Feb 20, 2022
Version 3
Introductory Hydra Activities V.3
- 1University of San Diego
- USD Hydra
Protocol Citation: Callen Hyland 2022. Introductory Hydra Activities. protocols.io https://dx.doi.org/10.17504/protocols.io.b5byq2pwVersion created by Callen Hyland
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 18, 2022
Last Modified: March 20, 2024
Protocol Integer ID: 58456
Abstract
This is a series of introductory lab activities for BIOL309-03: Research Methods. The purpose is to master vocabulary related to Hydra anatomy, become familiar with compound microscopes and dissecting microscopes, and practice microdissections and micropipette use. The second section sets up a head and tentacle regeneration experiment that will be documented a week later to provide quantitative data for a statistics activity.
Guidelines
Microscopes are delicate and expensive instruments. Please handle them with care at all times and follow these guidelines:
- Hold the microscope by its base and carry it with two hands.
- Avoid touching any of the optical components.
- Only clean lenses with a lens paper and lens clearer or DI water.
- When you are on high power (20X, 40X, or 100X) only use the fine focus adjustment. On 4X or 10X, you can use the course focus adjustment.
- Don't adjust any components if you don't know what they do or try to fix anything you don't know how to fix. Alert your instructor if there are any issues with the microscopes.
- When you are done using the microscope turn off the light, move the objective to 4X, and cover it with the dust cover.
Everyone's eyes are a different distance apart, so it's critical to adjust the distance between the oculars every time you use a microscope that someone else may have used before. If the oculars are too far apart or close together you will see double images or nothing at all.
Materials
Equipment
Stereo dissecting microscope
NAME
lab equipment
TYPE
Meiji Techno
BRAND
unknown
SKU
LINK
Any stereo dissecting microscope will do. This model might be discontinued.
SPECIFICATIONS
Equipment
Compound upright binocular microscope
NAME
Lab equipment
TYPE
Olympus
BRAND
CX31
SKU
LINK
Any compound microscope will do.
SPECIFICATIONS
Equipment
Multi-well plates, not treated
NAME
Lab consumable
TYPE
Corning
BRAND
CLS3738
SKU
LINK
Equipment
60 x 15 mm plastic petri dish
NAME
Lab consumable
TYPE
CellTreat
BRAND
T9FB2782491
SKU
LINK
Can use any plastic petri dish
SPECIFICATIONS
Equipment
Disposable scalpel
NAME
Lab consumable
TYPE
Feather
BRAND
08-927-5A
SKU
LINK
Use blade shape #10. They can be cleaned and used multiple times.
SPECIFICATIONS
Equipment
100-1000 µl Micropipette
NAME
lab equipment
TYPE
MiniOne
BRAND
M2011
SKU
LINK
Optional. Only needed for dispensing rifampicin solution.
SPECIFICATIONS
Equipment
TipOne Pipette tips in racks, blue graduated 1000uL
NAME
Pipette tips in racks, 1000uL
TYPE
TipOne
BRAND
1111-2831
SKU
LINK
Equipment
Glass pasteur pipettes, 5 3/4 inch
NAME
glass consumables
TYPE
Fisher Scientific
BRAND
1367820B
SKU
LINK
Any brand of glass Pasteur pipette will do, just make sure you have bulbs that fit. Plastic Pasteur pipettes are not recommended because Hydra tend to stick to plastic.
SPECIFICATIONS
Equipment
Rubber bulbs for Pasteur pipette
NAME
Lab equipment
TYPE
Heathrow Scientific
BRAND
HS20622B
SKU
LINK
Look for 2 mL latex rubber bulbs
SPECIFICATIONS
Equipment
Hydra with Bud
NAME
Preserved organism
TYPE
Ward's Science
BRAND
470176-910
SKU
LINK
Equipment
Hydra, Nematocysts
NAME
Preserved organism
TYPE
Ward's Science
BRAND
470181-506
SKU
LINK
Equipment
Hydra, General Structure
NAME
Preserved organism
TYPE
Ward's Science
BRAND
470181-502
SKU
LINK
Can be cross-section or longitudinal section
SPECIFICATIONS
Equipment
Preserved cnidarian specimens
NAME
preserved organisms
TYPE
Any
BRAND
NA
SKU
LINK
Set of preserved cnidarians (sea anemones, jellyfish, corals, Velella, etc.), can be from any vendor.
SPECIFICATIONS
Safety warnings
Scalpels, microscope slides, and glass Pasteur pipettes should be handled with care. Dispose of used Pasteur pipettes in the broken glass bin and dispose of all blades in the red sharps container.
Do not put any living organisms down the sink. Dispose of all media that comes in contact with Hydra in the "Hydra Waste" container.
Before start
Please read through the entire protocol before beginning.
Familiarize yourself with the components of the microscope and its basic operation and review the basic anatomy of Hydra.
Observation activities
Observation activities
30m
30m
In this activity you will become familiar with the anatomy of Hydra including areas of the body, cell layers, and nematocysts, while practicing the new vocabulary we learned in lecture.
Learning objectives:
- Use a compound microscope to examine prepared slides
- Master vocabulary related to Hydra anatomy
- Correctly identify body parts and cell layers in fixed Hydra specimens
- Sketch biological structures under a microscope
- Identify similarities and differences in animal anatomy
Materials:
- Compound microscope
- Microscope slides with Hydra whole mount, cross section, and nematocysts
- Selection of preserved Cnidarian specimens
- Lab notebook, pen, and pencil (optional)
Note
In this activity, you will be sketching biological structures that you observe under the microscope. Your sketches don't have to be "good", they just have to show the general shapes and relative positions of the structures you are asked to examine. You probably know not to write in pencil in your lab notebook, but you can make an exception here. It may be convenient to sketch the structures first with pencil then copy over the final lines in pen.
Figure 1. Parts of the compound microscope, just what you need to know for this activity.
Basic Hydra anatomy
Examine the whole mount Hydra slides with the 4X objective. Adjust the distance between the oculars and focus on the specimen if necessary. Sketch the whole Hydra in your lab notebook and label the following body parts:
- Tentacles
- Hypostome
- Head
- Body column
- Peduncle
- Basal disk
- Bud
- Budding zone
- Label the hypostome and tentacles on the bud
Body wall structure
Examine the Hydra cross section (transverse section) with the 40X objective. The cross section should already be centered in the field, so don't move the stage. Adjust the distance between the oculars and focus on the specimen if necessary (use only the fine focus adjustment). In your lab notebook, sketch just a section of the body wall and label:
- Endodermal cells
- Ectodermal cells
- Mesoglea
Hydra nematocysts
Examine the smear of Hydra nematocysts with the 40X objective. Adjust the distance between the oculars and focus on the specimen if necessary. Feel free to move the stage around to view different parts of the smear. Try to find at least two of the four different types of nematocysts (Figure 1) and sketch them in your lab notebook.
Figure 2. Four types of nematocysts in Hydra.
Desmoneme: coiled filaments wrap around prey.
Stenotele: pierces prey cuticle and injects venom.
Holotrichous isorhiza: sticky filament with barbs used for defense.
Atrichous isorhiza: sticky filament without barbs used for locomotion..
Comparing Hydra to other Cnidarians
Examine the preserved specimens. In your lab notebook, make a list of the features that all of these animals have in common. What do they have in common with Hydra? What features make them appear different from each other and from Hydra?
Start your regeneration experiment
Start your regeneration experiment
30m
30m
Anyone who examines a large number of Hydra in a dish can clearly see that they do not all have the same number of tentacles. You may wonder, does each animal have a fixed number of tentacles? If we were to hypothesize that each animal has a fixed number of tentacles, it might follow that we cut a Hydra in half and allowed the foot-end to regenerate a head, this head would have the same number of tentacles as the head that was removed. We will keep the head-end of the Hydra to test whether the number of tentacles on an intact Hydra head spontaneously changes in the course of a week.
Learning objectives:
- Use a dissecting microscope to view live animals
- Become proficient with using an adjustable volume micropipette
- Practice moving Hydra with a Pasteur pipette
- Apply micro-dissection skills to create transverse sections of Hydra
Materials:
- Common dish with Hydra
- Dissecting microscope
- Scalpel
- 60 mm Petri dish
- Hydra medium
- 100-1000 µL micropipette and tips
- 24-well plate
- Glass Pasteur pipette and bulb
- Waste container
- Permanent marker
- Labeling tape
For Hydra medium recipe and and methods for maintaining Hydra in the lab, please see:
Protocol
NAME
Low cost methods for Hydra care
CREATED BY
Callen Hyland
Figure 3. Parts of the dissecting microscope, just what you need to know for this activity.
Check your workstation to make sure you have all materials listed above.
Fill your Petri dish halfway with Hydra medium.
Use your glass Pasteur pipette to transfer six adult asexual Hydra without buds, one by one, from the common dish to your Petri dish.
Set your 100-1000 µL adjustable volume micropipette to 1000 µL. Get a new tip.
Fill 12 wells of your 24-well plate with 2 mL (2 X 1000 µL) of Hydra medium using your micropipette.
Dispose of used tips in the waste container.
Note
12-well plates and 6-well plates can also be used, just adjust the volume of medium in each well.
Two students can share a 24-well plate.
Put a piece of labeling tape on the lid of your 24-well plate.
Use a permanent marker to label it with your name and date.
Place your Petri dish on the stage of your dissecting microscope and turn the light control knob to the "I" (incident light) setting.
Adjust the zoom to the lowest setting.
Adjust the distance between the oculars until you see a single field.
Adjust the focus knob to get a crisp image of one of your Hydra.
Try slowly zooming in on it, adjusting the focus as you go.
While doing the dissection, you'll have to find a magnification that works for you.
Count the number of tentacles that the Hydra has and record it in your lab notebook.
Since you are going to dissect six Hydra you may want to set up a table to record the following information for each Hydra:
- Initial number of tentacles
- Well containing head-end
- Number of tentacles on head-end after a week
- Well containing foot-end
- Number of tentacles on foot-end after a week
Use your scalpel to cut the Hydra transversely (horizontally) in two. This is called a mid-gastric bisection.
Try to cut it right above the budding zone. In some adult Hydra, you can see a visible "bulge" near the budding zone. If this is not visible, just try to cut it into two equal parts.
Note
Hydra are sometimes elongated and sometimes contracted. They are easiest to cut when they are elongated. If your Hydra is contracted, let it sit undisturbed until it elongates again before trying to make the cut.
Sometimes the Hydra will be floating on the surface of the water. Use your scalpel or the tip of your glass pipette to push them down to the bottom of the dish before cutting.
What happened to your Hydra right after you cut it?
Record any behaviors you observed in your lab notebook. Also record any "mistakes" that you make while cutting the Hydra (for example, cutting the same animal twice, or cutting in the wrong place).
You should not have a head-end and a foot-end. Transfer both into separate wells of your 24-well plate.
Record which well the head and foot ends were placed in.
Repeat steps 11 through 15 for five more Hydra.
When you are done, place the lid on your 24-well plate and give it to your instructor.
Turn your microscope off when you're done and cover with the dust cover.
Note
If the number of tentacles is not fixed for an individual Hydra, what factors do you think might effect the number of tentacles?
Are there any other control groups you think should have been included in this experiment?
Document results of your regeneration experiment
Document results of your regeneration experiment
30m
30m
We will allow the Hydra to regenerate for 7 days before counting the number of tentacles on the head and foot-ends. Get you 24-well plate that you used in the previous class session from your instructor.
Materials:
- Dissecting microscope
- Your 24-well plate
- Forceps (optional)
With the dissecting microscope, examine the Hydra in each well and record the number of tentacles in the table you started in step 12. If the Hydra are hard to see because they are stuck to the side of the well, you can use the forceps to push them into the center of the well.
Note
Working with a partner, you can have one person observe and the other record, then switch roles.
When you're done, turn off the light on your microscope and cover the microscope with dust cover.
We will use the data you have recorded for statistical analysis to determine whether individual Hydra tend to regenerate the same number of tentacles that they had before bisection.