Allies and Enemies: How the World Depends on Bacteria (30 page)

Samples containing several thousand to millions of cells would

create an almost contiguous sheet of colonies unless the microbiologist serially dilutes the sample before inoculating the plates. Serial dilution produces plates containing between 30 and 300 CFUs, most of which are spatially separated from each other and easy to count. Microbiologists prefer plates with this many colonies because CFU numbers of less than 30 do not give consistently accurate results, and plates with 300 or more colonies are too dense to count. On densely populated

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plates, bacteria begin inhibiting the growth of nearby colonies by using up nutrients and excreting antimicrobial substances.

To determine the number of bacteria in a liquid culture, the

microbiologist selects duplicate plates containing 30 to 300 colonies

each. In this example, the plates that had been inoculated with 0.1 milliliter of the 1:10,000 dilution look like they have between 30 and 300 colonies. After counting the number of CFUs on each duplicate plate, the microbiologist discovers one plate has 98 colonies and the second has 138 colonies. The average of the two plate counts equals 118. Now the microbiologist must account for the dilutions to

calculate the number of bacteria that were in the original sample.

In the first step, the microbiologist multiplies 118 by the dilution,

in this case, 1:10,000:

118 × 10,000 = 1,180,000 or 1.18 × 106

The aliquot volume was only 0.1 milliliter, which is equivalent to

diluting a milliliter by 1:10. To correct for this dilution, the microbiologist multiplies the above result by 10:

10 × 1,180,000 = 11,800,000 or 1.18 × 107

The original culture therefore held almost 12 billion bacteria. In

microbiology, such large microbial numbers occur often. Soil, marine

water, surface freshwaters, and the animal digestive tract all contain

similar high bacterial concentrations.

Logarithms

Numbers of several million or billion can be unwieldy for calculations. Furthermore, when a number as large as 1.18 × 106 is doubled to 2.36 × 106 or even tripled, the differences between these numbers are not meaningful in microbiology. Variability in nature can cause replicate cultures prepared exactly the same way to produce different concentrations of bacteria. Microbiologists therefore use logarithms

to make very large numbers easier to use in calculations and to help

discern significant differences between large numbers.

Understanding the definition of a logarithm (abbreviated to log)

can be difficult, but an example helps. For the number 1.0 × 105, the log is 5.00. The log for 1.0 × 106 equals 6.00. Numbers that fall

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in between whole numbers also can be converted to a log value. For

example, the log of 5.0 × 105 equals 5.699. All of these logs are called logarithms to base 10 because they are multiples of 10.

Expressed as log , whole numbers and fractions can be looked up

10

in tables, determined by a slide rule, or produced by a calculator.

Use a calculator!

Converting large numbers to their log value illustrates that for

10

huge numbers of microbes, doubling, tripling, and even quadrupling

does not mean much in microbiology. The log of 1.18 × 107 equals 7.07. Doubling 1.18 × 107 to 2.36 × 107 results in a log of 7.37, not 10

14.14 (2 times 7.07). The triple of 1.18 × 107 is 3.54 × 107 or log10

equal to 7.55; quadrupling the number gives a log of 7.67. This illus—

10

trates that bacterial numbers differing by a few multiples can be viewed as being of the same general magnitude. Only when bacterial numbers change by at least 100 times do microbiologists view this as

a real change beyond the normal variability of nature.

Anaerobic microbiology

Diluting and counting anaerobic bacteria resembles the steps used for aerobic bacteria except that anaerobes require sealed containers that exclude all air. Anaerobic microbiology calls for diligence that aerobic methods ignore, that is, the microbiologist follow aseptic techniques and keep air away from the bacteria.

Anaerobic bacteria grow only on agar plates placed inside a sealed

jar containing a chemical to remove all the oxygen from the jar once it has been sealed. As a second option, microbiologists can use an anaerobic chamber, which is a large plastic bubble filled with an inert gas lacking oxygen. One side of the chamber has arm holes built directly

into the plastic so that a microbiologist can sit outside the chamber,

put her arms into the arm holes, and dilute and perform other activities with the anaerobes inside the chamber. Some anaerobic chambers include a small incubator so that plates need never exit the anaerobic environment during an experiment.

I learned anaerobic microbiology by using a third method named

for Robert Hungate who advanced the techniques for growing strict

anaerobes in the 1950s and 1960s. The Hungate method developed

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almost exclusively by studying the anaerobes from the digestive tracts

of cattle, sheep, and goats. These bacteria have more stringent requirements for oxygen-free environments—they are often referred

to as fastidious anaerobes. The Hungate method thus grows the bacteria in test tubes instead of plates, which are impractical for airtight conditions.

Hungate tubes are prepared by pouring sterile molten agar into

each tube and then inoculating the agar while it is still a liquid. Microbiologists exclude air from the open tube during this step by directing a gentle stream of inert gas into the tube. The microbiologist must inoculate the agar quickly and then withdraw the gassing hose an instant before sealing the tube with a rubber stopper. Fastidious anaerobes require stoppers made of special rubber that prevents any molecule of air from seeping into the tube during incubation. A good practitioner of anaerobic microbiology can perform the one-two step of withdrawing the hose and stoppering the tube quicker than the eye can follow. The

microbiologist then rolls the inoculated tubes on a horizontal surface

until the agar has solidified into a uniform layer coating the inside of

the tube. After incubation, the microbiologist counts CFUs in the agar.

Aseptic technique

All microbiological procedures require aseptic technique, which

refers to all the activities microbiologists perform to keep unwanted

microbes out of pure cultures or sterile items. Aseptic means free from germs, and sepsis is a medical term for the presence of germs.

Media, glassware, and anything else that comes in contact with live

cultures must be sterilized in an autoclave. This piece of equipment

treats liquids and solids with pressurized steam to kill all microbes.

Items that have been sterilized and covered can be stored indefinitely.

In addition to sterilized laboratory supplies, microbiologists also

“flame” items over a Bunsen burner before handling bacterial cultures. Flaming works well for metal or glass items such as inoculating loops, forceps, and open test tubes.

All these activities require that the microbiologist imagine where

bacteria exist and predict the places most likely to suffer contamination. To reduce the chances of contamination by unseen and

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unwanted microbes, aseptic technique includes disinfection of laboratory surfaces before and after using them. Microbiologists also avoid coughing, sneezing, and breathing into open culture containers.

Surgery rooms exemplify aseptic technique because every action

performed there is done in a manner to prevent contamination of the

patient. Aseptic technique does not require sophisticated technology,

but neither does it tolerate shortcuts. Whatever scientific advances microbiology absorbs in the future, aseptic techniques will endure in much the same way they are practiced today.

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Resources for learning

more about bacteria

Internet resources on bacteria

Bacteria World: http://www.bacteria-world.com/.

Cells Alive: http://www.cellsalive.com/.

Dennis Kunkel Microscopy: http://www.denniskunkel.com/.

Infectious Diseases in History: http://urbanrim.org.uk/diseases.htm.

Microbe World: http://www.microbeworld.org/.

Todar’s Online Textbook of Bacteriology: http://www.

textbookofbacteriology.net/.

The Microbial World: http://www.microbiologytext.com/index.php?

module=Book&func=toc&book_id=4.

University of California Museum of Paleontology: http://www.ucmp.

berkeley.edu/bacteria/bacteria.html.

The Virtual Museum of Bacteria: http://www.bacteriamuseum.

org/cms/.

Book resources on bacteria

Biddle, Wayne. A Field Guide to Germs, 2002, Anchor Books,

New York.

Dyer, Betsey Dexter. A Field Guide to the Bacteria, 2003, Cornell University Press, Ithaca, NY.

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Lax, Alistair. Toxin: The Cunning of Bacterial Poisons, 2005, Oxford University Press, Oxford.

Maczulak, Anne E. The Five-Second Rule and Other Myths about

Germs, 2007, Thunder’s Mouth Press/Perseus Books, Philadelphia.

Meinesz, Alexandre. How Life Began, Evolution’s Three Geneses,

2008, University of Chicago Press.

Sachs, Jessica Snyder. Good Germs, Bad Germs: Health and Survival

in a Bacterial World, 2007, Hill and Wang, New York.

Schaechter, Moselio, John L. Ingraham, and Frederick C.

Neidhardt. Microbe, 2006, American Society for Microbiology Press, Washington, DC.

Spellberg, Brad. Rising Plague: The Global Threat from Deadly

Bacteria and Our Dwindling Arsenal to Fight Them, 2009,

Prometheus Books, New York.

Zimmer, Carl. Microcosm: E. coli and the New Science of Life, 2008, Vintage Books, New York.

Classic reading on bacteria

De Kruif, Paul. Microbe Hunters, 1926, Harcourt, Orlando, Fla.

History of bacteriology through biographies of the greatest

microbiologists.

Garrett, Laurie. The Coming Plague: Newly Emerging Diseases in a

World Out of Balance, 1994, Farrar, Straus and Giroux, New York.

Mainly about viruses but with eternal lessons on all germs.

Karlen, Arlo. Biography of a Germ, 2000, Pantheon Books, New

York. A unique introduction to bacteria by following the activities of

the lyme disease pathogen, Borrelia burgdorferi.

MacFarlane, Gwyn. Alexander Fleming: The Man and the Myth,

1985, Oxford University Press, Oxford. The story and intrigue

behind a historic scientific discovery.

Thomas, Lewis. The Lives of a Cell: Notes of a Biology Watcher, 1974, Viking Press, New York. Appreciation of biology for nonbiologists.

Bacteria rule references