Epilogue: How microbiologists grow bacteria
Spending a career working with invisible objects can stretch a person’s patience. Bacteria demand that a microbiologist wait several hours, overnight, or even several days before multiplying to high numbers. Mycobacterium takes three weeks to reach numbers high enough to study the organism. Finally, a person cannot call herself a microbiologist without mastering the art of aseptic technique. The technique truly is an art because no two bacterial cultures behave exactly the same way, and the avenues for contamination seem limitless. The standard practices described here avoid some of the pitfalls that students new to microbiology see when growing bacteria.
Microbiology samples may be patient specimens (blood, sputum, stool, and so on), foods, consumer products, soil, drinking water, untreated surface waters, or wastewater. Microbiologists usually take samples of 100 milliliters liquid or 10 grams of a solid to a laboratory for “processing,” which is the series of steps needed to determine if bacteria are in the sample, how many, and what kinds.
Microbiology employs two aids for working with the huge numbers common in this field. First, microbiologists dilute samples containing millions or billions of cells in a technique called serial dilution. Second, the microbiologist converts these large numbers to logarithms.
Serial dilution
A sample containing more than a million bacteria per milliliter or gram is too concentrated with cells for scientists to study a species and draw meaningful conclusions. Rather than juggle numbers of this magnitude, microbiologists dilute each sample sequentially to reduce the cell concentration to between 30 and 300 cells per milliliter.
The method called serial dilution consists of a set of tubes each containing 9.0 milliliters of sterile buffer (water with a small amount of salts to maintain a constant pH range). By taking one milliliter of the sample and adding it to one of the 9-milliliter tubes, the microbiologist has made a one-to-ten dilution, or 1:10. With this step, a sample containing three million cells per milliliter now contains 300,000. A milliliter of this new dilution transferred to another sterile 9-milliliter volume lowers the concentration to 30,000 cells per milliliter. The microbiologist continues diluting each new dilution until arriving at what he assumes is a much lower concentration than the original sample. This is tricky because the process is done on supposition. When microbiologists receive a sample from a patient, food, or the environment, they have no idea if the sample contains millions of bacteria or a few. Serial dilution helps span the range of possible concentrations to determine the actual concentration of cells in a sample.
Following the serial dilution, the microbiologist has a set of tubes before him, each tube containing one-tenth as many cells as the preceding tube. The next step involves inoculating agar plates with small volumes, called aliquots, from each dilution. The microbiologist might take a 0.1-milliliter aliquot from each dilution and put this amount onto individual sterile agar plates. For example, 0.1 milliliter of the 1:10 dilution goes onto a plate (microbiologists usually include duplicate or triplicate plates for each dilution), 0.1 milliliter of the 1:100 dilution does the same, and so on. After all of these transfers have been completed, the microbiologist has a set of inoculated plates each containing a subsample (the aliquot) from the 1:10, 1:100, 1:1,000, 1:10,000, and 1:100,000 dilutions.
Next, the microbiologist spreads each of the aliquots over the agar surface to spread out whatever bacteria may be there—remember, they are invisible. This spreading step requires a sterile glass or plastic rod about seven inches long with a bend at one end about an inch from the end of the rod. Visualize a hockey stick shape. These spreaders are in fact called “hockey sticks” by microbiologists. When the aliquot has been spread as a thin transparent film over the agar surface, the agar is called a spread plate. Each plate comes with a cover, which now goes onto the inoculated spread plate.
The scientist puts the entire stack of spread plates into an incubator set at a favorable temperature. Although a stack of agar plates in an incubator seems an obvious space-saving arrangement, this innovation of German bacteriologist J. R. Petri in 1887 changed microbiology. The stackable, compact Petri dishes enabled microbiologists to study more replicates and a wider variety of microbes than in previous experiments.
Most bacteria recovered from temperate environments grow at body temperature, so incubators can be set to about 98.6°F (37°C) for the incubation step. Many foodborne contaminants and almost all pathogens and native flora prefer this temperature. Soil and water microbes and some foodborne psychrophiles grow better at lower temperatures.
Incubation lasts overnight, a day or two, or several days to weeks, depending on the bacterium. After incubation of the plates, the microbiologist sees visible colonies, usually no bigger than one-eighth of an inch in diameter, each containing millions of bacteria.
Counting bacteria
A colony of bacteria growing on agar contains identical cells that have all descended from a single ancestor cell. When a microbiologist inoculates agar, individual bacteria disperse in the medium. During incubation, each cell from the inoculum doubles in number every half hour or so, depending on species, until they form the visible mass of cells known as a colony. Microbiologists call the colonies CFUs for colony-forming units and count them either manually under a magnifying glass or electronically by scanning the agar plate with a laser beam.
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 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 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 log10, whole numbers and fractions can be looked up in tables, determined by a slide rule, or produced by a calculator. Use a calculator!
Converting large numbers to their log10 value illustrates that for 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 log10 of 7.37, not 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 log10 of 7.67. This illustrates 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 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 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.