Analytical Food Microbiology. Ahmed E. Yousef

Analytical Food Microbiology - Ahmed E. Yousef


Скачать книгу
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.

Schematic illustration of an example of a dilution model, showing the dilutions and the dilutions selected for plating, presuming the targeted population in the food is 1.0×107 CFU/g.

       Pipetting

      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

      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


Скачать книгу