Shapes, colors and communities of microorganisms

Put a drop of sludge from an anaerobic reactor under a microscope, switch to UV excitation, and parts of the field light up blue-green. Nothing was stained. The glow comes from coenzyme F420, a molecule that methanogenic archaea use in their energy metabolism. Within a minute, and without extracting a single strand of DNA, the microscope has answered two useful questions: are methanogens there, and do they look active.

Sequencing has become the default first move in microbial analysis, and for good reason. A 16S profile gives names and relative abundances that no eyepiece will ever deliver. What it does not give is shape, size, arrangement, or any sense of whether the cells were sitting as free swimmers in the liquid or packed into a biofilm on a steel surface. Microscopy still answers those questions faster and cheaper than anything else, and in the kind of samples that come out of gas storage sites and geothermal wells, the answers are rarely boring.

Cell morphology

Most introductions to microbiology stop after two shapes. Rods and spheres, the cocci, cover an enormous amount of ground and describe the majority of organisms anyone meets in a teaching lab. Samples from brines, hot springs and the deep subsurface look considerably stranger.

• Irregular cocci. Methanofollis and Archaeoglobus form spheres that are not quite spheres, lobed and uneven, since neither has the rigid peptidoglycan wall that holds a bacterial coccus in a tidy ball.

• Sarcina packets. Methanosarcina divides in more than one plane and the daughter cells stay together, producing cubic packets of eight or more, and under some conditions much larger aggregates.

• Spirilla. Methanospirillum grows as curved, helical cells that line up end to end inside a tubular sheath, so a single filament can run across much of the field of view.

• Short rods. The intermediate form between a rod and a coccus, common enough that it has its own descriptive category, and awkward enough that two people looking at the same slide will sometimes disagree.

• Rectangles. Haloquadratum walsbyi is flat and square, closer to a postage stamp than to anything else in the microbial world, and lives in saturated brine. Its gas vesicles show up as bright refractile patches inside the cell.

• Cells in a mantle. Thermotoga carries a loose outer envelope, the toga, which balloons out beyond the ends of the cell like a sleeping bag several sizes too large.

Shape is not decoration. A flat square cell has an unusually high surface area for its volume, which helps in a habitat where nutrients are thin on the ground. Sheaths and togas are structural features that hold cells together or protect them. Organisms that form long filaments or dense packets also behave differently during filtration and sample preparation, which matters when the goal is to concentrate cells from a large volume of formation water.

Four morphologies of methanogenic archaea. Top left: autofluorescence of Methanospirillum hungatei (spirillum). Top right: transmission electron microscopy of Pt/C shadowed Methanofollis propanolicus (irregular cocci). Bottom left: transmission electron microscopy of negatively stained Methanobacterium cahuitense (rod). Bottom right: autofluorescence of Methanosarcina sp. (sarcina).

Autofluorescence under the microscope

Some cell components emit light of their own when illuminated at the right wavelength, with no stain involved. This autofluorescence is one of the fastest ways to narrow down what is in a sample.

Methanogens glow blue-green. The source is coenzyme F420, a cofactor central to methanogenic energy metabolism, and its brightness varies with the physiological state of the culture. Cyanobacteria fluoresce red, driven by chlorophyll a and the phycobiliproteins of their light-harvesting machinery, which makes photosynthetic cells easy to separate from everything else in a mixed sample. Clostridia have been described as showing a light yellow to green autofluorescence. 

The technique has limits worth knowing. Mineral particles, plant debris and some plastics fluoresce too, and a signal on its own confirms a group rather than a species. What autofluorescence does well is triage: it tells the analyst which organisms deserve a closer look before any molecular work begins.

Photograph of autofluorescence of a methanogenic archaeon. Left: epifluorescence microscopy, Right: phase-contrast microscopy. 

Colony morphology on agar 

Step back from the single cell and a population growing on solid medium has its own morphology. Escherichia coli produces round colonies with a smooth surface and a clean edge. Bacillus cereus does something quite different, spreading across the agar in swarming growth with feathery, irregular margins, driven by coordinated movement across the surface rather than by simple outward growth from a central point.

Colony morphology is one of the oldest diagnostic tools in microbiology and it is still in daily use, because it costs almost nothing and reveals a great deal about how an organism behaves. The catch is that most environmental microbes never form a colony at all. Only a small fraction of the community in a subsurface sample will grow under standard laboratory conditions, and for anaerobes from gas storage the plate has to be anoxic, sufficiently salty, supplied with the right electron donors and acceptors, and in some cases held under elevated pressure. Organisms that fail those conditions leave no colony to describe, which is one of the reasons high-pressure cultivation matters for this kind of work. 

Community composition

Cells also differ in how they arrange themselves relative to each other, and that arrangement is often more informative than the shape of any individual cell.

Escherichia coli separates cleanly after division and is normally seen as single cells. Other organisms stay in pairs, forming the diplo arrangement, where two cells remain attached after splitting. Streptococcus divides along a single plane and the daughter cells hold on to one another, producing the chains that give the genus its name. At the far end sits the biofilm, where cells embed themselves in a matrix of secreted polymers and stop behaving like individuals altogether. Desulfomicrobium aggregans is one example of a sulfate reducer that aggregates in this way.

Biofilms are where microbiology stops being an academic matter for infrastructure operators. Sulfate-reducing bacteria attached to a steel surface generate conditions directly at the metal that bulk liquid chemistry will not predict, and this is the setting in which microbially influenced corrosion develops. A liquid sample can be full of planktonic cells while the community actually driving damage sits in a film a few hundred micrometers thick on the pipe wall. Recognizing aggregates and biofilm fragments in a sample is a signal that the sessile population deserves attention, and it changes what should be sampled next.

Floating biofilm of the sulfate reducer Desulfomicrobium aggregans grown in a serum bottle. The dark biomass aggregates at the surface and on the glass wall while the medium remains clear.

Microscopy and sequencing

Morphology cannot replace sequencing. Unrelated organisms converge on the same shapes, a rod is a rod whether it reduces sulfate or ferments sugar, and no microscope will resolve a species-level identification from outline alone. The reverse is equally true. A sequence returns a name and no indication of whether the cells were alive, aggregated, filamentous, or attached to anything.

Running both is what makes the picture complete. Microscopy on a fresh sample shows what the community looks like and roughly how it is organized, autofluorescence flags the functional groups worth pursuing, cultivation shows what can still grow, and 16S profiling supplies the names.

Microbes are not a uniform gray mass of rods and spheres. They come as squares, spirals, packets and sheathed filaments, they glow in different colors depending on their metabolism, and they arrange themselves as loners, pairs, chains and dense communities. Reading those features takes minutes and shapes every decision that follows.