Equipment and Materials

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Chapter 4a
Last Updated
2026-06-01
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8 minute(s)
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A. Equipment and materials

  1. Balance, ≥ 2 kg with 0.1 g sensitivity
  2. Blender, Waring or equivalent model with low speed operation at 8000 rpm, with 1 liter glass or metal jar
  3. Incubators, 35 ± 0.5°C and 44 ± 1°C
  4. Petri dishes 20 × 150 mm
  5. Pipets, Pasteur
  6. pH test paper, range 6.0-8.0

B. Media

  1. Tryptone phosphate (TP) broth (M162)
  2. Brain heart infusion (BHI) broth (M24)
  3. Levine's eosin-methylene blue (L-EMB) agar (M80)
  4. MacConkey agar (M91)
  5. Triple sugar iron (TSI) agar (M149)
  6. Blood agar base (BAB) (M21)
  7. Tryptone (tryptophane) broth (M164)
  8. Bromcresol purple broth (M26) supplemented individually with 0.5% (w/v) of each: glucose, adonitol, cellobiose, sorbitol, arabinose, mannitol, and lactose
  9. Urea broth (M171)
  10. Lysine decarboxylase broth, Falkow (M87).
  11. Potassium cyanide (KCN) broth (M126)
  12. MR-VP broth (M104)
  13. Indole nitrite medium (tryptic nitrate) (M66)
  14. Acetate agar (M3)
  15. Mucate broth (M105)
  16. Mucate control broth (M106)
  17. Malonate broth (M92)
  18. Koser's citrate broth (M72)

C. Reagents: inorganic, organic, and biological

  1. Sodium bicarbonate solution, 10%, aqueous (sterile) (R70)
  2. ONPG (o-nitrophenyl-β-D-galactopyranoside) disks (R53)
  3. Phosphate buffered saline solution, (sterile) (PBS) (R60), or Butterfield's Phosphate-buffered dilution water (BPBW) (R11).
  4. Kovac's reagent (R38)
  5. VP reagents (R89)
  6. Oxidase test reagent (R54)
  7. Nitrite detection reagents (R48)
  8. Mineral oil, heavy sterile (R46)
  9. Gram stain reagents (R32)

L. Equipment and materials

  1. Balance, 1-500 g with 0.1 g sensitivity
  2. Sterile glass jars for enrichment
  3. Optional: Stomacher with appropriate plastic bags for 250 ml vol.
  4. Large resealable plastic bags to accommodate 200 g of product (for analysis of leafy produce)
  5. Incubators: 36°C ± 1°C and 42°C ± 1°C
  6. Petri dishes 20 × 150 mm
  7. Microcentrifuge tubes (0.5 to 2.0 mL)
  8. Conical centrifuge tubes (50.0 mL)
  9. Micropipettors (e.g., 0.5-20 µL, 20-200 µL, 200-1000 µL)
  10. Microcentrifuge (capable of spinning at 15000 × g)
  11. Pipets (1 to 10 ml volume)
  12. Pipet tips (0.2 to 1000 µl volume) (aerosol resistant tips)
  13. Vortex Mixer
  14. Filter Paper
  15. Waterbath or heat block capable of maintaining 100°C.
  16. SmartCycler II PCR thermolcycler (Cepheid, Sunnyvale, CA) capable of performing cycling parameters described below and simultaneous real-time sequence detection for FAM, TET, Texas Red and Cy5 dyes.
  17. SmartCycler PCR reaction tubes (minimum reaction volume of 25 µl) and racks compatible with PCR thermolcycler
  18. Applied Biosystems 7500 Fast Real-time PCR thermocycler (SDS version 1.4) (Applied Biosystems, Thermo-Fisher Scientific Brand)
  19. AB 7500 Fast Plates (AB# 4346906)
  20. Optical Adhesive Film for 7500 Fast Plates (AB# 4311971)
  21. 8-well strips; alternative tp plates (AB# 4311971)
  22. Appropriate ABI 7500 Fast Plate Holder (specific for 96-well tray or 8-strip well tubes)
  23. Ice bucket and ice
  24. Sterile tongs
  25. Gloves

M. Media and Reagents

  1. Modified Buffered Peptone water with pyruvate (mBPWp) and Acriflavin-Cefsulodin Vancomycin (ACV) Supplement (M192a)
  2. STEC/O157 primers and probes listed in Tables 2 and 3 are specific to real-time PCR platform being used. a. Primers - 10 µM working solution of each primer listed in Table 2 or 3. Stock and Working solutions can be prepared from commercially synthesized primers with Page | 10 basic desalt purification (Fisher/Genosys or equivalent) by rehydrating with sterile distilled water to appropriate concentrations. Store at -20°C to -70°C in a non frost-free freezer. b. Probes - 10 µM working solution of each probe listed in Tables 2 or 3. Dual Hybridization Probes should be purchased as RP HPLC-purified and labeled as indicated in Tables 2 and 3. Stock and working solutions can be prepared from commercially synthesized probes with molecular grade sterile distilled water. Working solutions should be aliquoted in small amounts and stored frozen (-20 to -70°C) and away from light until use to avoid fluorophore degradation. c. Exogenous Internal Amplification Control (IAC) Smart Cycler Platform: (IAC) Full length sequence as described in U.S. Patent Application 0060166232: cgcatgtggt cacagccctg acgaagctgt catcaagttc ataatgacat cgatatgggt gccgttcgag cagtttagcc ctaaatcacc ctaccggcag acgtatgtca cattcaccag ggagacgcat gagattggat gctgttgtgc gccctcaaca atgtaacgaa tggctgcatc gtctcgcgaa tattgtcgta ccatcatctg acttggctca tgtctgcaag aggcttcgca ctgggctttatg Prepare a stock solution of the IAC DNA sequence from commercially synthesized product and titer to a stock solution where a known amount will reliably provide a Ct between 25-35 PCR cycles. NOTE: Alternatively, the primers, probes and IAC internal control assay components are also available as a lyophilized bead product for use on the SmartCycler II orIAC full length template (BioGX, Birmingham, AL). Table 2. Primer/probe sequences for use on SmartCycler II platform. Primers1 GenBank # Stx1F934
    Bases 5' → 3' Sequence 26 Stx1R1042 gTg gCA TTA ATA CTg AAT TgT CAT CA M19473 21 gCg TAA TCC CAC ggA CTC TTC Stx2F1218 X07865 24 gAT gTT TAT ggC ggT TTT ATT TgC Stx2R1300 X07865 26 UidAF241 Tgg AAA ACT CAA TTT TAC CTT Tag CA AF305917 21 CAg TCT ggA TCg CgA AAA CTg UidAR383 AF305917 22 ACC AgA CgT TgC CCA CAT AAT T IAC55F2
    17 ATg ggT gCC gTT CgA gc IAC186R2,3
    19 Cga gaC gAT gCa gCC aTT C Probes1 GenBank # M19473 Bases 5' → 3' Sequence stx1P990 31 TxRd-TgA TgA gTT TCC TTC TAT gTg TCC ggC AgA T-BHQ2 stx2P1249 X07865 25 6FAM-TCT gTT AAT gCA ATg gCg gCg gAT T BHQ1 UidAP266 AF305917 15 TET-ATT gAg CAg CgT Tgg-MGB/NFQ ICP-Cy52
    26 Cy5- TCT CAT gCg TCT CCC Tgg TgA ATg Tg BHQ2 1 Primer/Probe name composed of target gene (stx1, stx2 or uidA), forward primer (F), reverse primer (R) or probe (P), 5' base position of oligonucleotide in the respective gene sequence specified in column 2. 2,3 IAC primers and probes are covered by U.S. Patent Application 0060166232 and sited in: Nordstrom, et al. 2007. Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio Page | 11 parahaemolyticus bacteria in oysters. Appl. Environ. Microbiol. 73:5840-5847 (14); and Angelo DePaola, et al. 2010. Bacterial and Viral Pathogens in Live Oysters: 2007 United States Market Survey. Appl. Environ. Microbiol. 76:2754-2768 (15). AB 7500 Fast Platform: TaqMan® Exogenous Internal Positive Control (Applied Biosystems, Carlsbad, CA. #4308323) Table 3. Primer and probe sequences for use on the AB 7500 Fast platform. Primers1 GenBank # Bases 5' → 3' Sequence stx1F934 M19473 26 stx1R1042 gTg gCA TTA ATA CTg AAT TgT CAT CA M19473 21 gCg TAA TCC CAC ggA CTC TTC Stx2F1218 X07865 24 Stx2R1300 gAT gTT TAT ggC ggT TTT ATT TgC X07865 26 wzyF1831 Tgg AAA ACT CAA TTT TAC CTT Tag CA AF061251 24 wzyR1936 CTC gAT AAA TTg CgC ATT CTA TTC AF061251 23 CAA TAC ggA gAg AAA Agg ACC AA Probes1 GenBank # Bases 5' → 3' Sequence stx1P990 M19473 31 Cy5-TgA TgA gTT TCC TTC TAT gTg TCC ggC AgA T-BHQ2 stx2P1249 X07865 25 TAMRA-TCT gTT AAT gCA ATg gCg gCg gAT T-BHQ2 wzyP1881 AF305917 25 6FAM - ACT TAg Tgg CTg ggA ATg CAT Cgg C – BHQ1 1Primer/Probe name composed of target gene (stx1, stx2 or uidA), forward primer (F), reverse primer (R) or probe (610LC, 670LC, 705LC, or FL P), 5' base position of oligonucleotide in the respective gene sequence specified in column 2.
  3. Additional Real-time PCR additional reagents depending on real-time platform being used: a. SmartCycler II Platform - OmniMix-HS or SmartMix HM PCR Reagent Beads (Cepheid, Sunnyvale, CA. Also available through Fisher). b. AB 7500 Fast Platform - Express qPCR Supermix Universal Taq (Invitrogen, Carlsbad, CA. #11785200)
  4. Tellurite Cefixime – Sorbitol MacConkey Agar (TC-SMAC) (M194)
  5. Chromogenic selective agar: R&F® E. coli O157:H7 agar (R&F Laboratories, Downers Grove, IL), prepared according to manufacturer instructions. Rainbow® Agar O157 (BIOLOG, Hayward, CA) prepared according to manufacturer instructions for high background flora (contains 10 mg/L novobiocin plus 0.8 mg/L potassium tellurite).
  6. Levine's Eosin-Methylene Blue (L-EMB) agar (M80) (Section R only)
  7. Trypticase Soy Agar with Yeast Extract (TSAYE) (M153).
  8. Butterfield's Phosphate Buffer(R11) (pH 7.2 ± 0.2. Sterilize by autoclaving)
  9. Sterile distilled water, molecular grade water or equivalent
  10. Physiological saline (0.85% NaCl) Page | 12
  11. Kovac's Reagent (R38)
  12. ColiComplete Discs - fluorogenic MUG substrate for GUD and chromogenic X-gal for GAL (BioControl, Bellevue, WA)
  13. Anti-O157 and anti-H7 latex reagent (ThermoScientific Remel 4, Lenexa, KS, or equivalent)
  14. API20E or VITEK GNI (BioMerieux, St. Louis, MO).
  15. STEC heart infusion washed blood agar with Mitomycin-C (SHIBAM) (M195)