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  1. Laboratory Methods (Food)

Environmental Sampling and Detection of Salmonella in Poultry Houses

Testing Methodology for Salmonella Enteritidis (SE) Main Page

Other Analytical Methods of Interest to the Foods Program

October 2008

This method, for the detection of all Salmonella serotypes in environmental samples, has been used primarily for finding Salmonella Enteritidis (SE) in the hen houses. Salmonella Enteritidis (SE) foodborne outbreaks in human beings have been associated predominantly with eggs, when a vehicle has been identified. Due to the characteristics of SE organisms shed into the egg from the laying hen, environmental analytical methods for detecting SE were developed for the houses in which the laying hens are maintained. Environmental testing is used to verify that an on-farm egg safety program is functioning in the reduction of SE in laying hen houses, thereby reducing the number of SE contaminated eggs. Environmental testing is also used in hen houses that have been implicated in tracebacks from foodborne SE outbreaks. For further information on SE refer to Salmonella enterica Serovar Enteritidis in Humans and Animals: The Epidemiology, Pathogenesis, and Control (6).

  1. Sampling

    1. Sampling for verification of an on-farm egg program

      Manure is the preferred sample type. Use a 10 cm × 10 cm (4 in × 4 in) 12 ply sterile gauze pad which is aseptically attached to a pole by clips or to a string. The clips should be disinfected before each use with 70% ethanol. The gauze pad and string, or gauze pad alone, should be sterilized by autoclaving. Moisten pad with canned evaporated milk, canned skim (fat free) evaporated milk or canned low fat evaporated milk. Disinfect the top of the canned milk with 70% ethanol before opening. The can opener should also be disinfected with 70% ethanol. Place a gauze pad over the opened can of milk to deter flies from contaminating the milk. Use sterile gloves when handling and moistening the pad.

      Drag the moistened gauze pad over the manure the entire length of one side of the row/bank. Take another gauze pad and drag the other side of the row/bank. Repeat this procedure on all rows/banks of the house.

      Place each pad in a separate whirlpak-type bag with sufficient milk to keep the pad wet (no more than a tablespoon or approximately 15 ml). If the collection time is greater than 6 h, the use of a cooler with frozen gel packs is recommended for storage of the samples prior to shipment to the laboratory.

    2. Sampling for outbreak traceback

      An analytical unit is a 10 cm × 10 cm (4 in × 4 in) 12 ply sterile gauze pad that has been moistened with canned evaporated milk, canned skim (fat free) evaporated milk or canned low fat evaporated milk. Disinfect can as directed above. This pad is used to sample a designated area of the laying hen house environment. Designated sampling areas are manure piles/ pits/scrapers, eggbelts and de-escalators, and fans or walkways.

      For manure sampling, follow the procedure for verification of egg program (see A.1 above).

      For eggbelt and de-escalator sampling, use aseptic technique to moisten a gauze pad with canned evaporated milk, canned skim (fat free) evaporated milk or canned low fat evaporated milk. Use a 70% ethanol solution to disinfect the top of the canned milk before opening. The can opener should also be disinfected using 70% ethanol. Place a gauze pad over the opened can of milk to prevent flies from contaminating the milk. Use sterile gloves when handling and when moistening the pad. With gauze pad in hand, which has been aseptically gloved using sterile gloves, swab multiple areas of approximately 10 to 15 cm (4 to 6 in) in length on all eggbelts from all reachable tiers/levels on the side of a bank/row of cages for at least 12 to 15 meters (40 to 50 ft). With the same gauze pad, swab multiple areas of all de- escalators (fingers, belt or baskets). Place one moistened gauze pad in each bag. Repeat the procedure for all sides of all rows/banks.

      For fans, using aseptic technique, moisten a gauze pad with canned evaporated milk, canned skim (fat free) evaporated milk or canned low fat evaporated milk. Use the same procedures as with eggbelts to disinfect the top of the canned milk and the can opener. Use sterile gloves when handling and when moistening the pad. With gauze pad in hand, which has been aseptically gloved using sterile gloves, swab areas on the left, right, top and bottom of the frame/housing of the fan. If the fan is off, swab the blades of the fan. For walkways, use aseptic technique to moisten gauze pad on a string or pole and drag the pad along the length of a walkway. Use the same procedures for handling of the gauze pad, for moistening, and for handling the milk, as for gauze pads used for manure sampling. Place each pad in a whirlpak-type bag.  For fans take two representive samples. For walkways take two samples per walkway.

      Place each pad in a separate whirlpak-type bag with sufficient milk to keep the pad wet (no more than tablespoon or approximately 15 ml). If the collection time is greater than 6 h, the use of a cooler with frozen gel packs is recommended for sample storage prior to shipment.

      The laboratory will analyze each sample for the presence of Salmonella Enteritidis according to methods described for Salmonella spp. from laying hen houses (see D-E, below). The analytical unit is used in its entirety so that there is no remainder.

    3. Shipment and receipt

      All samples should be labeled to indicate 1) farm, 2) house and 3) type (eggbelt, manure or fan/walkway). The samples should be placed in an insulated transport container with frozen gel packs to keep the samples cool, but not frozen. Shipment must contain appropriate collection reports for the laboratory.

      Upon receipt at the laboratory, the samples should be checked for completeness of information and condition of sample. If the samples can not be processed at the time of receipt, store samples in a refrigerator kept at 7 ± 3° C (45 ±5° F) for no more than 2 days.

  2. Equipment and materials
    1. Sterile 12 ply gauze pads, 10 cm × 10 cm (4 in × 4 in)
    2. Sterile gloves
    3. Incubator, 35 ± 2° C
    4. Water bath, circulating, thermostatically controlled, 42.0 ± 0.2° C and 43.0± 0.2° C
    5. Canned evaporated milk, canned skim (fat free) evaporated milk or canned low fat evaporated milk
    6. Can opener
    7. Whirlpak-type bags, 15 cm × 22.5 cm (6 in × 9 in)
    8. Sterile 12 ply gauze pad 10 cm × 10 cm (4 in × 4 in) on a string
    9. Poles for manure dragging
    10. Ethanol, 70% (R23)
    11. Transport container with gel packs
    12. Appropriate tyvek suits, bouffant caps, boots for biosecurity measures for entry into laying hen houses
  3. Media and reagents
    1. Buffered peptone water (BPW; M192)
    2. Tetrathionate broth, Hajna (TTH)
    3. Rappaport-Vassiliadis medium (RV; M132)
    4. Brilliant green novobiocin (BGN) agar
    5. Xylose lysine Tergitol 4 (XLT-4) agar
    6. Triple sugar iron agar (TSI; M149)
    7. Lysine iron agar (LIA; M89)
    8. Salmonella polyvalent somatic (O) antiserum
    9. Salmonella polyvalent flagellar (H) antiserum
    10. Salmonella somatic group (O) antisera:

    Refer to BAM Chapter 5 for any media and reagents mentioned in the procedure except those listed at the end of this chapter.

  4. Preparation of test portions

    Aseptically add 100 ml of buffered peptone water (BPW) to the whirlpak-type bag containing the environmental gauze pad. Shake the bag vigorously in an up-and-down motion at least 10 times in a 30 cm (1 ft) arc in approximately 30 sec. Incubate 24 ± 2 h at 35° C.

  5. Isolation
    1. After incubation, shake the bag containing the gauze pad and BPW.
    2. Transfer 1 ml of the incubated pre-enrichment into 10 ml of tetrathionate (TTH) broth.
    3. Transfer 0.1 ml of the incubated pre-enrichment to 10 ml of Rappaport-Vassilliadis (RV) medium.
    4. Incubate the TTH broth at 43.0± 0.2° C in a circulating water bath and the RV medium at 42.0± 0.2° C (water bath) for 24 ± 2 h.
    5. After incubation, streak a loopful of TTH broth onto brilliant green with novobiocin (BGN) agar and xylose lysine Tergitol 4 (XLT4) agar plates.
    6. Repeat this procedure with the RV medium.
    7. Incubate all plates for 24 ± 2 h at 35° C.
    8. Examine plates for presence of colonies that may be Salmonella.

      Typical Salmonella colony morphology
      1. Brilliant Green with Novobiocin: Typical colonies appear pink to white opaque colonies surrounded by red zones in the medium
      2. Xylose Lysine Tergitol 4: Typical colonies appear black or black-centered with a yellow periphery after 18-24 hours of incubation. Upon continued incubation, the colonies become entirely black or pink to red with black centers. Colonies of H2S negative Salmonella strains appear pinkish yellow.

      SUGGESTED CONTROL CULTURES

      In addition to the positive control cultures (typical Salmonella) 3 additional Salmonella cultures are recommended to assist in the selection of atypical Salmonella colony morphology on selective agars. These cultures are a lactose-positive, H2S-positive S. Arizonnae (ATCC 12325), a lactose-negative, H2S-negative S. Abortusequi (ATCC 9842), and a lactose-positive, H2S -negative S. Diarizonae (ATCC 29934). These cultures may be obtained from the American Type Culture Collection, http://www.atcc.org, PO Box 1549, Manassas,VA 20108.

    9. Pick at least 5 typical colonies from each plate. Lightly touch the very center of the colony to be picked with sterile inoculating needle and inoculate TSI slant by streaking slant and stabbing butt. Without flaming, inoculate LIA slant by stabbing butt twice and then streaking slant. Since lysine decarboxylation reaction is strictly anaerobic, the LIA slants must have deep butt (4cm). Store picked selective agar plates at 5-8° C.
    10. Incubate TSI and LIA slants at 35° C for 24 ± 2 h. Cap tubes loosely to maintain aerobic conditions while incubating slants to prevent excessive H2S production. Salmonella in culture typically produces alkaline (red) slant and acid (yellow) butt, with or without production of H2S (blackening of agar) in TSI. In LIA, Salmonella typically produces alkaline (purple) reaction in butt of tube. Consider only distinct yellow in butt of tubes as acidic (negative) reaction. Do not eliminate cultures that produce discoloration in butt of tube solely on this basis. Most Salmonella cultures produce H2S in LIA. Some non-Salmonella cultures produce a brick-red reaction in LIA slants.
    11. All cultures that give an alkaline butt in LIA, regardless of TSI reaction, should be retained as potential Salmonella isolates and submitted for biochemical and serological tests. Cultures that give an acid butt in LIA and an alkaline slant and acid butt in TSI should also be considered potential Salmonella isolates and should be submitted for biochemical and serological tests. Cultures that give an acid butt in LIA and an acid slant and acid butt in TSI may be discarded as not being Salmonella. Test retained, presumed-positive TSI cultures as directed in E 12, below, to determine if they are Salmonella species, including S. Arizonae. If TSI cultures fail to give typical reactions for Salmonella (alkaline slant and acid butt) pick additional suspicious colonies from selective medium plate not giving presumed-positive culture and inoculate TSI and LIA slants as described in E-9, above.

    12. Apply biochemical and serological identification tests to:

      1. Five presumptive TSI cultures recovered from set of plates streaked from RV medium if present and 5 presumptive TSI agar cultures recovered from plates streaked from TT broth, if present.
      2. If 5 presumptive-positive TSI cultures are not isolated from one set of agar plates, test other presumptive-positive TSI agar cultures, if isolated, by biochemical and serological tests. Examine a minimum of 10 TSI cultures for each unit.

  6. Identification of Salmonella

    1. Mixed cultures. Streak TSI agar cultures that appear to be mixed on BGN agar, or XLT4 agar. Incubate plates 24 ± 2 h at 35°C. Examine plates for presence of colonies suspected to be Salmonella.

      1. Brilliant Green Novobiocin: (BGN) agar. See E-8a, above.
      2. Xylose Lysine Tergitol 4: (XLT4) agar. See E-8b, above.

      Transfer at least 2 colonies suspected to be Salmonella to TSI and LIA slants as described in E-8, above, and continue as in E-10, above.

      If this method is used solely for the detection of Salmonella Enteritidis on environmental surfaces, then the analyst should proceed to section E.6.b of BAM's Salmonella Chapter and test each of the TSI isolates for Salmonella (O) group D1 activity. If the isolates do not display Group D1 activity, then the isolates should be considered non-S. Enteritidis and discarded. If the isolates do display Group D1 activity, then the analyst should proceed as directed in the Salmonella Chapter. If this method is being used to detect all Salmonella on environmental surfaces, then the analyst should proceed as directed in the Salmonella Chapter .

  7. Media Preparations
    1. Brilliant Green Agar with Novobiocin
      Proteose peptone 10 g
      Yeast extract 3 g
      Lactose 10 g
      Saccharose 10 g
      Sodium chloride 5 g
      Agar 20 g
      Brilliant green 0.0125 g
      Phenol red 0.08 g
      Distilled water 1 liter
      • Suspend ingredients in 1 liter distilled water.
      • Autoclave 121° C for 15 minutes.
      • Add l ml of 20 mg/ml novobiocin to 1 liter basal medium and swirl thoroughly,
        dispensing 20 ml portions into sterile 15 × 100 mm petri plates.
      • Final pH 6.9 ± 0.2.
      • Store plates at 2-8° C.
    2. Xylose Lysine Tergitol 4 Agar
      Proteose peptone 1.6 g
      Yeast extract 3 g
      L-lysine 5 g
      Xylose 3.75 g
      Lactose 7.5 g
      Saccharose 7.5 g
      Ferric ammonium citrate 0.8 g
      Sodium thiosulfate 6.8 g
      Sodium chloride 5 g
      Agar 18 g
      Phenol red 0.08 g
      Distilled water 1 liter
      • Suspend ingredients in 1 liter distilled water.
      • Add 4.6 ml XLT agar supplement (27% solution of Tergitol 4).
      • Heat to boiling to dissolve completely. Do not autoclave.
      • Cool to 45-50° C in a water bath and dispense 20 ml portions into
        sterile 15 × 100 mm petri plates.
      • Final pH 7.4 ± 0.2
    3. Tetrathionate Broth, Hajna
      Preparation of medium:
      Na2S2O3 38.0 g
      CaCO3 25.0 g
      Casein/meat peptone (50/50) 18.0 g
      NaCl 5.0 g
      D-Mannitol 2.5 g
      Yeast extract 2.0 g
      Glucose 0.5 g
      Sodium deoxycholate 0.5 g
      Brilliant green 0.01 g
      Iodine solution 40.0 ml
      • Add components, except iodine solution, to distilled/deionized water
        and bring volume to 960.0 ml.
      • Mix thoroughly.
      • Gently heat and bring to boiling.
      • Do not autoclave. Cool to 40°C and then add 40.0 ml iodine solution.
      • Mix thoroughly and distribute into tubes in 10.0 ml volumes.
      • Final pH is 7.5 to 7.8. Use medium the same day as prepared.
      Preparation of iodine solution:
      • Add iodine and KI to distilled /deionized water and bring volume to 40.0 ml.
      • Mix thoroughly
  8. References
    1. AOAC INTERNATIONAL. 1995. Official Methods of Analysis, 16th ed., methods 967.25-967.28, 978.29, 989.12, 991.13, and 994.04. AOAC INTERNATIONAL, Gaithersburg, MD.
    2. Ewing, W.H. 1986. Edwards and Ewing's Identification of Enterobacteriacae, 4th ed. Elsevier, New York.
    3. Federal Register. 1971. 36(93):8815 (secs d, e, and f).
    4. Federal Register. 1972. 37(191):20556 (sec. 173.388 (a).
    5. June, G.A., P.S. Sherrod, T.S. Hammack, R.M. Amaguana, and W.H. Andrews. 1995. Relative effectiveness of selenite cystine broth tetrathionate broth, and Rappaport-Vassiliadis medium for the recovery of Salmonella from raw flesh and other highly contaminated foods: Precollaborative study. J. AOAC Int. 78:375-380.
    6. Salmonella enterica Serovar Enteritidis in Humans and Animals: The Epidemiology, Pathogenesis, and Control. Editors: A. M. Sadeed, Richard K. Gast, Morris Potter, and Patrick G. Wall: Iowa State University Press, Ames, Iowa, 1998, 516 pps.
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