SY/T 0532-1993 Bacterial analysis of oilfield injection water - Extinction dilution method
Some standard content:
Petroleum and Natural Gas Industry Standard of the People's Republic of China SY/T 0532-93
Analysis Method of Bacteria in Oilfield Injection Water
Extinction Dilution Method
Published on January 6, 1994
China National Petroleum Corporation
Implementation on June 1, 1994
1 Subject Content and Scope of Application
Petroleum and Natural Gas Industry Standard of the People's Republic of China Analysis Method of Bacteria in Oilfield Injection Water
Extinction Dilution Method
$Y/T 0532-93
This standard specifies the terminology, treatment principle, reagents and materials, instruments and equipment, operation steps, bacteria counting method, precision and analysis results of bacteria in injection water. This standard is applicable to the analysis of putrefactive bacteria, acid-reducing bacteria and iron-correcting bacteria in oilfield injection water. The analysis of bacteria in oilfield water can refer to this standard.
2 Reference Standards
SY5329 Recommended Standards and Analysis Methods for Water Quality in Detrital Rocks and Magnetic Injection Plants. 3 Terminology
Dry heat sterilization: Place the items to be sterilized in an oven, gradually raise the temperature to 110-160°C, and maintain for 2-3 hours to achieve sterilization. Moist heat sterilization: Place the culture medium and other items that are not suitable for heat sterilization in a high-low sterilization pot, heat them, and make the steam pressure in the pot reach 0.1MPa, which lasts for 20-30 minutes to achieve sterilization. 4 Principle of the Method
The test of saprophytic bacteria, sulfur-reducing bacteria and iron bacteria is to dilute them. The water sample to be tested is injected into the test period or test stop for inoculation dilution until the sterile growth in the test bottle or test tube is positive after the drum, and then the number of bacteria in the water sample is counted according to the bacterial growth and the correction multiple. 5 Reagents and Materials
a. Chemical reagents (all analytically pure): sodium lactate, sodium sulfate, calcium chloride, ferric sulfate, phosphamide, hydroxide, sodium chloride, ammonium hydroxide, magnesium sulfate, ammonium sulfate, sodium ferric citrate, dipotassium phosphate. E, alcohol: concentration is 75%
c Biological reagents: beef shank, strong white chen, yeast, glucose d, materials: gauze, cotton wool, kraft paper, thread, No. 00 glue, No. 6 needle. 6. Instruments and equipment
a: Electric constant temperature curing box: Xia 30601b. Electric heating wind drying box: convenient irrigation temperature 30~300 (±5 (c. High pressure sterilizer;
d, Toku balance: maximum weighing 100g: sensitivity 0.1g#, Lu box
f. Meter: scale is 0.1pl1 unit
g Electric lamp: 1000~2000W;
h, Beaker: 100.500, 1000ml
|Approved by China National Petroleum Corporation on January 6, 1994 and implemented on June 1, 1994
.Triangle: 500, 1000m
j.Flute: 250, 500mls
k, Test tube: 150mm×15mm, several
1. Wave shift tube: 1ml;
m.Alcohol lamp;
n, Syringe: 1ml;
o. Fine bottle: 125m
SY/T 0532--93
P. Sulfur salt reduction tooth test bottle, saprophytic bacteria test bottle, iron-bound bacteria test bottle. 7 Sampling
7.1 The collected water sample is representative and prevents positive contamination. 7.2 After brushing the narrow-mouth bottle, wrap the bottle mouth with kraft paper. Dry heat or wet heat sterilization 7.3 Before sampling, make the sample flow freely at a flow rate of 5-61/min for 3 minutes, use a sterilized narrow-mouth bottle to sample to the bottle mouth, and add.
7.4 The time from selection to testing of the product shall not exceed 24 175 Caixiang Store shall follow the station label, indicating the sampling period, time, location, sampling conditions and sampler. 8 Analysis steps
8.1 Test bottle method
8.1.1 According to the amount of saprophytic bacteria, succinate-reducing bacteria and iron bacteria in the water sample, arrange several test bottles containing the corresponding bacterial culture into a group and number them in sequence.
8.1.2 Use 75% alcohol solution to disinfect the top blue and operating parts of the test bottle. 8.1.3 Use a syringe without a case to draw 1ml of water into bottle No. 1, shake 8.1.4 Use another sterile syringe to draw 1 ml of liquid from bottle 1 and inject it into bottle 2, then shake. 8.15 Describe the operating procedure and reduce the concentration to the desired final concentration according to the bacterial content. Place in a 30~37℃ incubator for incubation. After 5~7 days of saprophytic culture, if the liquid in the test bottle turns from red to yellow or muddy, it indicates the growth of saprophytes. After 14~21 days of sulfate-reducing bacteria culture, if the liquid in the test bottle turns black, it indicates the growth of sulfate-reducing bacteria. After 7[4cl of iron bacteria culture, if the liquid in the test bottle turns muddy or Reddish brown jelly indicates the growth of iron ions. 8.2 Liquid test tube method
8.2.1 Preparation of sterile water and physiological saline
8.2.1.1 Fill with water: Place sterilized test tubes (or triangular flasks) in a hot or heat-proof container, add 9 1/2 of steamed water and 1/2 of sugar to each container, wrap with kraft paper, and sterilize with wet heat. 8.2.1.2 Physiological hot water: Weigh 0.85 g of sodium hyaluronate, dissolve in 100 ml of natural water, take ten test tubes and distill them into 9 1/2 of physiological saline, and wash and sterilize.
8.2.2 Aseptic operation technique and inoculation method
Put the sterile saline solution used for the sterilized test sample and the diluted water sample into a sterile air chamber, turn on the UV lamp, sterilize for 30 minutes
Turn off the UV, insert the water sample, use a 7% alcohol cotton ball to observe, light the alcohol lamp, take the test tube, twist the cotton ball to loosen it by hand, so as to facilitate the removal of the test tube, take the pipette, and pipette the liquid into the test tube near the flame. After the water sample is sent in, plug it immediately.
8.2.3 Dilution of water sample
8.2.3.1 Use the light bacteria pipette to suck up 1 ml of water, put it in a No. 1 light bacteria water or saline tube, and get a sample with a dilution of 10%.
SY/T0532-93
8.2.3.2 Take a sterile pipette and take 1 ml of water sample from tube 1 and put it into tube 2 to obtain a water sample with a dilution of 10-, and so on until the required concentration is reached. 8.2.4 Preparation of culture medium
8.2.4.1 Saprophytic culture medium
Beef extract
Sodium chloride (SaCl)
Put the above reagents into a beaker, add 1000 ml of distilled water, dissolve it with 10% sodium hydroxide solution and adjust the pH value to 7.4.-7.6, and divide it into test tubes, 5 ml each, cover with cotton wool, wrap it with kraft paper, and sterilize it with damp heat for use. 8.2.4.2 Vegetable acid salt reducing bacteria culture medium
Disindium dihydrogen sulfate
Ammonium chloride
Magnesium sulfate
Sodium sulfate
Calcium chloride
Alcohol paste
Sodium lactate
(K21PO4)
(NHCI)
(MgSO4:71120)
(Na2SO)
(CaCi2)
Put the above reagents into a beaker, add 1000ml of distilled water, dissolve it in 10% sodium hydroxide and adjust the pI value to 7.4~7.6, pour it into a trivalent bottle, add cotton wool, wrap it with kraft paper, and sterilize it in reverse order of damp heat to obtain a culture medium. 8.2.4.3 Iron bacterial culture salts
Magnesium sulfate
Ammonium sulfate
Potassium diammonium phosphate
Calcium sulfate
Ammonium ferric citrate
(MgSOa: 7ll2O)
E(NH4SO4)
(CaCl2)
(NaNO4)
1 Put the above reagents into a beaker, add 1000ml of distilled water, adjust the pH value to 6.6~6.8 with 10% sodium hydroxide solution, divide into tubes, store in basket 5, put in cotton plugs, wrap with kraft paper, sterilize with heat, and wait. 8.2.5 Determination of saprophytic bacteria
Put the test tubes with saprophytic bacteria culture in the test tube rack, and add 1ml of water sample with different concentrations to the test tubes respectively. Among them, one test tube is not added with water sample as a control. After inoculation, put it in a 30-37 incubator and culture for 5-7l. If the liquid becomes turbid or white floccules appear or the liquid surface is thick, it means that saprophytic bacteria grow. If there is bacterial growth in the sample, the following should be done: 8.2.6 Determination of sulfate-reducing bacteria
Wipe the rubber stopper with alcohol cotton and put it into a porcelain plate. Heat it under ultraviolet light for 30min. Add 1.2. Spread out at 30°C from the UV lamp, sterilize 30% of the sample in the sulphur-reducing bacteria culture medium, and shake.
Put the sterilized test tubes in the test tube rack, and add 1 and 1 of the water samples of different dilutions respectively. Fill the test tubes with the sulphur-reducing bacteria culture medium that has been depleted of ammonium sulphate, and then plug them. Do not connect the water sample in the test tubes as blank samples. After inoculation, put them in a 30~37 humidity box for 14~21 days. If black precipitate is generated accompanied by sulphide gas, it means that sulphate-reducing bacteria grow. If there is no bacterial growth in the sample, record it. 8.2.7 Test of iron bacteria
Put the test tubes containing iron bacteria culture medium in the test tube rack. Add 1ml of water samples of different dilutions to the test tubes respectively, and 3
test tubes do not connect the water sample as blank samples. SY/T0532-:93
Inoculate the shield and culture it in a 30-37℃ incubator for 11 days. If a mixed oil or reddish brown jelly is produced, it means that iron bacteria have grown. If the blank sample does not grow, it should be treated as follows. Bacterial counting method
According to Appendix A (Supplementary) for the bacterial counting method, 10 Precision
The same author shall repeat the determination of the same sample in the same experiment according to the steps specified in this standard within a continuous time. If the sample has a high bacterial content, the result shall have an allowable error of not less than 10. If the sample has a low bacterial content, the result shall have an allowable error of not less than 10.11
Analysis results
Give the bacterial content range or accurate bacterial count as needed, cells/ml. SY/T 0532—93
Appendix A
Method for counting bacteria
(Supplementary)bzxZ.net
A1 To measure the range of bacterial content by the bacterial counting method of filling a group of test bottles (tubes) with one sample, it is advisable to use one sample to make five dilutions and arrange them in order of dilution. If bacteria grow in bottle (tube) No. 1 and the other four bottles (tubes) are sterile, it indicates that the range of bacterial content is 1 to 10/ml. If bacteria grow in bottle (tube) No. 2, the range of bacterial content is 1010/ml. By analogy, the range of bacterial content is shown in Table 41. Table A Bacteria Count Table
Bottles (tubes) with bacterial growth
A2 One sample multiple groups of test bottles (tubes) Bacteria count method Bacteria count, pieces/ml
10t～102
102~103
10'~1c
To more accurately measure the number of Newborn bacteria, the three-tube method, four-tube method or five-tube method should be used. The three-tube method means that each water sample is tested for one parallel test at each dilution factor, and the four-tube method means that each water sample is tested for four parallel tests at each dilution factor, and so on. The water sample should be diluted to the highest dilution factor where no bacteria grow.
A2.1 Example of bacteria count method 1: The saprophytic bacteria content of a water sample is analyzed using the three-tube method, see Table A2. Table A2
Dilution multiple
Number of tubes with bacterial growth
Select: The number of tubes with bacterial growth in three adjacent dilution multiples, the index is "332", the bacterial count in Table A5 is 110/ml, and then multiplied by the dilution multiple 10° on the first digit, the saprophytic bacteria content in the water sample is 110×10/ml. A2.2 Example 2 of bacterial counting method: Use the four-tube method to analyze the saprophytic bacteria feeding capacity of a water sample, see Table A3. Table A3
Dilution multiple
Number of tubes with bacterial growth
Select the number of tubes with bacterial growth in three adjacent dilution multiples, and the index is 321". Check Table A6 for the number of fine shadows of 30/m1, and then multiply it by the dilution multiple 10 on the first digit, and the total amount of saprophytes in the water sample is 3×103/rl. A2.3 Example of bacterial counting method In March, the five-tube method was used to analyze the saprophytic bacteria content of a water sample, see Table A4. 5
Dilution multiple
Number of tubes with bacterial growth
SY/T0532-93
Select the number of tubes with bacterial growth in three adjacent dilution multiples, and the index is " 531”, the bacterial count in Table A7 is 11.0/ml, and then multiplied by the dilution multiple 10° on the first digit, the saprophytic bacteria content in the water sample is 11×103/ml. The calculation method is expressed by the formula:
The number of bacteria in 1ml water sample = the number of bacteria/ml×the dilution borrowing number on the first digit of the index A2.4 The statistical table of the dilution method measurement is shown in Tables A5, A6, and A7. Table A5
Number of bacteria, pieces/m1
The maximum possible number of bacteria in three parallel tubes
Number of bacteria, pieces/ml
Number of bacteria, pieces/mi
Number of bacteria, pieces/ml
SY/T 0532—93
The maximum possible bacterial count of four parallel tubes
Residual count, pcs/ml
The number of bacteria, pcs/ml
The number of bacteria, pcs/ml
SY/T 0532—93
The maximum possible bacterial count of five parallel tubes
The number of bacteria, pcs/ml
Residual count, pcs/ml
Additional instructions:
SY/T0532—93
This standard was proposed by the Technical Supervision Bureau of China National Petroleum Corporation. This standard was prepared by the China National Petroleum Corporation Design and Planning Institute. This standard was drafted by Yat Kuaitian Construction Design Institute. The drafters of this standard are Shuzhen and Jiqin.0/ml, then multiply it by the dilution multiple 10° on the first digit, and you get the saprophytic bacteria content in the water sample 11×103/ml. The calculation method is expressed by the formula:
The number of bacteria in 1ml water sample = the number of bacteria/ml×the dilution borrowing number on the first digit of the index A2.4 The statistical table of the dilution method is shown in Table A5, A6, and A7. Table A5
The number of bacteria, each/m1
The maximum possible number of bacteria in three parallel tubes
The number of bacteria, each/ml
The number of bacteria, each/mi
The number of bacteria, each/ml
SY/T 0532—93
The maximum possible number of bacteria in four parallel tubes
The number of bacteria, each/ml
The number of bacteria, each/m1
SY/T 0532—93
The maximum possible bacterial count of five parallel tubes
Bacterial count, pieces/ml
Distance count, pieces/ml
Additional remarks:
SY/T0532—93
This standard was proposed by the Technical Supervision Bureau of China National Petroleum Corporation. This standard was prepared by the China National Petroleum Corporation Design and Design Institute. This standard was drafted by Yat Kuaitian Construction Design Institute. This standard was drafted by Shuzhen and Jiqin.0/ml, then multiply it by the dilution multiple 10° on the first digit, and you get the saprophytic bacteria content in the water sample 11×103/ml. The calculation method is expressed by the formula:
The number of bacteria in 1ml water sample = the number of bacteria/ml×the dilution borrowing number on the first digit of the index A2.4 The statistical table of the dilution method is shown in Table A5, A6, and A7. Table A5
The number of bacteria, each/m1
The maximum possible number of bacteria in three parallel tubes
The number of bacteria, each/ml
The number of bacteria, each/mi
The number of bacteria, each/ml
SY/T 0532—93
The maximum possible number of bacteria in four parallel tubes
The number of bacteria, each/ml
The number of bacteria, each/m1
SY/T 0532—93
The maximum possible bacterial count of five parallel tubes
Bacterial count, pieces/ml
Distance count, pieces/ml
Additional remarks:
SY/T0532—93
This standard was proposed by the Technical Supervision Bureau of China National Petroleum Corporation. This standard was prepared by the China National Petroleum Corporation Design and Design Institute. This standard was drafted by Yat Kuaitian Construction Design Institute. This standard was drafted by Shuzhen and Jiqin.
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