Analytical Food Microbiology. Ahmed E. Yousef
dust particles can carry microorganisms. Mold and bacterial spores are common contaminants of air since they survive dryness and other detrimental environmental factors. The microbiological quality of air in a processing facility impacts the quality and safety of perishable food processed in this facility. Improper filtration of air entering a facility or recycling air from the raw product area into the finished product area can result in food contamination. Air quality in the packaging area is particularly important for the control of post‐processing contamination. Therefore, determining the microbial load in air is an important task.
Sedimentation is a simple method to measure air quality. It involves exposing agar media plates to air by leaving these plates uncovered in the location to be sampled. Air contaminants will sediment by the force of gravity during the exposure time (e.g., 15 min). The plates are incubated, and the colony count may be considered proportional to air contamination level.
Air in a particular environment may also be forcibly impacted onto the surface of agar media plates using mechanical means. Jets of air are directed over the media plates so that air load collides and sticks to agar surface. After receiving a measured air sample, the agar plates are incubated, and colonies are counted. Air streams also may be filtered through a microfilter. Microorganisms are released from the filter using a suitable diluent and the microbial load is counted.
SELECTED REFERENCES
1 Dell, R.B., Holleran, S., and Ramakrishnan, R. (2002). Sample size determination. ILAR Journal 43: 207–213.
2 Food and Drug Administration (FDA). (2017). Draft guidance for industry: Control of Listeria monocytogenes in ready‐to‐eat foods. Docket Number: FDA‐2008‐D‐0096. Washington, DC: FDA.
3 International Commission on Microbiological Specifications for Foods (ICMSF). (1986). Microorganisms in Foods 2. Sampling for Microbiological Analysis: Principles and Specific Applications. 2e. Oxford, UK: Blackwell Scientific Publications.
4 Moberg, L. and Kornacki, J.L. (2015). Microbiological monitoring of the food processing environment. In: Compendium of Methods for the Microbiological Examination of Foods, 5e. (ed. Y. Salfinger and M.L. Tortorella), 27–43. Washington, DC: APHA Press.
PRACTICING SAMPLING AND SAMPLE PREPARATION
Accurate sampling, careful sample handling, and diligent preparation of sample for analysis are critical measures for successful recovery of microorganisms from food. Factors to consider during sampling and sample preparation have been described in the first section of this chapter. These factors should be reviewed carefully before starting this exercise. Students also should prepare, in advance, an exercise summary that includes a sample preparation approach specific to the food assigned to them. The single‐sheet summary will be used on the bench as a guide while executing the exercise.
OBJECTIVES
Practice sampling of food and preparing the sample for microbiological analysis.
PROCEDURE OVERVIEW
Students will work in groups (two students per group). Each group will be assigned a food sample to prepare for a typical microbiological analysis. Ahead of the laboratory session, each group will be informed of the food sample assigned and members of the group are asked to prepare an exercise summary that includes a sample preparation approach specific to the food assigned to their group. Examples of foods to be provided are heads of iceberg or Romaine lettuce, and whole fish (e.g., tilapia). Members of each group will prepare their laboratory sample for analysis, withdraw two 10‐g analytical samples, homogenize the samples, and prepare three decimal dilutions of each homogenate. Students will be evaluated on the basis of work efficiency, aseptic technique, accuracy of measurements, and ability to work cooperatively within the group.
MATERIALS
Per Group of Two
Sterile peptone water (diluent):Two bottles, 90 ml eachSix test tubes, 9 ml each
Food sample (laboratory sample)One of the following foods: iceberg lettuce, Romaine lettuce, whole fish, cantaloupe, etc.
PipettesOne 1000‐μl automatic pipetter and a box of compatible sterile tipsTwo 1‐ml individually wrapped sterile serological pipettes, and two pipetting aids
Group‐Shared
Top‐loading balance
Stomacher and stomacher bags
Sample preparation equipment (equipment is to be cleaned and sanitized with ethanol, from an alcohol squeeze bottle, between uses)
Cutting board
Knife and a pair of scissors
Spatula and pair of tongs
Two aluminum‐covered sterile beakers: 250‐ml or 400‐ml capacity, depending on sample assigned
METHODS
Important Notes:
Aseptic technique is expected in this and all other exercises. This includes, but is not limited to working in proximity of a lit Bunsen burner, flaming the mouth of the medium container after removing the cap, protecting the sample during processing against contamination from the work environment or the analysts, and using common sense in handling sterile and contaminated tools.
Wearing gloves and goggles is needed for personal safety.
1 Safety and Laboratory Etiquette ComplianceRead and follow laboratory safety guidelines described in Chapter 1.Give special attention to the “practical aspects” described in that chapter.
2 Sample InformationPick the food sample assigned to the group.Record any pertinent sample information.
3 Sample Cutting (group task)Using the plan prepared by the group in advance, cut the food sample (laboratory sample) into small pieces suitable for withdrawing 10‐g analytical samples. Use the appropriate sample preparation equipment provided.Note: The analytical sample should represent the laboratory sample accurately.
4 Withdrawing the Analytical Sample (individual task: one sample per group member)Using the sterile beaker, weigh out one 10‐g analytical sample.Aseptically transfer the analytical sample from the beaker into a sterile stomacher bag.
5 Homogenization (individual task)Add the 90‐ml diluent to the contents of the stomacher bag. Carefully push most of the air out of the bag and close the bag with the provided fastener before stomaching.Homogenize the sample in the stomacher for two min. The contents of the stomacher bag (i.e., the homogenized sample) is the 1/10 (10–1) dilution of the original analytical sample.
6 Preparing Decimal Dilutions (individual task, see Figure 2.3)Label three dilution tubes that will become the 10–2, 10–3, and 10–4 dilutions.Using the serological pipette, transfer 1 ml of food homogenate into the diluent tube labeled 10–2. Vortex tube contents briefly.Using the automatic pipetter, transfer 1 ml of the 10–2 tube into the tube labeled 10–3. Vortex tube contents briefly.Using the automatic pipetter, transfer 1 ml of the 10–3 tube into the tube labeled 10–4. Vortex tube contents briefly.
7 Recording Observations (group task)Report work progress to instructor.
QUESTIONS
1 How similar or different is the sample preparation scheme you wrote from the one you executed? If you encountered