Mar 20, 2025
Detection of Shigatoxigenic E. coli O157:H7 & Enumeration of Fecal Indicators SOP
- Justine C. Condon1,
- Lisa M. Durso2
- 1USDA-ARS;
- 2USDA - ARS
Protocol Citation: Justine C. Condon, Lisa M. Durso 2025. Detection of Shigatoxigenic E. coli O157:H7 & Enumeration of Fecal Indicators SOP. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbrewqlpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 17, 2025
Last Modified: March 20, 2025
Protocol Integer ID: 121933
Keywords: Immunomagnetic Separation, Enumeration, Quanti-Tray, Coliert, Enterolert, STEC O157, Enterococcus, E. coli, Total Coliforms, Escherichia coli, Fecal Indicators, Shiga Toxin
Disclaimer
The use of trade, firm, or corporation names in this publication (or page) is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the United States Department of Agriculture or the Agricultural Research Service of any product or service to the exclusion of others that may be suitable.
Abstract
To describe the procedures on how to set-up culture-based assays for the detection of Shigatoxigenic Escherichia coli O157:H7 (STEC O157) from a variety of animal and environmental sources; and the procedures for enumeration of generic E. coli, total coliforms, and enterococcus from those same sources.
Guidelines
Scope
This protocol describes the procedures for two commonly used culture-based laboratory assays. All culture work must be done on fresh samples that are kept cool and processed within 24-hours of collection (or 48-hours if sample is collected remotely and shipped).
The first procedure applies to immunomagnetic separation (IMS) of different serotypes of STEC. IMS is a technique that will isolate cells from an enriched sample containing background substance matter. IMS is used widely for many organisms; this SOP is specifically for STEC isolation. Dynabeads are used to separate the target cells from the rest of the sample. Target-specific antibodies attach to the surface of the magnetic Dynabead sphere, allowing the STEC cells to remain while the rest of the sample is discarded. The isolated cells are then plated onto selective media for incubation to determine the amount of E. coli cells in each sample. These grown colonies can then be saved for freezer isolation. Escherichia coli (E. coli) are a large and diverse group of bacteria. Although most strains of E. coli are harmless, others can make you sick. Some kinds of E. coli can cause diarrhea, urinary tract infections, respiratory illness and pneumonia. Some E. coli cause disease by making a toxin called Shiga toxin. The bacteria that make these toxins are called “Shiga toxin-producing” E. coli, or STEC for short. Still other kinds of E. coli are used as markers for water contamination—so you might hear about E. coli being found in drinking water, which are not themselves harmful, but indicate the water is contaminated.
The second procedure applies to the IDEXX Quanti-Tray/2000 test. The IDEXX Quanti-Tray Colilert and Enterolert kits count and detect E. coli, total coliforms, and Enterococcus in a 100 mL sample. The Colilert test shows positive total coliforms as a yellow color, and positive E. coli as yellow and fluorescent. The Colilert test uses a proprietary Defined Substrate Technology to detect and quantify both total coliforms and E. coli. This technology uses two nutrient indicators – ONPG and MUG – as the major sources of carbon in the Colilert packets. These nutrients are metabolized by the enzyme β-galactosidase (coliforms) and β-glucuronidase (E. coli). Coliforms grown in Colilert metabolize ONPG using β-galactosidase, which changes it from colorless to yellow. The same is true for MUG. β-glucuronidase metabolizes MUG and creates a fluorescence. The MPN of E. coli can be found from the table based on the number of large and small wells that are yellow and fluoresce. The MPN for total coliforms can be found based on the number of small and large wells that are yellow with no fluorescence.
Enterolert testing involves the same procedure and a different proprietary Defined Substrate Technology. β-D-glucoside present in Enterococci binds with 4-methyl-umbelliferone in Enterolert to show fluorescence in a positive sample. As with Colilert, the MPN can be found using the table and the number of large and small fluorescent wells. (IDEXX Water Academy, 2020).
Responsibility
This SOP applies to all staff members and students. These individuals must be knowledgeable about the requirements set forth within this document. The lab manager or designee shall ensure that all staff and students know the proper techniques.
Materials
STEC O157 Enrichment
- 1.5X BGB media (274000EA, BD, Sparks, MD) (Recipe in Appendix A Step 19)
- STEC O157 positive control culture plate
- 50% glycerol tubes or freezer beads (G153-1, Fisher, Waltham, MA)
- Whirl-Pak® Filter Sterilized BagsWhirl-PakCatalog #B01348 , Pleasant Prairie, WI)
- Plastic beaker
- 100mL Cylinder
- Bunsen burner
- Scale
- Timer
- 1 gallon or quart zip freezer bag
- Spatulas, DisposableVWR International (Avantor)Catalog #80081-188 , Radnor, PA)
- 2mL Microcentrifuge tubes (02-681-299, Fisherbrand, Pittsburgh, PA)
- Orbital Shaker (located in Durso lab OR Miller lab, 340 Filley Hall)
- Dynabeads™ anti-E. coli O157Thermo FisherCatalog #71003 , Waltham, MA)
- DynaMag-2 - Magnetic Bead Separator (Dynal, Invitrogen, Carlsbad, CA)
- Disposable Inoculating Loops and Needles, Flexible Needle and LoopFisher ScientificCatalog #22-363-600 , Pittsburgh, PA)
- TCA media plates (Chromagar O157 + Potassium Tellurite) (EE222, Chromagar, DRG International, Springfield, NJ) (Recipe in Appendix A Step 18)
- Potassium Tellurite (3.5%) Solution (100427, MPBio, Santa Ana, CA)
- Syringe Filters - SterileFisher ScientificCatalog #09-720-005 , Pittsburgh, PA)
- Water Bath (for tempering TCA agar)
- Appropriate pipettes and tips, ranging from 20µL to 1000µL
- Bead wash (Recipe in Appendix A Step 17)
- DrySpot™ E. coli O157 Latex Agglutination TestThermo Fisher ScientificCatalog #DR0120M , Oxoid, Hampshire, United Kingdom)
- 37oC incubator
Quanti-Tray/2000
- 15 mL PET Centrifuge TubesCorningCatalog #430055 ., Corning, NY)
- 1X Phosphate Buffered SalineFisher ScientificCatalog #BP3994 , Waltham, MA) (Recipe in Appendix A Step 20)
- Automatic Pipettor/disposable pipettes
- Sharpie Markers
- Colilert Packs (WP200IPK, IDEXX, Westbrook, ME)
- Enterolert Packs (98-21374-00, IDEXX, Westbrook, ME)
- Whirl-Pak® Filter Sterilized BagsWhirl-PakCatalog #B01348 , Pleasant Prairie, WI)
- Plastic Beaker
- 100mL Cylinder
- Bunsen burner
- Micro Pipettor and tips
- Quanti-Tray Sealer (89-10894-04, IDEXX, Westbrook, ME)
- Quanti-Tray/2000 MPN Table (IDEXX, Westbrook, ME)
- Quanti-Tray/2000 Rubber Insert (WQTSRBR-2K, IDEXX, Westbrook, ME)
- Quanti-Tray/2000 Trays (WQT2KPK, IDEXX, Westbrook, ME)
- Quanti-Tray/2000 Colilert Comparator (98-09227-00, IDEXX, Westbrook, ME)
- Sterile polystyrene bottle (1156Z41, Thomas Scientific, Swedesboro, NJ)
- 37oC and 42oC incubator
- UV lamp/viewing cabinet (98-21322-00, IDEXX, Westbrook, ME)
- Sterile water (Milli-Q, distilled, or autoclaved)
STEC O157 Enrichment Procedure
STEC O157 Enrichment Procedure
14h 38m
14h 38m
Day 1: Sample Enrichment
If 1.5X BGB enrichment media has been refrigerated, warm to Room temperature .
Weigh out the sample: Using a plastic beaker to hold the filter whirl-pak bag, tare the scale, and add 10 g of sample with a clean disposable spoon or spatula to each labeled assay bag. Record all weights on a data worksheet.
Using a sterile 100mL plastic cylinder or sterile pipet, transfer 90 mL of Room temperature 1.5X BGB to each bag and homogenate gently by hand. Flame-sterilize the pipet between each sample.
Make a positive control. Add 9 mL 1.5x BGB to a whirl-pak bag or 15mL centrifuge tube. Using a loop, take a colony from the STEC O157 positive control plate and swirl around in the BGB to mix.
Place BGB bags and control in 37 °C incubator for 06:00:00 , then chill to 4 °C until ready for next step (can be held overnight). Record the time into the incubator on a data worksheet.
6h
Day 2: Manual Immunomagnetic Separation (IMS)
Label and dry two TCA plates per sample and control. Keep labeled plates in an organized order.
Note
To dry plates: Turn on a BSL-2 hood. Place the petri dishes inside with their lids askew to allow moisture to evaporate. Let set for approximately 30 minutes and check. There should not be any visible moisture remaining on top of the agar. Agar needs to be at room temperature prior to using.
Using one 2mL microcentrifuge tube per sample, label the ID# on the top of the tube, and place tubes in DynaMag-2 holder without the magnetic bar.
Vortex Dynabeads Anti-E. coli Beads well to re-suspend beads into solution.
Add 20 µL of beads to each microcentrifuge tube.
Gently mix the sample bag and add 1 mL of enrichment sample into each labeled tube.
Vortex tubes to mix, and place tube back into the holder.
Still without the magnetic bar, put the holder and tubes in a one-gallon freezer bag and zip close. Place on shaker for 00:30:00 .
30m
After 30 minutes, remove holder from the shaker and bag. Put the magnetic bar in and wait 00:05:00 (lay holder flat on bench).
5m
Remove fluid with a pipette without disturbing the beads.
Remove magnetic bar and add 1 mL of bead wash to tubes.
Vortex, put magnetic bar back in, and wait 00:03:00 .
3m
Remove fluid and repeat washing steps 2.9 - 2.11.
Remove fluid with pipette without disturbing the beads.
Remove the magnetic bar and re-suspend beads in 100 µL of bead wash. Vortex.
Pipette 50 µL of re-suspended beads onto each labeled TCA plate. Spread all over the plate with a sterile spreader.
Invert plates and incubate Overnight at 37 °C . Record the time into the incubator on a data worksheet.
8h
Day 3: Read and Record Results
Check each plate for suspect colonies. For STEC O157, suspect colonies will be mauve. See Appendix B go to step #21.1 .
Pick and test up to three suspect colonies per plate.
- DrySpot tests can be used for immediate confirmation. See Appendix B go to step #21.2
- If plates are over-crowded, streak for isolation and do dry-spot after 24-hour incubation.
Record results on a data sheet.
Quanti-Tray/2000
Quanti-Tray/2000
Pre-prep: Set out Quanti-Tray trays and bottles for each sample and remove the shrink wrap from each bottle cap. Separate the ‘Enterolert’ (Ent) and ‘Colilert’ (TC/EC (Total Coliform/E. coli)) so that they don’t get switched around.
Label the bottle lids and trays clearly with sample number and ‘Colilert’ or ‘Enterolert.’ Labels can be color-coded to distinguish between Colilert and Enterolert, and either printed or handwritten legibly.
Note
Only use a marker to label the trays. Stickers can get caught in the sealer.
Fill each bottle with sterile water with the correct amount for the labeled dilution according to Table 2. For this project we used 99 mL .
| A | B | C | |
| Final Dilution | Amount from Dilution Tube | Quanti-Tray Bottle Amount | |
| 10^-1 | 100mL of dilution bag | From bag: 100mL PBS with 10g Sample | |
| 10^-2 | 1mL of dilution bag | 99mL sterile water | |
| 10^-3 | 1mL 10^-1 dilution | 99mL sterile water | |
| 10^-4 | 1mL 10^-2 dilution | 99mL sterile water | |
| 10^-5 | 1mL 10^-3 dilution | 99mL sterile water | |
| 10^-6 | 1mL 10^-4 dilution | 99mL sterile water | |
| 10^-7 | 1mL 10^-5 dilution | 99mL sterile water |
Table 2. Quanti-Tray Dilutions – amount from dilution tubes that go into snap-pack bottles.
For Enteroloert - Empty an Enterolert Snap-Pack into each bottle. Tightly put the cap back on and shake the bottle to dissolve the Snap Pack powder.
For Colilert - Empty a Colilert Snap-Pack into each bottle. Tightly put the cap back on and shake the bottle to dissolve the Snap Pack powder.
Weigh out the sample: Using a plastic container to hold the filter whirl-pak bag, tare the scale, and add 10 g of sample with a clean disposable spoon or spatula to each labeled assay bag. Record all weights on a record worksheet.
Using a sterile 100mL plastic cylinder or sterile pipet, transfer 90 mL of Room temperature PBS to each bag and homogenate gently by hand. Flame-sterilize the pipet between each sample. This will create a 10-1 dilution.
Turn on the Quanti-Tray Sealer. When the sealer is ready, the green light will turn on at the top of the sealer.
Dilution tubes will be labeled and pre-filled with 9 mL PBS. Massage the PBS enriched sample bag to mix.
Pipette 1 mL from the dilution bag (10-1) into the correspondingly labeled 9mL PBS dilution tube, creating a 10-2 dilution. Tighten the lid and vortex to mix.
Continue pipetting 1 mL , then vortex, from each consecutive tube until you reach the desired final dilution (Table 1).
| A | B | |
| Dilution Needed | Use | |
| 10° | 10g of Sample | |
| 10^-1 | 90mL of PBS + 10g of Sample | |
| 10^-2 | 9mL of PBS + 1mL of 10^-1 Bag | |
| 10^-3 | 9mL of PBS + 1mL of 10^-2 Tube | |
| 10^-4 | 9mL of PBS + 1mL of 10^-3 Tube | |
| 10^-5 | 9mL of PBS + 1mL of 10^-4 Tube | |
| 10^-6 | 9mL of PBS + 1mL of 10^-5 Tube |
Table 1. Dilution tube set-up for solid samples.
Keep dilution tubes cool. Place On ice or in 4 °C until ready for use.
Vortex each dilution tube right before use and add the correct amount of the dilution to corresponding sample bottle(s) (see Table 2), tightly screw on lid and gently shake or invert to mix well.
| A | B | C | |
| Final Dilution | Amount from Dilution Tube | Quanti-Tray Bottle Amount | |
| 10^-1 | 100mL of dilution bag | From bag: 100mL PBS with 10g Sample | |
| 10^-2 | 1mL of dilution bag | 99mL sterile water | |
| 10^-3 | 1mL 10^-1 dilution | 99mL sterile water | |
| 10^-4 | 1mL 10^-2 dilution | 99mL sterile water | |
| 10^-5 | 1mL 10^-3 dilution | 99mL sterile water | |
| 10^-6 | 1mL 10^-4 dilution | 99mL sterile water | |
| 10^-7 | 1mL 10^-5 dilution | 99mL sterile water |
Table 2. Quanti-Tray Dilutions – amount from dilution tubes that go into snap-pack bottles.
Pour contents of the inoculated bottle into Quanti-Tray/2000 Trays.
Place the tray in Quanti-Tray/2000 Rubber Insert and run through the sealer (well side down). You may have to push the tray until the sealer begins to pull the tray through.
Check that all wells have liquid in them, face the trays up (wells on top), and place in incubator no more than 10-trays high. Record the time into the incubator on a data worksheet.
- Colilert: 37 °C for 24-28 hours.
- Enterolert: 42 °C for 24-28 hours.
After 24 hours, the trays can be removed from the incubator, and the results read. Mark all positive E. coli wells with a permanent marker. See Appendix B go to step #22 for additional information on reading results.
- Positive TC – Yellow Well
- Positive EC – Yellow and Fluorescing Well
- Positive Entero – Fluorescing Well
Appendix A – Recipes
Appendix A – Recipes
45m
45m
Bead Wash Solution Recipe (1x PBS with 0.05% Tween 20)
Directions to make 1L:
- Add 500 µL Tween 20 to 1 L 1X PBS in a media bottle.
- Stir until mixed
- Autoclave 00:15:00 at 121 °C with lid on loosely.
- Cool to Room temperature before use.
- Label and store in the refrigerator.
15m
TCA (Chromagar O157 + Potassium Tellurite) Recipe
Directions to make Potassium Tellurite (3.5%) Solution:
- Weigh 0.35 g Potassium Tellurite. Clean the scale and turn it off.
- Add weighed Potassium Tellurite to 10 mL distilled water in a sterile beaker.
- Filter-sterilize using a 0.45µM filter
- Open and attach the filter to the syringe, using the luer lock
- Remove the syringe plunger and pour the Potassium Tellurite solution into the open end of the syringe.
- Place the filter over a new, sterile labeled 15mL centrifuge tube and depress the filter until all the solution passes through the filter.
- Refrigerate any extra for up to 30 days.
Directions to make TCA Plates:
- Weigh 14.6 g Chromagar O157 powder. Clean and turn off the scale.
- Add 14.6 g Chromagar O157 powder to 500 mL distilled water in a 1L bottle.
- Heat while stirring and bring just to a boil. DO NOT AUTOCLAVE!
- Cool to 45 °C -50 °C in a water bath.
- Add 18µL / L (or 9µL/500mL) of 3.5% Potassium Tellurite Solution
- Swirl to mix media.
- Pour or pipette in 15 mL increments into sterile petri dishes and let cool until solid.
- Label and store in the refrigerator and use within 30 days.
1.5X Brilliant Green Bile Broth (BGB) Recipe
Directions to make 1L: (Make in 500mL batches)
- Weigh 60 g BGB powder. Clean and turn off the scale.
- Add BGB powder to 1 L distilled water in a media bottle.
- Heat while stirring until mixed.
- Autoclave for 00:15:00 at 121 °C with lid on loosely.
- Cool to Room temperature before use.
- Label and refrigerate if not being used the same day.
15m
Note
BGB powder is an irritant to the lungs. Take care to avoid inhalation of the powder while weighing or it will cause coughing. Can wear a mask to minimize impact.
1x Phosphate Buffered Saline (PBS) Recipe
Directions
- Measure 1800 mL of distilled water and pour into a 2L bottle.
- Measure 200 mL of 10X PBS.
- Pour the 200 mL of 10X PBS into the bottle with the 1800 mL of water.
- Autoclave at 121 °C for 00:15:00 .
- Cool to Room temperature before use.
- Label and store in the refrigerator.
15m
Appendix B – Reading Results
Appendix B – Reading Results
STEC O157
Positive and suspect colonies are a mauve color.
Positive colonies will coagulate on the DrySpot test, while negative colonies will not react and be smooth.
Quanti-Tray/2000
Note
*Be sure to cover any exposed skin before working with the UV lamp, and wear UV-blocking glasses*
Colilert: Count and record both yellow and fluorescing wells.
- Yellow wells indicate the presence of total coliforms. The wells must be as yellow as the Colilert Comparator (See: Materials Quanti-Tray)
- Wells that are yellow AND fluoresce indicate the presence of E. coli.
- Do not count the wells that fluoresce but are not yellow.
Enterolert: Count and record the fluorescing wells.
- Fluorescing wells indicate the presence of Enterococcus.
- Do not count the wells that fluoresce bright yellow.
Using the Quanti-Tray/2000 MPN Table software program (IDEXX MPN Generator) or the hardcopy that comes with the trays. Record the number of large and small wells to reach the Most Probable Number.
- With any dilution other than 100, you must account for the dilution in the final answer.
- Example: 41 Large Positive, 6 Small Positive tested at a 10-2 dilution
9.33X101 → 9.33X103
Example of MPN Generator
Protocol references
IDEXX Water Academy. 2020. "Testing for Coliform and E. coli" and "Testing for Enterococci." www.idexxwateracademy.com