Indirect Methods

Several commonly used methods of estimating the number of microorganisms in a sample depend on culturing them in or on a medium that supports their growth. These methods require the use of aseptic technique, in which sterile materials (initially containing no organisms) are used along with a variety of measures to prevent contamination by other organisms (e.g., from the air, the analyst's hands, or the lab bench surface). They also involve a period of incubation (time for growth under designated conditions) of from one to several days for most common tests.

In one type of approach (used for most plate count and membrane filtration methods; see below), the organisms are grown on a solid surface until each initial cell produces a visible colony consisting of many millions of daughter cells. The number of colonies is then counted and gives an estimate of the number of colony-forming units (CFUs) originally present. It is usually assumed that each colony arose from a single cell (or spore), but in some cases it is likely that a small cluster of cells or a filament was present initially and gave rise to the colony. This is especially problematic for fungi and actinomycetes, whose counts can increase dramatically with sporulation even though little change in the actual biomass present has occurred.

Another approach (used in most probable number techniques and some plate count and membrane filtration methods) is to grow the cells in or on a medium until they produce some detectable change that indicates their presence. This might be a change in pH, increased turbidity, the disappearance of a substrate, or the appearance of a product. Often, color indicators are used to make the activity readily apparent.

One advantage of culturing techniques is that they often can be performed in a way that minimizes interferences from extraneous materials. They may also be used to count cells at lower densities than direct methods (without a concentration step). However, culturing requires a medium on which the organisms of interest can grow. Since no single medium is suitable for all prokaryotes, a true total count cannot be obtained. In fact, total culturable bacterial counts in many samples are one to two orders of magnitude lower than total direct counts.

On the other hand, media can be made intentionally selective, so that only certain organisms will grow. For example, a selective substrate can be used, or a chemical that is inhibitory to nontarget organisms can be incorporated. Similarly, selective temperatures, salinities, or pHs can be used. Additionally, media can be differential, so that among the organisms that do grow, the ones of interest can be told apart from others. Often, this is done by a color reaction.

Some common approaches are described briefly below. By varying the medium used and the incubation conditions (e.g., temperature, aerobic vs. anaerobic), a wide variety of microorganisms can be enumerated.

Plate Counts Probably the most common methods of estimating bacterial numbers are plate counts. These use agar, or occasionally, some other solidifying agent, to convert liquid medium (broth) into a solid. Agar is particularly useful because it does not melt in water until heated to boiling, but it does not resolidify until it is cooled below 45 to 48°C. However, because some bacteria can digest agar (an organic polymer produced by certain red algae), it cannot be used in some special applications.

In the spread plate technique (Figure 11.11), an agar medium (often, 10 to 15 mL) is added to a small (commonly 100 mm in diameter by 15 mm high) sterile glass or plastic petri dish and allowed to solidify. A small (0.1 mL) amount of sample or dilution is then placed on top and spread over the surface of the medium using a bent glass rod called a hockey stick. Visible colonies that grow on the surface of the medium after incubation are counted.

Example 11.3 If an average of 38 colonies is counted for plates containing 15 mL of agar medium on which 0.1 mL of a 10~4 dilution of a sample was spread-plated, what was the concentration of CFUs in the original sample?

Answer

CFUs/mL = 38 colonies/plate ^ 0.1 mL ^ 10~4 = 3.8 x 106 CFU/mL Note that the amount of agar is irrelevant.

Figure 11.11 Spread plating.

For pour plates, a 1-mL sample or dilution is put on the bottom of a petri dish. Then 10 to 20 mL of the desired molten agar medium is added, and the dish is covered and swirled to mix the contents. The medium solidifies with the microorganisms distributed throughout it. After incubation, the visible colonies are counted.

Aside from the other limitations of methods involving culturing, incubation time is also an important factor affecting the accuracy of plate counts. If insufficient time is allowed, of course, some colonies will not yet have grown to visible size. On the other hand, if too much time is allowed, some colonies may grow so large that they cover up others, again leading to underestimation. Alternatively, secondary colonies may form (e.g., from motile bacteria swimming away to start new colonies), giving overestimations.

Plate counts are typically suitable for 20 to a few hundred CFUs per plate. Lower counts have poor precision, and at higher numbers they crowd each other and interfere with growth. If cell density is too high, the plate is recorded as TNTC (too numerous to count), and it is recognized that a greater dilution factor should have been used. Since it is often difficult to know ahead of time how many organisms are present, usually a range of dilutions are plated; some may end up with no colonies, others TNTC, but hopefully those in the middle will have countable numbers.

Membrane Filtration Methods Microorganisms can be collected on the surface of a membrane by filtration (Figure 11.12). If the membrane filter is then incubated on the surface of a growth medium in a small (usually, ^50 mm in diameter) sterile petri dish, the colonies that form give an estimate of the original numbers present. The medium can either be a prepoured agar (similar to a spread plate) or a broth contained in an absorbent pad.

Figure 11.12 Membrane filtration.

Example 11.4 If an average of 41 colonies is counted per filter when 10 mL of a 10~3 dilution of the sample is filtered, what was the concentration of CFUs in the original sample?

Answer

CFUs/mL = 41 colonies/plate ^ 10mL ^ 10~3 = 4.1 x 103 CFU/mL

Membrane filtration can be especially helpful in enumerating microorganisms present at low concentrations in water. However, particulate material present in the sample can be an interference.

Most Probable Number Techniques Suppose a sample that originally contained five bacteria per milliliter was diluted 10- and 100-fold (Figure 11.13). Then five tubes of media were each inoculated with 1 mL of the sample, another five tubes were each inoculated with 1 mL of the 10-fold dilution, and another five tubes were each inoculated with 1 mL of the 100-fold dilution (a total of 15 tubes inoculated, five at each of the three different concentrations). Further, assume that each cell could grow if it is placed in a tube. Which of the tubes are likely to show growth? Since on average the tubes receiving 1 mL of undiluted sample receive five bacteria each, it is likely they will all grow, although there is a statistical possibility that one or more tubes may not get any bacteria (and will not grow), whereas others will probably get more than five. However, for the 10-fold dilution, on average each tube gets 0.5 cell. Of course, a tube cannot receive 0.5 cell, so that some tubes will get one or more cells and be "positive" (show growth), whereas others will not get a cell and will thus be negative. Probably two or three of the sample

10 x dilution

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