Measurement of in Situ Activities of ...

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Ann. Rev. Microbiol.

1985.39:321-46 Copyright © 1985 by Annual Reviews Inc. All rights reserved

Annu. Rev. Microbiol. 1985.39:321-346. Downloaded from by University of Washington on 09/30/11. For personal use only.


Department of Microbiology and Immunology, University of Washington, Seattle, Washington 98195 Allan Konopka

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

CONTENTS INTRODUCTION ... . . . . . . . ... .... . ... . . . ..... . . . . . . ..... . . . . . . . . ... ............. .....................


AUTECOLOGy. . . .. . . ................ . ......... . . . .. . . ....... . . . . .................................... Techniques Used in Assessment of Autecological Activity ... . . . . . .. . ... . . . . . . . . . .. . . . . . . . . Applications of Techniques Used in Assessment of Autecological Activity . . ........ . . . . SyN ECOLOGy.. . . .................... .............................................. . . . ....... . . . .... Problems in Interpreting the Measurement . . . . . . ......... . . . . . . """"""""". Radioisotopic Methods . . . . .... .. . . . . . . . . . . . . . . . . . .. . . ..... . . . . . . . . . . . . . . .. . . . . . . . . . . . . ... . . . . . . . . Measurement of Cell Division . . . . . . . ... . . . ...... . . . .......... ... . . . .. . . . ...... ........ . . ....... Comparisons Between Methods.. . . . . . . . .... . . . . . .. . . . .... . . . . . . . . . . . .... . . . . . . . . . . . . ..... . . . . . .

322 323 324 329 329 332 341 343


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. . . .


INTRODUCTION Microorganisms play major roles in biogeochemical cycles. Microbial ecolo­ gists studying these cycles use a variety of scientific approaches to assess the 321


Annu. Rev. Microbiol. 1985.39:321-346. Downloaded from by University of Washington on 09/30/11. For personal use only.



activities of indigenous microorganisms and their importance to specific trans­ formations . Research in this area can be separated into two broad categories: (a) autecology, i . e . investigations of the activities of individuals and species, and (b) synecology, i . e . studies of the activities of an entire natural community typically containing many species. Ideally ecological investigations will be attuned to both perspectives, inasmuch as the ultimate goal of microbial ecology is to fully understand both the rates at which a process occurs in nature as well as the identity and characteristics of the species that are responsible for the process . Indeed, the rate of a process will be dependent upon the species involved and the physical, chemical, and biological characteristics of the habitat. A thorough understanding of the process in one habitat will provide meaningful information that can be extrapolated to other habitats that are less well understood but have similar species compositions. This review concerns rapid processes, i.e. activities whose half-life is of the order of minutes, hours, or days. Intermediate-term processes such as seasonal successions or annual cycles and slow processes such as weathering will not be considered . Rapid processes are amenable to analysis by numerous laboratory techniques developed in microbial physiology and biochemistry that have been specifically modified for field situations. These procedures are sensitive and may provide much useful information about transformations.

AUTECOLOGY Autecological studies of microorganisms differ in several respects from the autecology of higher organisms (10). First, the small size of microorganisms makes the study of their activities in situ particularly difficult and challenging. Many microbes exist as unicells in their habitats, and their microenvironment is therefore measured in nanometer and micrometer distances from the cell surface. Even visualization of these microorganisms requires artificial amplification of their size by use of microscopy, with the possible unwanted adverse effects imposed by the process of observation ( 1 06) . A second manner in which microbial autecology differs from that of higher organisms is with respect to identification of individuals. The simple morpholo­ gy of many microorganisms, particularly bacteria, means that it is oftentimes impossible to identify them by direct microscopic observation to species, genus, or sometimes even kingdom! However, some bacteria can be identified morphologically at the genus or species level, and these provide excellent model organisms with which to study such processes as growth (8) and activity (110). Furthermore, fluorescent antibody tagging has been successfully used for the recognition of many microbial groups that can be rendered morphologi­ cally identifiable by this procedure (7). The identification of individuals by direct microscopy and fluorescent antibody approaches permits the investigator



to enumerat,e and determine the in situ distribution of strains or species in their habitats . Thus, a number of investigations have examined the distribution of ecologically important organisms ranging from nitrifiers and nitrogen fixers to Legionella using these procedures (22, 23, 102, 108, 109, 119).

Annu. Rev. Microbiol. 1985.39:321-346. Downloaded from by University of Washington on 09/30/11. For personal use only.

Techniques Used in Assessment of Autecological Activity

In order to determine the in situ activities of microorganisms, it is essential to go beyond their detection and identification. Listed below are the three primary techniques alvailable to the microbial autecologist who is interested in assessing the activities of individuals, identified or not, in the environment. Microautoradiographic procedures entail the use of radiolabeled substrates , principally carbon- 14 and tritium eH), for microbiological work. Technical details of the grain density procedure have been discus:,ed elsewhere (9). The stren:gth of the microautoradiographic technique is that it permits one to relate the uptake of a known labeled substrate to an individual microorganism. If the organism can be identified by direct microscopic observation, or if it can be rendered identifiable by immunofluorescence, then one can relate the activ­ ity directly to the species. It is important to recognize some of the potential problems associated with grain density microautoradiography before a specific application is used. First, the choice of label must be made carefully . The high energy of decay of the phosphorus-32 13 particle prohibits its use in microbial autecology because it results in the exposure of silver grains some distance from the metabolically active organism that has taken up the label. Thus, it is not possible with great fidelity to relate a specific exposed silver grain with a specific microorganism using 32P-Iabeled substrates . Even the 14C 13 particle is of sufficiently high energy that ilt can cause problems in resolution when used with small microor­ ganisms such as typical heterotrophic bacteria (64). A thin emulsion helps alleviate this problem of resolution if 14C is to be used. Tritium, which has the lowest energy 13 particle of the three, is therefore generally preferable for labeling of organic substrates. The type .of fixative used in autoradiography is also important. The fixative must adequately preserve the organism so that lysis does not occur during development of the autoradiogram. Furthermore, the fixative should not cause latent image erasure. Latent image erasure occurs when the exposed silver grain is chemically altered so that it does not appear as an exposed grain when the autoradiogram is developed. Formaldehyde and glutaraldehyde may cause latent image erasure when used as fixatives (64) . Lugol's iodine is generally found to be satisfactory . It is very important to prepare appropriate controls to guard against latent image erasure, chemography (chemical exposure of silver grains), and other possible artifacts . MICROAUTORADIOGRAPHY



Many bacteria are able to reduce the tetra­ zolium dye INT[2-(P-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride] during active metabolism. Even many bacteria that do not use oxygen during respiration are able to reduce this dye with the formation of an insoluble, dark red, intracellular granule of the reduced product, INT formazan ( 1 30) . The usual procedure for natural samples is to add the dye at the time of sample collection, then incubate the sample under in situ conditions for a period of 20 min. Metabolically active cells can be detected as those with formazan gra­ nules. One limitation of this procedure is that granules may not be detectable in extremely small cells.

Annu. Rev. Microbiol. 1985.39:321-346. Downloaded from by University of Washington on 09/30/11. For personal use only.


NALIDIXIC ACID CELL ENLARGEMENT PROCEDURE The principle of this procedure is to inhibit DNA replication and cell division, but allow metabolism to occur. The result is that organisms whose cell division is inhibited by nalidixic acid, as are many gram-negative bacteria, enlarge in size. This procedure is accomplished by adding a nutrient, such as yeast extract, to the sample along with the nalidixic acid at the time of collection, then incubating the sample for several hours (65). The number of enlarged cells after incubation represents the number that are metabolically active. One limitation for this procedure is that not all cells are inhibited by the antibiotic; thus they may divide during the incubation period. Then too, not all living cells would utilize the nutrients added.

Applications of Techniques Used in Assessment of Autecological Activity

The three autecological activity measurements discussed above have been applied to answer a variety of questions relating to the in situ activities of microorganisms (Table 1 ) . Below are listed several areas in which they have found application: Perhaps the first report of the plate count anomaly was that of Razumov (94) , who noted a large discrepancy between the viable plate count and total direct microscopic count of bacteria from oligotrophic to mesotrophic aquatic habitats . He found higher numbers (by several orders of magnitude) by direct microscopic counting than by the plating procedure. He further noted that this same trend held in eutrophic canal water, but the differences between the two procedures were not nearly so great. Similar results were subsequently reported for marine habitats (49). A contemporary illustration of the anomaly is provided in a seasonal distribu­ tion study of mesotrophic Lake Washington (32) . Figure 1 shows the direct microscopic count using the acridine orange procedure (42), and Figure 2 shows the viable plate count for the same period of time. Viable plate counts THE GREA T PLATE COUNT ANOMALY

IN Table 1


In situ autecological activity procedures and their applications Applications


assessing the proportion of active bacteria to total bacteria

(75, 1 1 5, 1 32)

assessing metabolic activity of identifiable species


nalidixic acid cell enlargement

assessing the proportion of active bacteria to total bacteria

(65, 66, 75, 115)


assessing the proportion of active bacteria to total bacteria

( 2 8 , 46, 76, 1 1 3-115)

Procedure tetrazolium dlye reduction

Annu. Rev. Microbiol. 1985.39:321-346. Downloaded from by University of Washington on 09/30/11. For personal use only.


growth rates

(11 )

physiological activities


nutrient uptake: qualitative

( 12, 24)


(25, 1 1 0)

were made on CPS medium (51), which was determined to be the best of several media for ,enumeration of heterotrophic bacteria in Lake Washington. As the figures illustrate, only approximately 0. 1-1 .0% of the total bacteria can be enumerated by the plating procedure. Indeed, as a general rule we have found that the maximum recovery of heterotrophic bacteria is 1 % of the total direct count using plating procedures or other viable enumeration methods from a variety of oligotrophic to mesotrophic aquatic habitats, whereas higher recover­ ies (approaching 80-90%) can be achieved from eutrophic habitats ( 100, 107). These results for oligotrophic to mesotrophic waters can be explained in either of two ways. First, it is possible many of the bacteria that cannot be grown are nonviable. Alternatively, they could be considered as alive, but unable to grow under the conditions provided in the plating procedure . Some suggestion of the plausibility of this latter hypothesis is provided by examina­ tion of even the direct count data (Figure 1 ) . If they are not living, barring transport of allochthonous bacteria, how could their numbers increase from 2 X 106 ml- 1 to 4 X 1 06 ml- 1 in the surface waters from February to late April? It is the autecological activity measurements, however, that provide the most convincing evidence indicating that many of the bacteria from oligotrophic and mesotrophic habitats are metabolically active and therefore probably able to reproduce. The first suggestion of this came from the work of Hoppe (46) and Meyer-Reil (76), who used microautoradiography, and Zimmermann et al ( 1 32), who used the INT reduction method, to detect metabolically active cells in the Baltic Sea. Using tritiated glucose, Meyer-Reil found that between 2.3% and 56% (average of samples 3 1 .3%) of the total direct microscopic count were




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