CN113667647A

CN113667647A - Method for culturing and observing plaques

Info

Publication number: CN113667647A
Application number: CN202110980519.0A
Authority: CN
Inventors: 张俊会
Original assignee: BAODING UNIVERSITY
Current assignee: BAODING UNIVERSITY
Priority date: 2021-08-25
Filing date: 2021-08-25
Publication date: 2021-11-19
Anticipated expiration: 2041-08-25
Also published as: CN113667647B

Abstract

The invention relates to a method for culturing and observing plaques, which takes escherichia coli as a host, separates phages, and cultures the plaques, and comprises the steps of water sample pre-detection, bacterial suspension preparation, water sample treatment and phage multiplication, phage multiplication liquid dilution, phage-host mixed liquid preparation, plaque culture by a sandwich plate method, staining observation and the like, and finally clear plaques are observed. The colibacillus lawn is light blue cloud, and the plaque is dark blue transparent small round plaque. Compared with the traditional double-layer plate method, the method obviously reduces the operation difficulty of the experiment, reduces the risk of polluting other environmental microorganisms, improves the success rate of the experiment, is a good teaching experiment method, and can be applied to the teaching of colleges and universities or the training of related technical personnel. The sandwich plate method of the present invention can also be used for culturing and observing plaques formed by other phages, but the relevant steps need to be adjusted.

Description

Method for culturing and observing plaques

Technical Field

The invention belongs to the technical field of microorganisms, and relates to a method for culturing and observing plaques.

Background

Phage (phase) is a virus infecting prokaryotes such as bacteria, actinomycetes and cyanobacteria, is a common experimental material in genetics, molecular biology and genetic engineering, and widely exists in various habitats in nature. Depending on whether the host is lysed after it has been infected, it can be classified into virulent phage and temperate phage. The virulent phage will infect the host to form individual plaques with certain shape, size, margin and transparency on the lawn. The plaque of each bacteriophage has a certain shape, so that the bacteriophage can be used as an identification index of the bacteriophage and can also be used for pure species separation and counting.

The separation and purification of the phage is one of the necessary projects in the courses of ordinary microbiological experiments, pathogenic microbiological experiments, environmental engineering microbiological experiments and the like. The culture and observation of the plaques are the core technology of the experimental project. At present, the two-layer plate method invented in 1959 by Adams is widely used in domestic and foreign textbooks and mainly comprises the steps of preparing a bottom layer culture medium and an upper layer culture medium containing 2% and 0.7% of agar in advance. First, a layer of flat plate is poured on a culture dish by using a bottom layer culture medium, and the flat plate is solidified. Mixing the upper layer culture medium which is pre-melted and cooled to 45 ℃ with a concentrated sensitive host and a certain volume of phage solution to be detected, uniformly mixing, and paving on a solidified bottom layer flat plate, wherein if phage capable of cracking host bacteria exists, after a period of culture, macroscopic plaques appear. This method has several significant disadvantages:

(1) the host and phage are mixed using a pre-thawed upper medium, and the temperature is difficult to control. When the temperature is lower than 45 ℃, the upper layer agar is solidified in the mixing process, and when the temperature is higher than 60 ℃, the bacteriophage has the risk of inactivation, so that beginners are often in disorder, and the upper layer agar is easy to form agar blocks due to solidification.

(2) The difficulty of visual observation is large. Although the bottom layer culture medium is used for filling and leveling, the unevenness of the bottom of the glass culture dish is avoided, but because the escherichia coli bacterial colony is colorless and semitransparent, the agar is transparent and is in faint yellow, and a beginner can hardly identify the plaque only by observing with naked eyes.

(3) The culture medium has rich nutrition, has higher requirements on experimental environment and operation technology, and is easily polluted by other microorganisms in the environment to interfere the experimental result.

(4) The observation time is severe. Although most courses prescribe observations 18-24h after incubation. In practice, however, the optimal observation time for the plaques is between 4 and 20 hours of culture, and beyond 20 hours, the plaque boundary is often blurred, probably because the upper culture medium is soft and facilitates the movement of hosts or other contaminated bacteria on the surface of the agar.

In addition, in the phage multiplication link in the early stage of plaque culture, the teaching material mostly selects large-capacity sewage (200ml) to directly multiply the phage. The step has two defects, namely, the experimental material has larger volume and is inconvenient to operate; secondly, the sewage contains a plurality of microorganisms simultaneously, and the microorganisms can be proliferated in a beef extract peptone liquid culture medium with rich nutrition, so that the step does not selectively proliferate coliphage.

In order to increase the contrast between plaque and background, researchers add dyes such as red tetrazole to the upper agar, but the dyes inhibit the growth of escherichia coli to a certain extent. Or after culturing, 2,3,5 triphenyltetrazolium chloride (TTC) is used for staining, the dye is removed after staining, and the dye is reduced by living cells in the lawn, so that the plaque and the lawn are in sharp contrast. However, the upper agar is soft (0.7%), the hardness is low, the combination of the Escherichia coli colony and the culture medium is not tight, the lawn is easy to be washed up or the upper culture medium is easy to be damaged in the dyeing process, and the plaques cannot be observed and counted.

Disclosure of Invention

The invention relates to an improved plaque culture and observation method, which is characterized in that on the basis of a double-layer plate method, bottom-layer agar is changed into water agar without nutrients, and simultaneously, the double-layer plate is changed into three layers, namely, a layer of semisolid agar is clamped between the two layers of water agar, and host bacteria and phage are limited in the middle layer, so that the risk of colonization of microorganisms on the plate in the environment is reduced, formed plaques are close to the same plane, and the upper and lower overlapping of the plaques is avoided. After the culture is finished, other mixed bacteria polluted on the surface water agar are washed away, and the plaques can be clearly observed after being dyed by low-concentration methylene blue, the plaques are close in size and clear in edge, and the phenomenon of overlapping of upper and lower plaques is avoided. The invention comprises the following steps:

step 1: and (3) water sample pre-detection, namely selecting an environmental water sample with positive total coliform flora as a phage separation culture material.

Step 2: preparing bacterial suspension, selecting a single colony of a deep purple red colony with metal luster from an eosin methylene blue culture medium through aseptic operation, inoculating 5ml of beef extract peptone liquid culture medium, and culturing at 37 ℃ and 150rpm for 1-3 hours.

And step 3: treating a water sample and proliferating phages, taking 2ml of sewage, centrifuging at 4 ℃ and 10000rpm for 1min, collecting supernatant, adding 0.2ml of chloroform into the supernatant, whirling for 1min, standing, collecting 1ml of supernatant liquid, adding the supernatant liquid into 5ml of beef extract peptone liquid culture medium, simultaneously adding 0.2ml of bacterial suspension of escherichia coli, and culturing at 37 ℃ and 150rpm overnight to proliferate the phages.

And 4, step 4: diluting the phage multiplication solution, taking 2ml of the phage multiplication solution, centrifuging at 4 ℃ and 10000rpm for 1min, collecting supernatant, adding 0.2ml of chloroform into the supernatant, performing vortex oscillation for 1min, and standing, wherein the supernatant is an enriched phage sample. The enriched phage sample was diluted 10 fold to 10 with 1% peptone water^-3～10^-7。

And 5: preparing mixed solution of bacteriophage and host, adding colibacillus suspension into the prepared bacteriophage gradient diluent, adding 50 mul bacterial suspension into each 1ml diluent, and reversing and mixing evenly.

Step 6: plaque was cultured by sandwich plate method, approximately 5ml of water agar was added to a sterile plate, and cooled to solidify. And pouring the mixed host-phage liquid onto the solidified bottom plate, immediately pouring a layer of heat-insulating semi-solid culture medium, mixing uniformly, wherein each dish is about 10ml, and standing for solidification. Finally, a layer of incubated water agar, approximately 10ml per dish, was poured, allowed to set and incubated overnight at 37 ℃ while inverted.

And 7: staining and observing plaques, slowly cleaning the surface of the interlayer flat plate with running water, removing the contaminated bacteria and dust on the surface, sucking the residual water, staining with 0.05% methylene blue water solution for 2min, pouring off the staining solution, observing, wherein the background is blue, the Escherichia coli lawn is opaque and cloudy, and the plaques are dark blue circular plaques.

The invention can also be used for culturing and observing plaques formed by other phages, but relevant steps need to be properly adjusted.

The invention has the beneficial effects that:

(1) and a water sample pre-detection link is added. As the bacteriophage generally coexists with a host thereof in a specific habitat, and Escherichia coli is an important component of the total coliform group, if the total coliform group of a water sample is detected to be positive, the existence of the Escherichia coli in the water body is indicated, and the bacteriophage of the Escherichia coli also exists in the water body. In addition, if no suitable escherichia coli strain exists in a laboratory, the step can be used for simultaneously separating environmental escherichia coli strains, and specific phage can be separated aiming at escherichia coli in the water body. However, the success of the experiment is not necessarily guaranteed if engineering bacteria of Escherichia coli, such as DH5 alpha, BL21 and JM109, are adopted.

(2) The host bacteria are maintained on the eosin methylene blue culture medium for generation all the time. Because the eosin methylene blue culture medium is an identification culture medium, the escherichia coli presents unique colony characteristics on the culture medium, namely, a dark purple-red smooth colony with metallic luster and a relatively wet surface, the purity of the colony of a host is at least ensured, and the prepared bacterial suspension is prevented from containing other mixed bacteria.

(3) Selectively multiplying the bacteriophage in the water body. In the traditional method, a large amount of sewage (200ml) is used for directly proliferating the phage, so that a large amount of reagents are consumed, and the operation intensity is high. In addition, since the beef extract peptone liquid medium is rich in nutrients, many microorganisms in the wastewater can grow therein, and thus the selectivity is not strong. The method uses a small amount (2ml) of sewage, simultaneously removes cells in the sewage through high-speed centrifugation and chloroform treatment, only uses non-cell components in the sewage to proliferate phage, and enhances the selectivity of the experiment.

(4) The solidification of the semi-solid medium during the operation is avoided. In the traditional method, after the phage gradient diluent is prepared, host bacteria and the phage diluent are mixed in a semi-solid culture medium, the operation is required to be accurate and rapid, otherwise, agar blocks easily appear on the upper agar, and the later observation is interfered. The invention adopts a casting plate method, firstly host bacteria and phage diluent are mixed in 1% peptone water, the mixed solution is directly poured on the solidified bottom layer culture medium, then the heat-preservation semi-solid culture medium is poured, the mixture is mixed and stood for coagulation, the operation is simple and convenient, and the coagulation of the culture medium in the mixing process is avoided.

(5) The sandwich plate is used for limiting the host bacteria and the phage to the middle layer of the culture dish, and the plaques can be formed in the middle layer through culture, so that the upper layer and the lower layer of the plaques are prevented from being overlapped. In addition, the bottom layer and the surface layer are both 2% of water agar, and even if environmental microorganism pollution is caused during operation, the water agar does not contain nutrients and can not support the growth of most microorganisms, so that the pollution risk is reduced. The water agar on the surface layer also plays a role of a protective layer for the formed lawn and plaques, and prevents the lawn from being greatly damaged due to the intensity of the dropwise adding staining solution in the later dyeing process.

(6) The sandwich plates were stained with 0.05% methylene blue in water to increase the contrast between plaques and lawn, and plaques could be clearly observed. The background is blue, the colibacillus lawn is opaque cloudy, and the plaque is deep blue plaque. In addition, methylene blue is a living coloring agent, is non-toxic and harmless, and does not influence subsequent experiments. The phage can be continuously separated from the formed plaques for subsequent experiments.

(7) The observation time is wide. The optimal observation time of the plaque by the double-layer plate method is short, and the plaque lysogenesis or the tolerant bacteria coverage plaque can occur after more than 20 hours. This is because the upper medium is soft and the movement of the host bacteria on the agar surface changes the shape of the plaque. At the same time, the contaminating tolerant bacteria also destroy the shape of the plaque. The observation after the culture for 24h and 48h by the method shows that the characteristics of the plaque, such as size, distribution, shape and the like, observed by naked eyes are not obviously different, probably because the movement of the escherichia coli is limited by the water agar with a hard surface layer, and meanwhile, the water agar also avoids the growth of mixed bacteria. Therefore, students can conveniently observe the experimental results in the after-class time to develop subsequent experiments to a great extent.

Drawings

FIG. 1 is a schematic diagram of a sandwich plate method, which comprises water agar, a beef extract peptone semisolid culture medium and water agar from bottom to top in sequence.

FIG. 2 is a typical E.coli colony isolated.

FIG. 3 is the observed E.coli plaques.

Detailed Description

1. Experimental Material

1.1 culture Medium and reagent

Lactose peptone liquid medium

10g of peptone, 3g of beef extract, 5g of lactose, 5g of NaCl, 1.0ml of 16g/L bromocresol purple ethanol solution, 1000ml of distilled water, pH 7.2-7.4, subpackaging into test tubes, 5ml of each tube, placing one small inverted tube in each tube, sterilizing at 115 ℃ for 30 min.

② eosin-methylene blue culture medium (EMB) culture medium

10g of peptone, 10g of lactose, 2g of dipotassium phosphate, 18-20 g of agar, 1000ml of distilled water, 7.2-7.4 of pH, sterilizing at 115 ℃ for 30min, cooling, adding 20ml of eosin aqueous solution 20g/L and 13ml of methylene blue aqueous solution 5g/L, uniformly mixing, pouring into a flat plate, solidifying and storing in a refrigerator at 4 ℃ for later use.

③ beef extract peptone liquid culture medium

3g of beef extract, 10g of peptone, 5g of NaCl and CaCl₂10mM, 1000ml of water, pH 7.0-7.2, and sterilizing at 121 ℃ for 20 min.

Fourthly, water agar

18-20 g of agar and 1000ml of water, and sterilizing for 20min at 121 ℃.

Semi-solid culture medium of beef extract peptone

3g of beef extract, 10g of peptone, 5g of NaCl and CaCl₂10mM, 10-12 g of agar and 1000ml of water, the pH value is 7.0-7.2, and the mixture is sterilized for 20min at 121 ℃.

Peptone water

Peptone 10g, CaCl₂10mM, 1000ml of water, pH 7.0-7.2, and sterilizing at 121 ℃ for 20 min.

Seventhly 0.05 percent of methylene blue aqueous solution

Methylene blue 0.05g and water 100 ml.

(iii) chloroform

Ninthly, gram staining solution set

1.2 materials:

environmental water sample, culture dish, test tube, centrifuging tube, rifle head

1.3 strains: escherichia coli

If the laboratory has no preserved Escherichia coli strains, the Escherichia coli can also be obtained by separating and purifying water samples during the preliminary examination.

2. Instrumentation and equipment

Super clean bench, shaking table, incubator, water bath, microscope, centrifuge, vortex oscillator, pipettor, alcohol burner, transfering loop, centrifuge tube rack, test-tube rack.

3. Carrying out the step

Day 1：

1. Preparation of media and materials: lactose peptone water liquid medium, 10 tubes, 5ml per tube.

2. Collecting a water sample: the water body possibly polluted by human and animal excreta is selected as the research object.

3. Water sample pre-detection: and (3) performing primary fermentation, namely injecting 10, 1 and 0.1ml of water samples into a lactose peptone liquid culture medium by aseptic operation, repeating the steps for each sample, simultaneously making a blank control, uniformly mixing, standing at 37 ℃ for overnight culture. The acid and gas production is positive. The negative person is cultured for 24h +/-3 h and then checked.

Day 2：

1. Preparation of media and materials: eosin-methylene plate, several.

2. Observing whether the lactose peptone water produces acid and gas, if not, changing the water sample to repeat day 1 work, if so, carrying out subsequent work.

3. Plate separation experiment: and (3) lightly shaking the primary fermentation test positive tubes, taking the culture of the positive tubes by using an inoculating loop in an aseptic operation, streaking and inoculating on an eosin methylene blue plate, inversely placing the plate in an incubator at 37 ℃ for culturing for 18-24h, and streaking and inoculating one eosin methylene blue plate on each positive tube.

Day 3：

1. Preparation of media and materials: lactose peptone water, several, 5ml per tube. Eosin methylene blue plate, 2 plates; gram staining solution, 1 set.

2. The results of the experiment were observed on eosin methylene blue plates, and the colonies with deep purple red color and metallic luster (FIG. 1) were typical coliform colonies, and were streaked on eosin methylene blue plates.

3. A portion of the culture of a representative colony was smeared, gram stained, and examined as gram negative Bacillus free.

4. Secondary fermentation test: another part of the culture of the colony was inoculated into a lactopeptone fermentation tube by aseptic technique, while a blank control was made, and the culture was left to stand at 37 ℃ for 24 hours.

Day 4：

1. Preparation of media and materials: beef extract peptone liquid medium, 5ml per tube, 2 tubes; chloroform; centrifuging a plurality of tubes.

2. And observing whether the lactose peptone water after secondary fermentation produces acid and gas. If the acid and gas production is carried out, and the gram stain is negative and no bacillus exists, the existence of total coliform flora in the water body is verified, namely, the bacteriophage of the escherichia coli is separated with higher success probability. Meanwhile, the strain which can be speculated and separated by combining gram staining, physiological and biochemical characteristics and colony characteristics is escherichia coli, and the strain can be used for subsequent phage culture, but related experiments need to be supplemented if the classification status is further clarified.

3. Preparing a bacterial suspension: sterile operation from eosin Meilan plate to take 1 single colony inoculated with 5ml beef extract peptone liquid medium, 37 degrees C150 rpm culture about 1 ~ 3 hours.

4. Treating a water sample: taking 2ml of sewage sample, centrifuging at 10000rpm for 1min, taking supernate, adding 0.2ml of chloroform, swirling for 1min, and standing, wherein the supernate is a bacteriophage protosample in a water body.

5. And (3) proliferation culture: to 5ml of meat extract peptone broth, 1ml of the prepared phage stock sample and 0.2ml of E.coli suspension were added, and cultured overnight at 37 ℃ with shaking at 150 rpm.

Day 5:

1. Preparation of culture medium and materials: water agar 500ml, sterilizing and preserving heat in a 50 ℃ water bath kettle; 400ml of beef extract peptone semisolid culture medium, sterilizing and then preserving heat in a 50 ℃ water bath kettle; 100ml of 1% peptone water; chloroform. Sterile centrifuge tubes, petri dishes, and the like.

2. Preparing bacterial suspension, taking 1 single colony from an eosin methylene blue plate by aseptic operation, inoculating 5ml of beef extract peptone liquid culture medium, and culturing at 37 ℃ for 1-3 hours.

3. Preparing a bottom flat plate: sterile plates were prepared, approximately 5ml of water agar was added to each dish, and cooled to solidify.

4. Preparing a lysate: centrifuging 2ml of the phage proliferation solution at 10000rpm for 1min, collecting supernatant, adding 0.2ml of chloroform into the supernatant, performing vortex treatment for 1min, and standing to obtain an upper layer of liquid which is an enriched phage sample.

5. And (3) diluting a lysis solution: taking a plurality of 2ml centrifuge tubes, adding 0.9ml peptone water into each tube, diluting the enriched phage sample 10 times to 10 times with 1% peptone water^-3～10^-7。

6. Mixed host bacteria: selection 10^-3～10^-7Diluting the gradient phage lysate to mix the host bacteria, adding 50 mul of prepared host bacteria suspension into each 1ml of phage diluent, and reversing and mixing evenly.

7. Preparing a sandwich flat plate: pouring the mixed host-phage mixture on the solidified bottom plate, pouring a layer of heat-preservation beef extract peptone semisolid culture medium, mixing uniformly, wherein each dish is about 10ml, pouring a layer of heat-preservation water agar, and each dish is about 10ml for solidification, in the upper layer after solidification.

8. Culturing: after the sandwich plate was completely solidified, it was inverted and cultured overnight at 37 ℃.

Day 6：

1. Preparing a reagent: 0.05% methylene blue in water.

2. Cleaning a flat plate: taking out the cultured sandwich plate, slowly washing away the surface-polluted mixed bacteria with running water, and sucking the residual water with absorbent paper.

3. Dyeing: and (3) covering the surface of the plate with 0.05% methylene blue aqueous solution, and dyeing for 2min, wherein each dish needs about 10-20 ml of dyeing solution.

4. And (4) observation: the staining solution was poured off, the residual water was removed by absorbent paper, and the plaques were observed. The background is blue, the colibacillus lawn is opaque cloudy, and the plaque is deep blue plaque.

Remarking: if the laboratory has Escherichia coli environmental strains and the total coliform of the water sample is confirmed to be positive, the method can be directly started from Day 4 work 2 and lasts for 3 days before and after the experiment.

Claims

1. The invention relates to a method for culturing and observing plaques, which is an experimental method applicable to teaching in colleges and universities or training of related technicians. The principle is as follows: the phage is widely distributed in nature, and can be found in places where hosts exist. Virulent phage infect on a solid plate and lyse a sensitive host to form plaques. The basic principle and the method for separating and purifying the phage can be mastered by students by culturing and observing the plaque, the characteristics of the phage can be known, and the method specifically comprises the following steps:

step 1: the water sample pre-detection is characterized in that an environmental water sample with positive total coliform flora is selected as a phage separation culture material.

Step 2: preparing bacterial suspension, and is characterized in that a single deep purple-red colony with metal luster is picked from an eosin methylene blue culture medium in an aseptic operation, 5ml of beef extract peptone liquid culture medium is inoculated, and the culture is carried out at 37 ℃ and 150rpm for 1-3 hours.

And step 3: a water sample treatment and bacteriophage multiplication method is characterized in that 2ml of sewage is taken, centrifuged for 1min at 4 ℃ and 10000rpm, supernatant is collected, 0.2ml of chloroform is added into the supernatant, vortex oscillation is carried out for 1min, standing is carried out, 1ml of supernatant liquid is collected, added into 5ml of beef extract peptone liquid culture medium, 0.2ml of escherichia coli bacterial suspension is added at the same time, and overnight culture is carried out at 37 ℃ and 150rpm to multiply the bacteriophage.

And 4, step 4: and (3) diluting the phage multiplication solution, which is characterized in that 2ml of phage multiplication solution is taken, centrifuged at 10000rpm for 1min, supernatant is collected, 0.2ml of chloroform is added into the supernatant, vortex oscillation is carried out for 1min, and standing is carried out, wherein the supernatant is an enriched phage sample. The enriched phage sample was diluted 10 fold to 10 with 1% peptone water^-3～10^-7。

And 5: the preparation of phage-host mixed liquid is characterized by that adding colibacillus bacterial suspension into the prepared phage gradient diluent, adding 50 microliter bacterial suspension into every 1ml diluent, and turning and uniformly mixing them.

Step 6: the sandwich plate method for culturing the plaque is characterized in that about 5ml of water agar is added into a sterile plate, and the mixture is cooled to be solidified. And pouring 1050 mu l of the mixed host-phage solution onto the solidified bottom plate, immediately pouring a layer of heat-preservation semi-solid culture medium, mixing uniformly, wherein each dish is about 10ml, and standing for solidification. Finally, a layer of heat-preserved water agar, about 10ml per dish, is poured into the upper layer, and after solidification, the mixture is cultured in an inverted manner at 37 ℃ overnight.

And 7: and (3) carrying out dyeing observation on the plaques, wherein the plaques are characterized in that the cultured interlayer flat plate is washed by water, the surface contaminated mixed bacteria and dust are removed, the residual water is sucked dry, then the interlayer flat plate is dyed by 0.05% methylene blue aqueous solution for 2min, then the dyeing solution is poured out, the background is observed to be blue, the escherichia coli lawn is opaque and cloudy, and the plaques are dark blue round plaques.

2. The method according to claim 1, wherein step 1 is characterized by selecting a water body having a positive total coliform group as a detection target, wherein the water body contains gram-negative bacillus-free bacteria having 37 ℃ lactose fermentation, acid-producing gas-producing, aerobic and facultative anaerobic properties.

3. The method according to claim 1, wherein the sandwich plate of step 6 comprises three layers from bottom to top, and the glass plate is filled by first spreading a layer of water agar without any nutrient on the bottom layer. And secondly, pouring the mixed solution of the host bacteria and the phage on the solidified bottom flat plate, then pouring the heat-preservation semi-solid beef extract peptone culture medium, and uniformly mixing. And finally, after the middle layer is solidified, a layer of water agar without any nutrient is laid on the middle layer.

4. The sandwich plate of claim 3 wherein the amount of agar in the semi-solid medium of the middle layer is selected to be 1% so as to facilitate pouring of the water agar after coagulation.