---
# System prepended metadata

title: Lab 3 Sampling and the Detection of Pathogens
tags: [Spring 2024]

---

# Lab 3 Sampling and the Detection of Pathogens
#### Estimated time usage: 60 min/45 min/5 days
## Background

Pathogenic bacteria are major food safety concerns. Common foodborne pathogens include Shiga-toxin-producing Escherichia coli, *Salmonella* spp., and *Listeria monocytogenes*. Taking Salmonella spp. as an example, different *Salmonella* species have been involved in various foodborne outbreaks and recalls. The related products ranged from low-moisture foods, e.g. peanut butter and almonds, to high-moisture produce, e.g. tomatoes.

Two kinds of diseases are caused by *Salmonella* species: “enteric fever” (typhoid fever) and gastroenteritis. Both types begin with the ingestion of food or water containing viable *Salmonella* cells. *Salmonella* typhi and *Salmonella* paratyphi are both adapted to human and are transmitted from one person to the next via direct fecal contamination. This generally happens in areas with poor sanitation. With *Salmonella*-caused gastroenteritis (salmonellosis), bacteria remain in the intestines, the duration of the disease is shorter, and the mortality rate is relatively low. The name of the disease is salmonellosis. Strains of *Salmonella* that commonly cause gastroenteritis are not adapted to any particular animal host, and can be identified in birds, wild animals, and other mammals. Some of these animals are asymptomatic carriers. It is recognized that all gastroenteritis-producing *Salmonella* are closely related and all belong to a single species, *Salmonella* enterica. 

A couple of important characteristics that can distinguish gastroenteritis-causing *Salmonella* from other bacteria of the same family (Enterobacteriaceae) are: (1), they produce hydrogen sulfide from thiosulfate, and (2), they cannot ferment lactose or sucrose. These two characteristics have been used to differentiate Salmonella from E. coli. In addition, *Salmonella* can synthesize lysine decarboxylase, which this differentiates *Salmonella* from *Citrobacter*. Its ability to ferment dulcitol helps distinguish *Salmonella* from Proteus. Most Proteus species produce the enzyme urease, and this can also be used to separate Proteus from *Salmonella* since *Salmonella* cannot produce urease. 

The prevalence and levels of *Salmonella* present in food products are low, which is why enrichment is needed in order to detect this pathogen. Results are typically presented as positive or negative, instead of actual counts. Testing for positive or negative may sound easier than obtaining an actual count, but the truth is that it can be more challenging. That is because when you are deciding whether this lot of sample or batch of a product is negative, you need to know if your ***sampling numbers*** (the numbers of samples you obtained) and ***sample sizes*** (the weight or volume of each sample) are statistically solid enough to support making final conclusions. In addition, you need to know the ***limit of detection*** of your testing method as well as ***the maximum allowed number of non-conforming samples***. An acceptance sampling is one of three major statistical areas that are used for quality control and improvement. An acceptance sampling is most useful when product testing is destructive, very expensive and time consuming, and when product liability risks are significant. (Starbird, 1994; Dumičić and Žmuk, 2012). 

The **International Commission on Microbiological Specifications for Foods (ICMSF)** is an organization that evaluates issues and provides suggestions/guidelines for issues that are related to microbial food safety and quality. Its microbial sampling plans (https://www.icmsf.org/publications/software-downloads/microbiological-sampling-plans/) contain guideline information needed when one needs to determine a sampling plan. 

In general, sampling plans are divided into two groups based on the categories involved in the sampling plans. One is called a “**two-class sampling plan**,” and the other is called a “**three-class sampling plan**.” Categories refer to the acceptability criteria; the three categories include conforming, marginal, and nonconforming. The “two-class sampling plan” is designed to decide on acceptance or rejection of a lot, typically for food-safety-related reasons. The two classes or categories are “conforming” and “nonconforming.” Three main factors are considered: “n” (number of sample units to be chosen independently and randomly from the lot), “m” (a microbiological limit that is allowed in the conforming sample, e.g. cfu/g), and “c” (maximum allowable number of sample units yielding a positive result or exceeding the microbiological limit m). The “three-class sampling plan” can be used for rating quality (the numbers of spoilage microorganisms). The three categories are “conforming,” “marginal,” and “nonconforming.” Four factors are considered: n, c, m, and M. The M is the maximum number of colony-forming units that are allowed in the sample, while the m is the minimum CFU counts allowed. Figure 3.1 aims to show the relationships between n, c, proportion defective, and probability of acceptance. Sampling is very critical for microbial analysis. The excel sheets containing more detailed information about sampling planning are uploaded in Canvas.

![image](https://hackmd.io/_uploads/S1TjJhOwp.png)
<p style="text-align:center;"><strong>Figure 3.1</strong> Three operational characteristic curves (OC curves) established based on different sampling numbers (n numbers) and c numbers. The Y axis is “probability of acceptance,” with 1 meaning 100% and 0.8 meaning 80%. The X axis is the defective ratio. As you can see, if our defective ratio is 0.2%, when our sampling size is 20 and the c is 0 there is almost no chance for us to accept a lot. If our sampling size is 5, then the chance of accepting a lot is ~25%. Created with BioRender.com.</p>


In addition to the two sampling groups discussed above, sampling can also be divided into single sampling plans and double sampling plans. In a single sampling plan, the lot will be sampled once. The lot will be accepted if the lot inspection finds less or no defective units than are allowed and will be rejected if the lot inspection finds more defective units than are allowed. In double sampling, the lot can be sampled twice before the final acceptance or rejection decision can be made. This applies to the “three-class sampling plans” for one lot. Figure 3.2 is made based on the paper by Dumičić and Žmuk, 2012.    

![image](https://hackmd.io/_uploads/BJZhx2_va.png)
<p style="text-align:center;"><strong>Figure 3.2</strong> Differences between the single sampling plan and the double sampling plan (adopted from Dumičić and Žmuk, 2012). Created with BioRender.com.</p>

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### Objectives
1.	Practice the procedures used for isolating *Salmonella* from food products.
2.	Be familiar with the typical biochemical and serological tests associated with *Salmonella* detection.
3.	Understand how to use the FDA Bacteriological Analytical Manual (BAM). 

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### Materials
**Cultures**:
One *Salmonella* Typhimurium (attenuated strain)
One *Escherichia coli* strain
**Food Sample**:
Cilantro and eggs 
**Media**:
Trypticase™ soy broth (TSB) (35 ˚C for 24 hours)
Universal Pre-enrichment Broth (35 ˚C for 24 hours)
Rappaport-Vassiliadis (RV) broth (42 ˚C waterbath, 24 hours, Check Rappaport-Vassiliadis R10 broth in the media manual)
Tetrathionate (TT) broth (35 ˚C 24 hours)
Bismuth sulfite (BS) agar plates
Xylose lysine desoxycholate (XLD) agar
Hektoen enteric (HE) agar
Triple sugar iron slant (TSI)
Lysine iron agar slant (LIA) 
Salmonella Latex Agglutination test kit (Catalog number DR1108A)
**Supplies**:
			Whirl-pak bags, pipettes, and tips

---
### Procedures [VP for lab 3](https://drive.google.com/file/d/1UY_ueldBB_O124W8p1cKIdpPeDgsNY08/view)
**The sequence of the lab is as follows**: 
Monday - pre-enrich cilantro or egg samples
Tuesday - transfer food samples to RV broth, transfer positive control pure culture to RV broth
Wednesday - streak food samples and pure cultures on XLD, HE, BSA
Thursday - transfer probable colonies / pure colonies to TSI and LIA slants
Friday - read plates

**STEP 1**: Pre-enrichment of food samples (Monday)
1.	Each group will pick one food item.
2.	For cilantro, weigh 10 grams of cilantro and transfer them into a Whirl-pak bag with 90 ml of Universal Pre-enrichment Broth. Blend the mixture for two min in a stomacher. The sample needs to be pre-enriched at 35 ˚C for 24 hours.
3.	For eggs, crack two eggs into a clean jar and beat the eggs. Transfer 10 ml of liquid egg into the Whirl-pak bag (make sure there is no shell in the bag) together with 90 ml of TSB broth. Pre-enrich the sample at 35 ˚C for 24 hours.

**STEP 2**: Re-activate the pure *Salmonella* and *E. coli* reference cultures (Tuesday)
1.	Transfer a couple of loopfuls of *Salmonella* into 9 ml of TSB broth. 
2.	Transfer a couple of loopfuls of *E. coli* culture into 9 ml of TSB broth.
3.	Enrich these culture at 35 ˚C for 24 hours. 

**STEP 3**: Selective enrichment (Tuesday)
1.	After 24 hours of pre-enrichment, transfer 1 ml of the pre-enriched broth (from the Whirl-pak bag for food items or from the TSB tube for pure E. coli or *Salmonella* cultures) into the RV broth. Incubate the inoculated RV broth in the 42 ˚C water bath for 24 hours. 
2.	Transfer 1 ml of the pre-enriched broth into the TT broth. Incubate the inoculated TT broth at 35 ˚C for 24 hours. TT broth is needed for BAM manual, however, for this experiment, we do not use TT broth. 

**STEP 4**: Streak onto selective agar for suspect *Salmonella* isolation (Wednesday)
Streak selective enrichment samples onto XLD, HE, and BS agar. Do the same for the pure *E. coli* and *Salmonella* cultures. They are used as the quality control. 

**STEP 5**: Read the results and conduct further analyses (Thursday)
1.	Check for suspect colonies
HE agar: Suspect *Salmonella* colonies on HE agar are blue-green to blue color with or without black centers. Some cultures of *Salmonella* may produce colonies with large, glossy black centers or may appear as almost completely black. 
XLD agar: Suspect *Salmonella* colonies on XLD agar are pink with or without black centers. Some cultures of *Salmonella* may produce colonies with large, glossy black centers or the colonies may appear as almost completely black.
BS agar: Suspect *Salmonella* colonies are brown, gray, or black; sometimes they have a metallic sheen. The surrounding medium is usually brown at first but may turn black in time with increased incubation, producing the so-called halo effect.

2.	Streak suspect Salmonella colonies onto TSI and LIA agar slants. 
Pick one suspect colony from HE or XLD or BS agar with a sterile inoculating needle and inoculate the TSI slant by streaking the slant and stabbing the butt. Without flaming, inoculate the LIA slant by stabbing the butt twice and then streaking the slant. Pick another suspect colony and do the same. Incubate the inoculated TSI and LIA slants at 35 ˚C for 24 hours. Make sure you cap the tubes loosely to maintain aerobic conditions in the tubes. 
Pick one pure Salmonella control colony from the HE or XLD or BS agar and inoculate one TSI slant and one LIA slant as described above and then incubate them at 35 ˚C for 24 hours. Use them as the control. 

**STEP 6**. Read the LIA and TSI results and conduct a *Salmonella* serological test (Friday)
1.	Read the LIA and TSI results
   *Salmonella* produces an alkaline (red) slant and an acid (yellow) butt, with or without the production of H2S (blackening of agar) in the TSI. In LIA, *Salmonella* produce an alkaline (purple) reaction in the butt of the tube. Most *Salmonella* produces H2S in LIA. Some non-*Salmonella* cultures produce a brick-red reaction in LIA slants.  
2.	Conduct a Salmonella serological test 
Pick some Salmonella culture from the LIA or the TSI slants and place it in the center of the circle. Follow the serological test instruction and add the reaction agents. Monitor the formation of agglutination (the following figure shows the positive and negative agglutination results).  
<p style="text-align: center;"><strong>End of the second section of week</strong></p>