From Last Time:
• What are the major classes of bioaerosol
sampler?
• What are the different types of filter
media?
Surface Sampling
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…..
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
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
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.
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
Food Sampling
Microbiological Sampling of Food
• Depends on:
– Food Type
– Type of Contamination
– Expected Location
– Organism/Contaminant Type
Types of Foodborne Disease
• Infection
– Foodhandler
– Food Concentration
– Direct Contamination
• Production
• Processing
• Storage
– Water-washed
• Intoxication (food poisoning)
– Inherent
– Introduced
Location of Contamination
• Food Surfaces
– Animal carcasses
– Raw produce
– Egg surfaces
• Non-Surface Contamination
– Processed foods
– Ground Meats
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
Oyster Anatomy
• 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
Photo: http://www.mdsg.umd.edu/oysters/anatlab/index.htm
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
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
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
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
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
Sampling Criteria
Sampling Criteria
• Sampling plans will depend on the
question that they are designed to answer
• Basic criteria
– Replication
– Representative
– Random
– Controls
– Method Validation
• Regulations??