Penitentiary Glen

Diatom slides from core sample

This week in lab we extruded sediment from the core we collected at Beach City on October 10^th and learned how to prepare and examine diatom slides. The process of preparing diatom slides from a sediment core occurs in five steps: extruding the core from the collection tube, cleaning the sediment in nitric acid, rinsing and centrifuging the sediment, drying cleaned sample onto coverslips, and fixing dried material onto microscope slides.

Step 1: Extruding the core

extruding a core

For the sake of time, we only collected 4 samples from our core, but ordinarily an entire sediment core would be extruded and collected in 1-cm or 1/2-cm intervals. To extrude the core, the sediment must be pushed from the bottom up through the top of the collection tube. To prevent loss of sample, a plastic “stage” is attached to the top of the collection tube (fig. 1). This stage also allows for the sediment to be scraped into Whirl-Paks at desired intervals. Each Whirl-Pak was labeled with its corresponding measurement in the core (i.e. the top centimeter of sediment is labeled “0-1”).

Step 2: ‘Cleaning’ the sediment

plastic "stage" attached to collection tube

Approximately 0.5 grams of each sediment sample was mixed with 20 mL of DI water and 10 mL of nitric acid in a beaker and boiled on a hot plate until the solution reached a volume of ~10 mL. This step is necessary to remove organic matter from the sample. Because the inorganic sediment and diatoms cannot be separated in core samples, removing organic material makes diatom identification and count data easier to collect. At the end of this step, we are left with inorganic material (sediment + diatoms) and nitric acid.

samples boiled in nitric acid

Step 3: Rinsing the sediment

We did not complete this step in class, but the samples must be rinsed with DI water in order to remove the nitric acid. To do this, the sample is transferred to a Falcon tube and centrifuged at 2000 rpm for 10 minutes. The supernatant nitric acid is decanted and the tube is refilled with DI water. Next, the tube must be shaken to resuspend the sediment pellet. This process is repeated 6 times to completely remove the acid.

Steps 4 and 5: Preparing permanent mount slides

A solution of ~0.1 mL of cleaned sample + ~0.9 mL DI water was pipetted onto a coverslip and allowed to dry for 24 hours. Three dilutions of each sample were made and prepared slides were examined for concentration of diatoms. Dry coverslips were fixed to microscope slides using Naphrax permanent mountant. For the remainder of class, we looked at previously made permanent mount diatom slides and identified different genera based on morphology. 

Some of the common genera found were also known indicator organisms. Acidophiles: Eunotia, Frustulia, and Actinella, as well as Pleurosira, an indicator of elevated conductivity. Below are examples of each morphological group of diatoms: Centrics, with radial symmetry (most often seen in valve view); Araphids, without a raphe and often found in chains or star-shaped colonies; Eunotioids, acidophiles with an abbreviated (short) raphe along the valve mantle; Monoraphids (not pictured), with a raphe on only one of the two valve faces, Biraphids, with raphes on both valve faces (pictured: a "Naviculoid" biraphid with symmetry to both the apical and transapical axes); Gomphocymbelloids, with asymmetry to one or both axes (or symmetrical to both!), Epithemioid, with its raphe in a canal, Nitzschoid, with its raphe in a keel along one side of the valve, and Surirelloid, with a single raphe in a keel around the entire periphery of the valve face.

Asterionella, an araphid, and two (separated) centric
Stephanodiscus valves
 

Pinnularia, a biraphid

Eunotia, with an abbreviated raphe along the valve mantle

Cymbella, a biraphid with asymmetry to
both apical and transapical axes 

Epithemia, with a raphe in a canal

Surirella, with a single raphe around the periphery

Nitzschia, with its raphe in a keel 

Diatoms from Soil Cores

 

Diatoms on a Slide from a Soil Core

Background:

                  Diatoms are single celled organisms that have shells composed of silica. These incredibly diverse tiny organisms can be found in both freshwater and marine ecosystems. They are often studied as important indicators of past ecological conditions, because when the diatom dies, the silica shells remain in the sediment.  Because they remain in the sediment for long periods of time, the composition of diatoms found at a specific location in a soil core can tell researchers what conditions were like when that layer was deposited. Diatoms can indicate water salinity, temperature, and pH, as well as show nutrient and pollution levels in a system. For example, imagine a system that experienced no pollution until humans developed the surrounding area. The diatom composition before and after human development would differ. You would expect to see more pollution tolerant diatoms in the soil after development, and fewer pollution sensitive diatom species.

 

Taking a Soil Core at Beach City

Extracting a Core

                   Sediment core extraction required several pieces of equipment. Two boats were used to navigate the body of water (lake) in order to find a desired location for an extraction. Once a desired location was found, the boat remained stationary for a minute to ensure no sediment agitation occurred before taking a sample. Then, the core tube was slowly lowered into the water and forcefully pushed into the sediment, reaching a desired depth. The extraction of the core was performed by careful lifting of the sediment core tube, followed by a quick placement of a rubber stopper at the end of the tube. Finally, both ends of the core tube were plugged and taped to insure preservation of the sample. While, performing a sediment core extraction, it was observed that the bottom of the core was fairly dark due to anoxic conditions that are associated with deeper sediment layers. The top however, observed a brown-dark greenish color, indicating higher levels of oxygen. While extruding the sediment core, the pH of water was tested using a pH meter, which indicated a fairly neutral and acceptable value within the range of 6.5 to 8.  

  

Removing the Bottom Stopper and Inserting Plunger

 Creating Samples
                  Samples from a sediment core are important in studying the history of a desired body of water. They are great indicator of historical biodiversity which can be used to recreate past environments conditions. This information can be compared to the present biodiversity and further analyzed in order to grasp a better understanding of the changing environmental conditions such as the pH, as well as nutrient and pollution concentrations within the body of water. 

Extruding the Soil Core

                  In order to create samples from the extruded core, whirlpac bags were obtained and labeled to ensure accurate tracking of samples. Then, the water layer present inside the sediment core tube was expelled using a plunger- like tool that pushed the sediment from the bottom to the top. A specially designed tray was fitted on top of the sediment core which was used to concentrate the sediment samples into 1 cm intervals. The intervals were collected (by scraping the top) and stored inside designated whirlpac bags. All of the samples collected were then further analyzed by preparing microscopic slides in order to observe diatom biodiversity. 

This Plate Helps Measure Out 1cm of soil

Samples are Scraped into Whirlpak Bags for Sampling

 Preparing Slides

                  To analyze the diatoms in the sample, they must be spread out onto microscope slides. The first step in slide preparation is to remove as much of the organic matter and soil from your samples. This can be done by boiling a small about of sample in nitric acid, to break down organic compounds. After the samples are boiled, they are rinsed with water several times to remove traces of acid. The finished diatom mixture is cloudy and will need to be diluted farther before it can be made into slides. It is recommended that you make three separate dilutions of each sample while making slides. 

Boiling Samples to Purify Them

A Purified Sample: Ready for Dilution and Slides

                  The slides themselves are made by placing the sample onto a clean cover slip.  The sample should cover the entire cover slip and form a mound in the middle. These will need to sit for at least twenty-four hours, as they dry.  Once dry, the cover slips can be permanently mounted to a clean microscope slide.  Attaching the cover slips permanently allows researchers to preserve samples for later analysis.

Alison Demonstrates How to Correctly Make A Slide

Make sure to get a mound of water in the middle of your cover slip. It shouldn't spill onto other cover slips.

 Some of the diatoms found include:

Cymatopleura

Stauroneis

Stephanodiscus

Navicula

Pinnularia

Cocconeis

Cymbella

Aulacosara

Ctenophera

Puncticulata

Nitzschia

Diatoma

Rhopalodia

Eunotia

Frustulia

Asterionella

Platessa

Surirella

Gyrosigma

Enopnema

Pleurosira

Cymbella Seen Under the Microscope

A Visit to Triangle Lake Bog and Herrick Fen

Triangle Lake Bog - November 2013

Herrick Fen - November 2013

 
Located an hour south of Cleveland, there are two beautiful wetlands: Triangle Lake Bog and Herrick Fen. Both of these properties are examples of endangered habitats. Most of Ohio’s wetlands have been destroyed by development, while the wetlands that are left are continually threatened by eutrophication and suburbanization. Eutrophication threatens wetlands with large increases in nutrients from fertilizer run offs. The large inflow of nutrients disrupts the delicate balance of these systems.

Triangle Lake Bog - Early September 2013

Triangle Lake Bog was formed when glaciers moved across Northern Ohio approximately 25,000 years ago. As the glacier retreated it left behind heavy ice blocks that broke off and settled into the soil. After the ice melted, the depression filled in forming a kettle lake. The remnants of this kettle lake can be seen in the middle of Triangle Lake Bog. Bogs are a unique type of wetland because they lack water inflow and outflow; they are fed only through precipitation. The lack of water flow causes bogs to be acidic and low in oxygen. Nutrient levels are low in bogs because decomposition is slowed by the acidity and anoxic conditions. These traits that are characteristic of bogs make them the home of a number of unique species. 

Herrick Fen - Early September 2013

Herrick Fen was formed at approximately the same time as Triangle Lake Bog on top of a large gravel deposit left behind by the glacier. Fens are continuously fed by groundwater and water drainage from the surrounding areas. Because the fen has constant water influx, the soil is considered hydria (saturated and sometimes anoxic). Nutrient levels in fens are higher than in bogs because the mixing water has higher oxygen content and encourages more decomposition. Fens are also home to some of the special species found in bogs.

Tamarack is a common tree found in bogs and fens

Grass of Parnassus: a rare plant found at Herrick Fen

The field trip to visit these Ohio wetlands was filled with excitement and new discoveries, ranging from algae/diatom collections to discovering and learning about unique plant species and their preferred environments. Plants that were the center of attention during the trip were carnivorous plants. Due to their low nutrient environment, these plants have developed unique methods of acquiring nutrients. These plants compensate by consuming and breaking down animals such as ants and spiders as well as different types of protozoa. They each have a different method of trapping their prey. The types of carnivorous plants that were observed at Triangle Lake Bog and Herrick Fen were pitcher plants, sundews and bladderworts.

Close-up of the digestive juices in the pitcher plant

One of the most common carnivorous plants found in Ohio bogs are pitcher plants (Family: Sarraceniaceae). They are characterized by folding their leaves like a cup and forming a pitcher. The pitcher contains a sweet nectar juice that attracts many victims but at the same time has digestive properties. These victims crawl into the pitcher and once trapped, are unable to climb out. Over time, they are digested by the enzymes in the nectar juice and consumed by the plant. 
 

Pitcher plants at Triangle Lake Bog

Sundews (Family: Droseraceae) are carnivorous plants that are characterized by having specialized tentacles. The tentacles contain a gel like substance that will attract prey such as fruit flies, gnats and ants. After a prey insect gets stuck on the tentacles, the tentacles will wrap around the insect to prevent escape. The trapped animals will be dissolved by the enticing mucus and then absorbed as nutrients. 
 

Sundews grow in beds of Sphagnum moss

Close-up of Sundew: Red tentacles are visible

Another type of carnivorous plants that can be found in Ohio are bladderworts (Family: Utricularia). These plants are aquatic and are characterized by having a unique trap mechanism, which involves actively pumping water in order to initiate the trap. During contact with prey, a trap mechanism is initiated which will cause the plant to swell up due to active intake of water from the environment and at the same time suck in the prey. Once the prey has been engulfed by the bladderwort, they will be dissolved by the digestive secretions produced by the plant. 

 

Bladderworts (photo credit: plants.usda.gov)

These wetland habitats are home to so many unique plants that this blog is only able to highlight a few of them.  There are more beautiful things to be seen at these nature preserves. It is well worth the drive from Cleveland to visit them. It is important that people  visit these wetlands and appreciate their biodiversity. The general public needs to continue to educate themselves about the importance of these habitats and why they should be protected. 

For more information on these locations, visit:
Herrick Fen 
Triangle Lake Bog

For more information on the plants species above, visit:
http://plants.usda.gov/java/