48
Methods for Setting Up Primary Cultures Specific to Animal Groups
Sterilization
of insects
Procedure
1. Surface sterilize the insects by immersing them in 70% ethanol for
approximately 10 min. Pupae oflepidopteran insects have no hairs on
their surface, and are easy to sterilize. If using adults, first remove their
wings. If the adults or larvae are covered with dense scales or hair, it is
better to wash out as many scales as possible or to cut the hairs with
scissors before sterilization. It is also appropriate to singe animals by
instant flaming before sterilization. If the possibility exists that larvae
or adults may regurgitate or excrete material after sterilization, apply
a ligation between the head and thorax with a fine cotton thread, and
also ligate the last segment of the abdomen. During sterilization, agitate the ethanol several times.
2. After sterilization, wash the insects in sterilized distilled water.
Dissection 1. Fix sterilized insects on a dissecting tray. The tray can be sterilized by
filling it with 70% ethanol for 5 min and then washing the tray with
sterile distilled water. Because immature ovaries are situated dorsally,
it is better to fix insects with the ventral side up.
2. If the insect is smaller than 5 em in length, fill the tray with a physiological solution, and fix the insect under the solution. Dissection
should be performed in the solution under a dissecting microscope.
This prevents desiccation of exposed tissues, and facilitates the recognition of tissues.
3. Make an incision along the center line.
4. Pull the cut edge of the integument outward, and fix with pins to open
the hemocoel (Fig. 1).
5. Using forceps and a needle, pull the ovary out. If the ovary is not fully
developed, the ovary may be taken out as a whole by grasping the
common oviduct with forceps and pulling it out.
Culture l. Place the excised ovaries in a physiological solution on a Maximov
set-up
slide or in small petri dish.
2. Remove as much attached tissue as possible. When the ovary matures,
many trachea are distributed over the surface of the ovarioles, and are
difficult to remove all of them.
3. Wash the attached tissue-free ovarioles in a physiological solution.
4. Transfer the ovarioles into the culture medium.
5. Cut the ovarioles into small fragments (shorter than 1 mm in length).
6. Transfer the fragments, with the culture medium, into culture vessels.
7. Culture at 25°C.
Comments
Isotonic solutions, such as Ringer's, Tyrode's, or Lock's solution, as well
as BSS or PBS, may be used as the physiological solution (see Appendix
Methods for Setting Up Primary Cultures Specific to Animal Groups
Sterilization
of insects
Procedure
1. Surface sterilize the insects by immersing them in 70% ethanol for
approximately 10 min. Pupae oflepidopteran insects have no hairs on
their surface, and are easy to sterilize. If using adults, first remove their
wings. If the adults or larvae are covered with dense scales or hair, it is
better to wash out as many scales as possible or to cut the hairs with
scissors before sterilization. It is also appropriate to singe animals by
instant flaming before sterilization. If the possibility exists that larvae
or adults may regurgitate or excrete material after sterilization, apply
a ligation between the head and thorax with a fine cotton thread, and
also ligate the last segment of the abdomen. During sterilization, agitate the ethanol several times.
2. After sterilization, wash the insects in sterilized distilled water.
Dissection 1. Fix sterilized insects on a dissecting tray. The tray can be sterilized by
filling it with 70% ethanol for 5 min and then washing the tray with
sterile distilled water. Because immature ovaries are situated dorsally,
it is better to fix insects with the ventral side up.
2. If the insect is smaller than 5 em in length, fill the tray with a physiological solution, and fix the insect under the solution. Dissection
should be performed in the solution under a dissecting microscope.
This prevents desiccation of exposed tissues, and facilitates the recognition of tissues.
3. Make an incision along the center line.
4. Pull the cut edge of the integument outward, and fix with pins to open
the hemocoel (Fig. 1).
5. Using forceps and a needle, pull the ovary out. If the ovary is not fully
developed, the ovary may be taken out as a whole by grasping the
common oviduct with forceps and pulling it out.
Culture l. Place the excised ovaries in a physiological solution on a Maximov
set-up
slide or in small petri dish.
2. Remove as much attached tissue as possible. When the ovary matures,
many trachea are distributed over the surface of the ovarioles, and are
difficult to remove all of them.
3. Wash the attached tissue-free ovarioles in a physiological solution.
4. Transfer the ovarioles into the culture medium.
5. Cut the ovarioles into small fragments (shorter than 1 mm in length).
6. Transfer the fragments, with the culture medium, into culture vessels.
7. Culture at 25°C.
Comments
Isotonic solutions, such as Ringer's, Tyrode's, or Lock's solution, as well
as BSS or PBS, may be used as the physiological solution (see Appendix