Studies on the Red Halophilic Bacteria from Salted Fish and Salt*

STUDIES ON THE RED HALOPHILIC BACTERIA FROM SALTED FISH AND SALT*

BY R. VENKATARAMAN AND A. SREENIVASAN (Fisheries Technological Station, Kozhikode) Received June 9, 1953 (Communicated by Dr. B. S. Bhimaehar, F.^.sc.)

INTRODUCTION PRESERVATION of fish by salting is as old as civilization and is still widely practised. A great deal of work has been done abroad on the bacteriology of salted fish, particularly on the halophiles. The terms "pink " or "pink eye" (Cloake, 1923 ; Shewan, 1937), " Rotwerdens "(Liebert and Deems, 1930), "der rote Hund" (Horowitz-Wlassowa, 1931), "Red Heat "(Lochhead, 1934), "Rouge "(Bothello and Jose, 1949), "Rusty, (Harrison, 1923) and "Rote-erhitzung" (Stather, 1930), all refer to the same condition in salted fish or salted hides. The loss due to this affliction is incalculable. Beckwith (1911) isolated Diplococcus gadidarum Beckwith from salted stock fish. Bitting (1911) in his extensive work noted that in all cases, a coccus, a bacillus and the cells of a fungus were associated with reddening, which he considered to be essentially a surface infection. The bacillus formed spores. Cobb (1927) found, that the infection was due to the solar salt, that salt from underground mines was not infected and that cold checks the growth of' red ' bacteria. Kellerman (1915) studied the bacteria causing the red deterioration of salted cod fish and considered Sarcina littoralis (Poulsen), D. gadidarum (Beckwith), Micrococcus litoralis (Poulsen), and Clathrocystis roseo percisina (Farlow) to be synonymous. Harrison (1923) found Pseudomonas ,?alinaria causing reddening of salted herring, while Gibbons (1937) isolated Serratia salinaria, Ser. cutirubra Lochhead, Serratia spp., Bact. trapanicum Petter, S. littoralis and Sarcina sp. from reddened salt fish. Horowitz-Wlassowa (1932) described Tetracoccus carneus halo- philus which was later found to be identical with Petrova's strains and Patrova (1935) showed that the highly pleomorphic organism from red salted fish was identical with Micrococcus roseus. Liebert and Deems (1930) isolated 3 different red microbes from brine, a coccus, a sarcina and a spore-forming bacterium. Shewan (1937) obtained from salted fish, a bacillus identical

* Published with the kind permission of the Director of Fisheries, Madras. 17 B 9 18 R. VENKATARAMANAND A. SREENIVASAN

with Klebahn's Bacillus halobus robur, Harrison and Kennedy's Ps. salinaria, and Lochhead's Ser, salinaria, and a coccus related to M. morhuce, Klebahn. Dixit and Vachha (1942) isolated a new strain, Serratia sambharianus from the salt lake Sambhar in Rajputana, India, which thrived even on dry gait. Similar isolation of Ser. salinaria from Aral sea region was made by Kurochkin and Emilyanchik (1937). Stuart et aL (1933) found that crude solar salts harboured red halophiles, while none of the 62 mined salts did. But the opposite view has recently been expressed by Mueller (1950). Thus it is fairly clear that reddening of salted fish is essentially due to cocci and rod forms of bacteria originating from solar salts. We have isolated a number of such red halophilic organisms from various salts and salted fish and the results are presented in this paper.

MATERIALS, METHODS AND RESULTS Isolations of red halophiles from salted fish were made by streaking the ' red' on plates of salt milk agar of Lochhead as modified by Hess (1942). Salt samples were plated out on the same medium after making decimal dilutions. This gav~ also the halophile count of the salts. A modified Gibbon's starch medium was also tried successfully for isolations from salts. Pure discrete colonies were subcultured on salt milk agar slants. The various media for studying the physiological and biochemical reactions were prepared according to Gibbons (1937) with the following exceptions: Potato slants were soaked for 48 hours in saturated brine and then sterilized at 15 lb. for 20 minutes. Starch hydrolysis was tested on salt milk agar plates incorporated with 0.2~ B.D.H. soluble starch, after inoculation and incubation till good growth was obtained. All cultures were placed in a desiccator maintained at an R.H. of 759/o with sulphuric acid, and incubated at 37 ~ C. Gelatin tubes were incubated separately at the same temperature and after 7 days were periodically tested for liquefaction. The following salt samples examined so far showed the presence of Red haiophiles: Tuticorin salt, common salt, crushed from Tata Chemicals, Mithapur, Vedaranyam salt, and salt from the Model Farm at Wadala, Bombay. Salted fish obtained over a wide geographical range also showed the ' Red' discolouration : Black pomfrets, white pomfrets, Mullets, Leiognathus, Sillago, catfish, Polynemus, Therapon, Pristepoma, the Dorab, and Hemi- rarnphus, all from Rameswaram area, were afflicted by red discolouration profusely. So also were the catfish, Polynemus, Sharks and Skates received from Adirampatnam. On the West Coast, salted mackerels obtained from Red Halophilic Bacteria from Salted Fish and Salt 19 Malpe, and salted soles, mackerels, silver bellies and sharks from Calicut market had an, abundance of'Pink' over the surface. Washed and gutted fish salted in this laboratory with commercial Tuticorin salt for certain experiments (Venkataraman and Vasavan, 1953) were seen to develop the 'red '--Sharks, skates, mackerels, caranx, ribbon- fish, seer, silver bellies, the jew fish, soles and oil sardines, all developed the red in varying lengths of time. Some of the organisms were obtained in pure cultures and a technical description of them is given below:- Strains 111, H~ and Hla.wColonies on salt milk agar, circular, red, moist viscid, glistening, entire; gram positive cocci in pairs mostly, in short chains and clusters, old cultures showing sarcinal tetrads; non-motile; liquefy gelatin; nitrites not produced from nitrates; indole not produced; H,S not produced; starch not hydrolysed; dextrose, sucrose, lactose and xylose fermented with production of acid; casein digested with foul odour; red ring and pellicle in 2070 NaC1 broth; growth on potato, vermillion red, moist, viscid in less than a week. Identity: Sarcina sp. Source: From salted catla (Mettur) and salted sole. Strain H3.--Colonies bright red, circular, glistening, entire; gram positive cocci, single and pairs, non-motile; gelatin not liquefied; nitrates reduced to nitrites; indole and H,S not produced; starch not hydrolysed; dextrose, sucrose and lactose not fermented; growth on potato raised, pink casein digested. Identity: Micrococcus sp. Source: Sole, salted in the laboratory. Dr. Shewan (private communication) thinks this is not akin to any species described so far and that it reduces trimethylamine oxide to the amine. Strain H,.--Colonies red, circular, glistening, entire; gram positive cocci mostly single and pairs, a few in clusters; non-motile; gelatin not liquefied; nitrates reduced to nitrites; indole and H2S not produced; starch hydrolysed; casein not digested; dextrose, sucrose and lactose not fermented; bright red circular colonies on salt potato. Identity: Micrococcus morrhute Klebahn. Source : Tuticorin salt. 20 R. VENKATARAMANAND A. SREENIVASAN

Strain Hv--Colonies orange-yellow to orange-red or pale-red, circular, glistening, entire; gram positive cocci mostly in pairs in salt milk agar and starch media but in packets of 4 or 8 in yeast extract agar; the diplococcus form appears to be motile when young; gelatin not liquefied; nitrates reduced to nitrites, indole produced; H2S not produced; starch hydrolysed; dextrose, sucrose, lactose and xylose not fermented; casein not digested; on salt potato, flat orange-red streaks. Grows in KC1 medium in place of NaC1. Identity: Micrococcus sp. Source: Salted mackerels. Strains H~, H 2 and H13 and also H s appear to us to be new species. Their exact systematic position is being examined by us and a report on it will be published later. Strains He and Ha.--Colonies vermillion red, circular, glistening, moist; gram positive cocci, sin#e, pairs and packets of four; non-motile; gelatin not liquefied; nitrates not reduced to nitrites; indole and HaS not produced; starch not hydrolysed; dextrose, sucrose and lactose not fermented; casein not digested; on salt potato, abundant wrinkled vermillion growth. Identity: Sarcina sp. Source.--Salted mackerels and sole. Strain Hg.mCircular, reddish, glistening, moist colonies; gram positive cocci in pairs and tetrads, non-motile; gelatin not liquefied; nitrates reduced to nitrites; indole and H~S not produced; starch not hydrolysed; dextrose, sucrose, lactose and xylose not fermented; casein not digested. Source: Common salt. ldentity.--Corresponds to the description of Sarcina littoralis (Lochhead, 1934). Strain Hlv--Colonies rose-red, moist, glistening, viscous; rods, non- motile, curved, single, pairs and short chains; gram variable, non-spore- iorming, gelatin liquefied; nitrates reduced; indole produced frbm tryptone; H# not produced; starch hydrolysed; dextrose, sucrose, lactose and xylose not fermented; casein peptonized; on salt potato, raised, rose-red viscid growth, grows on 3.5~ NaC1 media. Identity: Appears to agree with the description of Serratia species b (Gibbons, 1937) except for gram reaction. Source: Vedaranyam salt. Red Halophilic Bacteria from Salted Fish and Salt 21

It was observed that these red growths were viscous tending to draw out into fine threads on the needle, as experienced by Bitting (1911). Hence purification was rather difficult. In the salt milk agar medium, good growth covering the entire slant, or plate could be obtained in 7 days at 37 ~ C., while at 4 ~ C. there was no growth for the observed period of 4 months. At room temperature of 28 to 33 ~ C. growth was slower than at 37 ~ C. Incubation of the plates in the incubator without adjusting the humidity, resulted either in loss of moisture and dehydration of the medium, or absorption of too much of moisture, and growth was adversely affected. Cultures placed in a desiccator and maintained at an R.H. of 75~ developed well. 0.1~o acetic acid in the medium did not prevent the growth of these organisms. DISCUSSION Though a great deal of work was done during the first three decades on the morphology of the halophilic bacteria, it was Lochhead's work (1934) that established the identity and synonimity of some of the confusing list of organisms described earlier. Later the excellent work of Shewan (1937, 1939) and Gibbons (1937) cleared the confusion in nomenclature and also yielded valuable information on the physiological properties. We came across only one rod form so far though many cocci were encountered both in salts and salted fish. Direct examination of the ' Red' from the surface of afflicted salted fish, consistently showed the presence of three or four types of bacteria, viz., gram positive sarcina; gram variable non-sporing rods; short, stout gram positive rods; and gram positive rods with terminal spaces, or "racket forms ". Shewan (1937) has mentioned that the bacillus with "terminal spaces" which look like spores may be either gas vacuoles or granules of sulphur. This is probable because when the ' red' was streaked on salt milk agar, no sporeforming rods were ever encountered. It was supposed that only rod forms, which were highly proteolytic and putrefactive, affected salted fish and hides. In fact Schoop (1935) detected no proteolytic activity on gelatin or in production of indole by these organisms. Our strains show that three of the cocci are gelatinolytic, while one produced indole. The above three strains in fact produced putrefactive odour in milk agar. At least four of our cultures peptonized milk showing their caseolytic power. Some of these, on salted fish, in course of time reduced the flesh to a pulpy mass. Their role in the spoilage of salted fish is thus evident. As regards the effect of temperature, failure of growth at 4 ~ C. noted by us agrees with the findings of Shewan (1939), Castell (1950) and Hess (1942). 22 R. VENKATARAMAN AND A. SREENIVASAN

A starch medium with salt, starch, beef infusion and tryptone was found to be much better than Lochhead's salt milk-agar, the growth mani- festing itself in 48 hours in most of the cases. But chromogenesis was poorer and further work on this is in progress. The susceptibility of various species of fish to the attack of 'Pink' may be widely different. Shewan (1939) has in fact found that " of the four species used Hake was the least susceptible ". We found the susceptibility in the following order in fish salted in the laboratory: Sharks and skates were covered with red in 19 to 30 days, mackerels after 30 days, caranx, ribbon fish and seer in 45 days and silver-bellies after three months, while oil sardines were seen to have the attack after four months. Salted fresh water fish like catla, labeo and catfishes also were susceptible. The varia- tion in susceptibility of different species to red attack cannot be explained with any definiteness in the present state of our knowledge about these organisms. SUMMARY 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 grew in salt concentration of 3.5% while one coccus form grew in KCI medium in p!ace of NaC1. Use of sterilized salt for curing and storage at low temperatures are indicated for preservation of salted fish.

ACKNOWLEDGMENTS Our grateful thanks are due to Dr. James M. Shewan, Torry Research Station, Aberdeen, for kindly checking the identity of 3 of our strains, and for helpful suggestions. REFERENCES Beekwith, T.D. .. "Ein Halophytischer Diplocoecus," Zentralblatt f. Bakt., II Abt., 1911, 32, 193-94. Bitting, A.W. .. "Preparation of Cod and other salt fish for the market, including a bacteriological study of the causes of redden- ins," U.S. Dept. Agrie. Bur. Chem. Bull., 1911, 133, 40. Red Halophilic Bacteria from Salted Fish and Salt 23

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.~ "The reddening of salt fish," Ibid., 1938; (1939), 113-15. Stuart, L. S., Frey, R. W. and "Microbiological studies of salt in relation to the reddening James, L. H. of salted hides," U.S. Dept. Agric. Tech. Bull., 1933, 383. Stather, F. .. " 'Rote verfarbung' und 'Rote Erhitzung' " auf gesalzener

o. Roh-haute." Cited by Stuart et al. (Supra.) Venkataraman, R. and Vasavan, "Studies on the salting of marine fishes of Malabar Coast A.G. with special reference to the ratio of salt to be used," Prec. Indian Sci. Congr., 1953 (Abstr.).