# Lab 4 D- and Z-Values and the Concept of Validation

## BACKGROUND

The inactivation of pathogenic microorganisms by thermal or non-thermal treatments is critical in food processing. Different microorganisms have various degrees of resistance. Taking heat resistance as an example, some heat-sensitive pathogens or psychrotrophs can be killed by brief exposure to moderate temperatures, e.g. 45 ˚C, while spore-formers and spores require long exposures to higher temperatures (e.g. 100 ˚C for more than 15 min). The concept of “D-value” was created to reflect the differences in heat resistance (or other types of resistance) of the microbes. The definition of D-value is that it is “the time necessary to decrease a population of cells by 90% (or 1 Log) at a particular temperature” in the particular food matrix (Figure 4.1). When the same microorganism is heated in the same food matrix at different temperatures, a set of D-values at various temperatures can be obtained. If these D-values are plotted on a logarithmic scale, with temperatures plotted on a linear scale and the Log10 D-values plotted on the Y axis, a “Thermal Death Time Curve” can be formed. By using the “Thermal Death Time Curve,” a Z-value can be determined. The Z-value is defined as the change in temperature required to reduce the D value by 90% or 1 log (Figure 4.2). Please do note that D-value and Z-value can be determined for different non-thermal treatments as well, e.g. UV treatments or acid wash treatments.

The microbial heat resistance (or other types of resistance) is impacted by multiple internal and external factors, including nutrient availability, the status of the microbes, the moisture level of the food and the processing environment, the pH values of the products, and the potential presence of other microbes). Thus, the D-value changes when you change the target microorganisms and food items. Peng et al., published a review paper on thermal pasteurization of ready-to-eat foods (2017). Table 4.1 includes thermal treatment guidelines for milk. By looking at this table, you can see that thermal treatment conditions are different based on the different fat and sugar contents as well as texture differences. Government guidelines are also available for different food items. For example, USDA Food Safety Inspection Service provides guidelines for lethality performance standards for certain meat and poultry products ([USDA FSIS Guidelines](https://www.fsis.usda.gov/wps/wcm/connect/fsis-content/fsis-questionable-content/oppde/rdad/frpubs/95-033f/95-033f_appendix-a/default/ct_95-033f_appendix+a)).

**Table 4.1 Thermal treatment guidelines for different milk products (adopted from Peng et al., 2017; Enright et al., 1957 and 1961).**
| Products | Target bacteria | Processing requirement |
|----------|-----------------|------------------------|
| Milk | *Coxiella burnetii* | 63 ˚C (145 ˚F) for 30 min for batch process; 72 ˚C (161 ˚F) for 15 s for HTST. |
| Cream; chocolate milk | *Coxiella burnetii* | 66 ˚C (150 ˚F) for 30 min for batch process; 75 ˚C (166 ˚F) for 15 s for HTST. |
| Ice cream mix | *Coxiella burnetii* | 69 ˚C (155 ˚F) for 30 min for batch process; 80 ˚C (175 ˚F) for 25 s for HTST. |

*Coxiella burnetii was recognized as the most resistant bacteria of concern in milk.*

## OBJECTIVES

1. Practice conducting thermal treatment experiments.
2. Get familiar with the calculation of D-value and Z-value.
3. Learn the background and practice of spot plating.

## MATERIALS

### Cultures:
- Hemolysin-negative *Listeria monocytogenes* (incubated in regular BHI with 0.5% NaCl)
- Hemolysin-negative *Listeria monocytogenes* (incubated in BHI supplemented with 5.0% NaCl)

### Media:
- 0.1% peptone water (4.5 ml tubes) x 84 tubes
- Brain Heart Infusion (BHI) agar (**Squre agar**)
- 12 x 2 ml centrifuge tube

### Supplies:
- 53, 58, and 63 ˚C water bath
- Sterile 1.5 ml microcentrifuge tubes with racks and float racks
- Pipette and tips

## PROCEDURES
### Procedures [VP for lab 4](https://drive.google.com/file/d/11vlNuwlj07irpewUXQpwDypA1ITmJHP6/view?usp=drive_link)
### STEP 1: Assign treatment temperatures to individual groups.
Given that we have three treatment temperature and two strains, getting all groups to work with all three temperatures and all two strains will be a lot of work. In this case, each group needs to choose only one temperature. The TA needs to make sure we have all temperatures covered. Some temperatures may get tested by two groups, some temperatures may be tested once by one group. This arrangement changes for each year based on the final enrollment and team numbers.

### STEP 2: Transfer your cultures into 1.5 ml tubes for treatment.
1. Transfer 1 ml of L-BHI or L-BHI-HS into a 1.5 ml tube. Since you need to plate five time points, you need to prepare five tubes for L-BHI and five tubes for L-BHI-HS.
2. Label the top of the tube with a marker.
3. Place these tubes on a float rack.

### STEP 3: Conduct the heat treatment and plate the surviving cells by using spot plating method
1. Keep the “time 0” tubes (one tube for L-BHI and one tube for L-BHI-HS) and dilute the cell suspensions to obtain cell counts. For this time 0 point, plate -6 and -7 dilutions (1:1,000,000 and 1:10,000,000 dilutions). Plate 10 µl from each dilution onto each 1/4 BHI plate to obtain cell counts.
2. Place your tubes (with the float rack) in the water bath with the temperature you choose. The temperature you choose is ____________.
3. Once you place the tubes in the water bath, start a timer immediately.
4. Take out one L-BHI tube and one L-BHI-HS tube at each of the following time points: 5, 10, 15, 20, and 25 min.
5. Once you take out these tubes, make dilutions and plate these samples to obtain the counts of the surviving cells.
6. Place the plated BHI plates at 35 ˚C and incubate them for 48 hours.
Note: we may change the dilution factors based on the initial cell counts.

### STEP 4: Enumerate the surviving cell numbers
Take out your plates and enumerate your surviving cell numbers. Record your data in the RESULTS section. See the next page.

### STEP 5: Calculate the D-values for Lm in BHI and in BHI-HS
To do so, make a D-value table (like the one shown in below) for each culture.

### STEP 6: Calculate the Z-value for Lm in BHI and in BHI-HS
To do so, you need to construct the “Thermal Death Time Curve” as shown in Figure 2 on Page 29. For this section, you need to obtain the data from all three temperatures. The TA will summarize the data for you.