Analytical Food Microbiology. Ahmed E. Yousef

many dilutions should be prepared, and which dilutions should be selected for plating? In other words, what dilution scheme should the analyst prepare and follow? Preparing all possible dilutions and plating these dilutions is a waste of resources and effort. On the contrary, preparing a limited number of dilutions may lead to the failure of the analyst to accurately determine the population of the organism in the food.

To answer the questions just presented, the dilution scheme should be based on the projected concentration of the targeted microorganism in the product. A product expected to contain a high load of the microorganism should be diluted further than that expected to contain a small load. The microbial load is sometimes easy to predict if the analyst has prior experience in analyzing the same product. In most cases, however, the analyst should check published literature for microbial populations expected in the product. In other situations, the analyst may seek information about the product from the producer, manufacturer, processor, or retailer. Considering that determining a dilution scheme is often based on the analyst’s best guess, the scheme should accommodate a reasonable margin for error. For example, if the food is presumed to contain 10⁷ CFU/g of the microorganism subject to analysis, the dilution scheme should allow determination of populations in the range of 10⁶ to 10⁸ CFU/g. This can be achieved by the scheme shown in Figure 3.1, assuming that an ideal countable plate contains 100 colonies. This scheme can be verified using the population count equations described later in this chapter.

Figure 3.1 Example of a dilution scheme, showing the dilutions (prepared from a homogenized food) and the dilutions selected for plating, presuming the targeted population in the food is 1.0×10⁷ CFU/g.

       Pipetting

Pipetting is an important activity for completing dilutions successfully. Quantitative transfer of broth culture, diluent, homogenized food, liquid food, or similar materials requires accurate pipetting. This can be accomplished by using a pipette in combination with a pipetting device (pipette dispenser, pipetaid, etc.). The pipetting device is an essential tool in analytical laboratories and may vary from simple rubber bulbs to automated pipetters. Simple mechanical pipetting aids are often used with glass or plastic pipettes. These pipettes, often referred to as serological pipettes, are maintained sterile in canisters or individual wrappers. In this book, it is suggested that individually wrapped sterile serological pipettes, along with a hand‐held pipette pump, are used to make the initial dilution (from the homogenized food) in most exercises.

      For convenience and consistency, quantitative and aseptic transfer of liquid in microbiological laboratories is accomplished using variable‐volume semiautomatic micropipetters (e.g., Eppendorf pipettes or Gilson Pipetman), in combination with matching sterile pipette tips. These micropipetters are capable of handling specific ranges of liquid volume and the 1000 μl and100 μl are the most popular sizes in microbiological laboratories. Tips matching these sizes are packed in autoclavable box‐racks. The packaged tips are autoclaved before use and disposed of appropriately after use. Adjusting micropipetters to desired volumes and accurate pipetting and dispensing of pipetted volumes requires some practice before starting laboratory exercises. Micropipetters should be calibrated regularly to avoid errors in volume measurements.

      When transferring a homogenized food (or a culture) to prepare a set of dilutions, a new clean and sterile pipette or pipette tip should be used for each dilution made. Transferring these dilutions to agar plates can be done using one of two approaches. Starting with the lowest dilution (i.e., most concentrated) requires the use of a new pipette or tip for each dilution transferred. However, a single pipette or pipette tip may be used to transfer multiple dilutions provided the analyst starts with the highest dilution, proceeding to the lowest dilution (i.e., from the least to the most concentrated). If the latter approach is followed, caution should be exercised to avoid contaminating the pipette or the tip during this multistep use. Additionally, plates must be spread with no delay to prevent inoculum from being absorbed into agar before proper distribution across agar surface.

      Plating

      “Plating” refers to the process of transferring and incorporating the sample to be analyzed, or its dilutions, into a suitable agar medium in a Petri plate. When the agar medium is poured and solidified in the Petri plate in advance, incorporation of a small volume of the sample dilution is done by spreading and the process is described as “spread‐plating.” Alternatively, a larger amount of the sample, or its dilution, may be dispensed first in an empty Petri plate into which warm molten agar is poured, and plate contents are mixed. This process is known as “pour‐plating.” Analyzing a food for a given microorganism may necessitate using pour‐plating or spread‐plating, but in other circumstances the two methods can be used interchangeably. Note that these two plating methods require different dilution schemes.

       Spread‐plating

After a set of dilutions is prepared from a homogenized food sample or a culture, portions of these dilutions are deposited and spread over the surface of agar plates. Spreading inocula (ideally 0.1 ml) on an agar plate requires the use of a cell spreader. This device can be as simple as a bent‐end glass rod, made in the laboratory by a skilled technician. Alternatively, metal cell‐spreaders are used for their durability. Glass or metal spreaders are decontaminated (sanitized) immediately before use as follows. Dip the spreader into a jar of alcohol, remove the spreader, and pass it quickly through the flame of a Bunsen burner to allow remaining alcohol to catch fire. Notice that alcohol decontaminates the spreader and flaming does not heat the spreader enough to sterilize it; flaming is done only to remove excess alcohol (the spreader should never be held in the flame). Disposable sterile plastic spreaders are also available; these are preferred when the transferred inoculum is expected to contain bacterial spores, as the ethanol dipping (just described) inactivates cells but not spores. Using an alcohol jar, along with glass or metal spreader used to spread spores, is likely to result in contamination of subsequently spread plates.

      Calibrated sterile inoculation loops (usually disposable) may also be used to spread a specimen or its dilution on an agar plate. This requires scanning the agar surface with the loop repeatedly in a systematic fashion. This spreading technique is used when a limited number of spread‐plates are needed and the microbial load in the analyzed sample is relatively small. This technique may be used in conjunction with sterility testing.

       Pour‐plating

      Pour‐plating involves dispensing a portion of the sample or its dilution (commonly 1 ml) into a standard Petri plate, adding molten agar medium (10–15 ml, at 48–50°C), mixing plate contents carefully, and letting the mixture solidify. Using this technique requires that molten agar media be prepared ahead of the sample preparation and held in a water bath set at ~50°C until poured. The molten medium could be prepared in bulk in Erlenmeyer flasks or partitioned in test tubes. In the former case, a skilled analyst can pour the agar into multiple plates at quantities suitable for the analysis. In the latter case, the agar quantity in each tube should be sufficient to prepare one plate.

      Incubation

      Inoculated plates are incubated at a time‐temperature combination appropriate for the growth and colony formation by the microorganism being counted. Microorganisms vary in their ability to grow at different temperatures. While psychrophiles prefer refrigeration temperatures (1 to 10°C), mesophiles grow optimally at temperatures close to that of the human body (37°C), and thermophiles grow best at higher temperatures (e.g., 55°C). Psychrotrophic bacteria grow optimally in the mesophilic range, but they are also