Collecting and Maintaining Ciliates in the Laboratory

Observing Living Ciliates

Light Microscopy Stains

Electron Microscopy

Nucleic Acid Sequencing

1. Isolating Individual Ciliates in the Lab

a. General procedure

An individual species can be selected and removed from the finger bowl with a micropipette. The protist can be placed with culture media in a culture tube or in a petri dish.

b. Separating ciliates from sand samples

a. Field samples - samples taken directly from nature. Any collection of pond water will contain numerous small invertebrates and protists, including ciliates. If the water is kept in a container with plenty of plant material from the pond and is protected from too much heat and from drying out, many of the ciliates will survive for several days. But these cultures will not survive in this condition for more than a week. Eventually, through competitive exclusion and predation, all but a few species will disappear from the sample.

b. Agnotobiotic cultures - a "ripened" field sample. After a week or so in the lab, a field sample will contain only a few species of ciliates, other protists and an unknown bacterial flora. These bacteria are the food source for several of the ciliate and other protist species (which, in turn are eaten by the carnnivorous ciliates) These cultures can remain stable for several weeks, so long as a source of food for the bacteria and addition water are occasionally added to prevent the culture from drying out. Substances added to keep the bacterial populations thriving include wheat grains, millet, rice grains, dried split peas, flaked fish food, and dried grasses.

2. Pure cultures

a. Axenic cultures - cultures of a single species grown in the absence of living organisms or cells of any other species.

b. Monoxenic cultures - cultures of two known species of organisms; in this case a ciliate and its food source.

c. Clonal cultures - cultures that are initiated with an inoculum of a single ciliate.

d. Strains - Usually a clonal culture of a species from a single geographic location.

1. Natural filtered pond water containing unknown bacterial organisms to which protists may be added.

2. Wild infusion - a field sample that has been allowed to stagnate in the laboratory to give an agnatobiota culture.

3. Culture Infusion - a culture medium produced by extracting organic nutrients from organic material (usually plant material).

i. Unboiled infusion - dried, grains, grasses (such as hay) cut into short pieces, or soil is added to clean pond or spring water. If the orgainic material is sterile, the resulting water can be inoculated with protists. If the organic material is not sterile the protists, fungi and some microinvertebrates emerge from protective cysts that were dormant in the soil, grass, or on the grain. These organisms can be observed feeding on bacteria that grow on the rotting vegetation in the water.

ii. Boiled infusion - a small amount of grain, grasses, or soil is added to clean pond water and the material is bought to a boil in order to kill any organisms living on the grain. When cool, the liquid is filtered, placed in culture dishes and inoculated with a species of protist.

iii. Sterile infusion - a small amount of grain, grasses, or soil is added to clean pond water and the material is bought to a boil in order to kill any organisms living on the grain. When cool, the liquid is filtered, placed in flasks and sterilized under 15 lbs of pressure for 15 minutes in an autoclave. This results in a sterile medium to which may be added a single species to establish an Axenic culture. If the protist to be cultured is a heterotroph (either a bactivore or predator), a prey species must be introduced first, allowed to grow for 24-48 hours before adding the protist (this establishes a monoxenic culture).

5. Semidefined media - Media that contain a mixture of biochemicals and standardized organic extracts. The organic extracts cotain ingredients with many unknown factors. Standardized organic extracts include proteose petone, loefers blood serum, and many others.

a. Filtered pond water (for freshwater species) or filtered seawater (for marine species) - collect water from the same source as you get your ciliates in the field. Filter it with a millipore filtration system.

b. Spring water - available in area grocery stores (Don't buy the carbonated or flavored brands!).

c. Glass-distilled water - distilled water often works less well than pond or spring water for culturing protists probably because it lacks trace minerals and vitamins that enhance the growth of the organisms.

d. Deionized water - tap water can be run through columns that just remove ions (such as chlorine and fluoride ions). This is less expensive than distilling the water and often the resulting water is pure enough for culturing protists.

a. Grasses - Hay and other grasses can be collected from nature, dried, then later used to make infusions. Because pesticides are often applied to lawns and even parklands completely wash grasses before they are dried.

b. Grains - Rice, split peas, wheat, or oats all can be purchased from a grocery store and can be used to make infusions.

c. Soils - Soils contain a surprising amount of dead plant and fungus material and so make good infusions. Sterile potting soil can simply be added to water to make an unboiled infusion. If you collect your own soil, it must first be sterilized or used only in boiled or sterile infusions.

a. Buffers - If the protists to be cultured require a pH different from the media, buffers must be added to change the media pH.

b. Vitamins - Some protists require special vitamins to serve as cofactors in their metabolic pathways. Add these according to directions in media recipes.

a. Patience - Sooner or later the organisms slow down on their own. As a

b. SaranWrap coverslips

c. Methyl Cellulose (Methocel or Protoslo)

1. Sudan III and Sudan Black stain fats and other lipids.

2. Methylene Blue will stain will color the nuclei dark blue against a light blue cytoplasm. Because it adds contrast to the protist cell, the functioning contractile vacuoles will be more easily seen.

3. Dry Films of vital stains - Some stains are dried into a thin film on a slide or coverslip to which a drop of culture fluid containing protists is

later added.

1. Lugols Iodine will stain starches such as glycogen.

2. Methyl green- acetic acid mixture will stain nuclei stain bright green while the cytoplasm acquires a bluish-gray tinge.

3. Klein's "Dry" Silver will stain cilia, flagella and cell surface

1. Protargol

2. Chatton Lwoff

a. Modified Karnovsky's Fixation For Marine Organisms

b. Modified Karnovsky's Fixation For Brackish Organisms

c. Fixation for Fresh Water Organisms