of sterilized saline solution were added to each test piece every day. 3. Moderate condition The Petri dish has been kept in a dark room and allowed to stand at room temperature (with cover). The moisture contents of test pieces decreased gradually from the initial value 57% to 42%. 3. Dry condition Two dishes containing pieces of pine or oak respectively have been kept out door without cover and exposed to sunlight every day during October to December. Results obtained may be summarized as follows: Length of survival of the bacteria at the inner part of test piece varied according to the moisture contents. Apparently under wet condition, no appreciable decrease in bacterial number was observed, while the keeping temperature so far examined (0°C to 30°C) had a little effect on the rate of survival of the organisms (Figs. 1, 2 and 3). Under the dry condition, the bacteria on the surface of test pieces were diminished markedly within 15 days owing to the exposure to direct sunlight and loss of moisture in the piece (Figs. 5 and 6). From these experimental results, the author concludes that the most practical and effective method for treating a large number of wooden fish container at one time to diminish such harmful organisms as pathogenic halophilic bacteria would be the drying under sunshine for more than 10 days.

The solid content of several halophilic bacteria was estimated by immersion refractometry. Values for extreme halophiles ranged from 28 to 32%, and for moderate halophiles from 31 to 45%. The solid content of these organisms changed with variation in the salt content of the environment. It is thought that these differences are the result of changes in the water content of the cell. Spheres (spheroplasts) had a slightly lower solid content than the parent rods. Nuclear and other inclusion bodies, mostly of lower refractive index than the cytoplasm, were noted.

Much attention has been paid on the Takikawa's so-called pathogenic halophilic bacteria, a new type of bacteria suspected to cause food poisoning. It has been only a short period since the investigation started and there are still many problems to be solved, in which pathogenicity of the organisms is still obscure due to the unavailability of susceptible experimental animals. In the present work, effect of various culturing conditions of the organisms upon the lethality for mice has been studied. The results may be summerized as follows: 1. The organisms grew well and gave a high lethality for mice when cultured in a 3% NaCl containing broth which was prepared with casein peptone and fish meat extract. The organisms grew rapidly in such 3% NaCl containing liquid media as the broth composed of meat peptone and fish meat extract, plain peptone water, or peptone-yeast extract medium, nevertheless, the lethality of the cultures were rather low as compared with the above-men-tioned broth culture. 2. As to the NaCl requirement of the organisms, the fastest growth rate was observed in a 3% NaCl broth, while there was no appreciable difference in the lethality for mice between the concentration 2 to 4%. 3. Growth of the organisms and their lethality for mice were varied according to the reaction of medium. Whithin the range of pH 6-8, the higher the pH value, the more rapid in growth or higher in lethality. 4. Presence of glucose in the medium enhanced the growth of the organisms. It was noted that the addition of more than 0.5% of glucose in the medium resulted in decrease in the lethality. 5. In the case of Matsuda strain (serotype XIII), higher lethality was observed in the early stage of culture of the organisms. The organisms lost their lethal toxicity for mice gradually by prolongating the incubation period. 6. Within a range 24° to 37°C., the highest lethality was recorded when the organisms were incubated at 24°C.

Summary Four autopsy cases of food poisoning caused by pathogenic halophilic bacteria (Pseudomonas enteritis Takikawa) have been reported for the first time.

In the previous work by Yasunaga and Zenitani, the halophilic bacteria belonging to Miyamoto's so-called euryhaline type were obtained in a great number from river water not mixed with sea water in Nagasaki City, and it was shown that some of the isolates in summer were almost similar in biological characters to so-called pathogenic halophilic bacteria, being considered to be responsible for the most of food poisonings caused mainly by fishes and shells.The present work was undertaken to ascertain the existence of the organisms in city river water, presumed from the previous results. The characters of the river waters collected as samples for detecting the organisms are shown in Table 1.The organisms were isolated from the samples by using a peptone water with 3% NaCl as final concentration and a nutrient agar medium with 3% NaCl, 0.1%, bile salts, sucrose and BTB. The incubations were performed at 37°C for 20-24 hours. The rate of isolation of the strains identified as the pathogenic halophilic bacteria, the classification of their biotypes by V.P. test and salt requirment, and grouping of O serotypes are presented in Tables 2, 3 and 4.From these results, it was confirmed that upper stream water than estuary in Nagasaki City were considerably contaminated with the pathogenic halophilic bacteria in summer, and this may explain to some extent the predominant distribution of the organisms in coastal area, especially in estuary. Furthermore, the facts that 10 of 24 strains isolated were belonging to biotype 1, and that the majority of them were classified into O1-O5 groups suggest possibility of the infection by the organisms through city river water.

Bacto-oxgall has shown a differential effect on the red halophilic bacteria commonly found in solar salt and in contaminated salted fish. Pseudomonas salinaria was inhibited by low oxgall concentrations while Sarcina littoralis tolerated high ones. When this differentiating test is applied to a larger number of strains it will be possible to determine if Bacto-oxgall can be used as the basis of a selective medium to distinguish one genus from the other. The clearing of bacterial suspensions and microscopical examination have shown that the inhibition of Ps. salinaria was produced by the lysis of the cells. The inhibition of Ps. salinaria was effective and rapid. It was not affected by pH variations but was reduced by the presence of proteins. When compared similarly to other bile products, Bacto-oxgall was found to have the same inhibitory power as sodium taurocholate. Bacto-oxgall, diluted to the equivalent of fresh bile, was also found to be three times more effective than fresh cod bile. From the evidence gathered it was concluded that the inhibition of Ps. salinaria by Bacto-oxgall is due to a mechanical disruption of the bacterial cytoplasm.

A novel phosphosulfolipid has been isolated as the potassium salt from the extremely halophilic bacterium Halobacterium cutirubrum; its molecular formula was established as C46H93PSK2. It accounts for 3–4% by weight of the polar lipids and about 6% of the total lipid phosphorus. The phosphosulfolipid yielded inorganic sulfate and 1-sn-phosphatidyl-3′-sn-glycerol (diphytanyl ether analog) in equimolecular proportions after solvolysis in 0.005 n HCl in tetrahydrofuran, and it was thus a sulfate ester of phosphatidylglycerol. The position of the sulfate group was determined by comparison of the infrared and NMR spectra of the salt and the methyl ester of natural phosphosulfolipid with those of synthetic 1- and 2-sulfate esters of phosphatidylglycerol. The results obtained established the structure of the bacterial phosphosulfolipid as 2,3-di-O-(3′R,7′R,11′R,15′-tetramethylhexadecyl)-sn-glycero-1-phosphoryl-3″-sn-glycero-1″-sulfate.

Late in July 1961, the authors investigated on occurrence of TAKIKAWA's so-called pathogenic halophilic bacteria in the sea area around Izu Islands and Suruga bay aboard the Umitaka-maru, a research vessel of Tokyo University of Fisheries. Isolation of the organisms was carried out by the method according to SEKINE et al8). SEKINE et al8). had already isolated some strains of organisms having similar biological characteristics to those so-called pathogenic halophiles from natural environments, and showed that these organisms can be divided into two Groups, I and II. The present investigation revealed that a group of organisms having similar features to some extent to those afore-mentioned two groups are widely distributed in sea area. This group of organisms are easily differentiated from the Group I or II, and then they were classified as Group III. The distinctive characters between Group III and the other two groups were shown in Table 1. Throughout the sampling stations no organism belonging Group I in which so-called pathogenic halophiles are included could be detected, while Group II organisms were isolated only in coastal sea water. The Group III organisms were found to be widely distributed in both pelagic and coastal water irrespective of water depth. Group III organisms are regarded as marine inhabitant. The results obtained in the present survey may suggest that the so-called pathogenic halophilic bacteria is not distributed in the pelagic sea water around Kuroshio current. Regarding coastal sea area, however, further detailed studies on the distribution of the organisms is necessary.

Synthesis of 1-sn-phosphatidyl-3'-sn-glycero-1'-sulfate (phosphatidylglycero-1-sulfate) was achieved by monosulfation of 1-sn-phosphatidyl-3'-sn-glycerol (diphytanyl ether analog) with an equimolar amount of SO(3)-pyridine complex at room temperature; with excess sulfation reagent at 60 degrees C, the 1',2'-disulfate ester was obtained. The phosphatidyl-glycero-2-sulfate isomer was synthesized by an unambiguous route starting from the bacterial 2,3-di-O-phytanyl-sn-glycerol. The synthetic phosphatidylglycerosulfates were characterized by analytical, chromatographic, optical rotatory, and spectral (infrared and NMR) data and compared with the phosphatidylglycerosulfate isolated from Halobacterium cutirubrum.

A survey of Canadian Atlantic shellfish was carried out to determine the presence of Vibrio parahaemolyticus. Twenty-eight strains, closely resembling authentic strains from Japan, were isolated from unfrozen specimens of shellfish obtained at local retail outlets. Six isolates were obtained from shellfish collected and frozen by the Inspection Branch, Department of Fisheries and Forestry.

SUMMARY: The sodium and potassium contents of cells of 32 strains of non-halophilic bacteria were determined after growth in a standard medium of low salt content. The salt tolerance of each strain was assessed by determining the lowest water activity at which growth occurred in sodium chloride medium. Among the strains examined the potassium contents varied about fivefold and were positively correlated with salt tolerance.

Extremely halophilic bacteria, which are able to grow in, and require, saturated or near-saturated salt solutions are both of historical interest and of practical importance as food spoilage organisms. In addition, because of the environment in which they live, their membranes, enzymes, and ribosomes have unusual or unique properties that make them especially worthy of physiological study. A simple method for growing large quantities of extreme halophiles is described, and various growth media, both complex and synthetic are discussed.

The quantitative determination of total numbers of red halophilic bacteria in contaminated solar salts and discoloured salt fish has been made possible through the successful adaptation of the "drop plate" method. The maximum number of colonies is reached after eight days for pure strains of Ps. salinaria, and after 14 days for the red halophiles present in contaminated solar salts. A brief statistical study has shown that a fair degree of reliability can be obtained with the method. From series of counts, the total error expressed as coefficient of variation is shown to vary from 3.34 to 8.72 per cent. Good reproducibility is also obtained when random samples are taken from stocks of contaminated salt.

Red halophilic bacteria were isolated in pure culture from salted fish and solar salts. A description of one rod-shaped bacterium and a few cocci is given. Proteolytic tendencies were noticed both in the rod and in four of the cocci. While 37° C. and a relative humidity of 75% favoured good growth in laboratory media, at 4° C. there was no growth. Almost all the species of salted fish from Malpe, Calicut, Adirampatnam and Rameswaram, that were examined, developed red discolouration. Among the fishes salted in the laboratory, sharks and skates appeared to be susceptible to the attack earlier than the others and fatty fish like oil sardines developed it very late, if at all. The rod form was “trained” to grow in salt concentration of 3·5% while one coccus form grew in KCl medium in place of NaCl.

Rabbits were immunized with four strains of halobacteria, Halobacterium halobium NRL, H. halobium R-1, H. salinarium NRL-9, and H. cutirubrum NRL-10, that had been fixed with formaldehyde. The antisera obtained detected the presence of an antigen common to the Halobacterium genus and, after absorption, detected three distinct antigenic groups within the Halobacterium genus. A fourth group was agglutinated only by unabsorbed sera.

Extremely halophilic bacteria are able to survive in a natural environment exposed to high doses of ultraviolet irradiation. One such organism, Halobacterium cutirubrum, has been characterized to possess an extremely efficient photoreactivation from ultraviolet damage. A population irradiated to 1% survivors can photoreactivate to complete recovery of the original population in less than 1 h of illumination. Inhibition of carotenoid pigments by 10−6 M diphenylamine decreased photoreactivation efficiency. The carotene pigments have been implicated in an energy-transfer role to augment the photoreactivation mechanism

A simple method, based on oxgall tolerance, is proposed for differentiating red halophilic bacteria commonly found in solar salt and discoloured salted codfish. Tests carried out on 18 strains isolated from various sources have shown that the rod forms are inhibited by low concentrations of Bacto-oxgall and that the coccus forms tolerate relatively high ones. Bacto-oxgall can thus be used as the basis of a simple selective medium. This test has been found useful for the isolation, purification and partial identification of unidentified strains and also for determining the relative proportions of the two main types of red halophilic bacteria present in solar salt and salted fish samples.

The nonpolar (acetone-soluble) lipids of the extremely halophilic bacterium, Halobacterium cutirubrum, were found to consist of red carotenoid pigments (43%) and squalenes (48%) with a small amount of a vitamin K-type quinone. The squalenes were shown by n.m.r. and mass spectra to consist of the fully isoprenoid squalene (S; C3OH5o), dihydrosqualene (S2; CJ&152), and tetrahydrosqualene (S4; C30H54) in the ratio of 1.0:0.4:0.1. S2 probably has one re- duced internal isoprenoid group, and S4 has one internal and one terminal reduced isoprenyl group. The vitamin K-type quinone was shown by n.m.r. and mass spectra to have a Cho isoprenoid side chain, and is thus identified as menaquinone-8 (MK-8).

1. Polynucleotide phosphorylase was purified 200-fold from Halobacterium cutirubrum. 2. It is membrane-associated and can be solubilized by sonication. 3. The purified enzyme requires a high ionic strength for both stability and activity. 4. It is Mn2+-dependent, has all three typical polynucleotide phosphorylase activities and is specific for nucleoside diphosphates. 5. The enzyme is of low molecular weight.

Growth of Halobacterium cutirubrum, H. salinarium, and H. halobium was followed by viable counts and optical density measurements. The last species, which grows most vigorously, was most intensively studied.The increase of optical densities and the increase in viable counts corresponded through the exponential growth phase. Dead cells did not lyse, and viable counts were needed to show that after the stationary phase was reached some cultures died off rapidly. Others remained stable for several days. Potassium can limit growth in synthetic media; in all media abundant growth could be obtained only when about 1 mg/ml K+ was present. Growth was stimulated by glycerol, certain carbohydrates, related metabolites, and vitamins. The most effective growth-promoting vitamins tested were thiamine, folic acid, biotin, and vitamin B12. Metabolites that stimulated growth were galactose, glucose, sodium lactate, and glutamine. One-tenth percent glycerol, sodium succinate, and sodium pyruvate were stimulatory, although 2.0% concentrations were inhibitory. Though carbohydrates may stimulate growth, the medium does not become acid; this may explain why standard bacteriological tests indicate that many extreme halophiles do not "utilize" carbohydrates. H. halobium produced considerable acid only when grown in the presence of 2% glycerol.

Cells of the halophilic bacteria Micrococcus halodenitrificans, Vibrio costicolus, and Pseudomonas salinaria have a relatively high nitrogen content, indicating that the cell material is predominantly protein. They contain only small amounts of fat and no detectable amount of carbohydrate other than nucleic acid pentose. Cell walls are lipoprotein. Cells disrupted by shaking with water alone contained electron-dense material similar in composition to the cell walls. In a few preparations of the Micrococcus a polymer of β-hydroxybutyric acid was found.

The carotenoids of two yellow and four red halophilic bacteria were studied by chromatography on alumina. One yellow organism contained predominantly one pigment, or a group of closely related pigments similar to neurosporene, while the other contained the neurosporene-like pigments and appreciable amounts of an unknown pigment with absorption maxima at about 420, 450, and 485 mμ.The red organisms, three species of Halobacterium and one Sarcina, contained α- and β-bacterioruberin, and at least six minor pigments. All except two of these were very similar to carotenoids previously reported in Rhodospirillum rubrum. Methoxyl could not be detected in α-bacterioruberin, supporting the suggestion of others that it is demethylated spirilloxanthin. It is suggested that the pathway of carotenoid biosynthesis in red halophiles is very similar to that in Rhodospirillum rubrum, except that the pigments are not methylated.The carotenoid content of Halobacterium salinarium was not influenced by addition of various precursors to the growth medium, or by altering the physical conditions of growth, but increased with the age of the culture.

Refractile granules of poly-β-hydroxybutyric acid are produced by Micrococcus halodenitrificans during the exponential growth phase in a nitrogenous medium. The amount produced varied from about 1% of the salt-free dry weight during vigorous aeration to as much as 28% with less vigorous aeration. If glycerol, pyruvate, or acetate was added to the medium, the polymer level increased to 40–60%. The polymer was not found in a number of other halophilic bacteria.

Four previously unrecognized strains of extremely halophilic bacteria that utilize carbohydrates have been isolated. Gas production proved an unreliable index of carbohydrate metabolism; therefore, carbohydrate utilization was measured by determining acid formation and sugar disappearance during growth. By these procedures, carbohydrate utilization was readily detected. The results suggest that carbohydrate dissimilation by extremely halophilic bacteria may be more common than previously thought and that the apparent rarity of carbohydrate-metabolizing halophiles may be an artifact of the isolation procedures used.

1. An investigation was carried out of a case of reddening in salted herring, caught in August and landed in September 1951. 2. Cultivation experiments were started using a fish milk medium, containing 10, 16 and 23 wt% of NaCl; incubation was carried out at 37, 30, 24 and 15°C. and both under aerobic and under anaerobic conditions. 3. Growth of red halophiles was observed exclusively in platings on 23 wt% NaCl. Those incubated aerobically at 37°C. showed growth after 1 month and those at 30 and 24°C. after about 2 months. No growth of red halophiles occurred at 15°C. 4. The brine and the gonads present in the barrel, containing red herring, and a brine and gonads sampled from a barrel of unspoiled herring, turned out to contain the same red, halophilic sarcinae. 5. The numbers of these sarcinae in the red and in the undiscoloured brine both were of the order of 100/ml, being about ten times the numbers found in either the discoloured or the normal tissues. 6. The conclusion must be drawn that the type of red discolouration of herring described here is not caused by red halophilic bacteria.