From Last Time:

1. From Last Time: • What are the major classes of bioaerosol sampler? • What are the different types of filter media?

2. Surface Sampling

3. Surface Sampling • Current Methods (5-90% recoveries, generally poorly characterized) • Several methods have been described none fully standardized • Swabs, Swipes/wipes, vacuum filtration, rinse/elute, contact plates, other…..

4. Surface Sampling • Swabs (better for gram negatives?) • Cotton • Dacron • Calcium Alginate (may inhibit PCR and be toxic to cell culture) • Sponge (Polyurethane and Cellulose) • Swipes/Wipes • Cotton • Nitrocellulose membranes • Polyester bonded cloth • Velvet or Velveteen Yamaguchi, et al. 2003; Cloud, et al. 2002; Lemmen, et al, 2001; Poletti, 1999; Craythorn, et al. 1980; Osterblad, et al. 2003; Taku, et al. 2003

5. Surface Sampling • Vacuum Filtration • Hepa bag vac • Wet Vac • Rinse/Elute • Contact Plates and Paddles (RODAC) • better for gram positives? • New Methods • Adhesive Strips and Paddles • Scraping/Aspiration Yamaguchi, et al. 2003; Cloud, et al. 2002; Lemmen, et al, 2001; Poletti, 1999; Craythorn, et al. 1980; Osterblad, et al. 2003; Taku, et al. 2003

7. Surface Sampling From anthrax investigations, methods performed in parallel • Dry Swabs (<25%) • Wet Swabs (~50%) • Hepa Vac (~80%) • Wipe (~85%) Teshale, et al. 2002; Sanderson, et al. 2002.

8. Recovery from Surfaces • Factors that may affect the recovery of microbes from surfaces: • Method selection • Particle size bias • Surface composition • Surface topography/roughness • Organism type and Distribution • Sample size • Target of detection

9. Food Sampling

10. Microbiological Sampling of Food • Depends on: • Food Type • Type of Contamination • Expected Location • Organism/Contaminant Type

12. Types of Foodborne Disease • Infection • Foodhandler • Food Concentration • Direct Contamination • Production • Processing • Storage • Water-washed • Intoxication (food poisoning) • Inherent • Introduced

13. Location of Contamination • Food Surfaces • Animal carcasses • Raw produce • Egg surfaces • Non-Surface Contamination • Processed foods • Ground Meats

15. Food Sampling Approaches • Concentration not often not as important as for air or water; however separation/purification may be much more important • Surface methods similar to those previously described • Swabs • Contact Plates • Rinse/elution • Non-surface methods are more involved due to the added complexity • Very food type/Organism type dependent • Typically require homogenization • (May require prior dissection) • May include adsorption/elution • Includes a variety of purification separation methods

16. Concern over ingestion of contaminated shellfish arises because bivalves concentrate and bioaccumulate environmental pathogens in their tissues through filter feeding Large quantities of water are pulled in through an incurrent siphon, and then passed through the body cavity over the gills Potentially high concentration of pathogens combined with the tradition of eating shellfish raw or undercooked poses a potentially serious health risk for consumers Oyster Anatomy Photo: http://www.mdsg.umd.edu/oysters/anatlab/index.htm

17. Collection and Preparation of Shellfish • Upon receipt of samples, oysters are cleaned under potable running water with a scrub brush • Half of each sample of oysters is placed in a -80°C freezer for later viral analysis, while the other half is shucked immediately for bacterial analysis

18. Preparation for RT-PCR Detection of Virus • Oysters frozen to –80°C, then shucked w/ sterile knife • DD and stomach removed with sterile scalpel • Blended for 2 minutes at high speed in laboratory blender with .25N Glycine Buffer pH 10 • Chloroform extracted to reduce particulate matter and some potential PCR inhibitors • RNA extracted using Qiagen RNeasy Midi kit

19. Preparation of Shellfish for Bacterial Analysis • 3-6 fresh oysters were shucked and combined to form a 100g sample • Each sample was then homogenized in a laboratory blender • Following 2 minutes of homogenization, the oyster lysate was immediately plated on selective agars and added to broth for the MPN test

20. Detection of Indicator Organisms • Selective agars used to detect indicator species • mFC agar (Difco) used to detect Fecal Coliforms and E.coli • mEnterococcus agar (Difco) used to detect Enterococcus species • The protocol for detection of E.coli in shellfish tissue outlined by Donovan et al (1998) was used as well • Consists of a 5-tube, 3 dilution MPN assay with resuscitation in MMGB followed by confirmation on chromomeric agar (tryptone bile agar with .75g/L BCIG) to identify E.coli

21. Sources for Food Methods • Compendium of Methods for the Microbiological Examination of Food • Standard Methods for the Examination of Dairy Products • Bacteriological Analytical Manual • AOAC and ISO methods • Stand Methods for the Examination of Water and Wastewater

22. Sampling Criteria

23. Sampling Criteria • Sampling plans will depend on the question that they are designed to answer • Basic criteria • Replication • Representative • Random • Controls • Method Validation • Regulations??