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Evaluation of modified Zobell marine agar for differential isolation of histamine-forming bacteria from fresh fish.

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Title Evaluation of modified Zobell marine agar for differential isolation of histamine-forming bacteria from fresh fish.
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Creator Devivilla S, Stephen J, Lekshmi M, Kumar SH, Nayak BB*
 
Subject Histamine formersSeafoodMarine agarNiven's medium
 
Description Not Available
Histamine formed in fish due to the activities of histamine-forming bacteria is a health hazard. In this study, the modified Zobell marine agar (mZMA) containing histamine and a pH indicator dye helped in better discrimination and isolation of histamine-forming bacteria from fresh fish.

Histamine fish poisoning is one of the major health risks associated with the consumption of scombroid fishes like tuna, seer fish and mackerel. Exogenous histamine formed in fish due to bacterial activities is responsible for scombroid poisoning (Feng et al., 2016). Some groups of bacteria are capable of producing histidine decarboxylase enzyme which converts free histidine present in the fish tissue to histamine by decarboxylation reaction (Hungerford, 2010). Early detection of histamine forming bacteria is necessary for assuring the quality of seafood.

Many different selective media have been proposed for the isolation of histamine-forming bacteria from fish. Niven's medium, which is a modification of Moeller's decarboxylase medium, is routinely used for the isolation of histamine-forming bacteria (Niven et al., 1981). Subsequently, this medium was modified by replacing the indicator dye bromocresol purple (pH range 5.2–6.8) with cresol red (pH range 7.2–8.8) which resulted in modified Niven's medium (MNM) (Yoshinaga and Frank, 1982). MNM is widely used for the detection and quantification of histamine-forming bacteria. However, certain limitations are associated with MNM. The low pH (5.3) of the medium restricts the growth of some acid-sensitive histamine-forming bacteria (Bjornsdottir-Butler et al., 2011; Niven et al., 1981). The occurrence of false positive reaction with this media has been reported to be as high as 63% (López-Sabater et al., 1996) which could be due to the production of non-histamine alkaline compounds (Tembhurne et al., 2013). Compared to Moeller's medium, Niven's medium is devoid of glucose. The absence of carbohydrate can force the microorganisms to use simpler proteins like tryptones leading to the formation of ammoniacal compounds which increase the pH of the medium. Thus, the addition of glucose as a carbon source can reduce false positives (Kim et al., 2001). However, the reduction in pH due to the production of acid from glucose can mask the pH increase by histamine leading to false negative results (Maijala and Eerola, 1993). In some cases, high histamine producers were found to be negative on Niven's medium and its modified versions (da Silva et al., 2002; Roig-Sagués et al., 1997). Thus, the existing media and cultivation methods need constant improvement for accurate detection and quantification of histamine-forming bacteria in fish. In this context, the present study was carried out to investigate the efficiency of Modified Zobell marine agar (mZMA) for the isolation of histamine-forming bacteria from fish.

Fresh fish were collected from local markets and brought to the laboratory. About 30 g of fish muscle containing 10 g each from dorsal, ventral and caudal regions of an individual fish was mixed briefly in a blender. From this, 10 g was taken and homogenized with 90 ml of sterile physiological saline (0.85% NaCl) for two minutes in a stomacher (Seward Inc., UK), serially diluted and spread plated on Modified Niven's medium (MNM) (Mavromatis and Quantick, 2002). The plates were incubated 30 °C for 18–24 h. Bacterial colonies surrounded by a pink halo were considered as presumptive histamine producers, while those which did not produce the typical colony phenotypes were considered as histamine negative bacteria. The selected isolates were purified on trypticase soy agar (TSA) plates containing 0.1% L-histidine. The histidine decarboxylase activity of the isolates was determined as previously described (Tembhurne et al., 2013). The isolates were stored in glycerol broth at −80 °C until further use.

Histamine production by selected bacteria was estimated using an ELISA kit (Immunolab, GmbH, Germany). Bacteria were inoculated in tuna fish infusion broth prepared from tuna meat to simulate the seafood characteristics and incubated for 24 h at 30 °C. The genomic DNA from the isolates was extracted using GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, USA) and used in PCR amplifications. The hdc gene was amplified using primers and conditions as described previously (Takahashi et al., 2003). Morganella morganii MTCC-662 (hdc+) was used as the positive control. For identification of bacteria, the partial 16SrRNA gene sequences were amplified using universal primers and sequenced (Bioserve Biotechnologies, Secunderabad, India).

Zobell marine agar (Hi-Media, Mumbai, India) was selected for modification because of its ability to support a range of marine bacteria. The final pH of the medium was adjusted to 6.5. Compositions of mZMA and MNM are given in Table 1. MNM was prepared as previously described (Yoshinaga and Frank, 1982). ZMA (Zobell, 1941) was modified by adding bromothymol blue indicator and L-histidine hydrochloride (Table 1). L-histidine hydrochloride (Sigma-Aldrich, India) was used at three different concentrations of 0.25%, 0.5% and 1%, while bromothymol blue was used at 0.02%, 0.04% and 0.08%. Both media were sterilized by autoclaving at 121 °C for 15 min at 15 lbs. pressure. Three histamine-producers namely Morganella morganii, Klebsiella variicola, Staphylococcus capitis and two histamine negative bacteria Bacillus subtilis and Proteus vulgaris were spot inoculated on both MNM and mZMA. The plates were incubated at 30 °C for 24 to 48 h and the colony colors were recorded.
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Date 2022-06-17T05:21:52Z
2022-06-17T05:21:52Z
2019-06-01
 
Type Research Paper
 
Identifier Not Available
Not Available
http://krishi.icar.gov.in/jspui/handle/123456789/72738
 
Language English
 
Relation Not Available;
 
Publisher ELSEVIER