CN111494651B

CN111494651B - Nematode larva activity detection method and application

Info

Publication number: CN111494651B
Application number: CN202010470462.5A
Authority: CN
Inventors: 王文龙; 陆静; 吕旭; 翟帅; 刘春霞; 刘晓磊; 苏倩; 王腾宇; 林洋
Original assignee: Inner Mongolia Agricultural University
Current assignee: Inner Mongolia Agricultural University
Priority date: 2020-05-28
Filing date: 2020-05-28
Publication date: 2023-01-03
Anticipated expiration: 2040-05-28
Also published as: CN111494651A

Abstract

The invention relates to the field of larva activity detection, and particularly discloses a nematode larva activity detection method and application. The nematode larva activity detection method comprises the following steps: (1) Dyeing the nematode larvae by using methylene blue as a dyeing agent; (2) And after 5-7 min, judging the activity of the nematode larvae according to the color of the nematode larvae. When the nematode larvae are blue or light blue, judging the nematode larvae to be dead or low-activity larvae; and when the nematode larvae are in an unstained state, judging that the nematode larvae are high-activity larvae. The invention visually identifies the vitality of the larvae of the digestive tract nematode by using MB as a coloring agent according to the dyeing depth effect, is convenient, rapid and low in cost, can effectively avoid subjective judgment difference, provides help for researching the drug effect, drug resistance and the like of the anthelmintic, and has great significance for selecting sensitive drugs to guide clinical medication and the like.

Description

Nematode larva activity detection method and application

Technical Field

The invention relates to the technical field of larva activity detection, in particular to a nematode larva activity detection method and application.

Background

Nematodes are a common group of parasites, which are usually parasitic in the digestive tract of animals and adversely affect the health of the host. Therefore, a great deal of research is carried out in the prior art on the mode of removing nematodes, but the conventional anthelmintic generally has the problem of drug resistance, and troubles the fields such as livestock breeding and the like.

The nematodosis of the digestive tract of the sheep is one of important diseases which endanger the healthy development of the sheep raising industry, the infection is very common, the larger economic loss is easy to cause, and the important point of prevention and treatment during the breeding is provided. In the production practice, due to the long-term unscientific application of the anthelmintic, many digestive tract nematodes generate different degrees of drug resistance to the common anthelmintic, and the effective anti-parasitic drugs are less and less, so that the sheep parasitic diseases are in a flooding situation. Therefore, a great deal of manpower and material resources are required to be continuously invested to develop new antiparasitic drugs to meet the production requirements. Before a new anthelmintic is researched and applied to clinic, a convenient and quick drug resistance detection method is researched, and it is important to select a sensitive anthelmintic for timely and effectively expelling anthelmintics.

Most of the currently applied parasite drug resistance in vitro detection methods aim at parasite larvae, and judge the sensitivity of parasites to anthelmintics through the influence or mortality of drugs on the vitality of the parasite larvae, but the existing parasite larva vitality detection methods have the defects of subjective judgment difference, unstable result, complex judgment process and the like, and are difficult to realize the consistency and standardization of detection results and popularize and apply in production practice.

Therefore, there is a need to provide a new method for detecting nematode larva activity and application thereof to solve the problems in the prior art.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a nematode larva activity test mode which effectively avoids subjective judgment difference, is convenient and quick and has low cost.

In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:

a nematode larva activity detection method comprises the following steps:

(1) Dyeing the nematode larvae by using methylene blue as a dyeing agent;

(2) And after 5-7 min, judging the activity of the nematode larvae according to the color of the nematode larvae.

The method adopts the MB staining method, accurately judges the activity of the larvae by directly observing the coloration condition of the (digestive tract) nematode larvae, and has the advantages of stability, reliability, strong repeatability and applicability and simple operation. The nematode larvae referred to in the present invention may be first to third stage larvae. Generally, nematode eggs develop into first-stage larvae within 12-34 h, second-stage larvae within 37-96 h and third-stage larvae within 97-168 h at the temperature of 27 ℃.

The existing tests such as the morphological analysis of the larvae in the resting stage need to identify the vitality of the larvae, but the identification methods are different, and have the problems of subjective judgment difference, complex judgment process, unstable test result and the like, so that the consistency, reliability and standardization of the detection result are difficult to achieve, the accuracy and the application range of the test are influenced, and the popularization and the application in production practice are difficult. The MB dyeing method disclosed by the invention is applied to tests such as the morphological analysis of the larvae in the stationary phase, so that the result judgment process is simplified and the operation is standardized, the defects of subjective judgment difference, non-uniform laboratory data, complex judgment process and the like are effectively avoided, the practicability of the tests such as the morphological analysis of the larvae in the stationary phase is greatly expanded, and the MB dyeing method is expected to be popularized and applied in production practice.

A great number of dyes for biological dyeing are available, and insect egg dyeing schemes are reported, but a great number of researches show that the dyeing scheme of the invention can be used for better, simply and quickly judging the activity of nematode larvae (especially larvae of haemonchus contortus).

The method for judging the activity of the nematode larvae in the step (2) comprises the following steps: when the nematode larvae are blue or light blue, judging the nematode larvae to be dead or low-activity larvae; and when the nematode larvae are in an unstained state, judging that the nematode larvae are high-activity larvae.

The dead or low-activity nematode larvae of the present invention will be dyed blue or light blue without fading over time. The high-activity nematode larvae are dyed into light blue firstly, and gradually fade after 5-7 min to recover to an undyed state.

Preferably, the nematode larvae of the invention are larvae of haemonchus contortus.

The invention also provides application of the detection method in the in-vitro detection of the drug resistance of the nematode larvae.

In the invention, the nematode larvae are obtained by the following method:

(1) Preparing nematode egg suspension

Sequentially arranging three sieves of 500 mu m, 145 mu m and 20 mu m from top to bottom, pouring the suspension of the host excrement containing nematode eggs into the uppermost 500 mu m sieve, sequentially passing the suspension of the host excrement through the three sieves, collecting substances on the surface of the 20 mu m sieve, and performing first centrifugation on the substances in physiological saline; subjecting the precipitate obtained from the first centrifugation to a second centrifugation in saturated saline; pouring the supernatant obtained by the second centrifugation into a 20-micron sieve for secondary filtration, rinsing the secondary filtered substance on the surface of the 20-micron sieve by using physiological saline, and then carrying out third centrifugation on the secondary filtered substance by using the physiological saline; diluting the precipitate obtained by the third centrifugation with PBS buffer solution to obtain nematode egg suspension;

(2) And (2) culturing nematode eggs in the nematode egg suspension prepared in the step (1) into the nematode larvae.

In the invention, the number of nematode eggs per gram of the host excrement is more than or equal to 15000;

in the invention, the solid-to-liquid ratio of the suspension of the host excrement is 1: (4.5-5.5) g/mL, preferably 1:5g/mL.

In the invention, the rotating speed of the first centrifugation is 2800-3200r/min, and the time is 4-6min; the rotation speed of the second centrifugation is 2800-3200r/min, and the time is 2-4min; the rotating speed of the third centrifugation is 2800-3200r/min, and the time is 4-6min;

preferably, the rotating speed of the first centrifugation is 3000r/min, and the time is 5min; the rotating speed of the second centrifugation is 3000r/min, and the time is 3min; the rotating speed of the third centrifugation is 3000r/min, and the time is 5min.

In the invention, the PBS buffer solution contains amphotericin B and streptomycin, the concentration of the amphotericin B is 250mg/L, and the concentration of the streptomycin is 10mL/L; preferably, the concentration of penicillin in the penicillin streptomycin is 10000U/mL, and the concentration of streptomycin is 10000 mug/mL.

The preparation of the nematode egg suspension by the method is beneficial to isolated culture and provides guarantee for the accuracy of subsequent detection.

The invention has the beneficial effects that:

the invention visually identifies the vitality of the larvae of the digestive tract nematode by taking MB as a coloring agent according to the dyeing depth effect, is convenient, rapid and low in cost, can effectively avoid subjective judgment difference, provides help for researching the drug effect, drug resistance and the like of the anthelmintic, and has great significance for selecting sensitive drugs to guide clinical medication and the like.

Drawings

FIG. 1 is a photograph of nematode larvae stained with MB according to example 1 of the present invention; wherein, fig. 1A is dead larva, fig. 1B is low-activity larva, and fig. 1C is high-activity larva after dyeing for 5min;

FIG. 2 is a graph showing the variation of the number of larvae of nematodes stained with MB with the increase of the concentration of albendazole in example 1 of the present invention;

FIG. 3 is a photograph of the nematode larvae after TB staining in comparative example 1 of the present invention; wherein, fig. 3A is dead larvae, and fig. 3B is high-activity larvae after dyeing for 5min;

FIG. 4 is a photograph of GV-stained nematode larvae in comparative example 2 of the present invention; wherein, fig. 4A is dead larvae, and fig. 4B is high-activity larvae after dyeing for 5min;

FIG. 5 shows the results of eosin staining in comparative example 3 of the present invention;

FIG. 6 shows the results of borax dyeing in comparative example 4 of the present invention;

fig. 7 shows the results of m.e.b staining in comparative example 5 of the present invention.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The experimental materials involved in the embodiments of the present invention are as follows:

1. laboratory animal

50 small-tailed Han sheep of about 8 months old are bred by the Caucao Haerdong rear flag raiser in Wulan Chongbu City, and all suffer from digestive tract linear diseases with different degrees.

2. Main reagent and preparation

Dimethyl sulfoxide (DMSO), agar powder, both from colaber corporation; albendazole (also called albendazole), amphotericin B, streptomycin, 1640 culture solution, trypan blue (C) ₃₄ H ₂₄ N ₆ Na ₄ O ₁₄ S ₄ TB for short), methylene blue (C) ₁₆ H ₁₈ ClSN ₃ MB for short), gentian violet (C) ₂₅ H ₃₀ ClN ₃ GV for short), and the like, available from Gibco.

Preparing a albendazole stock solution: taking a proper amount of albendazole powder, dissolving the albendazole powder in DMSO to prepare solutions with the concentrations of 1 mug/mL, 2 mug/mL and 3 mug/mL.

Preparing an MB stock solution: MB 0.3g is weighed, purified water is added to 10mL, and 3% stock solution is prepared by mixing.

Preparing TB stock solution: 0.4g of TB is weighed, a small amount of purified water is added for grinding, double distilled water is added to 10mL, the obtained product is filtered by filter paper and stored at 4 ℃.

Preparing a GV stock solution: 0.1g of GV was weighed, purified water was added to 10mL, and the mixture was mixed to prepare a 1% stock solution.

Preparing eosin stock solution: 0.1g of eosin was weighed, and purified water was added to 10mL to prepare a 1% stock solution.

Preparing borax dye liquor: 0.2g of borax is weighed, boiled purified water is added to 10mL, and the mixture is mixed for standby.

Preparing a mixed dye liquor (M.E.B for short) of methylene blue, eosin and borax: adding 0.2g of methylene blue into 100mL of distilled water, boiling for 10min, adding 0.5g of borax, boiling for 10min, cooling, adding 0.1g of eosin, completely dissolving, and filtering for later use, wherein the pH value is 10-11.

3. Preparation of worm egg suspension

Collecting 50 small-tail han sheep excrement samples, and screening 5 small-tail han sheep excrement samples with EPG (electronic program guide) of more than or equal to 15000 for later use by excrement worm egg counting. The specific operation for preparing the worm egg suspension is as follows: placing three sieves of 500 mu m, 145 mu m and 20 mu m from top to bottom in sequence, adding 10g of fresh excrement into 50mL of tap water, fully stirring to prepare a suspension, pouring the suspension into the sieve of 500 mu m, standing for 5min, and enabling the diluted sample to sequentially pass through sieves with different apertures (so as to filter out large impurities and small suspended particles, and finally collecting nematode eggs on the surface of the sieve of 20 mu m). Washing worm eggs on the surface of a 20-micron sieve with normal saline, placing the worm eggs in a 50mL centrifugal tube, centrifuging for 5min at 3000r/min, discarding supernatant, adding saturated saline, centrifuging for 3min at 3000r/min, and collecting supernatant; pouring the supernatant into a 20-micron sieve and rinsing the worm eggs with normal saline; washing eggs from the surface of a 20-micron sieve by using normal saline, placing the eggs in a 50-mL centrifuge tube at 3000r/min for centrifugation for 5min, discarding supernatant, adding a proper amount of PBS (phosphate buffer solution) into the collected egg sediment, wherein the PBS contains 250mg/L amphotericin B and 10mL/L penicillin streptomycin (10000 Units/mL penicillin and 10000 ug/mL streptomycin), fully mixing, counting, diluting the solution by using the PBS to obtain a suspension of about 20000 eggs/mL, and performing anaerobic preservation at 4 ℃.

4. Preparation of third-stage nematode larva suspension

Weighing a proper amount of fresh sheep manure sample, adding a proper amount of water, mashing, filtering with double-layer gauze until the filtrate is clear, collecting manure residues, and autoclaving to prepare sterilized manure residues for later use. And (3) sucking 500 mu L of the insect egg suspension obtained by the preparation into a culture dish, adding a proper amount of excrement residue (with the thickness of 0.2-0.5 mm) into the culture dish, uniformly mixing the excrement residue with the insect egg suspension, dropwise adding 5mL of 1640 culture solution, placing the mixture into a thermostat at 27 ℃ for culture for 7d, and appropriately adding 1640 culture solution during the period to prevent the sample from being dried to influence the development of the insect body. After 7 days, nematode larvae are separated by a Bellmann method, larva suspension is collected, centrifugation is carried out for 5min at 1500r/min, supernatant is removed, PBS buffer solution is added, 250mg/L amphotericin B and 10mL/L streptomycin (10000 Units/mL penicillin, 10000 mu g/mL streptomycin) are contained in the PBS buffer solution, the mixture is diluted to every 1mL suspension containing 500 nematode larvae (mainly the larvae of the haemonchus contortus) after counting, and the mixture is kept at the dark room temperature.

Before the specific experiment of the invention is carried out, 1-time dose and 2-time dose of albendazole are administered to the same group of sheep in groups, and the Fecal Egg Count (FECRT) shows that the number of digestive tract nematode eggs is respectively reduced by 32.4% and 64.8%, which indicates that although the sheep group has drug resistance to albendazole, the anthelmintic effect is improved along with the increase of the administration dose. The ED50 of the benzimidazole medicine given by reference WAAVP to the worm eggs is less than or equal to 0.1 mu g/mL.

Example 1

This example provides a method for detecting nematode larval viability.

Preparing nematode larvae with different activities:

700. Mu.L (containing about 350 larvae) of the above nematode larva suspension and 200. Mu.L of 1640 medium were added to 5 wells of the 24-well plate, respectively.

These 5 wells were set as 1 blank control well and 3 drug gradient wells and labeled 1-1, 1-2, 1-3, 1-4, respectively. Add 100. Mu.L DMSO to wells 1-1 as a blank, 100. Mu.L (1. Mu.g/mL) albendazole stock solution to wells 1-2 (albendazole final concentration 0.1. Mu.g/mL), 100. Mu.L (2. Mu.g/mL) albendazole stock solution to wells 1-3 (albendazole final concentration 0.2. Mu.g/mL), and 100. Mu.L (3. Mu.g/mL) albendazole stock solution to wells 1-4 (albendazole final concentration 0.3. Mu.g/mL). Placing into an incubator at 27 ℃ for further culture for 12h.

And (3) activity detection:

after 10. Mu.L of the MB stock solution (the concentration of MB in the well is approximately equal to 0.03%) is dropped into each well, the mixture is allowed to stand for 5min (after standing for 7min, the observation result has no obvious difference), the color condition of the larvae is observed and recorded by a 50-fold visual mirror, and 3 times of repeated experiments are carried out. During observation, liquid in the holes can be properly sucked away or normal saline can be added for dilution so as to facilitate observation. The dyeing of this example was carried out at 27 ℃ and furthermore the dyeing results obtained with dyeing at 20 ℃ were not significantly different.

And (4) observing the results:

dead or low-activity larvae were stained blue or light blue, with no discoloration over time. The high-activity larva is dyed into light blue, gradually fades after 5-7 min and returns to an undyed state.

After dyeing for 5min, a picture of Haemonchus contortus larvae which are observed by a 50-fold visual mirror and dyed by MB is shown in figure 1, wherein figure 1A shows dead larvae, the color of the larvae is blue, the transparency is reduced, and the internal tissues cannot be seen clearly through a dyeing agent; FIG. 1B shows larvae with low activity, pale blue color, and staining agent deposition in the internal tissues; FIG. 1C shows high-activity larvae (faded) after 5min of staining, with the body being off-white in color, and the internal tissues of the clear body being clearly visible.

The data of 3 replicates were averaged for analysis and the results were as follows:

in this example, statistics of staining rates of the nematode larvae stained with MB on 4 different gradients of albendazole (the staining rates are calculated by the number of larvae still stained after standing/total number of larvae 100%), which are 1.14% (1-1 well), 15.74% (1-2 wells), 33.99% (1-3 wells), and 44.69% (1-4 wells), respectively, are shown in table 1.

TABLE 1 staining of nematode larvae with MB stain

From the results shown in table 1, it can be seen that the staining rate of the MB-stained nematode larvae is increased with the increase of the albendazole concentration, and the difference of the staining rate from the blank control group is more obvious with the increase of the albendazole action concentration, and the MB staining group test data is in positive correlation with the fecal ova reduction test (fect), as shown in fig. 2. The influence of the medicines with different concentrations on the nematodes can be evaluated through the change of the dyeing rate of the nematode larvae after MB dyeing.

Comparative example 1

This comparative example performed the nematode body viability assay of example 1 with TB as the stain. The same operation as in example 1 is not specifically described.

The 1 blank control well and 3 drug gradient wells are labeled 2-1, 2-2, 2-3, 2-4, respectively. The drug addition and larva culture methods were the same as in example 1, 10. Mu.L of the stock solution of TB (TB concentration in well ≈ 0.04%) was added dropwise to each well after 12 hours, and 3 times of repeated experiments were carried out in the same manner as in example 1 for observation and recording.

A photograph of the nematode larvae stained with TB, which is observed by a 50-fold visual mirror, is shown in FIG. 3, wherein FIG. 3A shows dead larvae and the larvae are gray, and FIG. 3B shows the high-activity larvae after staining for 5min, and the larvae are gray.

the nematode larva bodies after 4 different gradients of albendazole were stained with TB, and the results showed that neither dead nor high-activity larvae were stained with prolonged staining time. No obvious difference is caused between the drug group without drug and the acting group with different doses of albendazole in the aspect of larva staining rate, and specific results are shown in a table 2.

TABLE 2 dyeing rate of nematode larvae stained with TB stain

Trypan Blue (TB) is a common cell biological stain, can identify dead cells and live cells (the live cells are not stained, and the dead cells are blue stained), theoretically, the cell membrane structure of viable larva cells is complete, the trypan blue can be effectively prevented from entering the cells for staining, the permeability of the cell membrane of the dead larva cells is increased, the TB can enter the cells for staining, and the dead larvae and the viable larvae are obviously different. However, the test shows that no expected staining difference appears after staining TB, and TB is not suitable for detecting the activity of nematode larvae.

Comparative example 2

This comparative example was tested for nematode worm activity as in example 1 using GV as the stain. The same operation as in example 1 is not specifically described.

The 1 blank control well and 3 drug gradient wells are labeled 3-1, 3-2, 3-3, 3-4, respectively. The drug addition and larva culture methods were the same as in example 1, and 10. Mu.L of the above GV stock solution (GV concentration in the well ≈ 0.01%) was dropped into each well after 12 hours, and 3 times of repeated experiments were carried out in the same manner as in example 1 for observation and recording.

A photograph of GV-stained nematode larvae is shown in FIG. 4, wherein the dead larvae are shown in FIG. 4A, and the larvae are purple in color, and the high-activity larvae are shown in FIG. 4B, after being stained for 5min, and the larvae are also purple in color.

the GV is used for dyeing the nematode larva bodies after 4 different gradients of albendazole action, and the result shows that the dead larva and the high-activity larva are dyed purple with the time. No obvious difference in the larva staining rate is found between the drug group without drug and the acting group with different doses of albendazole, and the results are shown in a table 3.

TABLE 3 staining of nematode larvae with GV stain

Gentian Violet (GV) is a basic dye, slightly soluble in water (1 g/100 mL), soluble in ethanol, and commonly used as a biological stain to accurately identify sperm motility, but the test results show that GV stains dead and viable larvae purple, and no obvious difference indicates that GV is not suitable for nematode larva motility detection.

Comparative examples 3 to 5

This comparative example performed the nematode worm body staining of example 1 with eosin, borax stain and m.e.b as staining agents, respectively. The specific operation mode is as follows: sucking a drop of the nematode larva suspension, placing the nematode larva suspension on a glass slide, adding a drop of staining agent, covering the glass slide, standing for 5min, and performing microscopic examination.

As a result: neither eosin nor borax dye liquor nor m.e.b complex dye liquor can dye the major gut nematodes of sheep. The staining results are shown in FIG. 5, FIG. 6 and FIG. 7, respectively.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Claims

1. A nematode larva activity detection method is characterized by comprising the following steps:

(1) Dyeing the nematode larvae by using methylene blue as a dyeing agent;

(2) After 5-7 min, judging the activity of the nematode larvae according to the color of the nematode larvae;

the method for judging the activity of the nematode larvae in the step (2) comprises the following steps: when the nematode larvae are blue or light blue, judging the nematode larvae to be dead or low-activity larvae; when the nematode larvae are in an unstained state, judging that the nematode larvae are high-activity larvae; the nematode larvae are larvae of Haemonchus contortus.

2. Use of the assay of claim 1 for the in vitro detection of nematode larval resistance for non-disease diagnostic or therapeutic purposes.

3. Use according to claim 2, wherein the nematode larvae are obtained by:

(1) Preparing nematode egg suspension

Sequentially arranging three sieves of 500 mu m, 145 mu m and 20 mu m from top to bottom, pouring the suspension of the host excrement containing nematode eggs into the uppermost 500 mu m sieve, sequentially passing the suspension of the host excrement through the three sieves, collecting substances on the surface of the 20 mu m sieve, and centrifuging the substances in physiological saline for the first time; subjecting the precipitate obtained from the first centrifugation to a second centrifugation in saturated saline; pouring the supernatant obtained by the second centrifugation into a 20-micron sieve for secondary filtration, rinsing the secondary filtered substance on the surface of the 20-micron sieve by using physiological saline, and then carrying out third centrifugation on the secondary filtered substance by using the physiological saline; diluting the precipitate obtained by the third centrifugation with PBS buffer solution to obtain nematode egg suspension;

4. The use according to claim 3, wherein the number of nematode eggs per gram of faeces of said host is more than or equal to 15000;

and/or the solid-to-liquid ratio of the suspension of the host feces is 1: (4.5-5.5) g/mL.

5. The use of claim 4, wherein the suspension of host feces has a solid to liquid ratio of 1:5g/mL.

6. The use according to any one of claims 3 to 5, wherein the first centrifugation is carried out at a speed of 2800 to 3200r/min for a period of 4 to 6min; the rotation speed of the second centrifugation is 2800-3200r/min, and the time is 2-4min; the rotation speed of the third centrifugation is 2800-3200r/min, and the time is 4-6min.

7. The use according to claim 6, wherein the first centrifugation is carried out at a speed of 3000r/min for a period of 5min; the rotating speed of the second centrifugation is 3000r/min, and the time is 3min; the rotating speed of the third centrifugation is 3000r/min, and the time is 5min.

8. The use according to any one of claims 3 to 5 and 7, wherein the PBS buffer contains amphotericin B and streptomycin, the concentration of amphotericin B is 250mg/L, and the concentration of streptomycin is 10mL/L.

9. The use according to claim 6, wherein the PBS buffer contains amphotericin B and streptomycin, the concentration of amphotericin B is 250mg/L, and the concentration of streptomycin is 10mL/L.

10. The use according to claim 8, wherein the penicillin concentration in said penicillin is 10000U/mL and the streptomycin concentration is 10000 μ g/mL.

11. The use according to claim 9, wherein the penicillin concentration in said penicillin streptomycin is 10000U/mL and the streptomycin concentration is 10000 μ g/mL.