Procedure ISO 11731:2017

Liquid Sample Preparation

Culture

Low concentration of Low concentration of High concentration of Extremely high concentration

interfering microorganisms interfering microorganisms interfering microorganisms of interfering microorganisms
High concentration of Low concentration of
Legionella species (>104 cfu/l) Legionella species (<104 cfu/l)

Heat treatment

Cool to RT
1st day

Filter + no Filter + treat Filter + No Concentrated Diluted 1:10

treatment with acid washing concentrated Acid treatment
procedure

Divide each subsample into 3 portions

No treated Heat Acid No treated Heat Acid No Diluted Diluted

treated treated treated treated concentrated 1:10 1:100
Direct placing of
membrane filter
on BCYE agar

Plate 0.1–0.5 ml Place the membrane Plate 0.1–0.5 ml

Plate 0.1–0.5 ml of each Plate 0.1–0.5 ml of each
on BCYE and filter on BCYE+AB or of each portion
portion on BCYE and portion on GVPC or MWY
BCYE+AB GVPC or MWY on GVPC or MWY
BCYE+AB or GVPC or MWY
11th day

Incubation
Incubation at 36 °C ± 2°C for 7 to 10 days. Check colonies on day 2, 3, 4, 5 and final incubation

Confirmation and results

Uncharacteristic growth Characteristic growth

Not detected Presumed
Legionella spp /x ml Legionella spp /x ml

Confirmation of Legionella spp

13th day

Re-inoculation and Incubation

at 36 ± 2 °C for 2 days

Growth + on BCYEx Other growth

Growth - on BCYE w/o cys combinations
(or Blood Agar or Nutrient Agar or TSA)

Confirmations at Presence (CFU) Not detected

the level of species Legionella spp /x ml Legionella spp /x ml