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 capable of growing under refrigeration. Choice of incubation temperature, therefore, depends on the microorganisms of interest and their natural habitat, as well as presence of competing microbes.
In food microbiology, several incubation temperatures are typically used. For potentially pathogenic organisms, such as Salmonella enterica, incubation occurs at 32–37°C, a temperature range suitable for mesophiles. A somewhat cooler temperature (e.g., 25°C) is more preferred by spoilage organisms such as yeasts, molds, and psychrotrophic bacteria. “Room temperature” is typically taken to mean 22°C, but this temperature may vary depending on the room used for incubation and even the season and area of the world. Refrigeration at 4°C is typically used to maintain cultures without allowing further growth. A refrigerated incubation can also be used for cold enrichments of psychrotrophic microorganisms such as Listeria monocytogenes.
Plates containing inoculated agar media are typically inverted before incubation. If plates are incubated with lids upward, water condensate falls on the agar surface causing the spreading of colonies. When plates are inverted, condensed water (from moist agar) accumulates on the plate lid. Excessive water condensation on the lid, however, is undesirable and should always be minimized. Pouring hot agar (> 50°C) aggravates this problem. In the case of spread plates, it is preferable to pour the agar in these plates 24–48 hours before use. Some microbiologists choose to “dry” the spread plates soon after preparation for several hours in a warm clean incubator. For the safety of the analyst, plate lids with excessive water condensation should be replaced with dry sterile lids.
Colony Counting
“Counting” in food microbiology refers to the determination of the size of a microbial population within a specific quantity of food (i.e., population concentration). Enumerating the number of colonies on agar plates may also be referred to as “counting,” therefore careful distinction between these two usages is urged. Throughout this manual, the former will be referred to as “population count” and the later as “colony count.”
Some enumeration techniques, such as the direct microscopic counting method, allow determination of the number of cells per unit volume or weight of the sample. The plate count technique, however, determines the number of cells or cell clumps capable of forming colonies on agar plates. Since it is impossible to distinguish colonies arising from individual cells and those from cell clumps, the final population count determined by this method is expressed as colony forming units per unit volume or weight, i.e., CFU/ml or CFU/g.
To begin the counting process, the analyst should lay out the incubated plates in order of dilution to evaluate the executed dilution scheme and technique. The lowest dilution
