#### Estimated time usage: 60 min/45 min
## Background
In the previous lab, we plated raw milk and pasteurized milk samples onto Plate Count Agar to obtain total aerobic bacteria counts. Unfortunately, the differences seen in PCA counts do not directly correlate with the safety of the products. For food products, there are three major safety concerns—biological, chemical and physical. Among these three, biological hazards cause the most illnesses and hospitalizations. Biological hazards include pathogenic bacteria and viruses that cause foodborne infections. Although pathogen contamination has been identified in different types of products, in the past decade we have seen increasing numbers of foodborne outbreaks and recalls associated with fresh produce.

The Food and Drug Administration Food Safety Modernization Act (FSMA) was signed into law in 2011. FSMA is “[transforming the nation’s food safety system by shifting the focus from responding to foodborne illness to preventing it](https://www.fda.gov/food/guidance-regulation-food-and-dietary-supplements/food-safety-modernization-act-fsma)” . Among these new rules and regulations, two of them apply particularly to fresh produce, including the “Current good manufacturing practice and hazard analysis and risk-based preventive controls for human food” and the “Standards for growing, harvesting, packing, and holding of produce for human consumption.” In both regulations, the microbial quality of agricultural water receives additional oversight. The levels of the indicator microorganism Escherichia coli need to be monitored for agricultural water in order to ensure that the water used for irrigating or washing the produce is safe. For these new regulations, the Western Institute for Food Safety & Security prepared a [video](https://www.wifss.ucdavis.edu/watersampling101videoseries/ ) to teach how to collect water samples.

Why indicator microorganisms? Pathogenic bacteria usually present in the product, if any, in low quantities and may not be counted by plating methods. To overcome this limitation, indicator microorganisms are monitored to facilitate evaluation of product safety. **Indicator microorganisms** present in higher numbers than pathogenic bacteria; their concentrations are correlated with the presence of pathogenic bacteria. There are different types of indicator microorganisms; some are directly correlated with the product quality and some are associated with product safety. For example, if we want to check the quality of raw milk products, *Lactococcus lactis* is used as the indicator microorganism. In this lab, we would like to use indicator microorganisms to evaluate the safety of water. Two indicator microorganisms can be used, ***Escherichia coli and total coliform***. See **Figure 2.1** for the relationships between total coliform, fecal coliform and E. coli.
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<img src="https://hackmd.io/_uploads/r1-PboRsn.png" alt="Figure" width="500">
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<p style="text-align:center;"><strong>Figure 2.1</strong> Relationships between coliform, fecal coliform, and E. coli. Created with BioRender.com.</p>

The coliform group of bacteria is defined as those aerobic and facultative anaerobic, gram-negative, non-spore-forming rods that can ferment lactose with acid and gas produced at 32-37 ˚C. They have been used extensively as a sanitary indicator, e.g. in water analysis. Not all coliform are from fecal origin; that’s why there is a “total coliform” group and a “fecal coliform” group. Fecal coliforms ferment lactose and produce acid and gas at higher temperatures (43-45 ˚C). Coliform includes species beyond *Escherichia coli*, such as *Enterobacter aerogenes* and Klebsiella peumoniae. Collectively, coliforms constitute an artificially defined group within the family of Enterobacteriaceae. While *Escherichia coli* and *Enterobacter aerogenes* are considered to be the primary coliforms, some species of Citrobacter and Klebsiella also ferment lactose and belong to the coliform group.

Detection and the enumeration of indicator microorganisms can be done in different ways. The first way is to plate samples onto coliform agar (Violet Red Bile Agar, VRBA) or CHROMagar™ ECC (E. coli and other coliform). The second way is to use pectin gel methods (e.g. Petrifilm™). The Petrifilm we will use today is 3M Petrifilm™ E. coli/Coliform Count Plates. The third way is to use the Most Probable Number (MPN) method. The MPN technique is a serial dilution test to estimate the concentrations of microbes of interest when their concentrations are too low for plating and direct enumeration. MPN has been commonly used to estimate coliform concentrations in food or environmental samples (e.g. water samples).

In an MPN determination, aliquots of the food sample itself or its homogenate as well as its serial dilutions are prepared and used to inoculate a number of tubes of media. Three or five replicate tubes are usually prepared and inoculated for each dilution. Lactose broth is used for estimating the MPN of coliform in this lab. After incubation, MPN tubes are examined for signs of microbial growth, and the presence or absence of growth is recorded for each tube. Depending on the type of broth medium used and the microbes of interest, tubes with positive microbial growth can be identified based on the turbidity of the broth, color change if a color indicator is added, production of gas, production of metabolites or reduction of chemicals. Based on the number of replicate tubes you use for each dilution, different statistical tables are used for result interpretation. The U.S. Public Health Service requires a minimum of five tubes per dilution for the testing of drinking water, while the three-tube series is considered acceptable for the testing of water used for other purposes.

As mentioned earlier, VRBA is one media used for enumerating the total coliform counts in water samples. This media contains a bile salts mixture and crystal violet as selective agents, lactose as the fermentable sugar, and neutral red as the pH indicator. The suspect coliform colonies are purplish-red because the coliforms produce acid from lactose and turn the pH indicator red. A reddish zone of precipitated bile surrounds the colonies. However, this agar will not tell us the coliform species. In order to characterize and **differentiate various coliform species**, additional biochemical tests are needed. Among these tests, one famous test is the “IMViC” test. It is a series of tests that can differentiate E. coli from other species, e.g. *Enterococcus aerogenes* and *Citrobacter freundii*. Please see **Table 2.1** for more information about “IMViC” tests.

| | | *Escherichia coli* | *Citrobacter freundii* | *Enterobacter aerogens* | *Proteus vulgaris* |
| --- | ------------------------------------ | ---------------- | --- | --- | --- |
| 'I' | Production of Indole from tryptophan | + | - | - | + |
| 'M' | pH after termination <4.5 (Methyl red turns red) | + | + | - | - |
| 'V' | Acetoin is a fermentative end-product (positive Voges-Proskauer test) | - | - | + | + |
| 'C' | Citrate can be used as the sole source of carbon and energy for growth | - | + | + | + |
---

### Objectives
1. Practice the use of VRBA and Petrifilm™ to determine the E. coli and coliform counts in surface and tap water samples
2. Understand and practice the use of the MPN table to estimate the numbers of coliforms present in surface water samples
3. Practice the use of the IMViC test to characterize different coliform species

---

### Materials

**Food samples**: Raw milk, tap water, and arboretum water (each team only needs to pick one sample, each room needs to have all three samples taken).
**Bacterial strains**: *Escherichia coli*, *Enterobacter aerogenes*, *Citrobacter freundii* and *Proteus vulgaris*. These cultures are provided to you in liquid culture format; they have been incubated in Tryptic Soy Broth for 24 hours at 37 ˚C.
**Media**:
Pre-melted liquid VRBA agar
Petrifilm™ *E.coli*/Coliform count plate **8** for 3M and **2** for CHARM
3 tubes of 0.1% buffered peptone water, (reason: 10-1 to 10-3 dilution)
12 tubes of regular lactose broth with Durham vials (9 ml of lactose broth in each tube) (reason: 10-0 to 10-3 dilution, 3 for each dilution)
3 tubes of double strength lactose broth in large tubes with Durham vials (9 ml of double strength lactose broth in each tube)

8 empty petri dish (double layer pourplating)

4 Eosin Methylene Blue (EMB) plates
4 MacConkey (MAC) plates
4 Brilliant Green Lactose Bile (BGLB) tubes with Durham vials
4 Tryptone broth (TB) (for Indole test, check Indole Nitrite Medium or
4 Simmons Citrate (SC) slants
4 Methyl Red-Voges Proskauer (MRVP) broth (check MR-VP medium in the media manual)

**Other Supplies**: Pipette and tips, incubators, Petrifilm™ spreaders

---
### Procedures [VP for lab 2](https://drive.google.com/file/d/1kwv5VQfchfw9Pgtb3Qv6cLV-Lm9DXFLn/view?usp=drive_link)
#### Part 1: Water quality examination
**STEP 1**: Prepare dilutions of your sample
- Please make serial dilutions of your samples first. Transfer 0.5 ml of the original sample into 4.5 ml of peptone water and vortex it. After vortexing, transfer 0.5 ml of the 1:10 diluted sample into the next 4.5 ml of peptone water and vortex it.

**STEP 2**: Plating samples with liquid VRBA (Pour-plate with an overlayer)
1. Get two empty plates for each dilution (including the undiluted original samples) and label them with sample names and proper dilution factors (e.g. Original or 1:10).
2. Pipette two 1 ml of a given dilution into two empty plates.
3. Make sure your liquid VRBA has cooled to about 45-50 ˚C.
4. Pour ~15-20 ml of melted VRBA agar into each plate and swirl gently to mix the sample and the agar.
5. After the agar is solidified in your plates, pour an additional top layer of VRBA over the surface and let it solidify again.
6. Incubate the plates at 35 ˚C for 24 hours.

**STEP 3**: Plating samples onto Petrifilm™ (both 3M and CHARM)
1. Lift up the cover layer of the Petrifilm.
2. Pipette 1 ml of the dilution on the media layer. Please only plate the 10-0, 10-1, 10-2, and 10-3 dilutions. Plate 2 Petrifilms for each dilution (3M).
3. For milk and tape water, use the 10-0 to plate the CHARM petrifilm. For pond-water, use the 10-3 to plate the CHARM petrifilm.
4. Gently place the cover film back and use the Petrifilm spreader to make sure the liquid is evenly distributed in the agar (circle).
5. Incubate the plates with the cover layer facing up at 35 ˚C for 24 hours.

**STEP 4**: Inoculating MPN tubes
1. Get three lactose broth tubes for each dilution of your sample. Three tubes double strength lactose broth and three tubes of regular strength lactose broth are need for your original sample. See Figure 2.2 for more details. Label your tubes.
2. Place the inoculated tubes in a tube rack and incubate them at 35 ˚C for 48 hours.

**STEP 5**: Result reading and calculation (after 48 hours)
1. Typical coliform colonies in VRBA are deep magenta color with fuzzy edges, as the colonies are surrounded by a cloud of bile salt precipitation. Count only the red fuzzy colonies that are over 0.5 mm in diameter in an uncrowded plate.
2. Typical coliform colonies on Petrifilm™ are red in color with gas bubbles around them. Typically, E. coli colonies are blue in color with gas bubbles around them. S[ee Petrifilm™ interpretation guide for more information and photos. ](https://multimedia.3m.com/mws/media/236246O/petrifilm-ecoli-coliform-interpretation-guide.pdf)
3. Positive lactose broth tubes are cloudy (increased turbidity), with gas bubbles trapped in Durham tubes. [Use the MPN table in the book or on the FDA website for a guide to more interpretation.](https://www.fda.gov/food/laboratory-methods-food/bam-appendix-2-most-probable-number-serial-dilutions) Please also read the supplement materials for Lab 2 for the computation of MPN.

#### Part 2: IMViC tests
**STEP 1**: Make Gram stains of each microorganism and examine them under the microscope.
| Culture name | Gram stain color | Cell shape | Draw a picture of what you see |
|------------------------|------------------|------------------|-------------------------------|
| *Escherichia coli* | | | |
| *Enterobacter aerogenes*| | | |
| *Citrobacter freundii* | | | |
| *Proteus vulgaris* | | | |

<p style="text-align: center;"><strong>End of the first section of week</strong></p>

**STEP 2**: Inoculate each medium with the cultures.
1. Label all your tubes with your group number and the microorganisms’ names.
2. Streak the liquid culture onto the agar plate to obtain discrete colonies.
3. Inoculate the broth tubes with a loopful of each respective organism, one organism per tube.
4. Inoculate the SC slant by streaking on the surface.
5. Incubate all inoculated media at 37 ˚C for 24-48 hours.

**STEP 3**: Read the results
1. Read and record the results for [EMB](https://asm.org/ASM/media/Protocol-Images/Eosin-Methylene-Blue-Agar-Plates-Protocol.pdf?ext=.pdf), [MAC](https://asm.org/ASM/media/Protocol-Images/MacConkey-Agar-Plates-Protocols.pdf?ext=.pdf), [BGLB](https://www.fda.gov/food/laboratory-methods-food/bam-media-m25-brilliant-green-lactose-bile-broth) and [LB](https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/409/736/l5285dat.pdf) media. Note that the characteristic reactions for each kind of medium are described in Wikipedia. When reading the results, it is important to consider only the differential reactions occurring in the uncrowded areas. When organisms die, they release degradative enzymes that can break down the amino acid in the meat or yeast extracts to form alkaline end-products. If an acid reaction constitutes a positive test, then a zone of dead cells can give a false negative result. The cells die sooner when they are more crowded. That’s why we need to avoid reading the results from crowded areas.
2. IMViC test.
**2.1** Indole tests: add 0.2-0.3 ml (4-6 drops) Kovac’s reagent to each TB tube and shake very gently.
**2.2** Watch the tube for 10 min to look for the appearance of a dark red ring at the top of the culture. The red ring is a positive indole test. It may form immediately after the addition of the Kovac’s reagent and then fade, or it may take ~10 min to form.
**2.3** Methyl red test: transfer 1 ml of the MRVP culture into a new empty tube and add several drops of methyl red. A red color is an indication of pH < 4.5 and is a positive test. A yellow color, indicating pH > 6.0, is a negative test. Some tubes give an intermediate reaction. If that happens, you just record “intermediate.” This “intermediate” result usually mean that the fermentation is not of the mixed acid type (e.g., a negative MR test), but you can’t always count on it.
**2.4** Voges-Proskauer test: transfer another 1 ml of MRVP culture into a new empty tube. Add 0.6 ml (12 drops) of the VP reagent and 0.2 ml (4 drops) of the 40% KOH reagent, shake gently to mix, and then let the tube stand undisturbed for up to two hours. Check it from time to time for the appearance of a pink or red color. The formation of the colored compound is indicative of the presence of acetoin. Acetoin is an intermediate in the formation of butylene glycol. If no change has occurred in two hours, or if the color is gray, record the results as negative.
**2.5** Citrate test: examine the SC slant for growth. Positive reactions are indicated by blue color on the slants. Bacteria which do not have the permease to take up citrate and/or require organic growth factors will not grow on this medium.
<p style="text-align: center;"><strong>End of the second section of week</strong></p>