INTRODUCTION

A simple medium using boiled wheat and a mixture of Giese salts and powdered non-fat milk in which Chilomonas paramecium is grown as a food organism has been suitable for maintaining cultures of Lacrymaria olor for over eight years. Due to the abundance of food organisms, the extensility of the neck of Lacrymaria is reduced, but this is easily remedied by transferring the organisms to small culture dishes containing only Giese salts and powdered non-fat milk.

PROTOCOL

Two kinds of cultures are maintained. The first is a method for keeping good numbers of organisms in a low-maintenance manner for 4 to 6 weeks. The second method produces active organisms for investigation.

TUBE CULTURES

These are used for long-term maintenance (4 to 6 weeks). Wheat grains are boiled for 5-7 minutes. The culture tubes used are 16x125 mm. A boiled wheat grain is placed in each tube and a solution of modified Giese salts is added until the tube is two-thirds full. The tubes are then inoculated with Lacrymaria from an existing culture or from specimens isolated from collected pond samples.

If one is starting cultures for the first time from wild specimens from pond samples, perseverance is required. Every medium used to start cultures from wild specimens has produced erratic results and it is only with patience that one can finally get productive cultures that are then relatively easy to subculture. Use ten or more tubes to try to get them started and put several specimens in each tube. Isolation can be done with a micropipet which can be made by drawing out a fine point on standard 5¾ inch disposable Pasteur pipet over the flame of an alcohol lamp. This takes a bit of practice. Make up about 50 such micropipets at a sitting for convenience.

Each culture is loosely plugged with a small ball of cotton to allow for some air circulation.

Modified Giese salt solution:

A standard solution of Giese salts is made up in its full dilution.(1)

NaCl 1.78 X 10^-3 M

KCl 3.08 X 10^-4 M

MgCl₂ 4.16 X 10^-4 M

MgSO₄ 1.62 X 10^-6 M

CaCl₂ 6.80 X 10^-6 M

This solution is then diluted again by 1/2 with artesian or distilled water. To 250 cc of this solution add one pinch of non-fat powdered milk and shake thoroughly. This mixture is used for both tube and dish cultures.

DISH CULTURES

Use small culture dishes (2½") and fill 2/3 full of the modified Giese salt solution (with the powdered milk). Inoculate with Lacrymaria from the tube cultures. After 2 or 3 days, the Lacrymaria will begin to be more active and their extensility will increase significantly. Also the cells which are often full of crystals in the tube cultures will begin to clear. These cultures need to be monitored regularly as they rarely last much more than a week.

1) With persistence a number of different strains or ecotypes of Lacrymaria olor can be obtained. The specimens used to maintain these cultures have been collected from a variety of locations. Generally Lacrymaria are considered detritus dwellers and are often found in substrates that have a high concentration of sulfur bacteria. It will be noticed that the tube cultures after several days will have a rather unpleasant odor. However, specimens of Lacrymaria have also been found in a moss sample collected in very clear, highly oxygenated water near a spring. Others have reported finding specimens in rivers, so there is no certain place to look for specimens. Cultures from five different environments have been maintained for over five years.

2) The tubes should be agitated about once a week. There will be a scum which will form along the surface and when using these cultures to inoculate others be sure to agitate the culture and include bits of the surface scum, especially from around the water line, in starting the new cultures. Use 1 to 2 cc of the culture for inoculation.

3) After using the tube cultures for inoculation, refill the tube to the 2/3 full level by adding modified Giese salt solution. If this is done every week to ten days, the cultures will last for 6 weeks or more.

4) New tube cultures should be started about every two weeks. These subcultures have to be checked about once a week to be sure that they are growing properly.

5) In the tube cultures, Lacrymaria olor will be relatively inactive and the cells will tend to get heavy and oblate showing little extensility. However, this soon corrects itself, when the Lacrymaria olor are transplanted into the culture dishes containing only the modified Giese salt solution.

6) Cultures may also be started in dishes by using the modified Giese salt solution and adding one grain of boiled wheat. The results with these cultures is fairly unpredictable and these cultures must be closely monitored.

7) In all of these cultures, the standard food organism is primarily Chilomonas paramecium. However, good success has also been achieved by raising Lacrymaria olor in cultures which contain large numbers of a very small amoeba (species unknown).

8) Lacrymaria olor is somewhat unpredictable and initial attempts to culture it in large numbers may be unsuccessful. However, using the combination of the two major methods described above should provide good results.

9) The cultures can be observed by tipping the tube, after agitating by hand, under a dissecting microscope at a magnification of about 20x. The organisms tend to concentrate around the water level and are often attached to the thin layer of scum which forms on the surface of the medium.

10) A drop or two of 0.001% solution of Acridine Orange has been found to be beneficial in maintaining the cultures.

COMMENTS

Lacrymaria olor is an organism which provides interesting research possibilities because of its extraordinary extensility and regenerative capabilities. It is difficult to start from wild cultures, but once established in the laboratory, stable cultures can be maintained for several years.

LITERATURE CITED

1. Giese, Arthur C 1973."Blepharisma:The Biology of a Light-Sensitive Protozoan", Stanford University Press, p.107.

Comments to the author Richard Howey welcomed.

Richard L. Howey, Adjunct Professor
Department of Zoology and Physiology,
Biological Sciences Building
The University of Wyoming,
Laramie, WY 82071
USA

Editor's note:
The author's other articles on-line can be found by typing 'Howey' in the search engine of the Article Library, link below.

Return to the article 'The Microscopic Loch Ness Monster' by Richard Howey.

Read the Micscape article 'Tear of a Swan' compiled by Maurice Smith and which presents the work of Richard Howey and features video clips / stills by Ken Jones.

 

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Published in the April 2000 edition of Micscape Magazine.

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