3
Preparation of conventional media
Conventional dehydrated media preparations were chosen for selection of H.
pylori from
mixed microbial populations based on the published clinical literature. The
five types of media
chosen for evaluation were BHI + 7% calf serum (Osaki, 1998), Brucella Agar
(Poms, 2001),
Campylobacter Agar Kit Skirrow (Corry, 1995), Columbia Blood Agar Base (Baronn,
1994;
Becton Dickinson, Sparks, MD), and HPSPA (Jiang, 2000; Stevenson, 2000).
Table 2. compares
ingredients of these media to demonstrate the common and unique features
of each. The
inclusion of either Helicobacter pylori selective supplement (Oxoid Limited,
Hampshire,
England) or Campylobacter selective supplement S (Becton Dickinson) provided
antibiotics to
prevent background while permitting H. pylori growth. Positive control consisted
of the five
conventional media without the selective supplements while negative controls
consisted of
uninoculated plates. All media were prepared according to manufacturer's
or authors'
instructions.
TABLE 2. Formulations of conventional media used to culture and select Helicobacter
pylori.
Brain Heart Infusion Brucella Agar Columbia Agar aHPSPA Campylobacter Agar
10 g proteose peptone 10 g peptamin 10 g pantone 15 g spec. peptone 15 g
proteose peptone
NA beef heart inf. 10 g tryptone 10 g bitone 2 g porcine mucin 2.5. ml liver
digest
NA calf brains inf. 2 g yeast extract 3 g beef heart dig. 5 g yeast extract
5 g yeast extract
2 g dextrose 1 g dextrose 1 g dextrose 5 g beef extract
5 g NaCl 5 g NaCl 5 g NaCl 5g NaCl 5g NaCl
15 g agar 15 g agar 15 g agar 15 g agar 12 g agar
2.5 g disodium phos. 0.1 g Na bisulfate 0.5 g ferrous sulfate
70 ml calf serum w/fe 70 ml calf serum w/fe
0.6 g urea
0.5 g Na pyruvate
bselective supplement selective supplement selective supplement selective
supplement cselective supplement
a Formula published by Jiang and Doyle. J. Clin. Microbiol. 2000.
b Vancomycin (10 mg/L), Cefsulodin (5 mg/L), Trimethoprim (5 mg/L), and Amphotericin
B (5 mg/L).
c Vancomycin (10 mg/L), Trimethoprim (5 mg/L), and Polymyxin B (2500 IU/L)
Initial conventional test media screen
Evaluation of the efficacy of the conventional media formulations used in
clinical microbiology
laboratories was carried out as follows. Each strain listed in Table 1 was
separately cultured on
each of the five media listed in Table 2 in order to evaluate the growth
and inhibition spectra of
individual formulations. First, pure colonies of each strain were picked
from solid growth media
(BHI agar plates) and homogenized in sodium phosphate buffer (4%). Serial
dilutions of each
pure homogenate were immediately spread (0.1ml/plate) onto each type of solid
media with and
without selective supplement added. All plates were incubated under microaerophillic
conditions at 37oC for up to seven days and examined to determine the presence
or absence of
colonies on the media compared to the positive control of a non-selective
media.
Selectivity of media formulations for H. pylori from samples spiked with
a cocktail of non
H. pylori bacteria.
Well water samples containing native flora (Table 5; identified using API
20 E
identification system; Biomerieux Vitek, Inc., Hazelwood, MO) were further
spiked with the
seven strains of background bacteria described above, and the H. pylori,
to represent a highly
contaminated water sample (10,000 cells per strain per 100 mls). The spiked
water sample was
then serially diluted and 0.1 ml of each dilution was spread onto duplicate
plates of each of the