Allies and Enemies: How the World Depends on Bacteria (4 page)

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 cross-links. This structure acts as a net to retain the large crystal chapter 1 · why the world needs bacteria

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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.

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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

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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.

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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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 asep-tically from a sterile bottle to a sterile Petri dish. (Courtesy of BioVir Laboratories, Inc.) The size of life

Bacteria need only be big enough to hold their vital enzymes, proteins, and genetic machinery. Evolution has eliminated all extraneous structures. Also, a small, simple architecture allows for rapid reproduction, which aids adaptation. Bacterial metabolism is a model of efficiency because of a large surface-to-volume ratio that smallness creates. No

part of a bacterial cell is very far from the surface where nutrients enter and toxic wastes exit. Eukaryotic cells that make up humans, algae, redwoods, and protozoa contain varied organelles each surrounded by a membrane. The surface-to-volume ratio in these cells is one-tenth that of bacteria, so shuttling substances across all those organelle membranes, the cytoplasm, and the outer membrane burns energy. Bacterial structure is less demanding and more efficient.

Finally, small size contributes to massive bacterial populations that dwarf the populations of any other biota.

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Large multicellular beings that produce small litters with long life

spans—think whales, elephants, and humans—take a long time to

make new, favorable traits part of their genome. Insects evolve faster

and can develop a new trait within a few years. In bacteria, evolution

occurs overnight. Often, the progeny contain a new trait that makes

them better equipped for survival.

No one knows the number of bacterial species. About 5,000

species have been characterized and another 10,000 have been partially identified. Biodiversity authority Edward O. Wilson has estimated that biology has identified no more than 10 percent of all species and possibly as little as 1 percent. Wilson’s reasoning would put the total number of bacterial species at 100,000, probably a tenfold underestimate. Most environmental microbiologists believe that less than one-tenth of 1 percent of all bacteria can currently be grown in laboratories so that they can be identified.

Microbial geneticist J. Craig Venter’s studies on microbial diversity have correctly pointed out that the number of species may be less important than their diversity and roles in the Earth’s biosphere. Ven—

ter concluded from a two-year study of marine microbes that for every 200 miles of ocean, 85 percent of the species, judged by unique genetic sequences, changed. The ocean appears to contain millions of

subenvironments rather than one massive marine environment, and

each milliliter holds millions of bacteria. The actual number of bacteria in the oceans alone may exceed any previous estimates for the entire planet. In future studies of Earth’s microbial ecology, the absolute number of species will probably never be determined.

Microbiologists begin defining the microbial world by taking samples from the environment and determining the types of bacteria found there. One of the first questions to answer is: Are any of these bacteria new, previously undiscovered species? To answer this, microbiologists must understand the species that have already been characterized, named, and accepted in biology, such as E. coli.

Taxonomists assign all living things to genus and species according to outward characteristics and the genetics of an organism. Until the late 1970s, microbiologists identified bacteria through enzyme activities, end products, nutrient needs, and appearance in a microscope. In 1977 Carl Woese at the University of Illinois proposed using

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fragments of a component of cell protein synthesis, ribosomal ribonucleic acid (rRNA). Cellular rRNA takes information contained in genes and helps convert this information into proteins of specific structure and function. Because the genetic information in rRNA is unique to each species, it can act as a type of bacterial fingerprint.

Woese’s method specifically used a component called 16S rRNA,

which relates to a portion of the ribosome, the 16S subunit. This analysis led to a new hierarchy of living things (causing considerable consternation among traditional taxonomists) with bacteria, archaea,

and eukaryotes comprising the three domains shown in Figure 1.4.