病原线虫在病组织中可通过挑针直接挑取、漏斗分离或将病组织捣碎分离等方法取得活线虫虫体，为了识别常见线虫，对其形态和结构做一般性观察，可以用活体线虫制作临时玻片，为了进一步仔细观察、认识其分类学特征，需将活体线虫杀死和固定，有时还需染色，制成永久性玻片标本，如需观察线虫与寄主组织之间的相互关系，还需要制作寄主和线虫组织的石蜡切片。
阿拉丁线虫染色液 (多色蓝法) 主要由次甲蓝、乙醇、甘油等组成，经过加热杀死并固定线虫，用多色蓝染色后观察，其染色原理是根据线虫在固定时体内各器官的氢离子浓度和生理状态的不同，本方法能改较好的研究线虫的不同器官结构。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
产品组分及储存条件：
N1510447
| Component
| 50mL
| Storage
|
N1510447A
| 线虫脱水剂 I
| 25mL
| RT
|
N1510447B
| 线虫脱水剂 II
| 25mL
| RT
|
N1510447C
| 多色蓝染色液
| 5mL
| RT |
自备材料：
1、蒸馏水、4%福尔马林固定液
2、载玻片、显微镜、酒精灯、水浴锅、恒温箱
操作步骤(仅供参考):
1、杀死：一般采用加热固定的方法，先在载玻片上滴加一滴蒸馏水，挑取一部分线虫在载玻片上，在酒精灯上加热5~6s，同时不断转动玻片，并随时在显微镜下观察；当线虫扭曲忽伸直时立即停止加热，否则会破坏线虫内部器官；亦可把线虫放入1滴水中，再加入0.5%热乙酸杀死线虫；也可以用0.1%碘液杀死线虫(线虫悬浮液与碘液等量混合，数秒钟即可杀死线虫)。
2、固定：采用TAF固定液或4%福尔马林固定液。
3、(备选)脱水：将固定好的线虫置于盛有0.5ml线虫脱水剂I的小玻皿中，将玻皿放在盛有过量95%乙醇的密闭容器中，放入35~40°℃恒温箱内，孵育12h以上，玻皿内加入少量线虫脱水剂，然后将玻皿放在半密闭培养皿中，置于40℃恒温箱，待脱水剂中的乙醇完全蒸发后 (一般需要2-3h)，将处理好的线虫置于无水甘油内，可长期保存。
4、将经固定的线虫,直接移入3mI蒸馏水和3~5滴多色蓝染色液的离心管中，在55~60°℃水浴加热染色3~5min。
5、挑取数条线虫置于放大镜或显微镜下观察，如果已经均匀染成暗紫色，其内部器官几乎看不清楚，则染色程度已经足够；如果还有空隙或染色不均，则要继续延长染色时间。
6、镜检：置于甘油中，加盖玻片观察。
染色结果：
生殖器官、卵原细胞、精原细胞
| 蓝紫色
|
| 细胞核 | 浅红色
|
染色体
| 蓝色-蓝紫色
|
肠
| 绿色(不定)
|
其他器官或细胞
| 深紫色或深蓝色
|
注意事项：
1、玻片应洁净，无油污。
2、线虫的染色标本置于无水甘油中可保存2~5个月，之后会逐渐褪色。
3、一般情况下线虫被染成蓝紫色，但并不是所有特征都能染出来。
4、固定时不宜用高热火焰固定。
5、为了您的安全和健康，请穿实验服并戴一次性手套操作。
6、试剂开封后请尽快使用，以防影响后续实验效果。
Live nematode individuals can be obtained from infected tissues through methods such as direct picking with a dissecting needle, Baermann funnel separation, or homogenization of infected tissues. For the general observation of the morphology and structure of common nematodes for identification, temporary slides can be prepared using live nematodes. For further detailed observation and identification of their taxonomic characteristics, it is necessary to kill and fix the live nematodes; staining is sometimes required to prepare permanent slide specimens. If it is necessary to observe the interaction between nematodes and host tissues, paraffin sections of host and nematode tissues also need to be prepared.
Aladdin Nematode Staining Solution (Polychrome Blue Method) is mainly composed of methylene blue, ethanol, glycerol, etc. Nematodes are killed by heating and fixed, followed by staining with polychrome blue before observation. The staining principle is based on the differences in hydrogen ion concentration and physiological state of various internal organs of nematodes during fixation. This method can be well applied to the study of the structure of different organs of nematodes. This reagent is only used in the field of scientific research and is not suitable for clinical diagnosis or other purposes.
Product Components and Storage Conditions:
N1510447
| Component
| 50mL
| Storage
|
N1510447A
| Nematode Dehydrant I
| 25mL
| RT
|
N1510447B
| Nematode Dehydrant II
| 25mL
| RT
|
N1510447C
| Polychrome Blue Staining Solution
| 5mL
| RT |
Self-prepared Materials:
1. Distilled water, 4% formalin fixative
2. Glass slides, microscope, alcohol lamp, water bath, incubator
Operating Procedures (For Reference Only):
1. Killing
The heat-fixation method is generally adopted. First, place a drop of distilled water on a glass slide, pick a portion of nematodes onto the slide, and heat the slide over an alcohol lamp for 5–6 seconds while rotating it continuously. Observe the nematodes under a microscope at all times; stop heating immediately when the nematodes twist and then straighten suddenly, otherwise their internal organs will be damaged. Alternatively, place the nematodes in a drop of water and add a drop of hot 0.5% acetic acid to kill them. Another option is to use 0.1% iodine solution (mix equal volumes of nematode suspension and iodine solution, and the nematodes will be killed within a few seconds).
2. Fixation
Use TAF fixative or 4% formalin fixative.
3. Dehydration (Optional)
Place the fixed nematodes in a small glass dish containing 0.5 mL of nematode dehydrant I. Put the glass dish in a sealed container filled with excess 95% ethanol, and incubate in a 35–40℃ incubator for more than 12 hours. Add a small amount of nematode dehydrant to the glass dish, then place the dish in a semi-sealed petri dish and put it in a 40℃ incubator. After the ethanol in the dehydrant evaporates completely (usually taking 2–3 hours), transfer the treated nematodes into anhydrous glycerol for long-term preservation.
4. Staining
Directly transfer the fixed nematodes into a centrifuge tube containing 3 mL of distilled water and 3–5 drops of polychrome blue staining solution. Heat the tube in a 55–60℃ water bath for 3–5 minutes to stain the nematodes.
5. Staining Evaluation
Pick several nematodes and observe them under a magnifying glass or microscope. The staining is sufficient if the nematodes are uniformly stained dark purple and their internal organs are almost invisible. If there are still unstained gaps or uneven staining, extend the staining time appropriately.
6. Microscopic Examination
Place the stained nematodes in glycerol, cover with a coverslip, and observe under a microscope.
Staining Results:
Reproductive organs, oogonia, spermatogonia
| Blue-purple
|
Cell nucleus
| Light red
|
Chromosome
| Blue–blue-purple
|
Intestine
| Green (variable)
|
Other organs or cells
| Dark purple or dark blue
|
Precautions:
1. Glass slides should be clean and free of oil stains.
2. Stained nematode specimens stored in anhydrous glycerol can be preserved for 2–5 months, after which they will gradually fade.
3. Generally, nematodes will be stained blue-purple, but not all morphological characteristics can be visualized by this staining method.
4. Avoid using high-temperature flames for fixation.
5. For your safety and health, please wear a lab coat and disposable gloves during operation.
6. Please use the reagent as soon as possible after opening to prevent affecting the results of subsequent experiments.
ZJ26F0433838
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