Materials and Methods in the Study of Protozoa

By Harold Kirby

This title is part of UC Press's Voices Revived program, which commemorates University of California Press’s mission to seek out and cultivate the brightest minds and give them voice, reach, and impact. Drawing on a backlist dating to 1893, Voices Revived makes high-quality, peer-reviewed scholarship accessible once again using print-on-demand technology. This title was originally published in 1950.
This title is part of UC Press's Voices Revived program, which commemorates University of California Press’s mission to seek out and cultivate the brightest minds and give them voice, reach, and impact. Drawing on a backlist dating to 1893, Voices Revived

• Language: English
• Publisher: University of California Press
• Release date: Nov 15, 2023
• ISBN: 9780520348141

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MATERIALS AND METHODS IN THE STUDY OF PROTOZOA

BY

HAROLD KIRBY

Professor of Zoölogy, University of California, Berkeley

COPYRIGHT, 1950, BY

THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Price: $2.50

UNIVERSITY OF CALIFORNIA PRESS

BERKELEY AND LOS ANGELES

CALIFORNIA

CAMBRIDGE UNIVERSITY PRESS

LONDON, ENGLAND

PRINTED BY OFFSET IN THE UNITED STATES OF AMERICA

In pursuing our researches we have become practically convinced of the importance of what we have theoretically assumed -- the absolute necessity for prolonged and patient examination of the same forms. Two observers, independently of each other, examining the same monad, if their inquiries were not sufficiently prolonged, might, with the utmost truthfulness of interpretation, assert opposite modes of development. Competent optical means, careful interpretation, close observation, and time are alone capable of solving the problem.’ -- Dallinger and Drysdale, 1874

PREFACE

Information given in this manual was originally compiled for use in instruction at the University of California, in order to have in readily accessible form some schedules of procedure. Among the data are techniques that are followed routinely in laboratory operations. But the compilation has been extended beyond provision of directions for routine procedure for a laboratory course. It deals with methods that are of value in providing and preparing materials for laboratory instruction and investigation, and with some techniques that may be useful in meeting special requirements, There is, however, no pretense to completeness in treatment of protozoo- logical methods or in presenting and evaluating advanced and refined techniques. The ways of collecting, cultivating, preparing, and studying particular protozoa must be adapted to each situation, and the most suitable methods must be learned by individual experience.

There must always be a beginning, and even an inadequate survey of established procedures is useful in laying a foundation and organizing a framework within which to work. The microscope reveals a world of living things and there are many ways of investigating that world. One person may feel that knowing the protozoa holds his interest no less than knowing the birds or trees or insects holds that of many persons. He may be content with collection and observation of living organisms. Another will study the relation of protozoa to one another and to the organic and physical environment. Another, again, will wish to preserve specimens for comparative studies, and to explore the details of cytology. The manifold types of life history, the problems of sexual differentiation and inheritance, the physiological activities, and the nutrition of protozoa are other matters for the concern of biologists. The student who continues to work with these organisms ultimately chooses a restricted phase of investigation. It may be a study of some aspects of the biology of protozoa, it may be a practical phase such as the diagnosis of protozoan infections of economic and medical importance, or the use of Foraminifera and Radiolaria in geological exploration. However it may be, he uses one set or another of standard techniques. The techniques dealt with here are mostly ones for securing, maintaining, and making preparations of protozoa for observation.

One of the useful features of this book should be the presence of blank pages opposite the printed pages. On them can be entered the corrections, additions, and modifications that will undoubtedly be necessary.

Acknowledgment is humbly and gratefully made to the authors and publishers of many books and articles from which information about methods has been obtained; and to the persons, associated with this author at one time or another,from whom he has learned the results of their experience.

HAROLD KIRBY

Berkeley, California

June, 1950

CONTENTS

CONTENTS

I. COLLECTION AND CULTIVATION METHODS FOR FREE-LIVING PROTOZOA GENERAL COLLECTION METHODS

KINDS OF CULTURES

CULTURE VESSELS

ISOLATION TECHNIQUE

MATERIALS AND PROCEDURE

REFERENCES ON COLLECTION AND CULTIVATION OF FREE-LIVING PROTOZOA

ADDENDUM SOIL-CHEESE MEDIUM

II COLLECTION AND CULTIVATION METHODS FOR SYMBIOTIC PROTOZOA MATERIALS AND PROCEDURE

REFERENCES ON COLLECTION AND CULTIVATION OF SYMBIOTIC PROTOZOA

III. TECHNICAL METHODS OF STUDY AND PRESERVATION SCHEDULES AND PROCEDURE

REFERENCES ON TECHNICAL METHODS

ADDENDUM REGAUD HAEMATOXYLIN

INDEX

I. COLLECTION AND CULTIVATION METHODS FOR FREE-LIVING PROTOZOA

GENERAL COLLECTION METHODS

Plankton forms are collected by means of a net of the finest grade of bolting silk. In lakes, the net can best be used from a boat. The forms collected should be brought to the laboratory in bottles (thermos bottles if the air temperature is high) and examined as soon as possible.

Plankton forms may be preserved by adding formalin to the water to make about a 5% solution; or they may be concentrated, fixed and stained by appropriate techniques.

The smallest forms will not be taken by a plankton net. To secure them the water must be filtered or centrifuged.

Samples of water from lakes, ponds, streams, water troughs, and other places may be collected in jars. Merely dipping into the water usually will not yield very much. Water samples should generally be taken where there is plant growth or organic debris. The water to fill the jar may be in part expressed from the plants, and some of the plant material should be taken along, but the jar should not be filled with it. The collection may also include samples of any slimy growths that are present on the bottom or on objects in the water, dead leaves, some sand from the bottom, pieces of sticks from the water, and surface scum.

After reaching the laboratory, the collections should be put out in crystallizing dishes or refrigerator jars. The bottom may be covered by sand or soil and some plant material included. In an aquarium of this sort the protozoa originally active at the site where the collection was made may live for some time; they usually will not become especially abundant. If more organic material is added, there will be an increase in numbers of some forms, but others will perish. In order to obtain large populations, cultivation methods are used.

Many protozoa may be captured on slides or cover glasses immersed in water. One method of obtaining some of them is to float cover glasses on the surface; this method can be used successfully only in indoor containers where the water is quiet. Slides immersed in the water in aquaria, or in the natural environment, will provide many forms for observation or for making preparations. Slides can be held in grooves in a piece of wood, which is fastened to a support under water or to a stake inserted in the bottom. Fauré-Fremiet (1931) recommended supporting them in grooves in a wooden frame similar to a slide box without top or bottom. The slides are separated by about 1 centimeter, and are held in the frame by strips of wood. The frame may be floated in the water, or fixed vertically. Attached organisms occur in the best condition on the lower side when the slide is horizontal. Immersed slides may be used for collection of protozoa in fresh water or in sea water. Various rhizopods, attached peritrlchs, and thigmo- tactic hypotrichs may be captured, among other forms.

Marine plankton protozoa may be collected by filtering or centrifuging samples of sea water, or for the larger species a fine plankton net may be used. Lackey (1936) secured a variety of bottom-dwelling marine protozoa on Syracuse dishes enclosed in wire cages to exclude larger organisms and suspended from a float at Woods Hole. Within 24 hours many thousands of individuals of many species of amoebae, flagellates, dilates, and one species of suctorian had accumulated on a square centimeter of an immersed dish. Enrichment cultures may be prepared by adding to filtered sea water nutrient materials and the sample to be studied.

KINDS OF CULTURES

A raw sample, obtained from the field, contains a mixture of organisms which are not under control, and as the culture ages the population may rapidly change in character.

An impure species culture of a protozoan contains one species as the dominant form,.and by suitable methods of feeding and transfer it can be maintained. Along with the species are unknown bacterial populations and other organisms. Most protozoa for general laboratory use are maintained in cultures of this sort.

A greater degree of control is obtained when a species is kept free of other organisms except those that are added for food. This method requires more refinement in original isolation and maintenance.

A pure culture is one in which the protozoan is the only organism. The culture is maintained bacterla-free, and the medium is a rich organic one which serves for nutrition of the protozoan. These media usually cannot be used when multiplying bacteria are present.

CULTURE VESSELS

Cultures may be kept in containers of various sorts, according to the requirements and to the degree of control that is used.

Vessels of ordinary glass, such as refrigerator dishes, drinking glasses, and jars, may be adequate for maintaining many general cultures. The glass may have unsuitable qualities, and more refined glass of the type used in making laboratory apparatus is often better.

For cultures with small numbers of the protozoa and for frequent transfer depression slides are used. — These may be kept in a closed dish in a saturated atmosphere.

Syracuse watch glasses are useful for larger populations and for cultures following isolation when mass cultures are being built up. Small Petri dishes may also be used.

Finger bowls (laboratory type) are useful for maintenance of mass cultures. They can be stacked one on another to provide covering and save space. Cultures of amoebae and other forms may be kept in such containers, and observations can be made directly under a stereoscopic microscope.

When more fluid is used, moist chambers of larger size are suitable containers. A greater volume of fluid is an advantage in cultures that are to be maintained for a long time with repeated feeding.

Erlenmeyer flasks are good for maintenance of mass cultures of many forms, but not for bottom-dwelling types such as rhizopods, which are best kept in moist chambers without great depth of water. The liter-size flask is useful for mass cultures of such protozoa as Euglena, Paramecium, and other free-swimming dilates. The flasks can readily be sterilized while they are stoppered with cotton, and evaporation is hindered, especially if a small beaker is inverted over the top. Samples can be taken and transfers made simply by pouring from the flask. The flask should be filled to not more than half its height.

Cotton-stoppered test tubes can be used for cultures of various sorts, and may be employed in the maintenance of pure cultures. Agar culture media may be made in slants in test tubes or placed in Petri dishes.

Unless crude mixed cultures are all that is desired, the glassware should be well cleaned and sterilized before use. It should not have been cleaned with sulfuric acid and dichromate cleaning solution. Wichterman (1949) recommended immersing in 10% nitric acid solution. It should never have been in contact with formalin, fixatives, or poisons. Even formalin fumes In the same room are detrimental to cultures.

ISOLATION TECHNIQUE

It is desirable for many purposes to begin cultures from single specimens of protozoa. When this is not necessary, but it is desired to maintain only the one species, cultures may be started from several specimens together; often the group does better than a single individual would.

Wild cultures brought into the laboratory may be examined under a stereoscopic microscope for the desired forms. These are then picked out with a pipette drawn out to a small end. The glass tubing used to make the pipette should not be too thin. Mouth pipettes are frequently used; a long rubber tube is held in the mouth and aspiration regulated by means of the breath. A rubber nipple on the pipette, regulated by pressure of the fingers, is often satisfactory enough. Transfer through a series of two or three watch glasses may facilitate freeing the forms desired of other species of protozoa.

In beginning a culture, use of a depression slide with as large an amount of culture medium as possible may be advantageous.

Acclimatization is often necessary; direct change from the original fluid to the culture medium may be unfavorable. The culture medium to be used may be diluted with the original fluid and the volume of fluid increased with higher proportions of culture medium as the organisms multiply.

Certain protozoa may serve as food for certain other protozoa. To maintain the latter, the food forms are cultivated separately and given as required.

It may be desirable to free the protozoa of bacteria, in order to keep them in pure culture or feed them on known bacteria. Some methods of doing this are described by Trager (1937), Kidder (1941), Hetherington (1934a), Phelps (1934), and Claff (1940).

In the migration method described by Trager, pipettes 14 inches long,, with 1/4 inch bore at one end and tapering at the other end are used. The large end is plugged with cotton and a rubber tube attached. The apparatus is sterilized and sterile water drawn up to within 2 inches of the top. 2 cc. from a rich culture of the protozoan are then sucked in, and the tapered end is sealed by heat. The apparatus is kept with the larger end up, and protozoa may migrate to the top in 5 to 30 minutes, leaving most on all of the bacteria behind. The procedure may be repeated once or twice, taking into a fresh sterile pipette, with sterile water to within 2 Inches of the top, about 2 inches of fluid from the top of the previous pipette.

When the protozoa that are being freed of bacteria are positively geotropic, the sample from the culture is placed on top of the column of water in the first pipette. After migration has taken place the sealed lower end is cut off and fluid from it used for inoculation, or if not yet bacteria-free, is added to the top of the column of water in a second pipette.

Hetherington (1934a) sterilized dilates by repeated migration in successive transfers in watch glasses. Seven watch glasses are enclosed in Petri dishes. Transfer pipettes are cotton- stoppered at the bulb end. Glassware and pipettes are sterilized. Sterile medium is placed in each of the series of watch glasses. A concentrated mass of the protozoa is placed at the left margin of the fluid of the first dish, and after migration to the opposite side (observed by a stereoscopic microscope) some of the protozoa are removed to the left margin of the fluid in the second dish. Similar migrations are allowed to take place in all the dishes of the series in succession, washing the organisms in the third and fifth dishes by leaving them in each for three hours. After the final migration, the organisms are introduced directly into the culture medium to be used.

The apparatus devised by Claff (1940) consists of a series of 6 small flasks, the top of each joined by tubing to the bottom of the next, the first connected to a large flask reservoir from which fluid is received, the tube from the top of the last flask opening into a test tube. The protozoa are introduced by a hypodermic syringe through a vaccine port near the bottom of the first flask. They migrate to the top and are washed by release of fluid from the reservoir into the bottom of the second flask; as the migration and dilution process continues they may be freed of bacteria.

A critical review of methods used in sterilizing Paramecium was given by Wichterman (1949). He pointed out that it is necessary to provide against contamination of cultures by spores of bacteria carried through the washing and migration processes within the body. When the dilates remain for several hours in sterile water, it is likely that the spores will have been eliminated from food vacuoles.

Seaman (1947) reported sterilization of dilates