Presence of Pathogenic Bacteria in Raw Meat Dog Food

“Presence of pathogenic bacteria in commercially available raw meat dog food”
SAMPLES:
No of samples: 4+4=8
Natures Menu
• poultry hearts
• Beef chunks
• chicken
• lamb
Natural instincts
• pure chicken
• Pure Beef
• Natural Lamb
• Pork
No of Brands: 2 (Natures menu, Natural instincts)
No of sampling=3
SELECTIVE AGAR PLATES:
 Listeria Brilliance- for isolation, enumeration and presumptive
identification of Listeria species and Listeria monocytogenes from food
samples
 Salmonella chromogenic- for the identification of Salmonella species from
other organisms in the family Enterobacteriaceae.
 Brilliance E. coli agar- for the detection and enumeration of Escherichia
coli and other coliforms from food and water samples.
 Plate count agar- for the enumeration of viable organisms in food, animal
feed and water samples
Table 1: Controls
Sample Negative controls Positive controls
Listeria Brilliance Enterococcus feacalis Listeria monocytogenes
Listeria innocua
Salmonella
chromogenic
Escherichia coli
Pseudomonas
aeruginosa
Salmonella enteritidis
Salmonella poona
Brilliance E. coli agar Staphylococcus aureus Escherichia coli
Klebsiella pneumoniae
Plate count agar Uninoculated medium Escherichia coli
Bacillus subtilis
Staphylococcus aureus

1) Prepare 60 agar plates for each of the selective media a day before
the actual experiment for determination of pathogenic bacteria in the
food samples
For 8 samples/per agar media=20
8 sample in duplicate (8*2=16) + positive controls duplicate (1*2=2) + Negative
controls duplicate (1*2=2)
No of sampling (3) = 20*3=60
2) Prepare 80 (8*10) agar plates of Plate count agar for calculation of
COLONY FORMING UNITS (CFU) and GROWTH RATE
Determination of pathogenic bacteria in the food samples
A) LISTERIA in Listeria Brilliance agar 1
1. Add 25g of food sample to 225ml of ONE Broth-Listeria (CM1066 &
SR0234) and stomach for a minimum of 30 seconds to mix the sample on
day1.
2. Incubate the broth at 30ºC for 24 ± 2 hours.
3. On day 2 Gently agitate the bag then, using a microbiological loop,
remove 10μl and inoculate onto an Brilliance Listeria Agar plate and
incubate at 37ºC for 24 ± 2 hours. Examine the plate for blue colonies
with and without opaque white halos.
4. Confirm presumptive colonies on the agar plate as Listeria
monocytogenes or Listeria species by appropriate methods e.g. Gram
stain, catalase, Oxoid O.B.I.S. mono ID0600M, Oxoid Listeria Latex Test Kit
DR1126A, Microbact Listeria 12L MB1128A.
Positive and negative controls
1. Streak a plate of nutrient agar with a glycerol stock of organisms listed as
positive and negative controls in Table-1 on Day 0.
2. On day 1 add one colony to 100 ml of nutrient broth and incubate the broth at
30ºC for 24 ± 2 hours.
3. On day 2 Gently agitate the culture, using a microbiological loop, remove 10μl
and inoculate onto an Brilliance Listeria Agar plate and incubate at 37ºC for 24 ±
2 hours. Examine the plate for blue colonies with and without opaque white
halos.
CFU/ml= (No of colonies X dilution factor)/ volume of culture on plate
B) Salmonella in Salmonella chromogenic agar plate2