Small Things Considered

Pictures Considered #4. Koch’s Development of Early InstaGram Positive Photography

by Daniel P. Haeusser

Figure 1A. Koch’s photograph of B. anthracis, one of several photomicrographs in his 1877 paper, the earliest published bacteria photos. Source.

Robert Koch is one of the key figures in early bacteriology, helping develop culture techniques (e.g. solid media), critical reasoning (e.g. Koch’s postulates), and disease etiology (e.g. cholera and tuberculosis). He also published the first photomicrographs of bacteria (Figure 1A) in his 1877 paper Verfahren zur Untersuchung, zum Conservieren und Photographiren der Bakterien.

Discontent with communicating microscopic observations with hand-drawn illustrations, Koch pioneered the photography of bacteria. On suitable days, Koch would set up to shoot outdoors. In his 1877 publication Koch explains how to take photographs outside through a basic microscope:

“Clean all the lenses, screw them in completely, and place the illuminating mirror on the sunny side of the microscope. With a dark cloth over your head, look through the ground glass and adjust the light and focus the specimen… Once the image is in focus… go inside to prepare the photographic plates. In the darkroom…remove a clean glass plate with forceps and pour over its surface the iodized collodion solution, making sure the film spreads evenly and completely. Once the collodion film is ready, close the darkroom door and carefully lower the plate into the silver bath… Allow it to drain and put it in the cassette… Go back outdoors to the photomicrographic apparatus. Remove the black cloth…and check to be certain that the proper image is still in focus… Then carefully place the cassette, being careful not to move anything. After the exposure… push the slide back in the cassette, remove the cassette from the microscope, and cover the microscope again with the black cloth. This whole procedure must be done quickly! Run back to the darkroom with the closed cassette, develop the plate, and fix the negative. If the photographic image is not completely sharp, or if there are imperfections in the emulsion… it is necessary to repeat the whole process, since nothing is more disheartening in the photographic technique than to try to make prints from unsatisfactory negatives.¹

Quite a process! Following this protocol, Koch reports it took at least three hours to obtain four to six good pictures. Note that this time does not include setting up the equipment, preparing the bacterial sample, or freshly mixing the photographic chemicals. Yet with skill, patience, and perseverance during his early studies on anthrax, Koch obtained photos of the Gram-positive B. anthracis in a quality that rivals images taken today. Indeed, as an exercise of curiosity, members of my department² took an Instagram picture of B. anthracis using a cell phone camera. The image took seconds to capture with modern technology, yet doesn’t come close to rivaling Koch’s.

Figure 1B. A 1982 stamp from Zimbabwe celebrating the centennial of Koch’s discovery of M. tuberculosis (DPH personal collection).

Koch’s realization of bacterial photography was not merely a pretty picture. The communication of accurate images of bacterial shape was vital to the understanding that multiple species of bacteria existed, and Koch’s practice in imaging relatively easy-to-cultivate organisms like B. anthracis allowed his eventual rapid success in discovering the extremely fastidious M. tuberculosis in diseased tissue. It is this attention to detail and scientific resolve that led Koch to the discoveries that have immortalized him in scientific, medical, and indeed general human history (Figure 1B).

Notes:

¹ Brock, Thomas D. (1999) Robert Koch: A Life in Medicine and Bacteriology. ASM Press, p. 61.
² Thanks are given to Katie McCallum for her Instagram account and to Lori Horton, Malik Raynor, and Michelle Swick of the Kohler Lab for the microscopy equipment and B. anthracis sample.

Putting Redundancy to Work

by Katrina Nguyen

Schematic diagram of the iMAD technology. Click image to view larger.

Genetic redundancy, when two genes encode for the same function, is widespread among many organisms. Redundant genes confer an advantage: if one gene is lost, its partner can substitute and the phenotype of the organism will not change. It is unclear how redundancy is maintained during evolution since selection against unneeded genes should eventually wipe out a truly redundant gene. In addition to posing this evolutionary question, redundant genes also pose a tangible problem for researchers. It would be nice if knocking out a single gene resulted in a specific phenotype that revealed the function of the gene. However, if a redundant gene is mutated or otherwise knocked out, its colleague can still perform the function, and no phenotypic effects will be seen. Therefore, assigning a function to redundant genes is often a real challenge.

A recent approach to understanding host-pathogen interactions provides a solution to the problems of redundancy. O’Connor et al. developed a novel genetic screening method, called iMAD for insertional Mutagenesis And Depletion, which enables the investigation of complicated genetic interactions. In iMAD, the bacterial pathogen is mutated and a host function needed to support growth of the pathogen is depleted via RNA interference. The idea is to produce an aggravating genetic interaction, where the combination of defects in host and pathogen produces a stronger phenotype than either would alone. Based on this interplay, it is possible to determine which genes are redundant, as well as their function.

A Day in the Life: Eavesdropping on Marine Picoplankton

by Heather Maughan

ESP being tested by divers at the Monterey Bay Aquarium Research Institute. Source.

Observing microbes in nature is a challenge. Compared to what goes on in the lab, there is not much one can do with them out there. So, instead of bringing the bacteria to the lab, why not bring the lab to the bacteria? Imagine being able to capture the expression of genes of a community of microbes in situ, and over multiple time points. This movement of the microbial stage to natural environments has been done for microbial niches that are easily accessible, such as agricultural soil, hot springs, or mine washes. But inhospitable sites far from a lab, sites such as hydrothermal vents and the open ocean, pose a bigger problem.

The solution? A steadfast robot designed and dispatched by researchers at MIT and the Monterey Bay Aquarium Research Institute. Known as the Environmental Sample Processor (ESP), this robot gathers samples of seawater and stores them temporarily so as to preserve the RNA transcripts for subsequent retrieval and analysis. ESP is also able to perform DNA and protein hybridization to identify and quantify specific molecules.

The Gender Bias of Science Faculty

by Vincent Racaniello

If you were a science professor, and you received two equally strong applications for the position of laboratory manager, one from a female, one from a male, which one would you pick? The answer may surprise you.

It is well known that women are underrepresented in many fields of science. Whether or not this disparity is a result of gender bias by science faculty has not been investigated. To answer this question, a randomized, double-blind study was conducted in which science faculty from research universities were asked to rate the application of a male or female student for a laboratory manager position. Identical applications were sent to all participants in the study, except that half (n=63) received materials from a male student, John, and the others (n=64) received materials from a female student, Jennifer. The faculty were then asked to rate the student’s competence and hireability, and the amount of salary and mentoring that they would offer.

The Gram Stain: Its Persistence and Its Quirks

by Elio

What is more emblematic of our science than the Gram stain? Since its invention 130 years ago, it has been in frequent and continuous use. It conveniently places most bacteria into one of two groups, the Gram-positives or the Gram-negatives. Gram staining is cheap, effective, quick, and relatively easy to interpret. Its most useful application is in the clinical setting. When examining a smear of, say, pus from an abscess, this stain often allows to include for consideration roughly half the clinically relevant bacterial species while excluding the others. Or consider a patient with meningitis. Here, speed is of the essence because treatment must be initiated right away. A Gram stain of the spinal fluid may reveal within minutes the presence of Gram-positive cocci, (probably pneumococci), Gram-negative cocci (almost certainly meningococci), or Gram-negative slender rods (most likely Haemophilus influenzae). This can make a critical difference in the choice of antibiotics that have to be administered in great haste. However, when it comes to characterizing the bacteria in an environment, its usefulness diminishes, in part because it is not always in step with taxonomy, which I’ll discuss below. And there yet is another side to this story. Quite a few bacterial species that stain positive early in the growth of a culture become Gram-negative later on. Does this detract from the value of Gram’s method? It may, but not in the hands of a person experienced in its use.

Who Would Have Thought It?Which Would You Bet Are Easier to Cultivate, Abundant Bacterial Species or Rare Ones?

by Elio

Surprises are the stuff of science, but some discoveries are more surprising than others. We are starting a new column, its aim being to highlight findings that, in our view, lie outside the norm for being markedly unexpected and unforeseen. We plan to post notices of such items periodically. You are invited to submit your own choices.

Surprise!

Although only a small fraction of the bacteria on Earth can be cultivated, the existence of many others has been inferred from the presence of their DNA in environmental samples. This two-fold approach sounds innocuous enough, but it has occasionally resulted in acrimonious controversies. This is puzzling because even a moment’s reflection should lead one to conclude that these strategies are complementary and that both are needed. But putting that aside, consider that bacterial species are far from uniformly abundant in the environment. Some are found in large numbers, others are exceedingly rare. Now, which do you think would be easier to culture, the abundant ones or the rare ones? If you bet on the abundant ones—surprise, surprise—you’d be wrong, even if your answer feels intuitively obvious.

Requiem for a Machine

by Elio

Beckman analytical ultracentrifuges, then (the Model E)...

Here’s a challenge for present-day systems biologists. Say you wanted to find out how many ribosomes are present in cells growing under different conditions. How would you do it? You might think of using quantitative PCR to measure the amount of rRNA inside the cell. However, you could end up with an overestimate because some of the rRNA may not yet be assembled in mature ribosomes. For instance, add chloramphenicol, a protein synthesis-inhibiting antibiotic, to a culture and you will measure increases in the cellular levels of rRNA due to the accumulation of non-functional, immature ribosomes. Perhaps, then, you would roll up your sleeves and run a sucrose gradient to separate the mature ribosomes from their precursors and immature forms by size fractionation, but this is a labor intensive method that not many people like to do these days. We’ll agree that making such measurements may not be as easy as it sounds. So let me reminisce about how we carried this out in the old days. All it took was a fancy apparatus and some chutzpah.

Cell Division Through DNA Curtains

by Gemma Reguera

Despite the apparent simplicity of bacterial cells, their cell division cycle is a complex developmental program that couples cellular growth to the replication and segregation of chromosomes and the division of the cell’s cytoplasm (aka cytokinesis) (Fig. 1). The bacterial cell division cycle starts with the commitment of the cell to reproduce. This is the step in which, forgive the pun, size truly matters. During active growth, the cell’s size changes to accommodate increases in mass, volume, and biosynthetic capacity. When the cell reaches a critical size, essential cellular functions such as intracellular transport and nutrient uptake are constrained and optimal growth can no longer be supported. Provided conditions are adequate for growth, cell division is undertaken to allow the bacterium to regain its individual cellular fitness.

Fig. 1: As the cell grows in size, the chromosome is replicated and segregated. A constricting septum forms at midcell to divide the cell’s cytoplasm and produce two daughter cells. Source.

Where Mathematicians & Biologists Meet

by Joe Mahaffy

Source.

Mathematics and Biology have a long history together. It goes back to early studies on epidemiology (such as John Snow‘s on cholera and the Broad Street pump), and includes Ross’s quantitative studies that show how malaria can be controlled by careful analysis of data. And, of course, there are many others. In the early 20^th century, population models with differential equations were developed to describe the dynamics of populations, such as the studies of Alfred Lotka, who felt that natural selection could be quantified by physical laws, and Vito Volterra, who created a model to explain the predator-prey ratios in the Italian fish markets. These early models provide excellent tools because in their simplicity they show biologists how mathematics can help explain noteworthy biological phenomena. Mathematicians enjoy such models because the examples themselves make it easier to explain what the equations are describing.

Polar Enchantment

by Elio

NASA's Polar spacecraft captured the first-ever movie of
auroras dancing simultaneously around both of Earth's
polar regions. Source.

All bacteria, even those that are spherical, display geometric asymmetries during at least part of their life cycle, and it appears that cells take advantage of these asymmetries to localize cellular components to specific sites. So begins a paper by Li and Young that describes a novel approach for detecting E. coli proteins that preferentially localize to the cell poles. There is already a considerable catalog of “polar” proteins in various bacteria, some involved in chemotaxis, signaling, regulation of septation, flagella synthesis, phage attachment, and a raft of other phenomena. Pretty soon you may be tempted to ask which proteins do not localize to the poles. This may sound facetious, but it illustrates the recent changes in mindset regarding the structural complexities of the prokaryotic cell. But many questions remain. We readily subscribe to the notion that bacteria are not bags of enzymes but this begs the broad question “what are they?”