ISOLATION TECHNIQUES

Diatom culturing was initially done with the natural light as the source of illumination (Miquel, 1892-93; Allen and Nelson, 1910). Later, it was Warburg (1919) and Hartmann (1921) who contributed significantly to use of electric lights as a source of illumination. Use of a screen of cold water between the lights and the cultures to avoid heating was also contributed by them (https://ccmp.bigelow.org/accessed on 20^th June 2011, 19:00 hrs). To provide light which nearly matches the natural light full spectrum, fluorescent bulbs are used (Andersen and Kawachi, 2005).

The maintenance of sterile technique was first adapted from microbial research (Beijerinck, 1890, 1891, 1893; Miquel, 1890/92a-e). These were then replaced by the Laminar Air Flow (Price et al., 1989) and sophisticated microwave sterilization (Keller et al., 1988). “Isolation” of an organism (or multiple organisms at a time) describes the process by which individual cells are physically separated from each other and/or from matrix material, such as water, air, soil particles, or eukaryotic tissues. Isolation therefore represents the most crucial step during the process of obtaining pure cultures (Zengler, 2009). Isolation based culturing gained impetus with Pasture’s work on bacteria and fungi. A pure culture consists of one species whose identity is known and contains progeny of that species alone. Attempts of Beijerinck, a Dutch microbiologist in obtaining axenic (“pure”) culture from cyanobacteria (Beijerinck, 1901) and diatoms (Beijerinck, 1904) were allegedly fruitful. Miquel (1893d) was however the first one to obtain axenic cultures of diatoms followed by Lockwood, Karsten, Stenft, (Eppley et al., 1977), Richter (1903) and Chodat (1904). Invariably to acquire pure culture of diatoms, isolating techniques are very important. Isolating specific freshwater and marine diatoms into culture was primarily done by Miquel (1893a-e) with contribution of Macchiati (1892a, b, c) for obtaining axenic cultures of diatoms. The isolation techniques are broadly grouped into:

1. Manual isolation technique.
2. Automated isolation technique.

1. Manual isolation technique:

The Pasteur pipette technique could be a viable method due to its narrow mouth and fine sized nozzle which is useful for the passage of most of the diatoms. However, the laborious technique has limitation in its inability to be used for the sample which has less of bacillariophyceae members as other members might pass through the opening. The above three mentioned pipettes (micropipette, capillary pipette and Pasteur pipette) have their own role to play in eliminating bacteria or other algal forms (except diatoms) to a certain extent. Depending on the opening of the pipettes they can be used for the sample ranging from a higher diatom population to a lower one.

Therefore, use of all the three techniques in complementary to each other could give an axenic culture, although, automated microinjectors could be a viable replacement of these three techniques.

b.    Agar plate method combined with antibiotic treatment: Agar plating method is used for the isolation of diatoms infested with bacteria, algae, etc thereby acquiring axenic culture of diatoms. Generally, higher concentrations of antibiotics combined with short-term incubations were more efficient than using low concentrations for longer periods.

Algal contamination: Diatoms are first concentrated by continuous centrifugation or sonication to avoid clumping and then isolated by a micropipette onto an agar plate containing the required media. This is then followed by repeated subculturing and streaking the colonies onto agar plates (Knuckey et al., 2002). Streaking of smaller fast-growing diatoms (1–5 µm) on agar plates is followed to separate the organisms without the need of antibiotics (Bruckner and Kroth, 2009).

Bacterial contamination: Microscopic observation of the larger benthic diatoms during exponential growth phase is suggested due to low population of bacteria (Bruckner and Kroth, 2009). Spreading the diatoms after ultrasound treatment (for 10 s, at an amplitude of 40 ℅ at 0.5 s intervals) or by vortexing (10 mins) on agar plates containing high concentration of antibiotics (Penicillin G, Streptomycin and Chloramphenicol) followed by removal of single cells by a suitable micropipette is recommended.

Co-culture with E. coli is also recommended since many diatoms in coculture with bacteria grew denser and faster than while being axenic (Bruckner et al., 2008). Often, such bacterial effects on diatom growth were inducible by E. coli. This was followed by antibiotic treatment (Penicillin G, Streptomycin and chloramphenicol) at higher concentration. Three diatom cultures (Achnanthes linearis (W.Sm.), Gomphonema clavatum Ehr., Navicula cincta (Ehr.) Ralfs.) were purified by substituting the associated bacteria with E. coli. Purification of diatoms from unialgal cultures usually was more difficult and less successful than from biofilm samples (Knuckey et al., 2002, Bruckner and Kroth, 2009).

These methods use combinations of most of the techniques and eliminate bacteria assuring high susceptibility of acquiring axenic cultures.

c.    Serial dilution method: Serial dilution technique was developed in late 19^th Century as an isolation technique to obtain axenic cultures of diatoms (Miquel, 1890/92d, e; Allen and Nelson, 1910), which later led to its exhaustive description (Kufferath, 1930; Droop, 1969 and Throndsen, 1978).  However, axenic isolates are not often obtained with this dilution technique, because bacteria are usually more abundant than algae (Andersen and Kawachi, 2005).

A centrifugation technique to isolate algae was introduced by Mainx (1927). Centrifugation was done at 1000 revolutions per minute (rpm) for 10 minutes, (Price et al., 1978) to separate mixed cultures of diatom species like Thalassiosira pseudonana, Skeletonema costatum (Grev.) Cl., Cyclotella cryptic Reimann, Lewin & Guillard, Pheodactylum tricornutum and Nitzschia species with the help of density gradients (Peroll, silica solution) (Price et al., 1978).  Gentle centrifugation for a short duration can be implemented for the isolation of dinoflagellates and diatoms (Andersen and Kawachi, 2005). Centrifugation technique with minimal speed ranging from 1000 – 1500 rpm for 10 minutes is apt as high speed would lead to clumping of diatom cellular mass.

2. An automated isolation technique like flow sorting was also attempted successfully to isolate diatoms (Reckermann and Colijn, 2000).  Production of cultures of Thalassiosira, unidentified diatoms and pico-eukaryotes from mixed natural assemblages has also been done (Reckermann, 2000).  The main advantage of the flow cytometric sorting is the simultaneous use of multiple cell characteristics to identify the cells enhancing much needed accuracy and speed in analysis (Ueckert et al., 1995).  If the sorting is done carefully, purity of the sorted cells could be as high as 98℅ (Hoffman and Houck, 1998). However, the disadvantages are the relative complexity, cost of the instrumentation and requirement of relatively longer time to obtain large numbers (millions) of sorted cells (Hoffman and Houck, 1998). This is not a serious concern in recent time due to the applications of the isolated diatoms in various fields.

Sophisticated instrumental techniques for the analysis and characterization of microorganisms are becoming more common. Although these newer, often experimental approaches will not replace traditional methods involving cultures, microscopy, etc. in the immediate future, their development will continue to grow (Isolation, purification, techniques, etc).

Combining techniques like flow sorting, Pasteur pipette and agar plating methods, would improve the possibility of pure isolated cultures. The former technique aids in primary isolation to quantify the diatom population from an algal sample and the latter isolates the required diatom species from the concentrated mass.  Combinations of various isolation techniques are responsible for the establishment of many axenic cultures of diatoms in collections like The Provasali-Guillard National Center for Culture of Marine Phytoplankton (https://ccmp.bigelow.org/), UTEX The culture collection of Algae (http://web.biosci.utexas.edu/utex/), etc.