Evaluation of Microbiological Test Kits for Hydrocarbon Fuel Systems

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1979, p /79/5-871/7$2./ Vol. 37, No. 5 Evaluation of Microbiological Test Kits for Hydrocarbon Fuel Systems C. A. BAILEY AND M. E. MAY* Naval Research Laboratory, Washington, D.C Received for publication 23 February 1979 Commercially available kits were tested for their ability to detect bacterial and fungal contamination in hydrocarbon fuel systems. The handling ease of the kits was evaluated, and their sensitivity was compared with that of conventional methods. Most kits in both laboratory and field studies compared well with laboratory methods and were sufficiently sensitive to determine contamination in shipboard fuel tanks. Microbial growth in hydrocarbon fuel systems arises because of the impossibility of keeping storage facilities sterile and the inevitable presence of water from condensation, poor maintenance, or its deliberate addition as ballast in shipboard tanks. The two-phase fuel-water system supports microbiological growth (4, 5, 8, 1, 11), which may use the fuel as an organic carbon source and manifest itself as slime and fungal mats causing line and filter blockages and gauge malfunctions. Metabolic products can also be deleterious; for example, sulfate-reducing bacteria produce sulfides which degrade the fuel and also accelerate metallic corrosion. Consequently, there is a need to monitor the buildup of microbiological contamination in shipboard fuel tanks. Test kits are commercially available for use as monitors of microbial contamination in fluids where bacteria and fungi are present. The hazards of such contamination in food processing and the medical and industrial fields have been well documented (3, 9), and test kits have the potential capability of exposing early signs of contamination (2, 3). Each kit is designed to detect a specific type of infection. Only the API broth vials (Difco) and the Microb Monitor (Boron Oil Co.) kits are specifically designed for use in fuel tanks, but others are adaptable for this purpose. A comparison of the sensitivity and specificity of various kits was made with standard laboratory methods by using laboratory cultures of common fuel contaminants. Selected kits were used to assay contamination of field samples taken from water-ballasted shipboard fuel tanks. MATERUILS AND METHODS Microbiological test kits. The kits, listed with their sources, shelf life, and type in Table 1, are used for detecting and enumerating (i) total bacteria, (ii) fungi and yeasts, and (iii) sulfate-reducing bacteria. Those that use dipslides or dip-pads (Fig. 1A, B, and C) consist of sterile containers with either plastic dipslides covered with a suitable agar medium or pads enriched with a nutrient. The slide or pad is immersed in the test liquid and returned to its container to be incubated for the appropriate time and temperature. To ease surface colony observation, some dipslides contain an indicator dye which turns the bacterial colonies red as the medium is used. The two test tube kits (Fig. 1D) designed for sulfatereducing bacteria detection consist of nutrient agarfilled tubes into which capillaries containing the test fluid are inserted. Mineral oil and a tablet generating CO2 are added to the S-R Deeps before capping, whereas the Easicult-S is simply capped. Sulfate reducers generate sulfide which reacts with ferrous ions in the medium, forming black ferrous sulfide. The medium turns black around and within the capillary tubes, indicating a positive test. For these kits, as well as the dipslides and the dip-pads, the quantity of growth appearing within a specific time can be compared with calibration photographs included in the kits to determine the approximate number of microorganisms in the original test liquid. Two vial-type kits tested are shown in Fig. 1E. Both require a syringe for inoculation. The API broth vials turn black as a positive test for sulfate reducers. By serial dilutions, it is possible to use the vials for an estimation of the numbers of sulfate-reducing bacteria. The Microb Monitor consists of two 12-ml vials containing both fuel and nutrient-enriched aqueous phases. After inoculation and incubation, the presence of microorganisms may be observed as turbidity, slime, mat formation, or color change in the fuel phase and compared with a similarly inoculated control vial containing a biocide. Laboratory microorganisms. A fungus (Cladosporium resinae) and a yeast (Candida sp.) commonly found in fuel tanks were isolated from a contaminated JP-5 storage tank. Sulfate-reducing bacteria mixed with other bacteria were from a semicontinuous laboratory culture originally isolated from infected fuel tanks (6). Pseudomonas marina (American Type lture Collection) was used for total bacterial count 871 Downloaded from on October 9, 218 by guest

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3 VOL. 37, 1979 because it is a ubiquitous marine bacterium commonly present in ballast water. ltures of C. resinae and Candida sp. were grown in 25 ml of mineral salts solution (1) enriched with.5% peptone and yeast under JP-5. The P. marina MICROBIOLOGICAL TEST KITS 873 was grown in marine broth 2216 (Difco) under JP-5. After inoculation the two-phase systems were incubated at 27 C long enough to yield copious growth. Laboratory test samples. The flasks containing the aerobic test organisms were shaken to disperse the Downloaded from on October 9, 218 by guest FIG. 1. Kits. (A) Dipslides showing 1-fold dilutions of bacterial growth. (B) Various designs of dipslides (left to right): Grotan, Keimindikator, Millipore. (C) Biostix and Mycostix dip-pads showing (left to right) bacteria, yeast, and fungi. (D) (Left) S-R Deep test tube kits showing the first trace of sulfate reduction. (Right) Easicult kit showing the first trace. (E) (Left) Microb Monitor control vial and vial supporting fungal growth. (Right) API control vial and vial showing sulfate reduction.

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5 VOL. 37, 1979 growth throughout the aqueous phase, after which a sample was removed. One hundred-fold dilutions were prepared as far as 1' to determine the end point sensitivity of the kits. Parallel measurements of the microorganisms were carried out by using the standard plate count method with potato dextrose agar (Difco) +.5% yeast extract for the C. resinae and the Candida sp. and marine agar 2216 (Difco) for the P. marina. The samples of sulfate reducers were diluted to 1' under heptane (to maintain anaerobic conditions). A reducing agar medium developed by Sisler and ZoBell (12), and modified by Klemme and Neihof (7), was used in the standard test for sulfate-reducing bacteria. Kit instructions were followed carefully. The samples were incubated at 27 C and read after the allotted times. Field test samples. Two naval ships were selected MICROBIOLOGICAL TEST KITS 875 for field testing. Samples of sludge from a centrifugal purifier were taken. Dewatering tubes were used to obtain samples of marine diesel fuel with entrained water and of seawater ballast. In an attempt to adapt the dip-pads for sampling the fuel, the Biostix pad was first hydrated with.1 ml and the Mycostix pad with.4 ml of distilled water before immersion in the fuel. Parallel tests were done by using standard laboratory methods to determine the microorganisms in these field samples. RESULTS AND DISCUSSION Laboratory studies. Most of the kits for testing aerobic bacteria, fungi, and yeasts compared favorably with the standard pour plate technique (Table 1). The Millipore Total Count TABLE 2. Field tests of microbiological kits used on samples from two naval ships' Bacterial/fungal growth Kit or laboratory medium Ship A (purifier)b" Ship A (diesel fuel)' Ship B (seawater ballast)' Biostix (bacteri <1 bacteria/ml <5 bacteria/ml 21' bacteria/ml' Grotan (bacteri Heavy fungal growth <13 bacteria/ml 15 bacteria/ml' Easicult TTC (bacteri Heavy fungal infection, Slight bacterial infec- 1/1 dilution tion, 1/2 dilution yielded 1 bacterial colony Millipore Total Count 12 bacteria/ml' Microb Monitor Fungal growth at inter- No growth at 4 days; Slime at interface; color face and throughout fungal growth at inter- change to pink solution face after 3 weeks of incubation Millipore (fungi) 12 fungi/ml Mycostix (fungi & -14 fungi/ml 51 fungi/mil 12 fungi/ml yeasts) Easicult M (fungi) Heavy fungal infection, No growth, 1/2 dilution 1/1 dilution PDA + Yf + 5% seawa- Heavy fungal growth No growth, 1/1 dilution 2 fungi/mil ter (spread plate) (fungi) Mineral salts overlaid Heavy fungal growth at No growth at 4 days; Fungal growth at interwith JP-5 (fungi) interface fungal growth at inter- face after 1 week of inface at 3 weeks of in- cubation cubation Sisler's (triple strength) No growth No growth Very heavy growth (sulfate-reducing bacteri S-R Deeps (sulfate-re- No growth No growth Very heavy, 216/ml ducing bacteri Easicult-S (sulfate-re- No growth No growth Very heavy ducing bacteri API broth (dilution 216/mi vials) (sulfate-reducing bacteri a Samples taken from ship A were sludge from a centrifugal purifier and fuel with entrained water from a fuel tank. For ship B, samples were taken of ballast water from a fuel tank. b The sludge was suspended using 1 part sludge to 9 parts sterile distilled water. c Results are recorded according to kit instructions. d Entrained water was also taken from this tube. Several tests made showed: Mycostix-12 to 13 fungi/ml; potato dextrose agar +.5% yeast extract + 5% seawater-15 fungi/mil; mineral salts overlaid with JP-5- fungal growth but no mat formation at interface. 'Total bacteria on marine agar 2216 (Difco)-15 to 16. f PDA + Y, Potato dextrose agar (Difco) +.5% yeast extract. Downloaded from on October 9, 218 by guest

6 876 BAILEY AND MAY kit for bacteria required at least a 1 log higher count for a positive test. This may be due to the *age of the kit; however, the Millipore fungus and yeast kit gave results comparable to the pour plate method. The Microb Monitor did not support the growth of P. marina, but the fungus C. resinae grew well. In the tests for sulfate reducers, Easicult and the API broth vials did not give positive results in the detection time specified, and the Biosan kit was 1 log less sensitive than the standard laboratory method. After 2 months, positive indications of growth were detected in the former two kits. Although it had been noted in this laboratory that the API broth vials were not as sensitive as the modified Sisler medium, the vials have been used effectively to determine the presence of sulfate reducers in fuel tank ballast water (5). Field tests. Table 2 gives the results from the fuel tanks on the two naval ships. The kit results agreed very well with the laboratory methods. With slight fungal contamination, as in the diesel fuel sampled from the dewatering tube, an increase in the incubation time may be required to give a positive test. In ship A the heavy fungal growth from the purifier was almost entirely C. resinae, as was the fungal growth from the diesel fuel. The predominant fungus in the entrained water from this ship was identified as a Fusarium sp. Previous studies in this laboratory on the spores of C. resinae found in fuel have shown them to be very hydrophobic, and they are rarely picked up in entrained ballast water. It was necessary to test the fuel for this contaminant. This was also evident in the studies on ship B; no C. resinae was found in the seawater ballast, but it was present in the fuel. The predominant feature in the seawater ballast of ship B was the heavy sulfate reducer contamination. Although negative or delayed-positive tests were obtained by using the laboratory cultures with the sulfate reducer kits, this laboratory has usually found that sulfate-reducing bacteria have a more vigorous activity in the field than under laboratory continuous culture conditions (this can be observed in the comparable growth responses in Sisler medium). The difference in activity could account for the disparity in the laboratory and field tests. It should be noted that since the dipstick kits were not designed for immersion in fuel, the counts of organisms in the diesel fuel cannot be considered accurate. Further studies need to be done to correlate the actual numbers per milliliter of fuel with what is evident on the test kits. Evaluation of test kits. The dipslides could be handled very easily by one person and were APPL. ENVIRON. MICROBIOL. convenient to use. Those kits requiring the use of syringes, e.g., the Microb Monitor, were more cumbersome. The capillary tubes in the sulfatereducing bacteria kits were difficult to handle, especially in field situations as on board ship. The directions were easy to follow, and sampling could be done by personnel with minimal training. If used properly, only three special precautions should be emphasized in the instructions: (i) avoid touching the sampling device to anything but the test liquid; (ii) reinsert the dipslides into their tubes immediately; and (iii) refrain from opening the incubated tubes. The kits showing positive results were suitable for indicating changes in microbial populations in shipboard fuel tanks. There is a good possibility that they could be adapted for use in fuel, as well as in water, and more work will be done on this aspect. Even though the kits were designed primarily for detection of freshwater organisms, as found in industrial fluids (3), with one exception the kits were also usable for marine organisms. The Microb Monitor did not support growth of the marine bacterium probably because the aqueous phase has a low salinity. In the case of the other kits, enough seawater was carried with the sample to provide the necessary salt concentration. Since there were no fuel-related problems evident on either of the sampled ships, the numbers given for microorganisms in the fuel and water (Table 2) could be considered normal background; however, it is not known what quantitative level of microorganisms can be tolerated before problems arise. Since the kits are sensitive enough for the background level, they would be acceptable for detecting higher levels of contamination (2, 3, 13). ACKNOWLEDGMENTS We are grateful for the encouragement and advice of Dorothea E. Klemme and Rex A. Neihof. LITERATURE CITED 1. Bushnell, L. D., and H. F. Haas The utilization of certain hydrocarbons by microorganisms. J. Bacteriol. 41: Cotton, R. A., K. J. Sladek, and B. I. Sohn Evaluation of the coli-count and total count samplers in a variety of source waters, p In World Congress of Environmental Medicine and Biology, Colloq. no. 1, Paris, France. 3. Genner, C Evaluation of the "dipslide" technique for microbiological testing of industrial fluids. Proc. Biochem. 11: Hendy, N. I Some observations on Cladosporium resinae as a fuel contaminant and its possible role in the corrosion of aluminum alloy fuel tanks. Trans. Br. Mycol. Soc. 47: Klemme, D. E Sulfate reducing bacteria in fuel tanks of aircraft carriers. NRL Memorandum Rep Naval Research Laboratory, Washington, D.C. Downloaded from on October 9, 218 by guest

7 VOL. 37, Klemme, D. E., and J. M. Leonard Inhibitors for marine sulfate-reducing bacteria in shipboard fuel storage tanks. NRL Memorandum Rep Naval Research Laboratory, Washington, D.C. 7. Klemme, D. E., and R. A. Neihof An evaluation in large-scale test systems of biocides for control of sulfate-reducing bacteria in shipboard fuel tanks. NRL Memorandum Rep Naval Research Laboratory, Washington, D.C. 8. Leonard, J. M., and D. E. Klemme Fungi in fuel, p In Report of NRL progress. Naval Research Laboratory, Washington, D.C. 9. Mossel, D. A. A., I. Eelderink, H. devor, and E. D. Keizer Use of agar immersion, plating and contact (AIPC) slides for the bacteriological monitoring of MICROBIOLOGICAL TEST KITS 877 foods, meals and the food environment. Lab. Pract. 25: Parberry, D. G; 1971,Biological problems in jet aviation fuel and the biology of Anorphotheca resinae. Mater. Org. 6: Sheridan, J. E., and J. J. Soteros A survey of fungi in jet aircraft fuel systems in New Zealand. Int. Biodeterior. Bull. 1: Sisler, F. D., and C. E. ZoBell Hydrogen-utilizing, sulfate-reducing bacteria in marine sediments. J. Bacteriol. 6: Thomas, C. J., T. A. McMeekin, and C. Balis Retention of bacteria in liquid films at agar surfaces. Appl. Ind. Microbiol. 34: Downloaded from on October 9, 218 by guest