NB/SH/T 0860-2013 Standard test method for viable bacteria and fungi counts in liquid fuels — Filtration and culture procedures

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NB/SH/T 0860-2013 specifies a membrane filtration method for the detection and counting of heterotrophic bacteria and fungi in liquid fuels with a kinematic viscosity not greater than 24 mm²/s at ambient temperature; it can be carried out in the field or in the laboratory.

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Scope: Applies to liquid fuels. Specifies a method for the detection and counting of heterotrophic bacteria and fungi in liquid fuels with a kinematic viscosity not greater than 24 mm²/s at ambient temperature, using membrane filtration. It can be carried out in the field or in the laboratory. Note: the quantitative test portion is mainly derived from IP 385 and ASTM D5259. Limits on interpretation of results: the ability of an individual microorganism to form a colony on a particular culture medium depends on the taxonomic and ecological state of the species counted, on the chemical properties of the medium and on the culture conditions; test results should not be interpreted as absolute values, but should be used together with other parameters as part of diagnosis and environmental monitoring in accordance with ASTM D6469. Counting results may be used as part of diagnosis or routine monitoring, and are not to be used as a fuel quality index. Not applicable: no clause explicitly listing "not applicable" is provided in the available material.

Principle: The membrane is placed on (the filtration apparatus); if the sample contains water, the water is first removed. A certain volume of the dehydrated fuel sample is filtered aseptically through the membrane, and the filtered (membrane) is placed on two agar media, MEA and TSA, incubated at a specified temperature for 3 d and 5 d to detect the presence of CFU, and the colonies are counted with a manual or electric counter. Where conditions permit, colonies are identified on the medium by colony colour, morphology and microscopic characteristics. (Note: this paragraph is [source unclear] and noisy in the original; text appears to be missing after "the membrane is placed on" and after "the filtered", [to be verified] ([source unclear]).)

Apparatus: Separatory funnel: glass, capacity 500 mL. 7.2 Graduated cylinder: glass, capacity 100 mL and 1 L. 7.3 Pipette: glass or pre-sterilized disposable plastic, or an adjustable-volume pipette with disposable plastic tips, volume 10 mL. 7.4 Filtration membrane: cellulose mixed ester, pre-sterilized, gridded preferred, diameter 47 mm, pore size 0.45 μm. Note: although the recommended membrane material is cellulose mixed ester, the choice of membrane material will depend on individual preference and fuel type. 7.5 Filter, either of the following two filters may be used: 7.5.1 Sterile syringe: of suitable capacity, with a pre-sterilized filter. 7.5.2 Filter assembly: pre-sterilized single or multiple filter holder assemblies, of glass, stainless steel or polypropylene. Note: if a vacuum filtration device is selected (7.5.2), a vacuum source with a vacuum not exceeding −66 kPa is required. 7.6 Tweezers: flat-tipped tweezers. 7.7 Suction flask: of sufficient capacity to hold the filtered sample and rinsing solvent. 7.8 Petri dishes: pre-sterilized disposable plastic or glass, diameter ≥50 mm. Note: pre-poured culture dishes can be purchased commercially and used in place of the dishes listed here. 7.9 Bacterial incubator: capable of controlling 25 °C±2 and other temperatures (temperature adjustable from room temperature to 60 °C), selected as appropriate. 7.10 Water bath: temperature controlled at 47 °C±2 °C, able to hold 500 mL flasks, and to hold at least one bottle of the agar medium to be used. 7.11 Glass bottles: screw-cap, airtight, nominal capacity 500 mL. 7.12 Culture tubes: glass, 16 mm×125 mm, with stoppers. 7.13 Autoclave: able to hold 500 mL glass bottles placed upright. Note: if commercially pre-poured culture dishes are used, clauses 7.10–7.13 may be omitted. 7.14 Colony counter: manual or automatic. 7.15 0.2 μm filter membrane: generally made of cellulose acetate, cellulose nitrate, polycarbonate or similar materials.

Reagents and reference materials: Reagent purity: analytical grade. 4.2 Agar used for culture media: biochemical reagent grade. 4.3 Water: unless otherwise specified, shall conform to the requirements for grade 3 water in GB/T 6682—2008. 4.4 Chlortetracycline (chlorotetracycline) aqueous solution: mass concentration 1 g/L. Dissolve 0.1 g of chlortetracycline in water and dilute to 100 mL; sterilize by filtration through a 0.2 μm filter membrane. 4.5 Detergent solution: mass fraction 1%. Dissolve 10 mL of polyoxyethylene (20) sorbitan monooleate in 990 mL of water; sterilization may be achieved by filtration through a 0.2 μm filter membrane or by autoclaving at 121 °C for 15 min. 4.6 Hydrochloric acid solution: concentration 1 mol/L. 4.7 Lactic acid aqueous solution: mass concentration 100 g/L. Dissolve 10 g of lactic acid in water and dilute to 100 mL; sterilize by filtration through a 0.2 μm filter membrane. 4.8 Preparation of malt extract agar (MEA): Composition: malt extract 30 g; peptone 5 g; agar 15 g; water 1 L. Preparation: boil the malt extract, peptone and agar in 1 L of water until dissolved. Adjust the pH to 5.4±0.2 with hydrochloric acid (see 8.6) or sodium hydroxide (see 8.10). Transfer 250 mL of this to a 500 mL glass bottle (see 7.11). Sterilize in an autoclave at 121 °C±2 °C for 10 min, remove, cool and keep in a water bath at 47 °C±2 °C. Alternatively, when the agar has cooled to 47 °C±2 °C, add 1 mL of chlortetracycline aqueous solution to each 100 mL of MEA solution and shake to mix evenly. If a pH of 3.5 is required for the medium, adjust by adding 10 mL of lactic acid aqueous solution. Once acidified, MEA must not be reheated. Prepare agar plates by pouring MEA into Petri dishes to form a film about 4 mm thick and allowing it to cool and set. Note 1: MEA is available from various manufacturers as a dehydrated medium or as pre-poured plates, with or without antibiotics. When sterilizing purchased dehydrated MEA medium, follow the instructions for use and avoid overheating. Note 2: for culture media other than MEA, it shall be demonstrated that the growth counts of bacteria in the test sample on the alternative medium are comparable. Note 3: other antibiotics may be selected, but it must be confirmed that the antibiotic can inhibit the growth of bacteria other than yeasts and moulds. 4.9 Preparation of quarter-strength Ringer's solution: Composition: sodium chloride 2.25 g; potassium chloride 0.105 g; calcium chloride 0.12 g; sodium bicarbonate 0.05 g; water 1 L. Preparation: dissolve the above salt components in 1 L of water, then dispense 10 mL into culture tubes and sterilize in an autoclave at 121 °C for 15 min. Note: quarter-strength Ringer's solution can be purchased commercially in tablet form. 4.10 Sodium hydroxide solution: mass concentration 100 g/L. Dissolve 10 g of sodium hydroxide in water and dilute to 100 mL. 4.11 Preparation of tryptic soy agar (TSA): Composition: tryptone 15 g; soy peptone 5 g; sodium chloride 5 g; agar 15 g; water 1 L. Preparation: add the above components to 1 L of water and heat to dissolve. Transfer 250 mL of this to a 500 mL stoppered glass bottle (see 7.11), sterilize in an autoclave at 121 °C±2 °C for 10 min, remove, cool and keep in a water bath at 47 °C±2 °C. Take a sample for pH measurement; if the pH is not within the range 7.3±0.3, discard this solution and prepare a fresh batch. Medium agar plates are made by pouring a sufficient amount of TSA into Petri dishes to form a film 4 mm thick, and allowing it to cool and solidify. Note 1: TSA medium is available commercially in dehydrated and pre-poured forms. Note 2: for culture media other than TSA, it shall be demonstrated that the growth counts of bacteria in the test sample on the alternative medium are comparable. 4.12 Alcohol solution: methanol or isopropanol solution of mass fraction (value [to be verified] ([source unclear]) in the original), used for disinfection of equipment and utensils in the field or laboratory.

Key conditions: Culture temperature: 25 °C±2 °C (TSA dishes incubated inverted). If the temperature of the system in which the sample is collected is 5 °C higher than the standard bacterial growth temperature, adjusting the incubator temperature to match the ambient temperature can improve the recovery of viable bacteria. Although 25 °C is suitable for most microbial systems, a higher growth temperature may also be used when the temperature of the system from which the sample is taken exceeds 30 °C; incubate within a range such that the culture temperature differs by not more than 5 °C from the sample collection temperature, and the incubator shall be able to control the temperature within ±2 °C of the set temperature. Culture time: check bacterial growth in the TSA and MEA dishes after 3 d, and check fungal growth in the TSA and MEA dishes after a further 24 h of incubation (i.e. bacteria 3 d, fungi 5 d). Water bath temperature: 47 °C±2 °C. Vacuum source vacuum: not exceeding −66 kPa (see note to 7.5.2).

Calculation and results: Quantitative index: CFU/L (colony-forming units per litre of sample). Calculation formula: N = CC × 1000 / V N: number of CFU per litre of sample; CC: number of colonies in the Petri dish (see 11.4.1); V: volume of sample filtered, mL. When there are no colonies: set CC=1, calculate N, and report bacteria <N CFU/L or fungi <N CFU/L. Multiple tests: calculate the mean and standard deviation of the results. Abbreviations: CFU (colony-forming unit), HPC (heterotrophic plate count), MF (membrane filtration), MEA (malt extract agar), TNTC (too numerous to count), TSA (tryptic soy agar). Symbols: CC (number of colonies on membrane filter); V (sample volume filtered, mL).

Normative references: GB/T 4756 Petroleum liquids — Manual sampling (GB/T 4756—1998, eqv ISO 3170:1988) GB/T 6682—2008 Water for analytical laboratory use — Specification and test methods (ISO 3696:1987 MOD) ASTM D3508 Standard test method for evaluating water-miscible metalworking fluids for coliform bacteria ASTM D5245 Standard practice for cleaning laboratory glassware, plasticware, and equipment used in microbiological analyses ASTM D5259 Standard test method for isolation and enumeration of enterococci from water by the membrane filter procedure ASTM D6469 Standard guide for microbial contamination in fuels and fuel systems ASTM D7463 Standard test method for adenosine triphosphate (ATP) content of microorganisms in fuel, fuel/water mixtures, and fuel-associated water ASTM D7464-08 Standard practice for manual sampling of liquid fuels, associated materials and fuel system components for microbiological testing ASTM E1326 Standard guide for evaluating nonconventional microbiological tests used for estimating bacterial populations IP 385 Determination of aerobic bacterial content of fuels and distillate fuels with a boiling point below 390 °C — Filtration and culture method Note: the first item in clause 2 of the original, "CB/T 4756", is suspected to be a [source unclear] error for "GB/T 4756", [to be verified] ([source unclear]).

Evidence note: [to be verified] and [source unclear] mark specific points that still require checking against the original text of the standard and do not indicate that they have been verified. Do not use these values or steps directly as a basis for work.

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