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Under the microscope

For the two centuries following van Leeuwenhoek's studies, microscopes improved, but microbiologists still needed a way to distinguish cells from inanimate matter in a specimen. They tested a variety of chemical dyes on bacteria with usually unsatisfactory results. In 1884, Danish physician Hans Christian Gram formulated through trial and error a stain for making bacteria visible in the tissue of patients with respiratory infection. On a glass slide, Gram's recipe turned some of the bacteria dark purple and others pink. The new method served Gram's purposes for diagnosing disease, but he had no notion of the impact the Gram stain would have on bacteriology.

The Gram stain divides all bacteria into two groups: gram-positive and gram-negative. This easy procedure serves as the basis for all identifications of bacteria from the sick, from food and water, and from the environment. Every student in beginning microbiology commences her education by learning the Gram stain.

Bacteria with thick cell walls of peptidoglycan retain a crystal violet-iodine complex inside the wall. These cells turn purple and are termed gram-positive. Other species cannot retain the stain-iodine complex when rinsed with alcohol. These gram-negative cells remained colorless, so Gram added a final step by soaking the bacteria in a second stain, safranin, that turned all the colorless cells pink. All bacteriologists now use the Gram stain as the first step in identification, monitoring food and water for contamination, and diagnosing infectious disease.

In the more than 100 years since Gram invented the technique, microbiologists have yet to figure out all the details that make some cells gram-positive and others gram-negative. The thick peptidoglycan layer in gram-positive cell walls has an intricate mesh of crosslinks. This structure acts as a net to retain the large crystal violet-iodine aggregate and might keep the alcohol from reaching the stain and washing it out. By contrast, the gram-negative cell wall is more complex. The thin peptidoglycan in gram-negatives lies in between membranes on both the outer and inner surfaces of the cell. The thinness of the layer has been proposed as one reason why gram-negative cells cannot hold onto the stain.

Few hard and fast rules can be attributed to gram-positive and gram-negative populations. Gram-negative bacteria were once thought to be more numerous than gram-positives and have a higher proportion of pathogens, but these generalizations probably hold little merit. The Gram reaction nevertheless helps gives clues to microbiologists about potential trouble. Food, water, consumer products such as shampoo, and skin with high concentrations of gram-negative bacteria signal possible fecal contamination. That is because E. coli and all other bacteria in its family come from animal intestines. But gram-positive bacteria are not totally benign. Gram-positive bacteria recovered from a person's upper respiratory tract might indicate strep throat (from Streptococcus) or tuberculosis. Skin wounds infected with gram-positives range in seriousness from Staph infections (from Staphylococcus) to anthrax. In the environment, the known gram-negative and gram-positive species distribute almost evenly in soils and waters.

During the time Gram worked out his new procedure, German physician Walther Hesse left his job of ten years tending to uranium miners in Saxony who were dying of lung cancer (although the disease had not yet been identified). After two years in Munich working in public hygiene, he became an assistant to Robert Koch who was second only to Louis Pasteur as the world's eminent authority on microbes. Originally a country doctor in a small German village, Koch had already immersed himself in the behavior of anthrax and tuberculosis bacteria in test animals. From these studies he began developing a procedure for proving that a given bacterial species caused a specific disease. In 1876, Koch established a set of criteria that a bacterium must meet in test animals to be identified as the cause of disease. The criteria to become known as Koch's postulates laid the foundation for diagnosis of infectious disease that continues today.

Medical historians have debated whether the criteria attributed to Robert Koch should be called the Henle-Koch postulates. Koch received his early training under German physician Jacob Henle who in 1840 published a list of criteria for confirming the cause of infectious disease. The criteria proposed by Koch were similar to Henle's, but the origin of Koch's postulates probably came by a gradual evolution of ideas with each new experiment on pathogens. I explain Koch's postulates here:

  1. The same pathogen must be present in every case of a disease.
  2. The pathogen must be isolated from the diseased host and grown in a laboratory to show it is alive.
  3. The pathogen should be checked to confirm its purity and then injected into a healthy host (a laboratory animal).
  4. The injected pathogen must cause the same disease in the new host.
  5. The pathogen must be recovered from the new host and again grown in the laboratory.

Some bacteria do not conform to Koch's postulates. For example Mycobacterium tuberculosis, the cause of tuberculosis, also infects the skin and bones in addition to the lungs. Streptococcus pyogenes causes sore throat, scarlet fever, skin diseases, and bone infections. Pathogens that cause several different disease conditions can be difficult to fit into the criteria for diagnosing a single disease.

In developing these criteria, Koch made another contribution to the fundamentals of microbiology by introducing a way to obtain pure cultures. For Koch's postulates to work, a microbiologist needed a pure culture of the potential pathogen. Without bacteria in pure form, no one would be able to prove bacterium A caused disease A, bacterium B caused disease B, and so forth. Koch used potato slices for growing bacterial colonies and for his studies used only colonies that were isolated from all other colonies. This concept seems elementary today, but it helped microbiologists of Koch's time rid their experiments of contaminants. To this day, prominent researchers have reported results only to make an embarrassing retraction months later because all of the data were collected on a contaminant.

When Hesse joined Koch's laboratory, Koch had stopped using potato slices and substituted gelatin as a handier surface for growing pure colonies. Soon both men were grousing about gelatin's flaws. In hot summers, the gelatin turned to liquid. Most other times, protein-degrading bacteria turned it into a useless blob. Hesse's wife, Angelina, often came to the lab to help—this was a period in Germany when women were taking their first steps into professions. Lina, as Hesse called her, was an amateur artist and helped Koch and Hesse by drawing the bacterial colonies they had grown in the laboratory. She soon understood why the two microbiologists needed something better than gelatin. Lina suggested that they try agar-agar, a common ingredient at the time for solidifying puddings and jellies. Wolfgang, the Hesses' grandson recalled in 1992, "Lina had learned about this material as a youngster in New York from a Dutch neighbor who had immigrated from Java." People living in the warm East Indian climate noticed that birds gathered a substance from seaweed and used it as a binding material in nests. The material did not melt and did not appear to spoil—bacteria cannot degrade it.

Hesse passed on to Koch the idea of replacing gelatin with agar-agar. Koch immediately formulated the agar with nutrients into a medium that melted when heat-sterilized and solidified when cooled (see Figure 1.3). Koch published a short technical note on the invention but mentioned neither of the Hesses. Lina lived for 23 years after her husband's death in 1911 and saved as many of his lab notes as she could find. A few of those notes showed that Hesse and Lina had originated the idea of agar in microbial growth media, and they have since been recognized for their part in microbiology.

Figure 1.3

Figure 1.3 Pouring molten agar. Agar melts when sterilized, and then solidifies when it cools to below 110°F. The microbiologist here pours the agar aseptically from a sterile bottle to a sterile Petri dish.

Three years after Koch and Hesse switched to agar-based media, another assistant in the laboratory, Richard J. Petri, designed a shallow glass dish to ease the dispensing of the sterilized molten media. The dishes measured a little less than a half-inch deep and 4 inches in diameter. This Petri dish design has never been improved upon and is a staple of every microbiology lab today.

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