Application of heterobacterium faecalis, aquatic feed and fish culture method

Abstract

The invention discloses an application of Allobacter stercolicis AS, an aquatic feed and a fish culture method, and relates to the technical field of feeds, in particular to the application of the Allobacter stercolicis AS as well as the aquatic feed and the fish culture method. The Alobacter faecalis AS can be used for preparing aquatic feed, the Alobacter faecalis AS is purchased from Leibniz Instrument DSMZ, and the serial number of the Alobacter faecalis AS is DSM 13633. The Alobacter faecalis AS can be used for preparing aquatic feed, and the Alobacter faecalis AS can be used for preparing aquatic feed. The heterobacter faecalis AS is added into the fish culture feed, so that the intestinal barrier health of fishes can be improved, and the number of goblet cells can be increased; meanwhile, the fish with the heterobacter faecalis can also reduce liver injury and reduce the content of cholesterol and free fatty acid in serum and liver; besides, by inhibiting expression of proinflammatory factors and improving expression of anti-inflammatory factors, resistance of fishes to aeromonas hydrophila is improved, and survival rate of fishes is increased.

Description

Technical Field

[0001] The invention relates to the technical field of feed, and in particular to an application of Allobaculum stercoricanis AS, an aquatic feed and a fish breeding method. Background Art

[0002] In recent years, with the steady growth of global aquaculture production, the demand for aquatic feed has also shown a rapid upward trend. In aquatic feed, soybean meal is a commonly used source of plant protein, which is popular due to its high protein content and palatability. However, my country's soybean meal production is low, heavily dependent on imports, and prices are rising year by year, posing challenges to the sustainable development of the industry. Therefore, the development of non-grain protein sources that can replace soybean meal has become one of the key means to promote the sustainable development of aquaculture.

[0003] Cottonseed meal is an important by-product plant protein resource in my country. It is considered to have application potential due to its rich protein content. However, due to the traditional production process, the quality of cottonseed meal is low, and the anti-nutritional factors in it seriously limit its wide application in aquatic feed. Cottonseed meal contains a variety of anti-nutritional factors such as free cottonpol, non-starch polysaccharides and phytic acid. These substances increase intestinal viscosity, interfere with the normal feeding and nutrient absorption of fish, damage liver function, induce inflammatory response, and significantly increase the susceptibility of fish to pathogens. In addition, the anti-nutritional factors in cottonseed meal may also damage intestinal epithelial cells and increase the permeability of the intestinal barrier; change the composition of the intestinal microbiota, inhibit the proliferation of beneficial bacteria, and promote the reproduction of intestinal pathogenic bacteria, thereby further threatening the health of fish. Therefore, how to effectively alleviate the negative impact of cottonseed meal anti-nutritional factors on fish has become an important issue to improve the application potential of cottonseed meal.

[0004] Studies have shown that intestinal flora can regulate and alleviate the harm of cottonpol in cottonseed meal to a certain extent. Exogenous addition of probiotics is considered to be one of the effective means to improve the utilization of cottonseed meal. At present, most of the probiotics commonly used in aquatic feed are aerobic bacteria, such as Bacillus and lactic acid bacteria, which have been widely studied and applied in regulating intestinal health and improving fish immunity. However, the role of anaerobic bacteria in fish and their potential benefits have not been fully explored.

[0005] Therefore, it is of great significance for technicians in this field to study whether anaerobic bacteria can improve the tolerance to cottonseed meal anti-nutritional factors by regulating the intestinal health of fish and improving the immune status. This will not only help develop new and efficient feed additives, but also be crucial to promoting the sustainable development of aquaculture. Summary of the invention

[0006] The present invention provides an application of Allobaculum stercoricanis AS, aquatic feed and a fish farming method, aiming to solve the problems existing in the above-mentioned background technology. The Allobaculum stercoricanis AS can improve the resistance of fish to pathogens, improve intestinal barrier function, and alleviate liver damage.

[0007] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0008] The invention discloses application of Allobaculum stercoricanis AS in preparing aquatic feed. The Allobaculum stercoricanis AS is purchased from Leibniz Institute DSMZ and is numbered DSM 13633.

[0009] In a preferred embodiment of the present invention, the aquatic feed is used to reduce the levels of alanine aminotransferase ALT and aspartate aminotransferase AST in serum of aquatic animals, and / or reduce the content of cholesterol and free fatty acids in serum and liver, and / or alleviate liver damage.

[0010] In a preferred embodiment of the present invention, the aquatic feed is used to increase the number of goblet cells in the intestine of aquatic animals.

[0011] Furthermore, the aquatic feed increases the number of goblet cells in the intestine by increasing the expression level of the intestinal mucin secretion-related gene muc2, and / or the expression level of the agr2 gene, and / or the expression level of the cadherin gene.

[0012] In a preferred embodiment of the present invention, the aquatic feed is used to inhibit the expression of the pro-inflammatory factor il1β, and / or, increase the expression of the antimicrobial peptide reg3g, and / or, increase the expression of the anti-inflammatory factor il-22, and / or, increase the expression of the anti-inflammatory factor il-10.

[0013] In a preferred embodiment of the present invention, the aquatic feed is used to improve the ability of aquatic animals to resist infection by Aeromonas hydrophila, and to enhance the immunity and disease resistance of aquatic animals.

[0014] The present invention also discloses an aquatic feed, containing Allobaculum stercoricanis AS, and each kilogram of the aquatic feed comprises the following raw materials in parts by weight:

[0015] Soybean meal 80-120g, casein 80-90g, cottonseed protein concentrate 200-250g, flour 300-500g, soybean oil 40-60g, vitamin premix 4-6g, mineral premix 4-6g, sodium carboxymethyl cellulose 20-30g, choline chloride 4-6g, antioxidant 0.2-0.5g, calcium dihydrogen phosphate 10-20g, cellulose 80-85g, attractant 1-3g;

[0016] The concentration of Allobacterium faecalis AS in each kilogram of the aquatic feed was 0.8×10 9 -1.2×10 9 CFU / g.

[0017] In a preferred embodiment of the present invention, the vitamin premix contains the following raw materials in parts by weight per kilogram: 0.1-0.2 g vitamin A, 0.001-0.0015 g vitamin D 3 , 2-3g vitamin E, 0.5-1.5g vitamin K 3 , 4-6g vitamin B 1 , 4-6g vitamin B 2 4-6g vitamin B 6 , 4-6g vitamin B 12 , 20-30g inositol, 8-12g pantothenic acid, 80-120g choline, 20-30g niacin, 0.8-1.2g folic acid, 0.2-0.3g biotin, 8-12g vitamin C, and the balance is defatted rice bran; each kilogram of the mineral premix includes the following raw materials in parts by weight: 140-150g MgSO 4 -7H 2 O; 48-50g NaCl; 10-12g iron (II) gluconate; 3-4g manganese sulfate hexahydrate; 4-5g zinc sulfate heptahydrate; 0.5-0.7g copper sulfate pentahydrate; 0.12-0.2g potassium iodide; 0.05-0.1g cobalt chloride hexahydrate; 0.05-0.1g ammonium molybdate; 0.01-0.03g sodium selenate, the balance is zeolite powder.

[0018] The invention also discloses a method for fish farming, comprising the following steps:

[0019] S1. The ingredients of the aquatic feed as described above are mixed with soybean oil and distilled water, then extruded into hard pellets, dried at room temperature, and stored at -20°C until use;

[0020] S2, adding the AS strain of Allobacterium faecalis to the modified PYG medium, culturing anaerobically to obtain a bacterial suspension; then centrifuging the bacterial suspension to collect the AS bacteria, adding PBS to resuspend the AS bacteria, and adjusting the final concentration of the AS bacteria to obtain an AS bacterial suspension;

[0021] S3, spraying the AS bacterial suspension onto the feed particles in step S1 to obtain probiotic feed;

[0022] S4. After the fry are temporarily kept in a water tank and fed routinely for a period of time, healthy fish without external injuries or deformities are selected and fed with the probiotic feed described in step S3, twice a day, at 4% of their body weight per day.

[0023] In a preferred embodiment of the present invention, the improved PYG medium comprises the following components: 4-6 g tryptic peptone; 4-6 g peptone; 8-12 g yeast extract powder; 4-6 g beef extract powder; 4-6 g glucose; 1-3 g dipotassium hydrogen phosphate; 0.5-1.5 ml Tween 80; and 0.5-1.5 mg resazurin.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention adds Allobacterium faecalis AS to fish farming feed, which can improve the intestinal barrier health of fish and increase the number of goblet cells; at the same time, Allobacterium faecalis AS fish can also reduce liver damage and reduce the cholesterol and free fatty acid contents in serum and liver; in addition, it can also inhibit the expression of pro-inflammatory factors and increase the expression of anti-inflammatory factors, thereby improving the resistance of fish to Aeromonas hydrophila and improving the survival rate of fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0027] Figure 1 The effect of the faecal bacteria AS of the present invention on liver damage;

[0028] Figure 2 The effect of the faecal bacteria AS of the present invention on liver cholesterol and free fatty acids;

[0029] Figure 3 This is an intestinal PAS slice after adding the faecal bacteria AS of the present invention;

[0030] Figure 4 The effect of the present invention's Allobacterium faecalis AS on the expression of intestinal barrier-related genes;

[0031] Figure 5 The effect of the faecal bacteria AS of the present invention on intestinal proinflammatory factors;

[0032] Figure 6The effect of the faecal bacteria AS of the present invention on intestinal anti-inflammatory factors;

[0033] Figure 7 This is the effect of the faecal bacteria AS of the present invention on the survival rate of Nile tilapia challenged with Aeromonas hydrophila. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0035] Among them, the Allobaculum stercoricanis AS (hereinafter referred to as "AS") provided by the present invention was purchased from Leibniz Institute DSMZ and is numbered DSM 13633.

[0036] Example 1

[0037] This embodiment provides a method for fish farming using Allobacterium faecalis AS (hereinafter referred to as “AS”), which specifically comprises the following steps:

[0038] S1. Feed preparation: The feeds of each group (soybean meal group, cottonseed protein concentrate group, and fecal allelopathic bacteria group) were prepared according to the feed raw material formula shown in Table 1; casein, soybean meal, and cottonseed protein concentrate were the protein sources in the feed, flour was the main carbohydrate source, and soybean oil was the fat source. Various feed ingredients were mixed with soybean oil and distilled water, and then extruded into hard particles of 2 μm. The prepared feed was placed at room temperature to dry, and then stored at -20°C until use.

[0039] S2. Add the AS strain of Allobacterium faecalis to the modified PYG medium and culture it anaerobically at 37°C for 48 h to obtain a bacterial suspension. Centrifuge the bacterial suspension at 4000×g for 10 min at 4°C to collect the AS bacteria, add PBS to resuspend the AS bacteria, and adjust the final concentration of the AS bacteria to 1.0×10 9 CFU / mL to obtain AS bacterial suspension;

[0040] The improved PYG medium includes the following components: 5 g of tryptic peptone; 5 g of peptone; 10 g of yeast extract powder; 5 g of beef extract powder; 5 g of glucose; 2 g of dipotassium hydrogen phosphate; 1 ml of Tween 80; and 1 mg of resazurin.

[0041] The AS bacteria were sprayed onto the feed particles in step S1 at a ratio of 1:9 to obtain 10 9CFU / g of probiotic feed (AS); at the same time, PBS was sprayed on the soybean meal group and cottonseed protein concentrate group feed respectively; prepared and used immediately.

[0042] S3. Temporary rearing of fry: 300 Nile tilapia were purchased and temporarily reared in six 400L water tanks and fed with commercial feed for two weeks. 270 healthy Nile tilapia without external injuries or deformities were randomly divided into two groups, each with three tanks (30 fish per tank), and fed with soybean meal group (SM), concentrated cottonseed protein group (CM) or feed containing AS bacteria of Allobacterium faecalis (AS) respectively;

[0043] Table 1 Feed raw material formula (g)

[0044]

[0045]

[0046] in: 1 Mixed vitamins (g / kg): 0.15g vitamin A, 0.00125g vitamin D3, 2.5g vitamin E, 1g vitamin K3, 5g vitamin B1, 5g vitamin B2, 5g vitamin B6, 5g vitamin B12, 25g inositol, 10g pantothenic acid, 100g choline, 25g niacin, 1g folic acid, 0.25g biotin, 10g vitamin C, and the remainder is defatted rice bran.

[0047] 2 Mixed minerals (g / kg): 147.4 g MgSO4-7H2O; 49.8 g NaCl; 10.9 g iron (II) gluconate; 3.12 g manganese sulfate hexahydrate; 4.67 g zinc sulfate heptahydrate; 0.62 g copper sulfate pentahydrate; 0.16 g potassium iodide; 0.08 g cobalt chloride hexahydrate; 0.06 g ammonium molybdate; 0.02 g sodium selenate, the remainder is zeolite powder.

[0048] S4. Feeding: Feed the three groups of experimental feeds to two groups of fry, twice a day, at 4% of body weight per day; at the 8th week of culture, weigh the fish after starvation for 12 hours, randomly select 12 fish from each group, and anesthetize them with eugenol. Use a 1mL sterile syringe to draw blood from the tail vein of tilapia. After the blood is allowed to stand at room temperature for half an hour, transfer it to 4°C until the plasma and serum are separated. Then centrifuge the blood at 4°C at 3,000rpm for 10 minutes to separate the serum. Collect the whole intestine of Nile tilapia.

[0049] Test Example The efficacy of Allobaculum stercoricanis AS was evaluated:

[0050] (1) Allobacterium faecalis AS improves liver damage

[0051] Serum was collected and the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum were determined using Nanjing Jiancheng kits (C010-2-1, C009-2-1). The operation steps were performed according to the kit instructions.

[0052] like Figure 1 As shown in (a), the serum ALT level in the AS group was significantly lower than that in the CM group. Figure 1 (b) shows that the serum AST content in the AS group was significantly lower than that in the CM group, indicating that the addition of Bacillus faecalis AS can reduce the serum ALT and AST levels of tilapia and alleviate liver damage.

[0053] Note: Figure 1 “*” in the figure represents the difference between the two groups, and *: P < 0.05, **: P < 0.01.

[0054] (2) Allobacterium faecalis AS reduces the levels of cholesterol and free fatty acids in the liver

[0055] The liver was collected and the total cholesterol and free fatty acid levels in serum and liver were determined using Nanjing Jiancheng kits (A111-1-1, A042-2-1). The operating steps were performed according to the kit instructions.

[0056] like Figure 2 As shown in Figure 2, compared with the CM group, the levels of total cholesterol and free fatty acids in serum of the AS group decreased ( Figure 2 a, 2b); the contents of total cholesterol and free fatty acids in the liver of the AS group were also significantly lower than those of the CM group. In general, the addition of F. faecalis AS can alleviate liver damage by reducing the contents of cholesterol and free fatty acids in serum and liver.

[0057] Note: Figure 2 “*” in the figure represents the difference between the two groups, and *: P < 0.05, **: P < 0.01.

[0058] (3) AS improves intestinal health

[0059] Intestinal samples were fixed in 4% paraformaldehyde and then embedded in paraffin. The embedded samples were cut into 5 μm sections transversely and then stained with PAS. The sections were photographed using a Nikon microscope.

[0060] from Figure 3 It can be found that compared with the SM group, the number of goblet cells in the intestine of the CM group was significantly reduced, but after adding AS bacteria, the number of goblet cells in the intestine increased significantly. This shows that Fecal Allobacterium AS can improve intestinal health.

[0061] (4) Isochromatin increases the expression of intestinal barrier-related genes

[0062] Total RNA was extracted from the intestine of Nile tilapia using Trizol. The integrity and purity of RNA were detected by 1% agarose gel electrophoresis and Nanodrop 2000, and the A260 / A280 ratio was in the range of 1.9 to 2.1. PrimeScript TM The RT kit reverse transcribed RNA into cDNA, and primers for qRT-PCR were designed in NCBI (Table 2), with ef1α (Elongation factor 1-alpha 1) and β-actin as reference genes. The qRT-PCR reaction solution contained 1 μL cDNA template, 5 μL 2×ultra SYBR, 3 μL ddH2O, and 0.25 μL 10 mmol / L forward and reverse primers. The reaction conditions were 95°C, 30s; 95°C, 5s; 60°C, 20s, with a total of 40 cycles. The qRT-PCR primer amplification efficiency was between 90% and 110%, and the correlation coefficients of each gene were all above 0.98. The relative expression level of the gene was calculated using 2 -ΔΔCt Method to calculate.

[0063] Table 2 Primer sequences

[0064]

[0065]

[0066] from Figure 4 The results showed that compared with the CM group, the expression levels of muc2, agr2 and cadherin in the intestine of the AS group were significantly upregulated, which indicated that the addition of Allobacterium faecalis AS could upregulate the number of goblet cells in the intestine by increasing the expression of genes related to mucin secretion in the intestine.

[0067] Note: Figure 4 “*” in the figure represents the difference between the two groups, and *: P < 0.05, **: P < 0.01.

[0068] (5) Allergens from Fecal Bacillus AS relieves intestinal inflammation

[0069] The experimental method is the same as (4). The primer sequences are shown in Table 3.

[0070] Table 3 Primer sequences

[0071]

[0072]

[0073] Anti-nutritional factors contained in cottonseed protein concentrate often lead to increased intestinal inflammation. Figure 5As shown in Figure 2, compared with the CM group, the expression of antimicrobial peptide reg3g, anti-inflammatory factors il-22 and il-10 in the intestine of the AS group was significantly increased; Figure 6 As shown in Figure 3, the expression of the pro-inflammatory factor il-1β in the intestine of the AS group was significantly decreased compared with the CM group. These results indicate that the addition of Bacillus faecalis AS can inhibit the inflammatory response in the intestine.

[0074] Note: Figure 5 and Figure 6 “*” in the figure represents the difference between the two groups, and *: P < 0.05, **: P < 0.01.

[0075] (6) Fecal Bacillus AS improves the survival rate of tilapia challenged with virus

[0076] After 8 weeks of feeding trials, 30 fish were randomly selected from each group and divided into 3 tanks. Aeromonas hydrophila was cultured in LB medium at 28°C for 16 hours and centrifuged at 3,000 rpm for 10 minutes to collect Aeromonas hydrophila. The bacteria were resuspended in phosphate buffered saline and the number of bacteria was counted by gradient dilution and plate method. The bacteria were diluted to 9.2×10 5 CFU / L, and injected intraperitoneally at a dose of 1g / μL. The mortality of Nile tilapia was recorded every day.

[0077] Note: Figure 7 “*” in the figure represents the difference between the two groups, and *: P < 0.05, **: P < 0.01.

[0078] The results are as follows Figure 7 As shown in the figure, the survival rates of the SM and CM groups were similar, while the survival rate of the AS group was the highest. As mentioned above, Allobacterium faecalis AS can effectively improve the fish's ability to resist infection by Aeromonas hydrophila, enhance the fish's immunity and disease resistance, and further improve the survival rate.

[0079] In summary, the faecal allergens AS of the present invention can be added to feed containing high plant protein as a safe functional feed additive. Faecal allergens AS can alleviate liver damage, reduce the content of free fatty acids in serum and liver, improve intestinal morphological damage caused by plant raw materials, reduce inflammatory response, and thus improve the body's ability to improve fish disease resistance.

[0080] The technical features in the above embodiments can be combined arbitrarily, and the combined technical solutions all belong to the protection scope of this application. The basic principles, main features and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may have various changes and improvements without departing from the spirit and scope of the present invention, and these changes and improvements all fall within the scope of the present invention to be protected. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. Application of Allobaculum stercoricanis AS in the preparation of aquatic feed, wherein the Allobaculum stercoricanis AS is purchased from Leibniz Institute DSMZ and is numbered DSM 13633.

2. The use according to claim 1, characterized in that: The aquatic feed is used for reducing the levels of serum alanine aminotransferase ALT and aspartate aminotransferase AST of aquatic animals, and / or reducing the content of cholesterol and free fatty acids in serum and liver, and / or alleviating liver damage.

3. The use according to claim 1, characterized in that: The aquatic feed is used for increasing the number of goblet cells in the intestine of aquatic animals.

4. The use according to claim 3, characterized in that: The aquatic feed increases the number of goblet cells in the intestine by increasing the expression level of the intestinal mucin secretion-related gene muc2, and / or the expression level of the agr2 gene, and / or the expression level of the cadherin gene.

5. The use according to claim 1, characterized in that: The aquatic feed is used to inhibit the expression of the pro-inflammatory factor il1β, and / or to increase the expression of the antimicrobial peptide reg3g, and / or to increase the expression of the anti-inflammatory factor il-22, and / or to increase the expression of the anti-inflammatory factor il-10.

6. The use according to claim 1, characterized in that: The aquatic feed is used for improving the ability of aquatic animals to resist infection by Aeromonas hydrophila, and enhancing the immunity and disease resistance of aquatic animals.

7. An aquatic feed, characterized in that: The aquatic feed contains Allobaculum stercoricanis AS, and each kilogram of the aquatic feed comprises the following raw materials in parts by weight: Soybean meal 80-120g, casein 80-90g, cottonseed protein concentrate 200-250g, flour 300-500g, soybean oil 40-60g, vitamin premix 4-6g, mineral premix 4-6g, sodium carboxymethyl cellulose 20-30g, choline chloride 4-6g, antioxidant 0.2-0.5g, calcium dihydrogen phosphate 10-20g, cellulose 80-85g, attractant 1-3g; The concentration of Allobacterium faecalis AS in each kilogram of the aquatic feed was 0.8×10 9 -1.2×10 9 CFU / g.

8. The aquatic feed according to claim 7, characterized in that: Each kilogram of the vitamin premix includes the following raw materials in parts by weight: 0.1-0.2g vitamin A, 0.001-0.0015g vitamin D3, 2-3g vitamin E, 0.5-1.5g vitamin K3, 4-6g vitamin B1, 4-6g vitamin B2, 4-6g vitamin B6, 4-6g vitamin B 12 , 20-30g inositol, 8-12g pantothenic acid, 80-120g choline, 20-30g niacin, 0.8-1.2g folic acid, 0.2-0.3g biotin, 8-12g vitamin C; per kilogram of the mineral premix, including the following raw materials in parts by weight: 140-150g MgSO4-7H2O; 48-50g NaCl; 10-12g iron gluconate (II); 3-4g manganese sulfate hexahydrate; 4-5g zinc sulfate heptahydrate; 0.5-0.7g copper sulfate pentahydrate; 0.12-0.2g potassium iodide; 0.05-0.1g cobalt chloride hexahydrate; 0.05-0.1g ammonium molybdate; 0.01-0.03g sodium selenate.

9. A method for fish farming, characterized in that: The steps include: S1. Mix the ingredients of the aquatic feed according to any one of claims 7 to 8 with soybean oil and distilled water, and then extrude into hard granules, dry at room temperature, and store at -20°C until use; S2, adding the AS strain of Allobacterium faecalis to the modified PYG medium, culturing anaerobically to obtain a bacterial suspension; then centrifuging the bacterial suspension to collect the AS bacteria, adding PBS to resuspend the AS bacteria, and adjusting the final concentration of the AS bacteria to obtain an AS bacterial suspension; S3, spraying the AS bacterial suspension onto the feed particles in step S1 to obtain probiotic feed; S4. After the fry are temporarily kept in a water tank and fed routinely for a period of time, healthy fish without external injuries or deformities are selected and fed with the probiotic feed described in step S3, twice a day, at 4% of their body weight per day.

10. The method for fish farming according to claim 9, characterized in that: The improved PYG culture medium comprises the following components: 4-6g of tryptic peptone; 4-6g of peptone; 8-12g of yeast extract powder; 4-6g of beef extract powder; 4-6g of glucose; 1-3g of dipotassium hydrogen phosphate; 0.5-1.5ml of Tween 80; and 0.5-1.5mg of resazurin.

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