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Chapter 11 : Enrichment and Isolation

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Abstract:

A section of this chapter focuses on the use of enrichment and cultivation procedures with molecular methods, such as wholecell fluorescent in situ hybridization (FISH) or denaturing gradient gel electrophoresis (DGGE), to monitor the progress of an enrichment, to evaluate the presence of contaminants, and to identify new isolates. Biophysical enrichments make use of such conditions as growth temperature, heat treatment, sonic oscillation, or UV irradiation to kill or inhibit the rest of the population. Biological enrichments may make use of specific hosts for selective growth of a particular organism, or they may take advantage of some pathogenic property, such as invasiveness, which the rest of the population does not possess. Bacteria are usually isolated from enrichment cultures by spatially separating the organisms in or on a solid medium and subsequently allowing them to grow into colonies. The chapter is designed to demonstrate the multiplicity and in many instances the considerable ingenuity of enrichment and isolation methods for bacteria by presenting specific selected examples. The buoyant density of bacteria in pure culture and in samples from natural aquatic environments has been studied by density gradient centrifugation in Percoll gradients, and the average density of a representative bacterium is 1.080 pg µm . Isolation of species, particularly those from the environment, can sometimes be facilitated by acidification of samples to pH 2.2, which kills contaminants more quickly than it does the species.

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11

Key Concept Ranking

Microbial Ecology
0.6236902
Bacteria and Archaea
0.614384
Denaturing Gradient Gel Electrophoresis
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0.6236902
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Image of FIGURE 1
FIGURE 1

Isoelectric focusing. Schematic (A) and photograph (B) of apparatus for isoelectric focusing of whole bacterial cells. The cathode (–) and anode (+) consist of platinum wires which are connected to a power supply ( ).

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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Image of FIGURE 2
FIGURE 2

Capillary enrichment method, based on chemotactic attraction of motile bacteria to different carbon and sulfur sources in glass microcapillaries ( ).

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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Image of FIGURE 3
FIGURE 3

FeS-based growth of Fe-oxidizing bacteria in gradient tubes. The four tubes on the left contain cultures of Fe-oxidizing bacteria, and the single tube on the far right represents an abiotic control. In culture tubes, a distinct band of cells develops at ca. 1 cm from the air-medium interface at the tops of the tubes. The milky region that can be seen clearly at the tops of the tubes is comprised principally of Fe oxide particles, which directly overlie the band of cell growth. In the control tube, Fe oxides develop from ca. 1 cm from the top of the tube to ca. 3 cm from the FeS-medium interface at the bottom ( ).

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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Image of FIGURE 4
FIGURE 4

Useful streak plate method for obtaining wellisolated colonies. (A) With a glass marker pencil, draw a “T” on the bottom of the petri dish to divide the plate into three sections. (B) Streak a loopful of culture lightly back and forth on the surface of the agar over section 1 as shown. Raise the lid of the dish just enough to allow the streaking to be done, and then replace it. Flame sterilize the loop, and allow it to cool (15s). (C) Draw the loop over section 1 as shown, and immediately streak back and forth over section 2. Flame the needle, and allow it to cool. (D) Draw the loop over section 2 as shown, and then streak back and forth over section 3. (E) Incubate the dish in an inverted position as shown, to prevent drops of condensed water on the lid from falling onto the agar surface. Section 1 will develop the heaviest amount of growth, and section 2 or 3 will usually have well-isolated colonies.

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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Image of FIGURE 5
FIGURE 5

Streaking an anaerobic roll tube ( ). (A) Loop needle (platinum or stainless steel; nichrome will cause oxidation of the medium). (B) Gassing cannula for continuous purging of the tube with oxygen-free gas. (C) Prereduced agar medium coating the inner wall of the tube. (D) Motor-driven tube holder for rotating the roll tube during streaking. Insert the needle with a loopful of inoculum to the bottom of the tube, press the loop flat against the agar, and draw it upward. After streaking in this manner for one-fourth of the way up the tube, turn the loop so that it is perpendicular to the agar (as shown), and continue to streak upward to the top. Remove the gas cannula, replace the rubber stopper in the tube, and incubate the culture in a vertical position.

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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Image of FIGURE 6
FIGURE 6

Membrane filter design and setups for aerobic and anaerobic incubation. Bacterial cells from precultures are filtered onto 0.2-μm-pore-size polycarbonate membrane filters and placed in PBS solution to avoid desiccation. Coverslips are covered with a thin layer of silicone oil. The membrane filters are mounted on the coverslip with the bacteria towards the silicone oil. The filter sandwich can be incubated either floating on a liquid surface or submerged within a liquid growth medium. For aerobic incubation, the filters can also be placed directly on a growth medium without being mounted on the coverslips ( ).

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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Image of FIGURE 7
FIGURE 7

Colonies of oxygen-sensitive anaerobes using the agar shake technique. A dilution series was established from right to left, eventually yielding well-isolated colonies. The tubes are sealed with a sterile mixture of paraffin and mineral oil to maintain anaerobic conditions (photo courtesy of Norbert Pfennig [ ]).

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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Image of FIGURE 8
FIGURE 8

(A) MPN dilution series, here shown with Hungate tubes in three parallel dilution series. The inoculum is transferred anaerobically using N2-flushed syringes (H. Cypionka).

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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Image of FIGURE 9
FIGURE 9

MPN counts for anaerobes with a microtiter plate. Inside an anaerobic glove box, four different samples are diluted down to 1:106 on a deep-well microtiter plate. For each sample, a row remains uninoculated as a control. The wells are sealed with a capmat, and the plate is stored in an anoxic bag with an oxygen-consuming catalyst such as Merck Anaerocult A and an oxygen indicator such as phenol red (H. Cypionka).

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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Image of FIGURE 10
FIGURE 10

Schematic illustration of the Microdrop® device for automated inoculation of 170-μl aliquots of a bacterial suspension into a 96-well microtiter plate ( ).

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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Image of FIGURE 11
FIGURE 11

Apparatus for washing of filamentous bacteria ( ).

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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Image of FIGURE 12
FIGURE 12

Isolation of a single bacterial cell of (arrow, larger cells) with the Bactotip method. The smaller cells are sp. (Left) Schematic drawing; (right) corresponding phase-contrast micrographs. Bar, 10 μm ( ).

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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Image of FIGURE 13
FIGURE 13

Optical tweezers for the isolation of bacteria. (a) Principle of the optical tweezers. A strongly focused laser beam on an object as small as a prokaryotic cell creates downward forces (Fa and Fb) on the cell, which allow the cell to be dragged in any direction as long as the beam force remains on it. (b) Isolation. The laser beam can lock onto a single cell present in a mixture in a capillary tube and drag the optically trapped cell away from the other cells. In the example used here, the desired cell is dragged from right to left. Once the desired cell is far enough from the other cells, the capillary is severed, the laser is turned off, and the cell is flushed into a tube of sterile medium ( ).

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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Image of FIGURE 14
FIGURE 14

Use of DGGE in the analysis of mixed cultures and enrichments. After DNA isolation from the sample or mixed culture, PCR, and DGGE, the sequences of the DGGE bands are determined and compared to the sequence information available from databases, to identify the components of the microbial mixture, and subsequently to select culture conditions to separate and isolate the bacterial species in pure culture. For verification, the DGGE bands of the pure culture are compared to the DGGE pattern of the original sample (A. Teske).

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
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References

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Tables

Generic image for table
TABLE 1

Examples of the use of antibiotics in selective media

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11
Generic image for table
TABLE 2

Some unusual carbon sources for free-living nitrogen fixers

See references , and .

Citation: Teske A, Cypionka H, Holt J, Krieg N. 2007. Enrichment and Isolation, p 215-269. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch11

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