第一室のトップへ戻る 海外の検査法との比較表
Detection of Campylobacter Species from Food
Product NIHS
BFR
|
PHLS method |
FDA-CFSAN (BAM) method |
ISO method (1st ed) |
検討した検査法試案 | |
|
Sample preparation |
Ｗeigh 25 g sample into a stomacher
bag ↓ Add 9 x weight (or volume) of Campylobacter enrichment
broth ↓ Homogenize by using a stomacher ↓ Transfer the suspension into a screw capped container (nominal volume 250 mL) leaving very little headspace |
Weigh 25 g most samples (50 g fruit or vegetables) into a
filter bag ↓ Add 100 ml of Campylobacter enrichment broth ↓ Rinse gently by shaking for 5 min or using a table-topshaker set at 25 rpm ↓ Hold 5 min ↓ Transfer the suspension into the
incubation bag or flask ↓ * The sample types listed below are prepared by
different procedure : (1) Lobster tail or crab
claws (2) Whole meat
carcass or sample that cannot be easily reduced to 25 g (e.g., whole rabbit,
lobster or large piece of game meat) (3) Liquid egg yolk
or whole egg mixture (4) Shellfish,
shucked (5)
Water (6)
Swabs (7) Milk, frozen daily products |
A quantity x (g or ml) of sample ↓ Add a 9x volume of Campylobacter enrichment broth (Preston broth or Park and Sanders) * Park and Sanders broth: For some physical treatment (e.g., freezing) received-sample or prduct |
（１）サンプルの採取 25 ｇ (25
mL) のサンプルを採取し、ストマックバッグに入れる。 （２）サンプルの調製 増菌培地225 ｍLを入れてホモジネートする。 増菌培養は通常の場合、Preston増菌培地を利用する。 また、菌損傷の可能性の高い場合はBolton培地を使用する。 |
|
Selective enrichment |
Incubate at 37℃ for 22 ± 2 h ↓ Incubate at 41.5℃ for 22 ± 2 h |
Pre-enrichment
:
Sample within 10 days of
production or
|
Direct plating out : * For those products suspected of containing a large quantity of thermotolerant Campylobacter Plate the non-incubated suspension onto Karmali agar and second selective isolation medium Modified Butzler agar, Skirrow agar,
Charcoal cefoperazonedeoxycholate agar (CCDA), or
Preston agar ↓ Enrichment : Incubate at 42℃ for 18 h under microaerobic conditions Park and Sanders broth Incubate at 32℃ for 18 h under microaerobic conditions ↓ Add the antibiotic solution B at a concentration of 5% (V/V). ↓ Incubate at 37℃ for 2 h under microaerobic ↓ Incubate at 42℃ for 40-42 h under microaerobic conditions
|
（Preston培地） 微好気条件にて42℃で 24 -
48時間培養する。 （Bolton培地） 微好気条件下にて37℃で4時間培養の後に、42℃で 24 - 44時間培養する。 微好気条件については以下の方法が推奨されており、微好気条件が保持される事を市販の指示薬などで確認して利用すること。 微好気条件とは酸素5％,二酸化炭素ガス10％,チッ素85％を基本とする。 ①培養室内を微好気条件に自動制御できるインキュベータ ②市販の微好気ジャーシステム（ガスキットシステムなど）を利用する方法。 ③微好気ガスで容器の気相を置換し、ガスを透過しない容器で密閉する。 |
|
Plate onto electiveagar |
Plate onto Campylobacter selective agar (modified CCDA) ↓ Incubate at 37℃ for 48 ± 2 h under microaerobic conditions |
Make a 1:100 dilution in 0.1 % peptone water of the enrichments ↓ Plate two loopfuls of undiluted and diluted portions onto either Abeyta-Hunt-Bark agar or modified CCDA agar ↓ Incubate at 42℃ for
24-48 h under microaerobic conditions * For C. fetus, incubate at 37℃ for 48-72 h |
Plate a loop of the enrichments onto Karmali agar and second selective isolation medium** **
Modified Butzler agar, Skirrow agar,
Charcoal cefoperazonedeoxycholate agar (CCDA), or
Preston agar Incubate at 42℃ for 24-72 h (more generally, 48 h)
and, if necessary, 5 days under
microaerobic
conditions |
分離培地に増菌培養した液を画線塗抹して42℃で24 -
48時間培養する。
分離培地はｍCCDA培地を用いる。また、必要に応じて、下記の二次培地を追加する。
Karmali 寒天培地、ModifiedButzlr培地、Skirrow培地、Preston寒天培地 |
| Identify Campylobacter |
Oxidase
test Oxidase negative colonies do not require further confirmatory
test ↓ Microaerobic
growth Incubate one plate microaerobically and the other plate aerobically
at 37℃ ± for 22 ± 2 h ↓ Cell morphology and
motility If cell morphology is in doubt then perform a Gram
staining
|
Cell morphology and
motility One typical colony/plate ↓ Restreak typical organisms onto Abeyta-Hunt-Bark agar without
antibiotics (two colonies/sub.)
and incubate at 42℃ for 24-48 h
under microaerobic conditions. For C. fetus, incubate at
37℃ ↓ Confirmatory test
: Confirm only one
plate/sub. Catalase
test Oxidase
test ↓ Biochemical tests
: Gram
staining Hippurate
hydrolysis TSI
reaction Glucose utilization
test “Dryspot Campy
test” or “Alter for
Campylobacter” Antibiotic
inhibition Growth temperature tolelance Growth on MacConkey agar Growth in modified semisolid
media (added biochemicals
described below) 1) with 1% glycine 2) with 3.5% NaCl 3) with H2S
from cysteine 4) with Nitrate reduction |
5 typical and/or suspect colonies ↓ 1) Inoculate into 1 ml of Brucella broth 2) Examination described
below (using the selected colonies on plates) Cell morphology and
motility Gram
staining ↓ Inoculate with a loop of
each selected colony suspensions (in Brucella broth) indicating typical cell morphology and motility
onto a ↓ Incubate at 42℃
for 24 h under microaerobic conditions ↓ Growth at
25℃ Incubate at 25℃
for 2-5 days under microaerobic conditions Biochemical tests : Oxidase
test TSI agar Catalase test Nalidic acid and cephalothin sensitivity Hippurate hydrolysis |
（１）コロニーの観察 疑わしい形状のコロニーを5コロニー程度採取。 直径1 ? 2 ｍｍ程度の正円形でやや隆起したコロニー（時に扁平する）を対象とする。 （２）コロニーの同定準備 コロンビア血液寒天培地およびこれと同等の糖の含有が少ない非選択培地に塗抹して42℃で24 -
48時間培養。 （３）鑑別同定 ①グラム染色：ラセン状（球状［コッコイド］の場合もある）のグラム陰性かん菌 ②カタラーゼ陽性 ③オキシダーゼ陽性 ④ラテックス凝集テスト（DrySpotTestなど）で陽性 ⑤馬尿酸塩加水分解試験（C.jejuni ［＋］、C.coli ［－］） ⑥インドキシル酢酸塩加水分解陽性（C.jejuni ［＋］、C.coli ［＋］） |