CN112146949B - 一种刺参石灰环的完整提取方法 - Google Patents
一种刺参石灰环的完整提取方法 Download PDFInfo
- Publication number
- CN112146949B CN112146949B CN202010839724.0A CN202010839724A CN112146949B CN 112146949 B CN112146949 B CN 112146949B CN 202010839724 A CN202010839724 A CN 202010839724A CN 112146949 B CN112146949 B CN 112146949B
- Authority
- CN
- China
- Prior art keywords
- stichopus japonicus
- lime
- solution
- ring
- japonicus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000965254 Apostichopus japonicus Species 0.000 title claims abstract description 142
- 235000008733 Citrus aurantifolia Nutrition 0.000 title claims abstract description 84
- 235000011941 Tilia x europaea Nutrition 0.000 title claims abstract description 84
- 239000004571 lime Substances 0.000 title claims abstract description 84
- 238000000605 extraction Methods 0.000 title claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 84
- 239000000243 solution Substances 0.000 claims abstract description 49
- 230000018044 dehydration Effects 0.000 claims abstract description 46
- 238000006297 dehydration reaction Methods 0.000 claims abstract description 46
- 239000007853 buffer solution Substances 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 21
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 11
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims abstract description 10
- 210000002808 connective tissue Anatomy 0.000 claims abstract description 10
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000005708 Sodium hypochlorite Substances 0.000 claims abstract description 9
- 230000029087 digestion Effects 0.000 claims abstract description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 210000001015 abdomen Anatomy 0.000 claims abstract description 6
- 239000012153 distilled water Substances 0.000 claims abstract description 6
- 238000011010 flushing procedure Methods 0.000 claims abstract description 6
- 230000002040 relaxant effect Effects 0.000 claims abstract description 6
- 239000013535 sea water Substances 0.000 claims abstract description 6
- 229910001629 magnesium chloride Inorganic materials 0.000 claims abstract description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 4
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 34
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 17
- 210000000988 bone and bone Anatomy 0.000 claims description 15
- 241000208340 Araliaceae Species 0.000 claims description 14
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 claims description 14
- 235000003140 Panax quinquefolius Nutrition 0.000 claims description 14
- 235000008434 ginseng Nutrition 0.000 claims description 14
- 241000251511 Holothuroidea Species 0.000 claims description 13
- 206010002091 Anaesthesia Diseases 0.000 claims description 7
- 230000037005 anaesthesia Effects 0.000 claims description 7
- 238000005259 measurement Methods 0.000 claims description 7
- 239000002250 absorbent Substances 0.000 claims description 6
- 230000002745 absorbent Effects 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 238000011002 quantification Methods 0.000 claims description 6
- 238000010008 shearing Methods 0.000 claims description 5
- 206010020751 Hypersensitivity Diseases 0.000 claims description 4
- 208000026935 allergic disease Diseases 0.000 claims description 4
- 230000007815 allergy Effects 0.000 claims description 4
- 238000011161 development Methods 0.000 abstract description 4
- 230000000877 morphologic effect Effects 0.000 abstract description 3
- 208000005156 Dehydration Diseases 0.000 description 38
- 239000000872 buffer Substances 0.000 description 7
- 239000002585 base Substances 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 3
- 238000002224 dissection Methods 0.000 description 3
- 230000000172 allergic effect Effects 0.000 description 2
- 208000010668 atopic eczema Diseases 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 241000265433 Thyone Species 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000002419 base digestion Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000018678 bone mineralization Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000003800 pharynx Anatomy 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B16/00—Devices specially adapted for vivisection or autopsy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D1/00—Surgical instruments for veterinary use
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2873—Cutting or cleaving
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Surgery (AREA)
- Molecular Biology (AREA)
- Medical Informatics (AREA)
- Heart & Thoracic Surgery (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
一种刺参石灰环的完整提取方法,使用氯化镁或硫酸镁的海水溶液,松弛刺参,直至触手从刺参围口部完全伸展出来,固定在福尔马林溶液中,使用乙醇缓冲液交换福尔马林溶液,并于4℃冷藏静置脱水,使样品脱水率达到相对恒定50%~60%,用眼科剪、解剖刀、镊子工具对刺参进行解剖,首先切开腹部,排除体腔液,然后于体前1/5处切下口前端,再利用眼科剪和镊子从剥取石灰环;将所取石灰环浸入盛有次氯酸钠的培养皿中,闭合盖子在室温下消解残余结缔组织,消解完毕后,用蒸馏水吹洗石灰环,获得完整的刺参石灰环。采用该方法可以获得高度完整的刺参石灰环,从而满足研究刺参生活史不同阶段石灰环的形貌特征和生长发育规律的需要。
Description
技术领域
本发明涉及一种刺参石灰环的完整提取方法。
背景技术
石灰环是围绕海参咽部起支撑水管系统、神经环等重要作用的内骨骼,其形态大小常随种类而异,故一直以来是分类学上的重要依据。典型的石灰环通常由5对辐板和间辐板借结缔组织交替愈合而成。辐板前端有一凹陷或穿孔构成辐水管的通道,纵肌带和咽收缩肌也插于凹陷内。
石灰环各骨板并不完全对称,一般是背面的骨板较大,形状也略有不同。不同品种的海参其石灰环骨板不同,无足目海参的石灰环常多于10板。平足目海参的石灰环则大为退化,失去间辐板,只保留辐板。枝手目赛瓜参属(Thyone)的石灰环由许多小骨板类似马赛克一样愈合而成。还有一些复杂的石灰环,辐板常有成对的后延部。研究发现,仿刺参(Apostichopus japonicus)的石灰环结构较为简单,辐板和间辐板完整,呈方形或长方形,辐板大于间辐板,尺寸较小,其上附有大量的结缔组织,解剖过程中容易损坏石灰环;此外,仿刺参受到物理刺激时(如干露),触手将完全收缩进围口膜,致使石灰环深藏于体内,难以通过直接解剖或弱碱溶液消解而完整获得,阻碍了对石灰环整体形貌和各骨板精细结构的认知。因此,基于本发明的刺参石灰环完整提取方法,可以深入了解不同发育阶段的石灰环形貌特征,探究石灰环骨板的生长规律,为揭示海参内骨骼矿化机制提供方法学参考。
发明内容
本发明的目的在于提供一种刺参石灰环的完整提取方法,采用该方法可以获得高度完整的刺参石灰环,从而满足研究刺参生活史不同阶段石灰环的形貌特征和生长发育规律的需要。
为了实现上述目的,本发明通过如下技术方案实现:
一种刺参石灰环的完整提取方法,其具体步骤如下:
(1)刺参选取:适用于变态附着后的稚参、幼参及成参;
(2)体重测量:用吸水纸吸干步骤(1)所选取的刺参体表水分,以分析天平测量湿重;
(3)刺参麻醉:使用质量浓度为5%~10%氯化镁或质量浓度为5%~10%硫酸镁的海水溶液,松弛步骤(2)已知重量的刺参,直至触手从刺参围口部完全伸展出来;
(4)刺参固定:将步骤(3)麻醉的刺参固定在质量浓度为5%-10%福尔马林溶液固定液中1h~12h,每升固定液中加入5g~10g碳酸钙作为缓冲液保护刺参骨骼;
(5)刺参脱水:使用70%~75%的乙醇缓冲液交换步骤(4)固定液,所述70%~75%的乙醇缓冲液是每升质量浓度为70%~75%的乙醇溶液中加入5~10g碳酸钙配制而成,并于4℃冷藏静置脱水1-24h,43g以上的规格较大的刺参脱水24h后转入90%~95%的乙醇缓冲液继续4℃冷藏24h~96h,使样品脱水率达到相对恒定50%~60%,所述90%~95%的乙醇缓冲液是每升质量浓度为90%~95%的乙醇溶液中加入5g~10g碳酸钙配制而成;
刺参脱水率=(Mo–Mt)/Mo,其中Mo为步骤(2)所测刺参重量,Mt为步骤(5)刺参转入乙醇缓冲液后脱水时间t时测定的刺参重量;
(6)刺参解剖:用眼科剪、解剖刀、镊子等工具对步骤(5)的刺参进行解剖,首先切开腹部,排除体腔液,然后于体前1/5处切下口前端,再利用眼科剪和镊子从剥取石灰环;
(7)石灰环定量:将步骤(6)所取石灰环浸入盛有弱碱溶液的培养皿中,闭合盖子在室温下消解残余结缔组织15min~30min,消解完毕后用吸管将培养皿内弱碱溶液吸出,并用蒸馏水吹洗石灰环3次~5次,获得完整的刺参石灰环。
进一步的,所述步骤(6)中为得到完整石灰环,剖取的过程中确保从刺参前端反口面开始,不可出现撕毁、剪短等损伤石灰环的操作。
进一步的,完整的刺参石灰环以70%乙醇缓冲液保存;所述70%的乙醇缓冲液是每升质量浓度为70%的乙醇溶液中加入5g~10g碳酸钙配制而成。
进一步的,对于湿重0.4~0.8g稚参,脱水时间控制在1h~5h。
进一步的,对于4.2~5.6g的幼参,脱水时长为13h。
进一步的,对于湿重38.4~42.7g的成参,脱水时间控制在24h。
进一步的,所述弱碱溶液为质量浓度为5%~10%次氯酸钠(NaClO)。
本发明的有益效果是:
刺参石灰环的完整提取方法,包括麻醉与固定,低温脱水,石灰环解剖、消解与定量等步骤;通过麻醉使刺参的触手从刺参围口部完全伸展出来,用固定液维持刺参触手的伸展状态;然后通过脱水使刺参体壁纤维化或革质化,从而使刺参可以实现完整剥离石灰环;最后通过弱碱消解使包围石灰环内外及周边的结缔组织消解完全,石灰环的整体形貌清晰可见,能够明显辨别各块骨板的形态差异,达到石灰环几何参数与质量参数的定量标准。完整提取的石灰环既可以观察到整体形态及骨板精细结构,还可以测量石灰环的直径、周长、质量等重要参数,对于研究刺参骨骼生长发育与演化规律、刺参骨骼荧光标记与增殖放流等等方面具有基础应用价值。
附图说明
图1是本发明(对应实施例1)脱水处理后的刺参形貌照片;
图2是本发明(对应实施例1)刺参石灰环整体形貌及其随消解时间变化特征图;
图3是本发明(对应实施例2)刺参石灰环的周长测量照片;
图4是本发明刺参石灰环直接解剖效果图。
具体实施方式
下述非限定性实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何方式限制本发明。
以下结合实施例对本发明作具体的介绍。
实施例1
(1)刺参选取:适用于变态附着后的稚参;
(2)体重测量:用吸水纸吸干步骤(1)所选取的刺参体表水分,以分析天平测量湿重0.43g;
(3)刺参麻醉:使用质量浓度为5%氯化镁的海水溶液,松弛步骤(2)已知重量的刺参,直至触手从刺参围口部完全伸展出来;
(4)刺参固定:将步骤(3)麻醉的刺参固定在质量浓度为5%福尔马林溶液固定液中1h,每升固定液中加入5g碳酸钙作为缓冲液保护刺参骨骼;
(5)配制乙醇缓冲液
70%的乙醇缓冲液:每升质量浓度为70%的乙醇溶液中加入5g碳酸钙配制而成;
(6)刺参脱水:使用70%的乙醇缓冲液交换步骤(4)固定液,并于4℃冷藏静置脱水1h后,刺参的湿重为0.21g,完成脱水;
(7)刺参解剖:用眼科剪、解剖刀、镊子等工具对步骤(6)的刺参进行解剖,首先切开腹部,排除体腔液,然后于体前1/5处切下口前端,再利用眼科剪和镊子从剥取石灰环;剖取的过程中确保从刺参前端反口面开始,不可出现撕毁、剪短等损伤石灰环的操作;
(8)石灰环定量:将步骤(7)所取石灰环浸入盛有质量浓度为5%次氯酸钠溶液的培养皿中,闭合盖子在室温下消解残余结缔组织15min,消解完毕后用吸管将培养皿内次氯酸钠溶液吸出,并用蒸馏水吹洗石灰环3次,获得完整的刺参石灰环,以70%乙醇缓冲液保存。
实施例2
(1)刺参选取:适用于变态附着后的成参;
(2)体重测量:用吸水纸吸干步骤(1)所选取的刺参体表水分,以分析天平测量湿重45.2g;
(3)刺参麻醉:使用质量浓度为10%氯化镁的海水溶液,松弛步骤(2)已知重量的刺参,直至触手从刺参围口部完全伸展出来;
(4)刺参固定:将步骤(3)麻醉的刺参固定在质量浓度为10%福尔马林溶液固定液中12h,每升固定液中加入10g碳酸钙作为缓冲液保护刺参骨骼;
(5)配制乙醇缓冲液
70%的乙醇缓冲液:每升质量浓度为70%的乙醇溶液中加入10g碳酸钙配制而成;
75%的乙醇缓冲液:每升质量浓度为75%的乙醇溶液中加入10g碳酸钙配制而成;
95%的乙醇缓冲液:每升质量浓度为95%的乙醇溶液中加入10g碳酸钙配制而成;
(6)刺参脱水:使用75%的乙醇缓冲液交换步骤(4)固定液,并于4℃冷藏静置脱水24h,用分析天平测量湿重为24.3g,转入95%的乙醇缓冲液继续4℃冷藏48h,用分析天平测量湿重为22.6g,完成脱水;
(7)刺参解剖:用眼科剪、解剖刀、镊子等工具对步骤(6)的刺参进行解剖,首先切开腹部,排除体腔液,然后于体前1/5处切下口前端,再利用眼科剪和镊子从剥取石灰环;剖取的过程中确保从刺参前端反口面开始,不可出现撕毁、剪短等损伤石灰环的操作;
(8)石灰环定量:将步骤(7)所取石灰环浸入盛有质量浓度为10%次氯酸钠溶液的培养皿中,闭合盖子在室温下消解残余结缔组织30min,消解完毕后用吸管将培养皿内次氯酸钠溶液吸出,并用蒸馏水吹洗石灰环5次,获得完整的刺参石灰环,以70%乙醇缓冲液保存。
实施例3
(1)刺参选取:适用于变态附着后的幼参;
(2)体重测量:用吸水纸吸干步骤(1)所选取的刺参体表水分,以分析天平测量湿重4.3g;
(3)刺参麻醉:使用质量浓度为质量浓度为8%硫酸镁的海水溶液,松弛步骤(2)已知重量的刺参,直至触手从刺参围口部完全伸展出来;
(4)刺参固定:将步骤(3)麻醉的刺参固定在质量浓度为8%福尔马林溶液固定液中5h,每升固定液中加入8g碳酸钙作为缓冲液保护刺参骨骼;
(5)配制乙醇缓冲液
72%的乙醇缓冲液:每升质量浓度为72%的乙醇溶液中加入8g碳酸钙配制而成;
70%的乙醇缓冲液:每升质量浓度为70%的乙醇溶液中加入8g碳酸钙配制而成;
(6)刺参脱水:使用72%的乙醇缓冲液交换步骤(4)固定液,并于4℃冷藏静置脱水9h,以分析天平测量湿重2.1g,完成脱水;
(7)刺参解剖:用眼科剪、解剖刀、镊子等工具对步骤(6)的刺参进行解剖,首先切开腹部,排除体腔液,然后于体前1/5处切下口前端,再利用眼科剪和镊子从剥取石灰环;剖取的过程中确保从刺参前端反口面开始,不可出现撕毁、剪短等损伤石灰环的操作;
(8)石灰环定量:将步骤(7)所取石灰环浸入盛有质量浓度为8%次氯酸钠溶液的培养皿中,闭合盖子在室温下消解残余结缔组织20min,消解完毕后用吸管将培养皿内次氯酸钠溶液吸出,并用蒸馏水吹洗石灰环4次,获得完整的刺参石灰环,以70%乙醇缓冲液保存。
对照例
(1)刺参选取:适用于变态附着后的成参;
(2)体重测量:用吸水纸吸干步骤(1)所选取的刺参体表水分,以分析天平测量湿重44.5g,如图4所示;用眼科剪、解剖刀、镊子等工具对刺参直接剖取,刺参石灰环表面结缔组织较多,辐板与间辐板的形貌结构不清晰,其重量、周长等特征参数很难准确测量。
一、不同温度下刺参脱水率的测定:
采用实施例1的方法,在4℃和20℃不同温度下分两组对刺参脱水,每组刺参6个,检测初始平均质量,6h,检测终末平均质量,结果如表1所示。
表1温度为4℃和20℃时的刺参脱水率
温度(℃) | 初始平均质量(g) | 终末平均质量(g) | 脱水率(%) |
4 | 1.43±0.11 | 0.61±0.03 | 57.34±1.12 |
20 | 1.10±0.09 | 0.64±0.06 | 41.82±3.55 |
由表1可以看出,在温度为4℃的条件下,更有利于刺参的脱水,因此,本发明选用的脱水温度为4℃。
二、湿重0.4~0.8g的刺参4℃脱水率的测定
采用实施例1的方法,在4℃下分对刺参脱水,分别检测脱水1h、2h、3h、4h、5h、6h、7h刺参的湿重,结果如表2。
表2湿重0.4~0.8g的刺参4℃脱水率
由表2可以看出,对于湿重0.4~0.8g稚参,脱水时间控制在1~5h,刺参脱水率在即可满足50%~60%。
二、湿重4.2~5.6g的刺参4℃脱水率的测定
采用实施例3的方法,在4℃下分对刺参脱水,分别检测脱水7h、9h、11h刺参的湿重,结果如表3。
表3湿重4.2~5.6g的刺参4℃脱水率
由表3可以看出,对于4.2~5.6g的幼参,脱水时长为13h,大部分幼参的脱水率仍低于50%,此时,可以适当延长幼参脱水率至24h以内,以满足脱水率50%~60%的要求。
三、湿重38.4g~50.9g的刺参4℃脱水率的测定
采用实施例2的方法,在4℃下对刺参脱水,分别检测脱水7h、9h、11h刺参的湿重,结果如表4。
表4湿重38.4~50.9g的刺参4℃脱水率
由表4可以看出,对于湿重38.4~42.7g的成参,脱水时间控制在24h,刺参脱水率在即可满足50%~60%;而对于湿重45.2~50.9g的成参脱水时间控制在24h,刺参脱水率<50%,且湿重42.7g的成参脱水率50.8%,接近50%,因此,对于质量≥43g的成参,脱水24小时后,可以转入95%的乙醇缓冲液继续4℃冷藏脱水处理。
以上仅为本发明的具体实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (7)
1.一种刺参石灰环的完整提取方法,其特征是:
具体步骤如下:
(1)刺参选取:适用于变态附着后的稚参、幼参及成参;
(2)体重测量:用吸水纸吸干步骤(1)所选取的刺参体表水分,以分析天平测量湿重;
(3)刺参麻醉:使用质量浓度为5%~10%氯化镁或质量浓度为5%~10%硫酸镁的海水溶液,松弛步骤(2)已知重量的刺参,直至触手从刺参围口部完全伸展出来;
(4)刺参固定:将步骤(3)麻醉的刺参固定在质量浓度为5%-10%福尔马林溶液固定液中1h~12h,每升固定液中加入5g~10g碳酸钙作为缓冲液保护刺参骨骼;
(5)刺参脱水:使用70%~75%的乙醇缓冲液交换步骤(4)固定液,所述70%~75%的乙醇缓冲液是每升质量浓度为70%~75%的乙醇溶液中加入5~10g碳酸钙配制而成,并于4℃冷藏静置脱水1-24h,43g以上的规格较大的刺参脱水24h后转入90%~95%的乙醇缓冲液继续4℃冷藏24h~96h,使样品脱水率达到相对恒定50%~60%,所述90%~95%的乙醇缓冲液是每升质量浓度为90%~95%的乙醇溶液中加入5g~10g碳酸钙配制而成;
刺参脱水率=(Mo–Mt)/Mo,其中Mo为步骤(2)所测刺参重量,Mt为步骤(5)刺参转入乙醇缓冲液后脱水时间t时测定的刺参重量;
(6)刺参解剖:用眼科剪、解剖刀、镊子工具对步骤(5)的刺参进行解剖,首先切开腹部,排除体腔液,然后于体前1/5处切下口前端,再利用眼科剪和镊子从剥取石灰环;
(7)石灰环定量:将步骤(6)所取石灰环浸入盛有弱碱溶液的培养皿中,闭合盖子在室温下消解残余结缔组织15min~30min,消解完毕后用吸管将培养皿内弱碱溶液吸出,并用蒸馏水吹洗石灰环3次~5次,获得完整的刺参石灰环。
2.根据权利要求1所述的刺参石灰环的完整提取方法,其特征是:所述步骤(6)中为得到完整石灰环,剖取的过程中确保从刺参前端反口面开始,不可出现撕毁、剪短等损伤石灰环的操作。
3.根据权利要求1所述的刺参石灰环的完整提取方法,其特征是:完整的刺参石灰环以70%乙醇缓冲液保存;所述70%的乙醇缓冲液是每升质量浓度为70%的乙醇溶液中加入5g~10g碳酸钙配制而成。
4.根据权利要求1所述的刺参石灰环的完整提取方法,其特征是:对于湿重0.4~0.8g稚参,脱水时间控制在1h~5h。
5.根据权利要求1所述的刺参石灰环的完整提取方法,其特征是:对于4.2~5.6g的幼参,脱水时长为13h。
6.根据权利要求1所述的刺参石灰环的完整提取方法,其特征是:对于湿重38.4~42.7g的成参,脱水时间控制在24h。
7.根据权利要求1所述的刺参石灰环的完整提取方法,其特征是:所述弱碱溶液为质量浓度为5%~10%次氯酸钠。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010839724.0A CN112146949B (zh) | 2020-08-19 | 2020-08-19 | 一种刺参石灰环的完整提取方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010839724.0A CN112146949B (zh) | 2020-08-19 | 2020-08-19 | 一种刺参石灰环的完整提取方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112146949A CN112146949A (zh) | 2020-12-29 |
CN112146949B true CN112146949B (zh) | 2024-04-02 |
Family
ID=73888503
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010839724.0A Active CN112146949B (zh) | 2020-08-19 | 2020-08-19 | 一种刺参石灰环的完整提取方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112146949B (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103380768A (zh) * | 2013-07-05 | 2013-11-06 | 河南科技大学 | 一种成年公羊的神经系统标本制作方法 |
CN103868768A (zh) * | 2014-02-14 | 2014-06-18 | 河南省农业科学院植物保护研究所 | 一种昆虫触角及附肢的扫描电镜样品处理方法 |
CN104764624A (zh) * | 2014-01-07 | 2015-07-08 | 中国人民解放军军事医学科学院毒物药物研究所 | 动物肺组织的取材和固定方法 |
CN107648269A (zh) * | 2017-11-02 | 2018-02-02 | 山东好当家海洋发展股份有限公司 | 从刺参体腔液中提取抗菌组合物的方法、其组合物及用途 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5792886B2 (ja) * | 2012-03-09 | 2015-10-14 | 株式会社日立ハイテクノロジーズ | 試料観察方法および試料前処理方法 |
-
2020
- 2020-08-19 CN CN202010839724.0A patent/CN112146949B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103380768A (zh) * | 2013-07-05 | 2013-11-06 | 河南科技大学 | 一种成年公羊的神经系统标本制作方法 |
CN104764624A (zh) * | 2014-01-07 | 2015-07-08 | 中国人民解放军军事医学科学院毒物药物研究所 | 动物肺组织的取材和固定方法 |
CN103868768A (zh) * | 2014-02-14 | 2014-06-18 | 河南省农业科学院植物保护研究所 | 一种昆虫触角及附肢的扫描电镜样品处理方法 |
CN107648269A (zh) * | 2017-11-02 | 2018-02-02 | 山东好当家海洋发展股份有限公司 | 从刺参体腔液中提取抗菌组合物的方法、其组合物及用途 |
Also Published As
Publication number | Publication date |
---|---|
CN112146949A (zh) | 2020-12-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2004006808A3 (en) | Soft tissue implants and methods for making same | |
CN112146949B (zh) | 一种刺参石灰环的完整提取方法 | |
Reeve | Repair of large experimental perforations of the tympanic membrane | |
Kunoh et al. | Cytological studies of early stages of powdery mildew in barley and wheat. IV. Direct ingress from superficial primary germ tubes and appressoria of Erysiphe graminis hordei on barley leavest | |
Erbele et al. | Over-under cartilage tympanoplasty: technique, results and a call for improved reporting | |
Cho et al. | Correction of cleft lip nasal deformity in Orientals using a refined reverse-U incision and VY plasty | |
Rabie et al. | Use of tragal cartilage grafts in rhinoplasty: an anatomic study and review of the literature | |
MD2784G2 (ro) | Metodă de tratament al herniilor ventrale gigantice | |
Bodade et al. | Endoscopic thyroidectomy-axilla breast approach: the experience of 26 cases | |
Hellström et al. | The original description of Shrapnell's membrane reviewed in the light of recent experimental studies | |
Van Loon et al. | X Neurofibroma of the Larynx | |
CN107059466A (zh) | 一种易冲散的湿厕纸 | |
Hann | A case of osteitis deformans terminating with cerebral symptoms | |
Enroth | Contributions to the bryoflora of China. 2. Caduciella guangdongensis sp. nov.(Leptodontaceae, Musci) | |
Dirr | Rooting response of Photinia× fraseri Dress ‘Birmingham’to 25 carrier and carrier plus IBA formulations | |
CN107916594A (zh) | 一种抗菌生活用纸 | |
Pick | A rapid method of preparing permanent sections for microscopical diagnosis | |
Taylor et al. | Note brève | |
Roderick | The infection of white clover (Trifolium repens) by conidia of Cymadothea trifolii | |
Petrunkevitch | External reproductive organs of the common grass spider, Agelena naevia Walckenaer | |
Schumann | TRACHELOPLASTY A NEW OPERATION FOR THE RELIEF OF STERILITY DUE TO STENOSIS OF THE CERVIX UTERI. | |
RU94040113A (ru) | Способ лечения транссексуализма | |
Kümmell | Resorbable tampon | |
TR202101978A2 (tr) | Güve kokonunun ori̇ji̇nal yapisi korunarak bi̇youyumluluğu yüksek cerrahi̇ ağ üreti̇mi̇ ve kullanim alanlari | |
DE50210977D1 (de) | Verfahren zur herstellung eines biologischen gewebes unter verwendung einer kollagenunterlage und zugehöriges gewebekonstrukt |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |