A.
Equipment and materials
1. Covered water bath, with circulating system to maintain temperature of
44.5 ± 0.2°C. The temperature for water baths for the shellfish program is
44.5°C ± 0.2°C. Water level should be above the medium in immersed
tubes.
2. Immersion-type thermometer, 1-55°C, about 55 cm long, with 0.1°C
subdivisions, certified by National Institute of Standards and Technology
(NIST), or equivalent Incubator, 35 ± 0.5°C.
3. Balance with capacity of >2 kg and sensitivity of 0.1 g
4. Blender and blender jar (see Chapter 1)
5. Sterile graduated pipets, 1.0 and 10.0 mL
6. Sterile utensils for sample handling (see Chapter 1)
7. Dilution bottles made of borosilicate glass, with polyethylene screw caps
equipped with Teflon liners. Commercially prepared dilution bottles
containing sterile Butterfield's phosphate buffer can also be used.
8. Quebec colony counter, or equivalent, with magnifying lens
9. Longwave UV light [~365 nm], not to exceed 6 W.
10. pH meter
B. Media and Reagents
1. Brilliant green lactose bile (BGLB) broth, 2% (M25)
2. Lauryl tryptose (LST) broth (M76)
3. Lactose Broth (M74)
4. EC broth (M49)
5. Levine's eosin-methylene blue (L-EMB) agar (M80)
6. Tryptone (tryptophane) broth (M164)
C. MPN - Presumptive test for coliforms, fecal coliforms and E. coli
Weigh 50 g of food into sterile high-speed blender jar (see Chapter 1 and current
FDA compliance programs for instructions on sample size and compositing)
Frozen samples can be softened by storing for <18 h at 2-5°c, but do not thaw.
Add 450 mL of Butterfield's phosphate-buffered water and blend for 2 min. If
<50 g of sample are available, weigh portion that is equivalent to half of the
sample and add sufficient volume of sterile diluent to make a 1:10 dilution. The
total volume in the blender jar should completely cover the blades.
Prepare decimal dilutions with sterile Butterfield's phosphate diluent or
equivalent. Number of dilutions to be prepared depends on anticipated coliform
density. Shake all suspensions 25 times in 30 cm arc or vortex mix for 7 s. Using
at least 3 consecutive dilutions, inoculate 1 mL aliquots from each dilution into 3
LST tubes for a 3 tube MPN analysis (other analysis may require the use of 5
tubes for each dilution; See IV). Lactose Broth may also be used. For better
accuracy, use a 1 mL or 5 mL pipet for inoculation. Do not use pipets to
deliver<10% of their total volume; eg. a 10 mL pipet to deliver 0.5 mL. Hold pipet
at angle so that its lower edge rests against the tube. Not more than
15 min should elapse from time the sample
is blended until all dilutions are inoculated in appropriate media.
Incubate LST tubes at 35°C± 0.5°C . Examine tubes and record reactions at 24 ±
2 h for gas, i.e., displacement of medium in fermentation vial or effervescence
when tubes are gently agitated. Re-incubate gas-negative tubes for an additional
24 h and examine and record reactions again at 48 ± 3 h. Perform confirmed test
on all presumptive positive (gas) tubes.
D. MPN - Confirmed test for coliforms
From each gassing LST or lactose broth tube, transfer a loopful of suspension to a
tube of BGLB broth, avoiding pellicle if present. (a sterile wooden applicator stick
may also be used for these transfers). Incubate BGLB tubes at 35°C ± 0.5°C and
examine for gas production at 48 ± 3 h. Calculate most probable number (MPN)
(see Appendix 2) of coliforms based on proportion of confirmed gassing LST
tubes for 3 consecutive dilutions.
E. MPN - Confirmed test for fecal coliforms and E. coli
From each gassing LST or Lactose broth tube from the Presumptive test, transfer
a loopful of each suspension to a tube of EC broth (a sterile wooden applicator
stick may also be used for these transfers). Incubate EC tubes 24 ± 2 h at 44.5°C
and examine for gas production. If negative, reincubate and examine again at 48
± 2 h. Use results of this test to calculate fecal coliform MPN. To continue with E.
coli analysis, proceed to Section F below. The EC broth MPN method may be
used for seawater and shellfish since it conforms to recommended procedures (1).
F. MPN - Completed test for E. coli.
To perform the completed test for E. coli, gently agitate each gassing EC tube,
remove a loopful of broth and streak for isolation on a L-EMB agar plate and
incubate for 18-24 h at 35°C ± 0.5°C . Examine plates for suspicious E.
coli colonies, i.e., dark centered and flat, with or without metallic sheen. Transfer
up to 5 suspicious colonies from each L-EMB plate to PCA slants, incubate them
for 18-24 h at 35°C ± 0.5°C and use for further testing.
NOTE: Identification of any 1 of the 5 colonies as E. coli is sufficient to regard
that EC tube as positive; hence, not all 5 isolates may need to be tested.
