Use of a Chemical Sanitizer for Cleaning Fish Boxes J appl Bact 1975, 38, 107 113 Use of a Chemical Sanitizer for Cleaning Fish Boxes R BHADRA AND A N BOSE Department of Food Technology and Biochemica.
J appl Bact 1975, 38, 107-113 Use of a Chemical Sanitizer for Cleaning Fish Boxes R BHADRA AND A N BOSE Department of Food Technology and Biochemical Engineering, ladavpur University, Calcutta 700 , India Received M a y 1974 and accepted 14 December 1974 A number of organic, inorganic and surfactant substances were tested for their cleaning efficiency, ability to reduce bacterial load and deodorize fish boxes A 0.1 % iodine solution had good bactericidal and deodorizing properties, whilst a number of surfactants, especially the quaternary compound Quat-LTM, had good cleaning and bactericidal properties None of the substances used possessed all three desirable properties sought for in this study WASHINGWITH water alone has not been found to be a satisfactory method for cleaning fish containers (Spencer, 1959, 1960) Hypochlorite solutions and quaternary ammonium compounds significantly reduced the bacterial load by up to 90-99 ;( of the initial count, but the remaining numbers still amounted to millions/cm2 of surface of fish holds (Spencer, 1960) For cleaning fishing vessels and fish holds, the Fisheries Research Board of Canada (Bulletin, 1967) recommended the use of alkalies such as sodium silicates and trisodium phosphate and a proprietary surfaceactive agent Alkaline salts mixed with detergent sanitizers are generally used for cleaning fish holds and fish containers The present work studied the cleaning of fish containers using criteria of effectiveness;reduction of bacterial load, deodorizing efficiency and visual cleanliness A number of chemical sanitizer, surfactant and quaternary compounds were tested using experimental fish boxes made from plywood, moulded expanded polystyrene or plywood lined with polystyrene Materials and Methods Containers The returnable containers were of types, (a) 60 x 37.5 x 22.5 cm, 2.5 cm thick expanded polystyrene boxes (BASF India Ltd); (b) 40 x 40 x 35 cm, 7-ply plywood boxes; (c) 40 x 40 x 35 cm, 7-ply plywood boxes lined with expanded polystyrene (2 cm thick) with an inner lining of polythene In India such boxes are used to transport fish by rail or road during periods which may extend to 72-96 h The boxes are opened if necessary and re-iced to maintain the temperature Sampling Swabs were made of absorbent cotton (1.5 cm diam., cm long) on a 15 cm glass rod, 2.5 mm diam The swab, contained in a tube, was autoclaved at 121O for 20 [lo71 108 R BHADRA AND A N BOSE The area swabbed was cm wide and cm long using a tinplate swab guide which was sterilized by alcohol and flaming before use The area was sampled by rotating the swab on it, firmly against the direction of wiping, and then transferring it immediately into 100 ml of normal saline; the handle was then pulled free from the swab and discarded The bottle containing the swab was shaken thoroughly Decimal dilutions were prepared and plated on tryptone-glucose agar (TGA) The plates were incubated for days at 37" or days at 22" before counting Cleaning materials The cleaning materials were aqueous solutions of commercial grades of a chemical sanitizer (Hindustan-Lever Sanitizer), inorganic and organic compounds and detergents (see Tables) The commercial preparations were: Teepol, a blend of sodium alkyl aryl sulphonate and alkyl phenol ethylene oxide condensate (National Organic Chemical Industries Ltd, Bombay-18) ; Idet-SL Spl, an anionic liquid detergent (Sarabhi Sons Private Ltd, Ahmedabad-4); Genteel, a liquid detergent for textile washing (Swastik Oil Mills, Bombay-31); Hindustan-Lever Sanitizer and NP-IOE (Hindustan-Lever Ltd, Bombay-69); Quat-LTM, alkyl trimethyl ammonium chloride (Hico Products Private Ltd, Bombay-16); Acinol (anionic) and Ahuralan (non-ionic) detergents (Ahura Chemical Products Private Ltd, Bombay-22) Cleaning methods A solution of the substance being tested was prepared in tap watcr The contaminated surface was then rubbed for a few minutes with a hand brush wetted with the solution after which the containers were washed with water either under a tap or by hosing The boxes were then allowed to drain and stacked Examination of cleaned boxes The total bacterial count/cm2 of surface of the boxes was determined before, and 4-5 days after, washing The load reduction was calculated by standard statistical methods and expressed as percentage of initial load The persistance of fishy odour was judged by a 6-member panel using an arbitrary scale (Hedonic scale), in which represents an extremely strong odour of spoiled fish and 0, the absence of any fishy odour The visual appearance of the container after washing was categorized as thoroughly, moderately or poorly, cleaned The overall efficiency of a cleaning solution was determined by the reduction of bacterial load, the removal of fishy off-odours and the appearance of the washed surface of the box Results In the first series of experiments inorganic substances were used as cleaning agents Sodium triphosphate and iodine gave maximum reduction of bacterial load, while sodium hydroxide and sodium sulphate were also fairly effective in reducing the bacterial load (Table 1) Despite a reduction in the bacterial load of 90-99%, the residual count was still of the order of I O ~ - I O ~ /which C ~ ~ is fairly high Some of the experimental trials with iodine showed a bacterial load reduction to c 10-102/cm2 CLEANING OF FISH BOXES 109 TABLE1 Effect of various cleaning solutions on the reduction of bacterial load of the fish boxes ~~ - Reduction of bacterial load (%) in box A I A* I * * * 37"t 22" 37" 22 37" 22" Cleaning solution (% w / Sodium sulphate (2.5) 97&+0.12" Sodium triphosphate (2.5) 99+0-89 Sodium chloride (2) 9450.92 Sodium carbonate (2) 97k0.49 Sodium hydroxide (1) 98k0.12 Hydrochloric acid (1) 95f0.91 Nitric acid (1) 9520.2 Iodine (0.1) 99kO.9 Sodium lauryl sulphate (0.1) 9420.81 Control 92k0.321 972 0.58 98kO.91 95k0.32 97k0.56 9720.12 97k0.26 9520.21 9850.99 96k0.29 9050.97 B 95L0.33 9710.12 89 0.3 9150.14 94 3.0.11 94 3.0.92 93 0.77 97+ 0.32 93 rt 0.17 87 0.93 + + C 96A0.16 97+0-89 89+0.99 92 f 0.35 95 0.13 94k0.92 94 0.11 952 0.89 94k0.21 85 3.0 11 + + 9820.1 99+ 0.92 95 k0.73 97fO-32 98k 0.95 95 k 0.39 98 k 0.82 99 k0.85 97+ 0.18 93 f0.19 982 0.80 99k0.12 9650.73 96k0.88 9820.92 961t0.01 97k0.31 98 k 0.27 98k0.91 9020.21 * A , B, C, denotes fish boxes as described in the text t Incubation temperatures 8, Mean; b, standard deviation TABLE Evaluation of residual $shy odour and surface appearance of the washedfish boxes - Fishbox L I- A Cleaning solution ( % wlv) Sodium sulphate (2.5) Sodium triphosphate (2.5) Sodium chloride (2) Soaium carbonate (2) Sodium hydroxide (1) Hydrochloric acid (1) Nitric acid (1) Iodine (0.1) Lauryl sulphate (0.1) Control Residual fishy odour score 3 3 4 l B Surface cleaning efficiency Moderate High Moderate High High Moderate Moderate Moderate High Moderate Residual fishy odour score 4 4 4 C Surface cleaning efficiency Residual fishy odour score Surface cleaning efficiency Poor Moderate Poor Moderate Moderate Poor Poor Poor High Poor 3 3 3 Moderate High Moderate High High Moderate Moderate Moderate High Moderate Table gives the results of the evaluation of washed boxes for the removal of fishy odour and for their surface appearance Iodine was best for removing odour, a result consistent with the use of iodine as a deodorizing agent in iodophors Caustic soda and sodium triphosphate removed odour fairly well, but the best cleaning of containers was achieved by using sodium lauryl sulphate, though this failed to remove fishy odour to any significant extent Thus none of the substances tested met all the experimental requirements of this study A few commercial sanitizers (hypochlorite, bleaching powder extract, benzalkonium chloride) and organic compounds (phenol and formalin) were also tested in a similar R BHADRA AND A N BOSE 110 TABLE3 EfSect of certain selected substances as cleaning agent for fish boxes - Reduction of bacterial load (%) in box f A Cleaning solution Commercial hypochlorite (15 % v/v) Bleaching powder extract (20 % wiv) Formalin (4 % v/v) (40 % formaldehyde) Phenol (1 % w/v) Benzalkonium chloride (O.l%w/v) Control * 37"t 22" B * 37" 22 a C' 37" 22 97a+_0.12b98k0.92 95a0.35 97k0.38 98k0.29 98k0.12 9750.32 97k0.99 93k0.91 9550.82 981-0.23 98k0.12 9850.18 9850.82 96k0.28 97k0.39 97k0.51 9850.21 98k0.81 98k0.31 9550-89 97k0.12 95a0.58 97k0.88 94k0.82 95k0.26 97k0.51 98k0.21 98k0.29 98k0.91 9250.81 9150.98 92k0.81 9340.91 90k0.31 89k0.31 * A, B, C, denotes fish boxes as described in the text Incubation temperatures 8, Mean; b, standard deviation TABLE4 Evaluation of residualfishy odour elimination and surface appearances of the washedfish boxes Fishbox h r Cleaning solution Commercial hypochlorite (15 % v/v> Bleaching powder extract (20 % w/v) Formalin (4 % v/v) Phenol (1 % w/v) Benzalkonium chloride (0.1%W/V) Control > A B C & & & Residual Residual Residual Surface Surface fishy fishy Surface fishy odour cleaning odour cleaning odour cleaning score efficiency score efficiency score efficiency Moderate Poor High Moderate Poor Moderate 4 Moderate Moderate Moderate 5 Poor Poor Moderate 4 Moderate Moderate High Moderate Poor Moderate way for their ability to reduce bacterial load of fish boxes (Table 3) It is evident (Table 3) that none of these materials was more effective than sodium triphosphate or iodine in reducing bacterial counts of the fish boxes Their cleaning efficiency and ability to eliminate fishy odour were judged as before and the results are given in Table The fishy odour was almost removed by hypochlorite and bleaching powder just after washing, but significant odour retention occurred after stacking the washed boxes for 3-4 days (Table 4) Thus odour removal efficiency of these materials was not usefully effective because it was of very short duration None of the materials in Table was superior to iodine or sodium triphosphate in cleaning efficiency, CLEANING OF FISH BOXES 111 TABLE5 EfSect of surfactants on the cleaning of the fish boxes Reduction of bacterial load (%) in box A r Cleaning solution* ( % vlv) Teepol (2) Idet 5L Sp (2) Genteel (2) Hindustan Lever-sanitizer (2) NP-1OE (2) Quat LTM (2) Laurylbenzyldimethyl ammonium chloride (2) Acinol(2) Ahuralan (2) Octylphenoxy poly (ethelenoxy ethanol) ethoxylated alkyl phenol (2) Suspension (1 %) of phenylethyl alcohol Control > * * * A C B 37"t 22" 37" 22" 37" 22" 9850.21 9950.12 96kO-19 9850.29 9750.31 9950.72 9850.26 99kO-32 9950.91 97k0.19 9950.12 9820.23 99k0.98 9850.81 9750.35 9850.92 9550.28 9650.39 9650.21 9850.35 9750.21 9750.92 9850.88 9650.38 97k0.12 9750.71 98+0-41 9850.92 99k0.91 9920.91 9750.21 9850.91 9850.29 9920.91 9950.92 99+0-97 9950.98 9950.12 99k0.12 9850.36 9950.99 9950.94 96k0.85 9750.32 9850.52 98k0.88 9850.25 9950.12 9650.25 9750.35 97k0.32 9850.12 9550.18 9650.28 9250.91 9420.73 9350.12 9250.21 - - 8850.91 8950.31 98k0.41 9750.98 9720.29 98k0.92 9850.29 98k0.91 9150.81 9350.81 9450.21 9550.28 * Information on proprietary surface active substances are given in Materials and Methods t Incubation temperatures odour removal or bactericidal action Therefore they were regarded as unsatisfactory for use in cleaning fish boxes A number of commercial surfactants was then used for cleaning the fish boxes The data on the bacterial load reduction (Table 5) show that Quat LTM, Idet-5L Sp and Teepol reduced the bacterial load to almost 102/cm2 Their cleaning efficiencies and deodorizing properties (removal of the fishy odour) are summarized in Table This shows that odour reduction is substantial with Hindustan-Lever sanitizer, Quat LTM, Idet-5L Sp and Ahuralan but not as effective as with the 0-1% iodine solution (Table 2) Indeed when the boxes, after washing with these surfactants, were stacked for a few days, they gave off smells other than fishy odours Although these substances were satisfactory for cleaning they were not effective deodorizing agents Discussion Simply washing fish boxes with tap water, although substantially reducing the surface bacterial load, leaves numbers of c 103-104 organisms/cm2, a residual inoculum likely to cause spoilage of fish placed subsequentlyin the boxes Washing with cleaning agents like iodine solutions, sodium triphosphate and Quat LTM reduced the bacterial load by as much as 99.9% in some trials; iodine solution being most effective The absorption of iodine by Escherichia coli and staphylococci (Hugo & Newton, 1964) strongly inhibits their growth and multiplication Other organisms may be similarly affected which probably explains the role of iodine as a sanitizer Hotchkiss (1944) and Pethica & Schulman (1953) gave evidence that membrane damage at the biochemical level occurred in staphylococci and leakage of nitrogen 112 R BHADRA AND A N BOSE TABLE Evaluation of the residuaIfishy odour and surface appearance of the washed Jish boxes Fishbox h r A > B C & & Cleaning solution (% v/v/) Teepol (2) Idet-5L Sp (2) Genteel (2) Hindustan Lever Sanitizer (2) NP-1OE (2) Quat-LTM (2) % Laurylbenzyldimethyl ammonium chloride (2 %) Acinol (2) Ahuralan (2) Octylphenoxy poly (ethelenoxy ethanol) ethoxylated alkyl phenol (2) Suspension (1 %) of phenylethyl alcohol Control Residual fishy Surface odour cleaning score efficiency 3 3 High High High High High High Moderate 3 Residual Sshy Surface odour cleaning score efficiency Residual fishy Surface odour cleaning score efficiency 2 2 High High Moderate High Moderate High Moderate High High High High High High High Moderate High High 3 Moderate Moderate Moderate 2 High High High Moderate Moderate High Moderate Poor Moderate 3 3 3 and phosphorus-containing compounds from cells together with some dissociation of proteins was observed According to Baker, Harrison & Muller (1941a,b) cationic detergents inhibited respiration of Staphylococcus aureus, Staph albus, E coli, Proteus vulgaris, Salmonella paratyphi and Shigella dysenteriae, while some of the anionics, at sub-inhibitory concentration stimulated bacterial growth This may explain the effectiveness of materials such as Quat LTM Anionic detergents, like Idet-SL Sp and Teepol may remove bacterii simply by cleaning Arguably, the cationic Quat LTM is better than anionic preparations like Idet-5L Sp or Teepol It is concluded that the use of iodine, surfactants and sodium triphosphates are highly effective as cleaning constituents of washing solutions for fish boxes in order to reduce the bacterial load However, iodine solutions are the best for removing ‘off fishy odours and such solutions fulfil of the criteria set in the present study On the other hand, Quat LTM was an effective cleaner and showed high bacterial load reduction but did not adequately remove fishy odours Accordingly none of the materials used met all criteria The authors wish to thank Indian Council of Agricultural Research, New Delhi for financial support for this work Grateful thanks are due to Dr S Varadarajan of Hindustan-Lever Limited, Bombay for kindly supplying the chemical sanitizer and to Hico Chemicals, Bombay, Ahura Chemicals, Bombay and Sarabhi M Chemicels, Bombay for their co-operation and supply of commercial detergents CLEANING OF FISH BOXES 113 References BAKER,S., HARRISON, R W & MULLER,B F (1941~).Action of synthetic detergents on the metabolism of bacteria J exp Med 73,249 BAKER, S., HARRISON, R W & MULLER, B F (19416) The bactericidal action of synthetic detergents J exp Med., 73,249 BuLLETriV (1967) Bulletin No 160 of the Fisheries Research Board of Canada HUGO,W B & NEWTON, J M (1964) The adsorption of iodine from solution by microorganisms and serum J Pharm Pharmac 16, 49 HOTCHKISS, R D (1944) Gramicidin, tyrocidine and tyrothricin, Adv Enzymol 4, 153 PETHICA, B A & SCHULMAN, J H (1953) The physical chemistry of haemolysis by surface active agents Biochem J 53, 177 SPENCER, R (1959) The sanitation of fish boxes I The quantitative and qualitative bacteriology of commercial wooden fish boxes J appl Bact 22, 73 SPENCER,R (1960) The sanitation of Fish boxes 11 The efficiency of commercial wooden fish boxes J appl Bact 23, 10 ... for use in cleaning fish boxes A number of commercial surfactants was then used for cleaning the fish boxes The data on the bacterial load reduction (Table 5) show that Quat LTM, Idet-5L Sp and... the chemical sanitizer and to Hico Chemicals, Bombay, Ahura Chemicals, Bombay and Sarabhi M Chemicels, Bombay for their co-operation and supply of commercial detergents CLEANING OF FISH BOXES. .. temperatures 8, Mean; b, standard deviation TABLE4 Evaluation of residualfishy odour elimination and surface appearances of the washedfish boxes Fishbox h r Cleaning solution Commercial hypochlorite