Marine Botany

BIOLOGY 4429

LABORATORY

PROCEDURES

AND

PREPARATIONS
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Dr. Roy L. Lehman

Texas A&M University-Corpus Christi
TABLE OF CONTENTS

TABLE OF CONTENTS.....................................................................................................2
ALGAL IDENTIFICATION CARDS..................................................................................3
METHODS OF ALGAL COLLECTION............................................................................4
Soil Algae.................................................................................................................4
Phytoplankton..........................................................................................................4
Marine Algae (Other Than Plankton Tow)...............................................................5
HISTOCHEMICAL AND GENERAL METHODS............................................................6
Temporary Fixation..................................................................................................6
General Staining.......................................................................................................6
Arresting Movements Of Algae................................................................................6
Flagella.....................................................................................................................6
Cell Walls..................................................................................................................6
Gelatinous Envelopes and Sheaths...........................................................................7
Chloroplasts..............................................................................................................7
Nuclei And Chromosomes........................................................................................7
Starch And Other Reserve Materials........................................................................7
CLEANING AND MOUNTING OF DIATOM FRUSTULES...........................................8
ALGAL CULTURE TECHNIQUES.................................................................................10
Materials.................................................................................................................10
Procedures..............................................................................................................11
Culture Media.........................................................................................................11
ISOLATION AND PURIFICATION.................................................................................12
Methods of Isolation..............................................................................................12
Sterile Pasteur-Type Pipette...................................................................................12
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Capillary Pipette (Simplified).................................................................................13

Streak Plating.........................................................................................................14
Spray Plating..........................................................................................................14
PURIFICATION................................................................................................................15
Centrifugalization...................................................................................................16
Ultrasonic Treatment..............................................................................................16
Antibiotic Treatment...............................................................................................16
Potassium Tellurite Treatment................................................................................17
PROBLEM AREAS...........................................................................................................17
Alternative Techniques - Isolation..........................................................................18
PRESERVATION...............................................................................................................18
PERMANENT PREPARATIONS.....................................................................................19
CELL COUNTING AND GROWTH CURVES................................................................22
GROWTH CURVES..........................................................................................................24
PREPARATION OF HERBARIUM MOUNTS................................................................26
REFERENCES..................................................................................................................29

ALGAL IDENTIFICATION CARDS

It is recommended that each student construct and maintain a set of index cards
(recommend 5” X 8”) which will provide the following:

1. A record of each alga identified

2. It’s phylogenetic relationship
3. Identification and morphology of internal and external structures and
components.

PROCEDURE: (side A)

List the phylogenetic relationship of the alga.

DIVISION: (-phyta) _________________________

CLASS: (-ophyceae) _________________________
ORDER: (-ales) _________________________
FAMILY: (-aceae) _________________________
Genus: _________________________
species: _________________________

PROCEDURE: (side B)

List the following information:

1. Name of the alga
2. Habitat type/thallus type
3. Pigment type and chloroplast shape and location
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4. Type of locomotion (Organism and/or reproductive cells)

5. Reproduction (asexual or sexual) and Type of Life Cycle
6. Food storage substances
7. Diagram of a cell, colony or filament. Identify and label internal and external
morphology especially taxonomic criteria.
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METHODS OF ALGAL COLLECTION

SOIL ALGAE:

1. Dry Surface Layer

Cut a soil section with a small sharp bladed shovel. The section should be about 10
cm X 10 cm in size. Place the sample into a container for transport to the laboratory.

2. Damp Surface Layer

If the soil is damp, less material is necessary. Use a scalpel to cut a small block (about
2 cm X 2 cm) of the soil. Place the sample in a container that will retain the moist nature of
the soil until laboratory procedures can begin.

3. Other Surface Material

Algae may be careful scraped from tree bark, damp walls, flower pots, etc. with a
scalpel and placed into a vial or other suitable container.

4. How to Obtain Clean Algal Specimens from Soil Samples:

A. Place the soil block (wet or dry) into a petri dish and add enough distilled water to
saturate the sample.
B. Place a few cover glasses on the soil block and leave the dish open until the excess
water has evaporated.
C. After evaporation, cover the dish with a lid and place in a north window.
D. Within one to two days, motile algae found on the soil sample will move up to the
undersides of the cover glasses and begin to multiply.
E. During subsequent days, additional algal species may begin to grow on the cover
glasses. Material can be removed repeatedly for examination. A small amount of
algal material should be placed into a drop of water on a glass slide, covered with a
cover glass and observe under microscopic power.

PHYTOPLANKTON:

1. Plankton Net Tow

The plankton net consists essentially of a cone of nylon (or equivalent material)
mounted on a ring or hoop to which are attached three thin rope bridles spliced onto a smaller
ring by means of which the net can be shackled to a towing rope. The end of the cone is left
open and reinforced by strong material. Tapes or cords are sewn in place so that small glass
or plastic jars can be put into position at the end of the net. The jar receives most of the
plankton as the net is towed although some always remains on the wall of the net and is
removed by turning the net inside out and washing it in a wide-mouth receiving jar. This
container should hold about a liter of sea water or freshwater. The sample is maintained in a
cool environment until examination in the laboratory can take place. The sample may also be
preserved or fixed at this time with an appropriate preservative.

2. Filtration Technique
Water may be collected in a clean container or Nansen Bottle and returned to the
laboratory for examination. Small algae may be collected in this manner by filtering through a
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Millipore Membrane of 0.45 m or a 0.80 m pore size. Water from a plankton tow may also
be filtered in a likewise manner. A standard sample size is 100 ml.

Fig 1. Membrane Filter

Apparatus

Fig. 2 Filter Assembly Procedure.

3. Physical/Mechanical Collection:
Reed stems, twigs or submerged weeds, bottom soil samples, pieces of tree bark and
stones or other objects may be collected and placed into plastic bags. Add just enough water
in the container to ensure a saturated atmosphere when it is closed. On return to the
laboratory, the bottles should be opened and examined as soon as possible. If it is intended to
keep the algae alive for any length of time, there should be no overcrowding. The larger the
mass of algae the sooner they will die. Also be cautious, never add tap water!

Marine Algae (other than plankton tow)

1. Damp sand and other material may be collected from the intertidal area of the seashore.
Particles of the sample are added to a culture media for marine algae and allowed to grow.
2. The macro algae along the Texas coast are found growing attached to the rocks that make
up the jetties or as epiphytes on other algae attached to hard substrates. Algae are also
found attached to shells, rocks and debris of various kinds and as epiphytes on seagrasses ,
other algae and animals. This is especially true of those algae found in the bays and
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lagoons. Algae that are attached to the bottom or other substrates (including jetties) are
termed benthic. Free-floating forms that may spend their entire lives at sea are termed
pelagic. Equipment consists of a pail or two for carrying specimens, a quantity of plastic
bags for separating the larger species and a number of small. screw-cap vials with and
without a preservative into which important specimens may be preserved from loss or
mixing. For removing small plants from rock surfaces, a heavy knife or other scraping
tool is used, while encrusting forms which adhere too firmly may be obtained by using a
geologist’s hammer for cracking off small pieces of the supporting rock.
HISTOCHEMICAL AND GENERAL METHODS

Temporary Fixation
Add a drop of dilute Potassium Iodide (IKI) solution or FAA to a 1-2 ml sample of
algae. This will also act as a temporary preservative. Algae can also be fixed and temporarily
preserved by a 1-2 % solution of Gluteraldehyde.

General Staining
Different algal structures vary in their affinity for stains. More frequently employed
stains are Methylene Blue, Gentian Violet or Acid Fuschin (up to 1% aqueous solutions). The
following simple procedure is recommended. Mount algae in a drop of water on a slide and
apply a coverglass. Add a drop of stain to one edge of the coverglass and let it diffuse to the
opposite edge by removing water from the opposite side with a piece of dry blotting paper. In
this way a spectrum of staining is obtained, the algae near one side of the coverglass are
intensely stained whereas those on the opposite side are weakly stained.

Arresting Movements Of Algae

Certain algal species and reproductive forms are actively motile or may exhibit a
gliding movement. To slow down such movements:
1. Add a tiny drop of IKI solution to a drop of algal suspension.
2. Add a pinch of powdered gum arabic, methyl cellulose or dextran to a drop of
algal suspension and apply a coverglass,
3. Mix a drop of chloroform water (i.e. a drop of chloroform in 5 ml of distilled
water) with a drop of algal suspension and apply a coverglass, and
4. Place a drop of algal suspension directly in the center of the slide or petri dish
containing a thin sheet of 2.5% aqueous agar, apply a coverglass and observe
under the microscope. This method will also stop Brownian Movement.

Flagella
Most algal flagella cannot be seen under an ordinary light microscope without special
staining. Two commonly used methods to render flagella visible are listed.

1. Add a few drops of India Ink to a drop of algal suspension, apply a coverglass and
examine. -or-
2. Fix algae in dilute IKI solution, apply a coverglass and examine. If overstained,
decolorize to desired degree by adding dilute sodium thiosulphate.

Cell Walls
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The presence or absence of a true cell wall may be determined by microscopic

observation of algal cells that had been placed in a plasmolysing solution, e.g. 10% sucrose,
for a few minutes. Mix a drop of dilute IKI solution with a drop of algal suspension on a
slide, followed either by treatment of the mixture with a drop of concentrated sulfuric acid or
by irrigation with 70% sulfuric acid. Cellulose will turn a blue color. The IKI solution is
prepared by dissolving 2 gm of KI in 20-30 ml of distilled water with one gm of dissolved
iodine. A solution is made to a total 100 ml by adding distilled water.
Cell walls are composed primarily of pectic substances which acquire a red color when
stained with a fresh, dilute aqueous solution of Ruthenium Red (one or two crystals in a small
watchglass containing 2-3 ml of water).

Gelatinous Envelopes And Sheaths

These structures may be rendered perceptible by staining with a very dilute aqueous
solution of either Ruthenium Red or Methylene Blue. Alternatively, a small drop of India Ink
may be used for the same purpose.

Chloroplasts
The chloroplast may assume a distinct appearance when viewed through a microscope
which has a blue filter inserted between its light path. In visual work, this filter has been
reported to produce a surprisingly clear image and in microphotography the use of this filter
yields sharp and brilliant negatives. A corresponding advantage of the blue filter is that it can
be used equally well with different kinds of algae since most of them have a strong yellow
component (i.e., the complementary color to blue) in their chloroplast pigments. In addition,
careful focusing through the cell is necessary for viewing the shape of the chloroplast.

Nuclei And Chromosomes

These can be demonstrated by the application of the iron acetocarmine or aceto-orcein
technique. In temporary preparations, nuclei may sometimes acquire a bluish color when the
cells are immersed into a very dilute aqueous solution of Methylene Blue and then washed in
water to remove the stain from the cytoplasm.

Starch And Other Reserve Materials

If a blue or violet color develops after treatment of the material with an IKI solution,
the presence of starch is indicated. Glycogen or cyanophycean starch generally will give a
brownish color on treatment with iodine, whereas floridean starch gives a reddish color with
the reagent.
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CLEANING AND MOUNTING OF DIATOM FRUSTULES

Rinsing
1. Pour sample into test tube and centrifuge ( you may wish to kill cells first to fix
membranes).
2. Pipette supernatant liquid off the top.
3. Refill tube with distilled water and vortex sample.
4. Centrifuge for ten (10) minutes at 80-90.
5. Repeat three times - Vortex and centrifuge X 3.
6. When the sample is rinsed, view one drop with the microscope to determine the
proper density of cells.
7. Place the appropriate number of drops (determined in step 6) on the coverslip.
Dry under a cover.
8. Add an equal amount of KMnO4 to the remaining rinsed sample and allow to stand
overnight under the hood.

Cleaning
1. Add HCl to the KMnO4 rinsed samples while slowly heating over alcohol lamp.
Two pipettes of HCl are usually enough to reach the endpoint. A light brown,
yellow or greenish color is indicative of the endpoint.

CAUTION!!! Heat slowly, add acid slowly and always point the tube away from
yourself!

2. Centrifuge test tubes and refill with distilled water six times (as in rinsing step).
3. View under microscope to determine cell density.
4. Place drops of cleaned sample on cover slips and dry under a cover.
5. The remaining sample is retained in a small vial, labeled, sealed with parafilm and
stored.

Mounting
1. Cover slips, with dried material are mounted to glass slides using FloTexx,
Eupheral or Hyrax.
2. Finished slides are placed in their proper storage box and a card is made for the
indexing of the slide collection.

Permanent Mounting Of Diatoms

1. Place diatom sample (cleaned and rinsed) on a coverslip. Allow to dry.

2. After sample is dry, place one drop of mounting medium in the center of a microscope
slide.
3. Position the coverslip sample side down onto the mounting medium. Light pressure may
be applied to the coverslip to ensure even spreading of the medium.
4. Heat the slide (coverslip side up) over a hot plate until the mounting medium begins to
bubble. Allow the medium to bubble a short time period and quickly remove when the
bubbling stops.
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5. Remove the slide from the hot plate and place on a cool surface. The bubbles will be
reabsorbed; however, large bubbles may require the use of a probe to apply light pressure
to expel them before cooling is complete.
6. Trim excess mounting medium from edges of the coverslip with a razor blade and seal the
edges with clear fingernail polish.

Slide Label Format

Slide label format:

Cruise Information:

Fig. 3. Slide label format.

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ALGAL CULTURE TECHNIQUES

Introduction
If algae are to be used as experimental organisms, they often have to be maintained in
the laboratory in good physiological condition for extended periods of time. Certain
morphological and reproductive stages can best be demonstrated by suitable manipulation of
live cultures of algae. This will require that you become competent in at least a few algal
culture techniques.

MATERIALS:

Glassware. Borosilicate (e.g., Pyrex, Kimax) glassware is preferred, although ordinary flint
glass and plastics are often satisfactory and may be necessary for large-volume cultures.
Culture tubes, bottles or erlenmeyer flasks are used for smaller volumes, jars and carboys for
larger volumes.

Temperature Control. Temperature should be controlled to within a few degrees Celsius in

order to obtain and maintain consistent results. A controlled-temperature chamber or room is
best, but an air conditioned laboratory will often suffice. The optimum temperature for a
given species is the idea temperature for a culture, but a great many species have been found
to grow well at temperatures ranging from 15° to 25° C.

Light. Fluorescent lighting is normally used, although in the northern hemisphere, a north-
facing window often works well. A light intensity of about 60-80 E m-2 s-1 (3,000-4,000
Lux) is often used and can as a rule be obtained within 20-30 cm of standard daylight or cool-
white fluorescent tubes. It should be noted that the light output of fluorescent tubes declines
with age, however cultures are probably given excess light in many cases. Some species
benefit from a dark period each day, so a photo-period of 18 hours:6 hours dark is often used.

Mixing. As the density of cultures increases, agitation of the medium is usually

advantageous. Shakers, magnetic stirrers or aeration can be used. CAUTION! Air supplies
must be free of oil vapors from pumps.

Media. No single medium can be expected to support good growth of all freshwater and
marine species. Ideally one should use a medium that has been found to support good growth
of the species to be cultured. Otherwise, one should use a medium that is known to be
suitable for a large number of species, such as Chu Number 10 or Bold’s Basal Medium.
Media for initial isolations are normally enriched with soil extract.

Water. Glass-distilled and deionized water should be used in preparing freshwater media, salt
solutions and soil extract. Seawater media should be made with water obtained from an
unpolluted area, preferably offshore. Seawater should be filtered. Newer marine water mixes
when prepared with deionized water are good substitutes.

PROCEDURES
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Sterile Techniques. Bacteriological-quality sterile techniques must be used for axenic

(bacteria-free) culturing and they are usually required for unialgal culture of freshwater
species, as contaminating fungi and bacteria are often airborne. Less exacting techniques may
suffice for marine species, especially if culturing takes place away from the ocean.
All glassware and implements should be autoclaved at 121C for 20 minutes.
Disposable plasticware is usually sterilized by the manufacture. Metallic implements may be
flame-sterilized between repeated uses.

Basic Inoculation:

1. Agitate stock culture to ensure uniform distribution of cells in each sample.

2. If culturing axenically, remove the cap from the stock culture vessel and briefly
flame the opening.

3. Use a sterile pipette to remove a measured aliquot of the stock culture. Flame the
opening of the stock vessel containing sterile medium.

4. Cap culture vessel aseptically and place in culture appliance.

CULTURE MEDIA

Chu No. 10 (Modified), for common freshwater and soil algae:

Calcium Nitrate [Ca(NO3)2] 0.04 gm/l

Dipotassium hydrogen phosphate (K2HPO4) 0.01 gm/l
Magnesium sulfate (MgSO4*7H2O) 0.025 gm/l
Sodium carbonate (Na2CO3) 0.020 gm/l
Sodium silicate (Na2SiO3) 0.025 gm/l
Ferric chloride (FeCl3) 0.008 gm/l
pH = 6.5-7.0
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ISOLATION AND PURIFICATION

Introduction.

This section deals with selected methods for the isolation and purification of
freshwater or marine microscopic algae. Microscopic algae as defined for this section are
non-swimming, unicellular or filamentous eukaryotic algae from freshwater, brackish, marine,
soil or atmospheric environments. It is also possible to use methods as discussed in this
section to isolate blue-green algae. Application of the following methods to natural
collections of microscopic algae produces unialgal or axenic cultures. Unialgal cultures are
considered as clonal cultures with bacteria. These methods are not difficult, although the
preparation of axenic (bacteria-free) may require patience and perseverance. The methods of
isolation include the following: 1) capillary pipette, 2) capillary pipette (simplified), 3) streak
plating, and 4) spray plating. Four methods are described for purification: 1) washing, 2)
ultrasonics, 3) antibiotics, and 4) potassium tellurite treatment.

Methods of Isolation

The isolation of a single algal unit into a medium suitable for growth is required to
establish a clonal, unialgal culture. The term unit, as used in this section, denotes any unialgal,
colony, filament, thallus piece or reproductive body. A reproductive body must produce only a
single individual when it develops to be considered a clonal culture. Established unialgal
cultures may be the source of material for preparing axenic cultures.
Four methods of isolation are described. The capillary pipette and streak plate
methods are recommended for most isolations. Additional methods are described to show
variations of the basic methods but does not include all known methods.
The method of isolation depends on thallus type and size. Algal units less than a m in
diameter are difficult to see, and, therefore to isolate with a capillary pipette. However, larger
thalli, such as filaments, may be isolated by the capillary pipette method. Sometimes it is
easier to isolate newly-released reproductive cells (zoospores, aplanospores, etc.) rather than
the vegetative thallus into unialgal cultures. It might be possible to establish axenic clones
from these reproductive cells or after suitable growth has occurred, axenic cultures may be
established.

Sterile Pasteur-Type Pipette

A. Heat-sterilize isolation dishes and Pasteur-type pipettes in an autoclave following standard

techniques.
B. Place 6-8 drops of liquid culture medium in a watch glass or 3-4 drops in the depressions
of the spot plate portion of each isolation dish. Keep isolation dishes covered except
when in use.
C. Place 2-3 drops of a natural collection into the medium on a watch glass or a single drop
into a depression in a spot plate. The dilution of the natural collection may be modified,
depending on the number of units of the desired species present.
D. Prepare a fine capillary pipette by aseptically taking a sterile Pasteur-type pipette and
putting a rubber bulb on the wide end. Use forceps to hold the narrow end of the pipette
in a low flame of a bunsen burner. As the glass softens and reddens, gently and with a
smooth lengthwise pulling action, remove the pipette from the flame. A sudden jerking
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and/or pulling while the pipette is in the flame does not result in a properly formed
capillary pipette. Break off the tip of the capillary pipette near the point where the glass
bends under its own weight. The bore of the pipette should be several times the diameter
of the algal units being isolated.
E. Locate desired algal assemblages for isolation while looking through the stereomicroscope
or low power of a compound microscope.
F. Manipulate the tip of the fine capillary pipette to a position just out of the liquid and above
the algal unit. Gently dip the pipette into the liquid and by capillary action the liquid with
the desired cells should flow into the pipette. This flow of liquid may be controlled by
slight pressure on the rubber bulb, although this is generally not necessary. Only one algal
unit should be drawn into the pipette at each dip. Depending on size of algal unit, several
may be picked up in one pipette.
G. Place the tip of the capillary pipette into the liquid of a second isolation dish. Gently
squeeze the rubber bulb of the pipette until an air bubble is released. The pipette should
be re-pulled between successive transfers.
H. Wash the units by transferring them singularly with a capillary pipette to the liquid in the
remaining two isolation dishes.
I. Transfer single algal units by a capillary pipette to tubes of liquid culture medium. As a
precautionary step, the single algal unit in a droplet may be transferred first to a small
piece of cover glass resting on a microscope slide; the droplet may then be examined with
the compound microscope. The small piece of cover glass with the algal unit is then
transferred to the tube of culture medium. This precaution ensures that a single algal unit
is isolated and that it goes into the culture medium. Also, isolation dishes should be
changed between successive transfers. Never use the same washing dish twice.
J. Place tubes of isolated algal units under conditions suitable for growth.

Capillary Pipette (Simplified)

A single inverted plastic petri dish top is used as an isolation dish.

A. Place 10-15 drops of a natural collection in the center of an inverted plastic petri dish top.
B. Place 6-8 drops of suitable medium in six positions encircling the natural collection. Mark
one position as number one.
C. Using a newly pulled, sterile, capillary pipette, transfer the desired algal units from the
natural collection to one of the six drops of liquid medium as outlined in the preceding
“Capillary Pipette” method.
D. Transfer a single algal unit from the first drop to a second drop of the liquid medium
moving in a clockwise direction.
E. Repeat the transfer process through the remaining drops of liquid medium until you are
certain that only a single algal unit is present in a drop.
F. Transfer the single algal unit to tubes of liquid culture medium and incubate.

Streak Plating

When algal units are 10 µm or less in diameter, they are isolated more easily by streak
or spray plating. Either method serves to isolated an algal unit and may produce an axenic
culture.
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A. Prepare petri dishes containing growth medium solidified with 1-1.5% agar medium. The
agar should be 0.5 to 0.66 the depth of the dish. Plastic or glass (100 x 15 mm) petri
dishes are suitable.
B. Place 1-2 drops of a natural collection near the periphery of the agar. Flame sterilize
either a wire loop or a bent-glass rod (dip rod in 70 % ethanol, flame and remove from
flame; repeat several times, being careful that the flame is extinguished before dipping in
alcohol each time). Use aseptic technique, a sterile loop or glass rod is used to make
parallel streaks of the suspension on the agar. If desired, a wax pencil outline may be
drawn on the outside bottom of the plate.
C. Cover, invert and incubate the plate for 4-8 days under suitable growth conditions.
D. Observe with a stereomicroscope and select desired colonies that are free of other
organisms for further isolation. Remove a sample of a colony with a fine capillary pipette
or fine wire needle and place in a sterile culture medium on a cover glass. Observe under
high power of a compound microscope to ensure that the desired species has been isolated
and is unialgal.
E. Repeat the streak plate procedure with algal units from a single colony and again allow
colonies to grow and develop. This second streaking reduces the possibility of bacterial
contamination and of colonies originating from more that one algal unit.
F. Transfer algal units from a desired colony to liquid or agar medium.

Fig. 4 Streaking Plate Method.

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Spray Plating

This method is similar to streak plating except that the algal units are sprayed onto the
surface of the agar in a petri dish rather than streaked with a wire loop.

A. Prepare petri dishes as in streak plating.

B. Draw a drop of the natural collection into a Pasteur-type pipette. Depending upon the
spray-size desired, the pipette may be pulled in a manner to that of a capillary pipette.
C. Fasten the Pasteur-type pipette on a table so that the thin end is free. An air source
(compressed air, pump, etc.) should be attached to the blunt end of the pipette so a jet of
air may be used to create the spray.
D. Hold the petri dish with solidified agar about 200 mm perpendicular to the pipette.
E. Place the tip of the Pasteur-type pipette with the algal units into a jet of air passing
through a drawn pipette attached to an air source. The algal suspension will atomize and
spray onto the agar surface. The degree of separation of algal units is controlled by
varying the diameter of the Pasteur-type pipette or by varying the velocity of air flow.
F. Follow steps C-F in the preceding section on streak plating.

Figure. 5 Atomizer apparatus.

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PURIFICATION

Physiological and biochemical studies of microalgae require axenic cultures. Streak

and spray plating may yield axenic cultures directly; however, algal units isolated with a
capillary pipette may have associated contaminants. Repeated washings and transfer of algal
units with a capillary pipette may yield axenic cultures. If contaminants adhere to the algal
units, they may be physically separated by ultrasonic vibration. But for those algal units with
tenaciously attached contaminants, it may be necessary to kill or inhibit the growth of
contaminants in situ by chemical methods. Often it is easier to rid a newly-isolated unialgal
culture of contaminants rather than work on xenic cultures that have been maintained for a
period of time.
Four methods yielding axenic cultures are described here. They may be used singly or
in combination. To test that an algal culture is bacteria-free, standard microbiological media
may be employed. In addition, putative axenic cultures should be examined using phase
contrast microscopy or light microscopy at high magnification.
The methods of purification described omit reference to the use of sterile equipment.
It should be assumed that all equipment, media and other materials are STERILE!

Centrifugalization

Purification by washing is accomplished by repeated transfer of algal units through a

liquid medium and centrifuging.
A. Place algal units in centrifuge tubes half-filled with distilled water or culture medium.
Filamentous fragments or small thallus pieces may be used. The thallus may be cut on a
glass surface with a scalpel or a series of razor blades bound 1.5 mm apart prior to use.
B. Centrifuge until algal units are loosely packed; decant supernatant. Time and speed of
centrifugalization have to be determined by trial and error.
C. Suspend algal units in fresh liquid and centrifuge. Repeat the washing process and
centrifuge at least five times.
D. Using a Pasteur-type pipette, transfer single algal units to test tubes containing fresh
culture medium as in the preceding capillary pipette method.
E. When the tubes show algal growth, test for bacterial contamination.
F. Streak or spray plating methods may be used after repeated washing and centrifuging if
bacterial contamination is not eliminated.

Ultrasonic Treatment

This method uses a low intensity (90 k cycles/s) ultrasonic water bath in which
contaminants are physically separated from algal units. The algal units freed of contaminants
yield axenic cultures with repeated washings and centrifugalization.
A. Place algal units in a centrifuge tube half-filled with distilled water or culture medium.
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B. Place centrifuge tube in an ultrasonic water bath for 5 seconds to 20 minutes, depending
on algal units. Time is determined by viewing algal units microscopically for evidence of
cell damage and by testing them for viability. Death may occur before visual evidence is
available.
C. Centrifuge until algal units are loosely packed, decant supernatant and add fresh liquid.
D. Repeat sonication and centrifugalization.
E. Wash algal units at least five times, centrifuging and resuspending them in liquid between
washes.
F. Streak or spray the material onto dishes of suitable agar medium.
G. Test mature cultures for bacterial contamination.

Antibiotic Treatment

It may be difficult to free algal units of certain contaminants by repeated washing

and /or ultrasonic treatment. In these instances use of a chemical rather than a physical
method is preferred, but physical methods such as washing and ultrasonic treatment may
precede. One such chemical method uses antibiotics individually or in combination to kill or
inhibit the growth of tenaciously attached contaminants.
A. Prepare the antibiotic solution as follows: dissolve 100 mg penicillin G (K or Na Salt) and
50 mg streptomycin SO4 together in 10 ml distilled water; add 10 mg chloramphenicol
dissolved in 1 ml 95% ethanol to the penicillin-streptomycin solution; mix well.
B. Filter the triple antibiotic solution quickly, using a membrane or Seitz filter apparatus.
C. Place 1 ml of algal suspension to be purified in each of six 125 ml Erlenmeyer flasks, each
containing 50 ml culture medium.
D. Add one of the following volumes of antibiotic solution to each of the flasks: 3.0, 2.0,
1.0, 0.5, 0.25, 0.125 ml. This provides penicillin levels ranging from approximately 20 -
500 mg/l and corresponding levels of the other two antibiotics.
E. Place the flask cultures under conditions suitable for growth.
F. After 24 and 48 hours, aseptically transfer some algal units from each flask to tubes of
sterile, antibiotic-free culture medium. Prepare tubes in triplicate at each time interval.
G. Place tube cultures under conditions suitable for growth.
H. After 2-3 weeks check tube cultures for bacterial contamination.

Potassium Tellurite Treatment

This compound is a bacteriostatic agent, allowing algal units to grow away from
regions of bacteria. The clean algal units are used to establish axenic cultures.
A. Prepare 1-1.5% agar medium petri dishes containing 10 mg/l potassium tellurite (K2TeO3).
B. Place the algal units in centrifuge tubes half-filled with distilled water or culture medium.
C. Streak or spray a suspension of the algal units to be purified onto the agar medium plates.
D. Cover, invert, incubate dishes under conditions suitable for growth.
E. Using stereomicroscope examine the dishes after 4-8 days for bacteria-free algal units. If
bacteria-free units are present, isolate them into liquid or onto agar medium.
F. Test mature cultures for bacterial contamination.
In addition to the method described above, the potassium tellurite may be used in
liquid medium. Use the method outlined for antibiotic treatment substituting 10 mg/l
potassium tellurite for the antibiotic.
 19

PROBLEM AREAS

Sometimes unialgal cultures are difficult to achieve due to contamination by other

algae such as the Cyanophyceae and Bacillariophyceae (Diatoms). Some success has been
achieved in controlling diatom growth with 1-10 mg/l of germanium dioxide. Blue-green
algae can be reduced or removed by using antibiotics. Medium containing 25 mg/l
streptomycin is effective. The success of a purification treatment is dependent upon the
critical factor of timing and, in the case of chemical treatments, also upon the concentration of
chemical agents. When purification fails, isolates are either killed or the cultures produced are
not axenic. In ultrasonic treatment, too long an exposure is lethal whereas too short a time
period the contaminants are not removed from the algal units. The use of antibiotics or
potassium tellurite requires careful attention to the concentration of these chemical agents and
to the length of time of exposure to them have been suggested, but experimentation is usually
required for each alga. If a method does not work the first time, it is appropriate to Try, TRY,
and TRY AGAIN! Do not hesitate to try modifications of a method! Conditions are never
identical even on successive trials.

Alternative Techniques - Isolation

1. Glass Hook. This method is identical to the capillary pipette method except a glass hook
is used instead of a fine capillary pipette to isolate the algal units. This method is
especially useful for isolating simple and branched filaments. The glass hook is made by
passing a fine capillary pipette through a flame. The capillary bends to form a hook with
the end sealed in the form of a glass ball. This prevents algal units from slipping off the
glass hook during the steps of isolation.
2. Cover Glass Attachment. In this method, algal units from a sample of a natural
collection become attached to pieces of cover glass placed in petri dishes or other suitable
containers of liquid culture medium. Algal units may become established on pieces of
cover glass from structures such as motile and nonmotile reproductive bodies. These units
should then be isolated with a capillary tube or suitable instrument.

PRESERVATION

When it is intended to store algae in the laboratory for subsequent morphological

studies, they may be killed and preserved in a 2-4% solution of formalin (prepared by adding 4
ml of 40% formalin to 96 ml of distilled water; 40% commercial formalin is regarded as 100%
for purposes of calculation). For preserving aquatic algae, an appropriate quantity of 40%
formalin may be added directly to the sample so as to get a final concentration of 4%.
Many terrestrial or subaerial algae can be preserved dry and stored in paper envelopes
for long periods without any apparent damage or loss of viability.
For maintaining the algae in their natural (green) color, any one of the following
solutions may be employed. The algae are immersed in the preservative for a few days and
then transferred to Formalin Acetic Alcohol (F.A.A.) solution.

Formalin Acetic Alcohol (F.A.A.) Solution

1. 50% ethyl alcohol 90.0 ml

 20

40% formalin 4.0 ml

Glycerol 3.0 ml
Glacial acetic acid 3.0 ml
Cupric chloride (CuCl2) 9.5 gm
Uranium nitrate (UNO3) 1.5 gm

Although suitable for most green algae, this preservative can also be used for blue-
green algae if 10 gm of copper acetate is substituted for the cupric chloride and uranium
nitrate.

2. Cupric sulfate (CuSO4 * 5H20) 0.25 gm

Water 38.00 ml

When completely dissolved, add:

Glacial acetic acid 4.00 ml

40% formalin 8.00 ml
95% ethyl alcohol 50.00 ml

3. Potassium chrome alum 10.00 gm

40% formalin 6.00 ml
Distilled water 500.00 ml

These solutions can also be used when certain rare algae have to be exhibited in their
natural colors in a museum.

PERMANENT PREPARATIONS

Permanent slides of algae have a limited use and observations of living specimens
should be preferred to making permanent preparations.

Procedure I

This method may be used for both freshwater and marine algae. It is suitable for
morphological study and identification, but not for cytological study.

1. Place algae on a clean slide.

2. Add a small drop of 40% formalin to fix and stain (if required) with the appropriate type
of stain. A 2-4% solution of Gluteraldehyde may be substituted for the formalin. Remove
excess liquid.
3. Place a small drop of preheated (melted) glycerine jelly on the algae. Glycerine jelly is
prepared by dissolving 5 gm of gelatin in 30 ml of water with gentle heat and adding 0.125
gm phenol and 35 ml of glycerol.
4. Apply coverglass. Place the slide on a slide warming table to dry. Wipe off excess jelly
from around the cover glass. Drying may take a few days
 21

5. When the slide is completely dried, clean excess material with a single edged razor blade
and a solvent (e.g. acetone or alcohol) and . When the glycerine has hardened, seal it with
clear fingernail polish and affix a completed slide label. Store flat.
6. Substitutes for Glycerine include Eupheral and commercial slide mounting preparations
such as FloTexx (Excellent if specimen is fixed in Gluteraldehyde. Remove the fixing
solution and allow the specimen to dry. A small drop of xylene is added and quickly
blotted away. Apply FloTexx and add cover slip).

Procedure II

1. Place alga on a slide in a tiny drop of water, fix by adding a drop of 40% formalin and then
add a small drop of 10% glycerol.
2. Leave the slide in a warm, dust-free atmosphere for a few days so that the glycerine may
concentrate.
3. Add a drop of glycerine jelly and apply a warm coverglass.

Procedure III

1. The fresh material should be fixed in any one of the following solutions.
A. F.A.A.
B. 2-4% Gluteraldehyde
C. Chrome-Acetic Fixative
10% aqueous chromic acid 2.5 ml
10% aqueous acetic acid 5.0 ml
Distilled water 92.5 ml
D. Dioxan Fixative
Dioxan 50.0 ml
40% formalin 5.0 ml
Glacial acetic acid 5.0 ml
Distilled water 50.0 ml
E. Chrom-Osmo-Acetic Fixative
Chromic anhydride 1.0 gm
Glacial acetic acid 3.0 ml
1% aqueous osmic acid 1.0 ml
Distilled water 100.0 ml

2. Wash the fixed material several times in tap water. Dehydrate gradually by passing the
material through a large number of grades of ethyl alcohol (3%, 10%, 25%, 50%, 75%,
100% absolute alcohol). In the lower grades the material may be kept for 3 minutes; in
the higher grades for about five minutes. After absolute alcohol, the algae is mounted
directly in a drop of Eupheral on the slide and a cover glass is applied.
3. If stained specimens are required, any one of the following stains may be used:
A. Aniline Blue (1% solution in 90% ethyl alcohol) used for filamentous green
algae; stain for five minutes after 85% ethyl alcohol stage.
B. Erythrosine Bluish (1% solution in absolute ethyl alcohol) used for staining
gelatinous envelopes or sheaths; stain for 30 seconds after 95% ethyl alcohol.
 22

C. Light Green (0.2% solution in ethyl alcohol) used for cellulose cell walls; stain for
30-60 seconds after 85% ethyl alcohol.
D. Congo Red (0.2% solution in absolute alcohol) used for staining mucilage sheaths
of Cyanophyta; stain for 60 seconds after 95% ethyl alcohol.

Procedure IV.

This method is suitable for filamentous marine algae.

1. Fix the material in the following fixative for 12-24 hours:

A. Absolute ethyl alcohol 75.0 ml
B. Glacial acetic acid 25.0 ml
C. Concentrated solution of Ferric Chloride 3-4 drops
2. Wash in three changes of water and store in 70% ethyl alcohol.
3. Wash in distilled water and arrange on a slide in a few drops of 5% aqueous solution of
sodium carbonate.
4. Warm the slide gently and then cover the material with an albumenized coverglass.
5. Squash between two thickness’ of filter paper.
6. Invert the slide in a ridged dish containing 20% acetic acid until the coverglass (along with
the material) floats off.
7. Transfer the cover glass to a watch glass containing aceto-carmine and heat to steaming
for 7-10 minutes.
8. Transfer cover glass to 45% acetic acid and keep in this for 1-2 minutes until material
becomes pale yellow.
9. Place in 95% ethyl alcohol and then give two changes of absolute ethyl alcohol.
10. Clear in Euparal essence and mount in Euparal.
 23

CELL COUNTING AND GROWTH CURVES

Introduction

Cell counting is a valuable and necessary technique for the study of phytoplankton
numbers in both laboratory culture and field work. Experiments completed on phytoplankton
in culture require knowledge of the age and health of the population. Cell density (determined
by cell counts) plotted against time yield a growth curve for the population. From this growth
curve, the investigator can determine the age of the population at the time of the experiment.
With cell density data, the experimenter can relate physiological processes to a per-cell basis.
While counting cells, one can assess the health of the population by monitoring the relative
numbers of dividing, broken or dead cells. Cell counting is a much used technique in
phytoplankton culture work.
To perform cell counts on cultured phytoplankton, you will use some type of counting
chamber and the compound microscope. The procedure involves: placing a known volume of
your culture in a hemocytometer or counting chamber; counting the number of cells that
appear in several microscope fields, so that you can obtain the mean number of cells per
known volume; and converting your replicated counts to number of cells per milliliter.
In the laboratory, you will be required to maintain cultures for a duration of time. It is
therefore important that you learn and follow proper techniques for keeping your cultures
alive and healthy. You should note, in particular, the following points:

1. Avoid contamination of your stock culture by ambient bacteria and fungi by using sterile
techniques. Be sure to flame the culture flask during transfer of cultures. When obtaining
a cultures sample, never stick a pipette into the culture flask. Instead, pour a small
amount of culture (mix it first) into a sterile beaker and draw your sample from this supply.
Also, never place the stopper on the counter. Hold it in your hand, and be sure not to
touch the part of the stopper that will be reinserted into the flask.
2. Your culture may become too dense for its flask. If need be, split a culture that is too
dense by adding an equal volume of fresh medium, swirling and pouring half of it into a
new sterile flask. Remember to flame both flasks.

Procedure

1. Pour a well-mixed sample into a small beaker. Remember to use sterile technique!! Add
one drop of diluted Lugol’s Solution to kill the cells.
2. Determine which counting chamber is appropriate for your culture. We will use the
Improved Neubauer Hemocytometer. This is the best with dense cultures of small cells.
Use a Pasteur pipette to bubble the sample and mix it.

Hemocytometer Instructions.

1. The hemocytometer has two counting grids onto which you should place your sample.
Discard the first drop in the pipette, as it contains few phytoplankton cells. Then apply
one drop to each grid at the point shown by the arrow in the figure. The Improved
Neubauer grid has nine squares, each one mm along a side and is further divided into 20 to
25 smaller squares. The chamber is 0.1 mm deep, therefore each grid (9 mm2) holds 0.009
ml of sample.
 24

Fig. 6 . Hemocytometer. Fig. 7 Sedgwick-Rafter Chamber

2. Use the lower power to scan the grid in order to aquatint yourself with the setup and
to identify the plankton that you will be counting. Make sure that you can distinguish
cells from detritus. Decide if cells are IN or OUT. Are dividing cells counted as two
cells? These are questions that you must answer for yourself. Whatever you decide,
you must be consistent.

Fig. 8 Improved Neubauer Ruling.

1. Use the magnification that enables you to count your cells most easily. Be careful not
to crack the special hemocytometer cover slip. Always start with the ocular in the
lowest position and move it upwards until in focus.
2. Use a counter to keep track of your results. Individual counts of at least 30 cells per
unit are statistically desirable. Counts above 30 cells per unit area may become
 25

unmanageable. Choose the unit area to be counted accordingly. Select areas to be

counted randomly from both grids and do at least 7-10 replicate counts. Calculate a
mean, standard deviation and coefficient of variance from your counts. A basic
statistics book can help you to understand the statistical implications of these
measurements.
3. Multiply the means of your counts by the factor which will give the average number of
cells in the entire grid. Multiply this value by 1 ml divided by the volume of the sample
on the grid. Your calculations are:
A. Mean number of cell / unit area * area of entire grid/unit area counted = number of
cells/grid.
B. Number of cells/grid * ml/volume of sample on the grid = number of cells/ml

For example, with a Neubauer hemocytometer you might have counted cells in the
1mm2 squares. You would then multiply your mean count (let’s say it was 20 cells) by nine to
obtain the average number of cells over the entire grid, or 180 cells. The grid has a volume of
0.009 ml. Therefore, the number of cells per ml for your sample = 180 cells * (1ml/0.009 ml)
= 2.0 * 104 cells ml-1.

Fig. 9 Fuchs-Rosenthal ruling.

 26

GROWTH CURVES

You will count cells from your cultures during each laboratory period over the course
of several weeks (more often if necessary!). Once you have obtained the data set of sequential
cell counts, you are to construct a growth curve from your data. You should plot the number
of cells against time in days. From these curves, you can calculate two useful parameters of
population growth: Specific growth rate or growth constant (u) and the division time or
generation time (tg).
Your plot of cell number against time probably shows an S-shaped growth curve. At
first, the cells were not dividing. This is the lag phase. Within a few days, the cells started to
divide and your curve probably shows a rapid increase in cell number over time. This is the
exponential or log phase and here the rate of increase in cell number is proportional to the
number of cells present (which is increasing). Finally, your curve may show a plateau, or
leveling off, of cell density. This final stage is the stationary phase and is indicative of a
population that may have grown too dense for its container and that will be light or nutrient-
limited.

The growth rate shown by your phytoplankton cells during the exponential phase is
and important characteristic of your particular culture, as is the division time (or time for your
cells to divide). You should calculate both the growth rate and division time for your culture
using the following equations:

1. The growth rate can be calculated with the differential equation

dX/dt = uX

where X is the number of cells, u is the growth rate and t is time in days. Rearrangement of
this equation yields:

u = 1n X2 - 1n X1 / t2 - t1

where X2 and X1 are cell densities at times t2 and t1.

2. Division time, t g, is the time for cells to divide (in days) and can be calculated from the
growth rate:

tg = 0.6931 / u.

For example, you have two counts on day six and day eight (which coincide with the
exponential phase of growth). The counts are 5.0 X 10 3 and 1.5 X 104 cells ml-1 , respectively.
Your calculations for growth rate and division time are:

u = 1n X8 - 1n X6 / t8 - t6
= 1n 1,5 X 104 - 1n 5.0 X 103 / 2
= 1.0986 / 2
= 0.5493
 27

tg = 0.6931 / u
= 1.2618 days
 28

PREPARATION OF HERBARIUM MOUNTS

Materials:
Plant Press Wax Paper Nylon Cloth
Blotters Corrugated Ventilators Small Paint Brush
Newspaper Bulb Syringe (Pipette) Paper Towels
Herbarium Sheets Scissors (Small) Shallow Pan

Procedure: (Angiosperms)

1. Fold a single sheet of newspaper in half. On either the outside long edge or short edge,
write the collection number, and the date of collection.
2. Remove the plant from your collecting bag, leaving the rest of the plants covered so that
they will not wilt. Wash any remaining dirt off the roots and spread the plant out in a little
less than the space of a half newspaper sheet. It is important to take plenty of time,
DON’T HURRY!
3. Spread each leaf out flat. Turn over at least two leaves to show the underside, as the
hairiness of that underside is often a crucial part of identification. Carefully spread open
the flowers, making sure every petal lies smoothly. Try to press the flower in such a way
as to best show important characteristics. Try to show the interior of at least one flower
even if the important characteristics are unknown to you. Some of the flowers should lie
with the lower side uppermost, as did the leaves.
4. If the plant is too long for the sheet, fold it sharply in a “V”; if that is not enough fold it
into an “N”; and if that is not enough, fold it into a “W”. If it is still too big, cut it and
use only part, or press it in several sections. Be sure to describe the original size of the
plant in your field notebook.
5. Small strips of wet newspaper are effective in holding down thinner flowers and leaves and
does not have to be removed before pressing.
6. Open the plant press, lay a corrugated cardboard ventilator on one grid, then a blotter or
heavy thickness of newspaper on that, then the pressed plant on the paper. If heavy
newspaper is used as a blotter, slide in the folded single sheet of newspaper, with plant
inside, so that its open edge is against the closed edge of the thick folded newspaper. This
helps hold everything in and makes changing the heavier paper easier. If you are using
blotters, lay a second one on top of the thin newspaper, then a second corrugated sheet,
and you are ready to repeat the procedure with the next plant.
7. When all the plants have been pressed, place them, as described, in a series of sandwiches
in the plant press and strap the press shut, as tightly as possible. Having one student stand
on the press while the other pulls the straps tight helps produce flat specimens. Loose
strapping gives wrinkled leaves and flowers.
8. Set the press where a current of warm, not hot, air can flow up through the ventilators. A
large drying cabinet with light/heating element and a fan may be used.
9. Most plants dry in 2 or 3 days to a week. After 24 hours, go through and change or
remove the blotters. This is very important. The faster the plants dry, without high heat,
the better the flower color will be retained. After removing the blotters, return the press,
strapped shut again, to the warm drying area.
PROCEDURE: (Algae)
 29

1. Because these plants collapse when removed from the water, in order to press them you
must first float them out in a shallow pan of water (saltwater if they are marine plants).
Slide a piece of herbarium paper underneath the floating plant, use a piece only large
enough to take the entire plant. Arrange the delicate thalli and branches carefully with the
fingers, a narrow brush, blunt probe or use water from a pipette to aid in spreading the
fine branches and structures. Slowly withdraw the paper from the water, tilting it slightly
to allow the water to flow off gently.
2. Drying algae in a press is similar to drying land plants, except that the top of the plants
must be covered with something to prevent their sticking to the enclosing paper or blotter.
Either wax paper or a piece of unbleached muslin is good, but I prefer the use of white
nylon lining material. It is cheap, reusable and dries very quickly. Write the collection
number and date on the outside margin of the herbarium sheet. Care must be taken to
ensure that in every step in the process that accurate records are maintained. Some marine
algae grow to great size, in which case only part of the plant can be pressed while the rest
must be described. Some become very brittle and do not adhere well after drying. These
can be carefully removed from the herbarium sheet (after drying) with a scalpel or single
edge razor blade and re-glued with white glue to a clean sheet of herbarium paper.
3. Algae are also preserved in fluids such as 3-5% formalin in seawater, 70% ethyl alcohol,
FAA (Formalin-Acetic Alcohol) or my favorite, 2% Gluteraldehyde. The algae should be
transferred to new solution and into an appropriate size jar or vial upon returning to the
laboratory. Again do not forget an identification tag (mylar) inside the container and a
label on the outside. If the containers will remain on the shelf for a long period of time, it
is often necessary to dip the top of the container, cap and all into melted paraffin (wax)
and allowed to cool. This will help prevent evaporation of the preservative.
4. Preparation of permanent microscope slides as voucher specimens are sometimes
necessary and desirable. This is especially important in the collection of reproduction
structures from field and pressed specimens that are necessary for identification. The
specimen to be mounted must have been preserved earlier in the field or laboratory. The
alga or structure from an alga is placed on a microscope slide and arranged so that it is
one plane. A drop of 0.5 to 1.0% aqueous stain is added and fixed (if necessary) with a
drop of 1% aqueous hydrochloric acid. This is rinsed off by flooding with distilled water
and holding the slide at a slight angle over a paper towel to drain. The excess stain can
also be carefully removed by blotting along the edges of the thallus with a piece of paper
towel or bibulous paper. Add a drop of alcohol, whisk it away with blotter and allow to
air dry. Repeat the procedure with a drop of xylene. A drop of FloTex is added to the
specimen. A cover slip is added carefully, one edge resting on a probe or forceps, and it is
gently lowered over the mounting media surface. Gently work any air bubbles out from
under the cover slip. The slides must be kept on a flat surface. It is helpful (in our high
humidity) to place the slides on a slide warming table. When dry, add a proper label to the
slide. The slide should be stored flat for 2-4 weeks and then may be stored on edge in
standard slide boxes.
5. To complete the documentation, an adequate label must be prepared to accompany the
field or collecting number that was first assigned to the specimen, and necessary cross-
indexing must be added to the herbarium materials. Any preserved material or slides
containing a subset of the material, for example, should be noted on the herbarium sheet.
6. A voucher specimen is fully prepared when:
a. it is a dried, pressed, herbarium specimen that is adequately labeled
b. it is a dried or wet-preserved specimen that is labeled and in a container, or
 30

c. it is a small specimen that is mounted on a prepared microscope slide and labeled.

A label should contain:

 Genus, specific epithet and author of the species name
 Family
 Common Name (if it has one)
 Locality (Country, state, county, etc.)
 Habitat notes
 Miscellaneous observations
 Collector’s full name
 Collection number
 Date
 Name of the person identifying the plant

Fig. 10. Loading a plant press.

A = Binding strap, B = Plant press top, C = Corrugated cardboard ventilator, D = Newspaper or blotter, E =
Single sheet of newspaper folded or herbarium paper (algae) with nylon covering, F = Plant being pressed.
 31

Fig. 11 Screw type plant press

 32

REFERENCES

Fryxell, G. 1975. Morphology, taxonomy and distribution of selected species of the diatom
genus Thalassiosira Cleve. In the Gulf of Mexico and Antarctic waters. Ph.D.
Dissertation. Texas A&M University, College Station. 189 pp.

Kumar, H.D. and H.N. Singh. 1971. A Textbook on Algae. Van Nostrand Reinhold Co.,
New York. 200 pp.

Lee, R.E. 1980. Phycology. Cambridge University Press, New York. 478 pp.

Lobban, C.S., D.J. Chapman and B.P. Kremer (Eds). 1988. Experimental Phycology: A
Laboratory Manual. Cambridge University Press, New York. 295 pp.

Prescott, G.W. 1978. How to Know the Freshwater Algae. Wm.C. Brown Co. Publ.,
Dubuqne, Iowa. 293 pp.

Stein, J.R. (Ed). 1973. Handbook of Phycological Methods. Cambridge University Press,
New York. 448 pp.