Purpose
To determine the net growth of the indicator species of calcified algae.
Materials
Alizarin red stain
1.5 ml Eppendorf tubes
4 L plastic bags
Small rubber bands
Two 50 m transects
Sand anchors
Cable ties
Scissors (or Sea Snips®)
Mesh diving bag
Overview
Calcified macroalgae can be stained with Alizarin red stain in the field and gathered at a
later date (7 - 8) days to measure growth rate. This data can then be used to correlate
growth rate with other physical parameters, such as temperature, nutrients, light, and sedimentation.
(When correlating with other factors be sure to include controls). The method was
developed for a Halimeda growth study but this technique can be modified for most calcified algae.
Method
The method requires laboratory preparation, deployment in the field, later collection of
the stained plants and final processing. The number of plants to be stained is dependent upon
the goals of the research project. If the staining is part of a
Halimeda study in conjunction with the Nutrient Enrichment method, 60 plants are recommended: 30 with nutrients
(manipulation) and 30 without (control).
Preparation
Prepare a 1% Alizarin red stain by mixing 1 gm
of Alizarin red stain powder with 100 ml of water.
Place 1 ml of the stain into each of the 1.5 ml
Eppendorf tubes, one vial per plant to be stained.
Each Eppendorf tube is placed into a 4 L plastic bag.
Randomly select numbers for plant placement along
100 meters (2-50 m transects) and note the numbers on
a slate or waterproof paper.
Installation
Attach the end of a 50 m transect to the
substrate; use a sand anchor in areas with sand. Run
the transect out along a depth gradient. Note
compass heading and mark the beginning of transect (or leave sand anchors) as the transect will
be reeled up and redeployed the next day for bag removal. Swim to the first random number
and select the nearest mature plant. Place a plastic bag with an Eppendorf tube over the
complete plant and secure with a small rubber band at the base of the plant. The rubber band must
be tight enough to cut of water circulation in and out of the bag without
constricting the plant.
Once the plastic bag is securely tied over the plant, carefully open the Eppendorf tube
and gently massage the contents to mix with the seawater in the bag. Leave the bags with
the stain over the plants for 24 hours. Do the same for the second transect.
Bag Removal
Run the transects back along compass headings. Swim to the first plant and with a scissors
carefully cut the cable tie, releasing the plastic bag.
Gather cable tie pieces and plastic bag for disposal. Move to the next plant and repeat
the process until all bags are removed from the stained plants. Again, reel up transects but
leave sand anchors or markings for later
deployment for plant collection.
Plant Collection
Eight to nine days later the plants are
collected for processing. Run out the transects once again.
Swim to the first plant and collect the entire
plant (leaving only holdfast), and place in a
marked ziplock (plants with same
treatment/transect may be placed in same bag). Continue collection until all plants are collected. Reel
transects and remove markers/sand anchors.
Plant Processing
Back in the laboratory/work area, place the plant in a refrigerator for approximately 2
hours until chlorophyll retreats into segments, leaving the stained portions of the plant (old
growth) easily distinguished from the unstained (new growth) portions. Cut the stained portion
from the unstained and weigh each portion separately. If the plant is segmented as in
Halimeda spp., also count the stained and unstained portions. Normalize the data over the time
the stained plants were left in the field after the bags were removed. Fo example: segments
per day or grams per day of new growth, or percentage of new growth per day [(new growth /
old growth) / number of days].
References
Smith, J.E., C.M. Smith, P.S. Vroom, K.S. Beach and S. Miller. Nutrient dynamics of
Halimeda tuna along a depth gradient in the Florida Keys: possible influence of internal tides?
Submitted to Limnology and Oceanography.
Sources
Alizarin Red-S (C.I. 58005): Catalog NO. A5533
Sigma Chemical Company
P.O. Box 14508
St. Louis, MO 63178
(314) 771-5750
