CN113122484A - Freeze-drying protective agent for nontuberculous mycobacteria, and preparation method and preservation method thereof - Google Patents
Freeze-drying protective agent for nontuberculous mycobacteria, and preparation method and preservation method thereof Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 59
- 238000004321 preservation Methods 0.000 title claims abstract description 57
- 238000004108 freeze drying Methods 0.000 title claims abstract description 45
- 239000003223 protective agent Substances 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 241000186359 Mycobacterium Species 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000000843 powder Substances 0.000 claims abstract description 8
- 239000010703 silicon Substances 0.000 claims abstract description 8
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 8
- 235000020183 skimmed milk Nutrition 0.000 claims abstract description 6
- 239000000725 suspension Substances 0.000 claims description 26
- 230000001580 bacterial effect Effects 0.000 claims description 19
- 235000013336 milk Nutrition 0.000 claims description 16
- 239000008267 milk Substances 0.000 claims description 16
- 210000004080 milk Anatomy 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 8
- 239000001971 Middlebrook 7H10 Agar Substances 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 230000004083 survival effect Effects 0.000 abstract description 19
- 238000011084 recovery Methods 0.000 abstract description 14
- 238000011160 research Methods 0.000 abstract description 5
- 230000015556 catabolic process Effects 0.000 abstract description 3
- 238000006731 degradation reaction Methods 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 7
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 6
- 244000005700 microbiome Species 0.000 description 6
- 239000004156 Azodicarbonamide Substances 0.000 description 5
- XOZUGNYVDXMRKW-AATRIKPKSA-N azodicarbonamide Chemical compound NC(=O)\N=N\C(N)=O XOZUGNYVDXMRKW-AATRIKPKSA-N 0.000 description 5
- 235000019399 azodicarbonamide Nutrition 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 3
- 229930182912 cyclosporin Natural products 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 229920001296 polysiloxane Polymers 0.000 description 3
- 108010036941 Cyclosporins Proteins 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010024229 Leprosy Diseases 0.000 description 1
- 241000187480 Mycobacterium smegmatis Species 0.000 description 1
- 241001302239 Mycobacterium tuberculosis complex Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- ZHPNWZCWUUJAJC-UHFFFAOYSA-N fluorosilicon Chemical compound [Si]F ZHPNWZCWUUJAJC-UHFFFAOYSA-N 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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Abstract
The invention relates to the technical field of strain preservation, in particular to a freeze-drying protective agent for nontuberculous mycobacteria, a preparation method and a preservation method thereof. The freeze-drying protective agent comprises the following components: 1-10 g/L of Middlebrook7H9 broth base, 80-120 mL/L of ADC enrichment broth, 50-100 g/L of skimmed milk powder, 0.00001-0.0001 mL/L of organic silicon and water to make up to 1L. The invention improves the category and the concentration of the protective agent for preserving the mycobacterium, thereby solving the problems that the preservation and the preservation time of the existing protective agent for preserving the non-tuberculous mycobacterium under the freeze drying method is short, the strain is easy to generate variation and degradation, the recovery survival rate is low and the like; the preservation method has the advantages of simple operation, high survival rate of the strains, long preservation time, high stability of the strains, convenient transportation and wide application range, and is beneficial to the research and utilization of the nontuberculous mycobacteria.
Description
Technical Field
The invention relates to the technical field of strain preservation, in particular to a freeze-drying protective agent for nontuberculous mycobacteria, a preparation method and a preservation method thereof.
Background
The microbial preservation technology is a very basic but very important technology in the field of microbes, and no matter basic scientific research work or application research of biotechnology, in order to make isolated strains in the nature permanently preserve the original high-quality performance of the isolated strains, the isolated strains play a role to the greatest extent and serve human beings, correct strain preservation methods and technologies are needed to ensure the quality and the activity of the strains. Mycobacteria (Mycobacterium) is one of the most historically important groups of microorganisms in humans. The genus contains more than 250 strains, the nontuberculous mycobacteria refer to other mycobacteria except mycobacterium tuberculosis complex (human type mycobacteria, bovine type mycobacteria, African and hamster mycobacteria) and leprosy mycobacteria, are conditional pathogenic bacteria, have important function in hospital infection quality control, and secondary development of strain resources (strain metabolites, vaccines and the like) of part of the mycobacteria such as Mycobacterium smegmatis is more and more discovered and valued by more scientific research workers, so how to ensure that the mycobacteria strain can be effectively and stably preserved for a long time becomes the problem which needs to be solved at present urgently.
For mycobacteria, the traditional preservation method mainly comprises a regular transplantation method and a freeze-drying method, wherein the regular transplantation method is a method for short-term preservation of strains commonly used in a laboratory, and is a method for short-term preservation of strains by placing fresh strains cultured under specified conditions in a refrigerator at 4 ℃, the living conditions of the original strains can be changed due to accumulation of metabolites by using the method, so that individuals in colony groups continuously age and die, the method is simple to operate, but the preservation period is short, the strains are required to be transplanted again after 1-3 months, and the contamination to mixed bacteria or the variation of the strains is easy.
The freeze drying method adopts skim milk in a certain proportion as a protective agent for freeze drying preservation, and the metabolic activity of the strain is relatively static by using the method, so that an environment which is not suitable for the growth of the strain and can be preserved for a long time is created. At present, the freeze-drying method can achieve an effective long-term preservation effect on most strains, but for mycobacteria, the requirement on required nutrient components is high, nutrient substances are not easy to absorb, the growth speed of the mycobacteria is relatively slow, the survival rate of the strains is low when the strains are redissolved after freeze-drying, and the survival rate of the mycobacteria cannot be well improved if the known mycobacteria enrichment liquid (CN102471757A) is used as a protective agent for freeze-drying. There is therefore a need to establish effective long-term preservation methods for these particular cases of mycobacteria.
Disclosure of Invention
In view of the above, the present invention provides a non-tuberculous mycobacterium cryoprotectant, a preparation method and a preservation method thereof. The preservation method is simple to operate, easy to preserve, convenient to transport, long in preservation time and high in strain stability, and solves the problem that the survival rate of the mycobacteria revived by adopting the traditional preservation method is low.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a freeze-drying protective agent for nontuberculous mycobacteria, which comprises the following components:
the freeze drying preservation method is that the metabolic activity of the strain is relatively static under the conditions of low temperature, drying and oxygen deficiency of the microorganism. The method combines the characteristics of a plurality of strain preservation methods, thereby creating an environment which is not suitable for strain growth and can be preserved for a long time, and the protective agent is added before freeze drying, thereby greatly improving the success rate of the method, but the type of the protective agent needs to be added in a customized manner according to different microbial characteristics. The invention improves the category and the concentration of the protective agent for preserving the mycobacterium on the basis of a freeze-drying method, thereby solving the problems that the preservation and preservation time of the existing protective agent for preserving the non-tuberculous mycobacterium is short, the strain is easy to generate variation and degradation, the recovery survival rate is low and the like under the freeze-drying method.
Preferably, the silicone is a nonionic silicone surfactant.
In the specific embodiment provided by the invention, the organosilicon is fluorine silicon FY-4902.
Preferably, the lyoprotectant comprises the following components:
preferably, the lyoprotectant comprises the following components:
the invention also provides a preparation method of the freeze-drying protective agent, which comprises the following steps:
dissolving Middlebrook7H9 broth base dry powder and skimmed milk powder in water, and sterilizing to obtain milk suspension;
and (3) when the temperature of the milk suspension is cooled to 40-60 ℃, adding the ADC enrichment fluid and the organic silicon, and uniformly mixing.
In the specific embodiment provided by the invention, ADC enrichment liquid and organic silicon are added when the temperature of the milk suspension is cooled to 50 ℃.
Preferably, the sterilization temperature is 113-121 ℃ and the time is 10-30 min.
In the specific embodiment provided by the invention, the sterilization temperature is 113 ℃ and the sterilization time is 30 min.
The invention also provides a preservation method of nontuberculous mycobacteria, which comprises the following steps:
(1) under the aseptic condition, non-tuberculous mycobacterium is mixed with the freeze-drying protective agent to prepare bacterial suspension;
(2) the bacterial suspension is pre-frozen, freeze-dried and stored in an environment at the temperature of minus 20 ℃ or below minus 20 ℃.
In the present invention, the method further comprises the step (3): recovering the preserved nontuberculous mycobacteria.
Preferably, the resuscitation is: mixing non-tuberculous mycobacteria with water at 15-30 ℃ under an aseptic condition for redissolution to obtain a bacterial suspension; inoculating the bacterial suspension to a mycobacterium solid culture medium plate for culturing.
Preferably, the mycobacteria solid medium plate is a Middlebrook7H10 agar medium plate.
Preferably, the nontuberculous mycobacteria in step (1) are nontuberculous mycobacteria in log phase.
The invention provides a freeze-drying protective agent for nontuberculous mycobacteria, and a preparation method and a preservation method thereof. The freeze-drying protective agent comprises the following components: 1-10 g/L of Middlebrook7H9 broth base, 80-120 mL/L of ADC enrichment broth, 50-100 g/L of skimmed milk powder, 0.00001-0.0001 mL/L of organic silicon and water to make up to 1L. The invention has the following technical effects:
the invention improves the category and the concentration of the protective agent for preserving the mycobacterium on the basis of a freeze-drying method, thereby solving the problems that the preservation and preservation time of the existing protective agent for preserving the non-tuberculous mycobacterium is short, the strain is easy to generate variation and degradation, the recovery survival rate is low and the like under the freeze-drying method;
the preservation method has the advantages of simple operation, high survival rate of the strains, long preservation time, high stability of the strains, convenient transportation and wide application range, and is beneficial to the research and utilization of the nontuberculous mycobacteria. The preservation method is suitable for long-term preservation of nontuberculous mycobacteria.
Detailed Description
The invention discloses a freeze-drying protective agent for nontuberculous mycobacteria, a preparation method and a preservation method thereof, and a person skilled in the art can realize the freeze-drying protective agent by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The materials used in the preservation method of the invention are sterilized for standby at 121 ℃ for 15 min;
the preparation method of the non-tuberculous mycobacteria needing to be preserved comprises the following steps: non-tuberculous mycobacteria are inoculated on a Middlebrook7H10 agar medium plate, and fresh logarithmic phase colonies are cultured according to the proper temperature of the strain.
The reagents or apparatus used in the present invention are commercially available.
The invention is further illustrated by the following examples:
example 1:
the preparation method of the non-tuberculous mycobacterium freeze-drying protective agent comprises the following steps:
weighing 4.7g of Middlebrook7H9 dry powder and 50g of skimmed milk powder, adding into 900mL of purified water, sterilizing at 113 ℃ for 30min under high pressure, cooling to about 50 ℃, adding 100mL of ADC (azodicarbonamide) enrichment solution serving as a matched reagent of Middlebrook7H9 and 0.00005mL/L of organic silicon solvent, and obtaining 5% Middlebrook7H9 milk suspension containing organic silicon.
The preservation method of nontuberculous mycobacteria comprises the following steps:
(1) selecting a strain to be preserved: the strains selected in the experiment are 50 strains of nontuberculous mycobacteria purchased from German collection center of culture collection of microorganisms and are to be preserved.
TABLE 1 List of 50 non-tuberculous mycobacterial strains
(2) Strain preservation and freeze-drying: and (2) performing colony culture on the bacterial strain in the step (1), collecting cultured bacterial colonies of 30-100CFU/mL and sterilized Middlebrook7H9 milk suspension containing organosilicon to prepare bacterial suspension, subpackaging the bacterial suspension into sterile freeze-drying bottles in an amount of 0.5-1 mL per bottle, and freeze-drying after pre-freezing. The freeze-dried strain is stored in a refrigerator at the temperature of 20 ℃ below zero.
(3) Recovering the strain: when activating the strain, taking out the freeze-drying bottle from a refrigerator at the temperature of 20 ℃ below zero, placing the freeze-drying bottle at room temperature, inoculating 0.5mL of sterile water into the freeze-drying bottle for redissolution under the aseptic condition, inoculating 5-10 cyclosporins into a Middlebrook7H10 agar medium plate after redissolution, culturing at the proper temperature, and observing the recovery survival condition.
Example 2:
the preservation method is the same as the preservation method described in example 1, the milk concentration of the milk suspension is changed to 8%, the operation is carried out according to the steps of example 1, the culture is carried out at a proper temperature, and the recovery survival condition is observed.
Example 3:
the preservation method is the same as the preservation method described in example 1, the milk concentration of the milk suspension is changed to 10%, the operation is carried out according to the steps of example 1, the culture is carried out at a proper temperature, and the recovery survival condition is observed.
Comparative example 1:
the preservation method is the same as the preservation method described in example 1, the milk suspension is 10% Middlebrook7H9 milk suspension without organic silicon, the operation is carried out according to the steps of example 1, the culture is carried out at a proper temperature, and the recovery survival condition is observed.
Comparative example 2:
the preservation method of nontuberculous mycobacteria comprises the following steps:
(1) selecting a strain to be preserved: the strains selected in the experiment are 50 strains of nontuberculous mycobacteria purchased from German collection center of culture collection of microorganisms and are to be preserved.
(2) Strain preservation and freeze-drying: and (2) performing colony culture on the bacterial strain in the step (1), collecting cultured bacterial colonies and sterilized 10% milk solution to prepare bacterial suspension, subpackaging, pre-freezing and freeze-drying. The freeze-dried strain is stored in a refrigerator at the temperature of 20 ℃ below zero.
(4) Recovering the strain: when activating the strain, taking out the freeze-drying bottle from a refrigerator at the temperature of 20 ℃ below zero, placing the freeze-drying bottle at room temperature, inoculating 0.5mL of sterile water into the freeze-drying bottle for redissolution under the aseptic condition, inoculating 5-10 cyclosporins into a Middlebrook7H10 agar medium plate after redissolution, culturing at the proper temperature, and observing the recovery survival condition.
Comparative example 3:
the preservation method of nontuberculous mycobacteria comprises the following steps:
(1) selecting a strain to be preserved: the strains selected in the experiment are 50 strains of nontuberculous mycobacteria purchased from German collection center of culture collection of microorganisms and are to be preserved.
(2) Strain preservation and freeze-drying: and (2) performing colony culture on the bacterial strain in the step (1), collecting cultured bacterial colonies and sterilized 10% milk solution to prepare bacterial suspension, subpackaging, pre-freezing and freeze-drying. The freeze-dried strain is stored in a refrigerator at the temperature of 20 ℃ below zero.
(3) Recovering the strain: when activating the strain, taking out the freeze-drying bottle from a refrigerator at the temperature of 20 ℃ below zero, placing the freeze-drying bottle at room temperature, inoculating 0.5mL of sterile Middlebrook7H9 solution into the freeze-drying bottle for redissolution under the aseptic condition, inoculating 5-10 cyclosporine suspension onto a Middlebrook7H10 agar medium plate after redissolution, culturing at the proper temperature, and observing the recovery survival condition.
Test examples
The results of the experiments of examples 1 to 3 and comparative examples 1 to 3 are shown in tables 2 and 3, and it is understood from the results of table 3 that the survival rate of resuscitation after lyophilization preservation and resuscitation after preparation of bacterial suspension from 5% to 10% milk suspension of Middlebrook7H9 containing silicone is the highest, and the method is the most suitable freeze-drying preservation method for nontuberculous mycobacteria. In the invention, the recovery survival rate of the strain is obtained by dividing the recovery strain number by the experimental strain number, and the recovery standard is that the colony is formed in 21 days, can be observed by naked eyes and is identified as the colony. The scheme adopts an automatic ms1000 type full-automatic microorganism mass spectrum identification system of Zhengzhou Antu laboratory instruments (Zhengzhou) Limited company to carry out strain identification. And (3) no colony is observed beyond the recovery time, or the colony is identified to be different from the experimental standard strain and is judged to be dead, and the death rate is obtained by dividing the number of dead strains by the number of experimental strains.
Table 2: survival conditions of 50 kinds of non-tuberculous mycobacteria with different preservation and different recovery modes
Table 3: resuscitation survival rate and resuscitation death rate of 50 kinds of nontuberculous mycobacteria
| Item | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Survival rate of resuscitation | 100％ | 98％ | 100％ | 84％ | 80％ | 84％ |
| Resuscitation mortality | 0％ | 2％ | 0％ | 16％ | 20％ | 16％ |
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A freeze-drying protective agent for nontuberculous mycobacteria is characterized by comprising the following components:
2. the lyoprotectant of claim 1, comprising the following components:
3. the lyoprotectant of claim 1, comprising the following components:
4. a process for the preparation of a lyoprotectant according to any of claims 1 to 3, comprising the steps of:
dissolving Middlebrook7H9 broth base dry powder and skimmed milk powder in water, and sterilizing to obtain milk suspension;
and (3) when the temperature of the milk suspension is cooled to 40-60 ℃, adding the ADC enrichment fluid and the organic silicon, and uniformly mixing.
5. The method according to claim 4, wherein the sterilization temperature is 113 to 121 ℃ and the sterilization time is 10 to 30 min.
6. A preservation method of nontuberculous mycobacteria is characterized by comprising the following steps:
(1) aseptically mixing nontuberculous mycobacteria with the lyoprotectant according to any one of claims 1 to 3 to prepare a bacterial suspension;
(2) the bacterial suspension is pre-frozen, freeze-dried and stored in an environment at the temperature of minus 20 ℃ or below minus 20 ℃.
7. A preservation method according to claim 6, further comprising the step (3): recovering the preserved nontuberculous mycobacteria.
8. Preservation process according to claim 7, characterized in that said resuscitation is: mixing non-tuberculous mycobacteria with water at 15-30 ℃ under an aseptic condition for redissolution to obtain a bacterial suspension; inoculating the bacterial suspension to a mycobacterium solid culture medium plate for culturing.
9. The preservation method according to claim 8, wherein said mycobacteria solid medium plate is a Middlebrook7H10 agar medium plate.
10. A preservation process according to any one of claims 6 to 9, wherein the non-tubercular mycobacteria in step (1) are non-tubercular mycobacteria in log phase.
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