ISO 10705-4 pdf download – Water quality – Detection and enumerationof bacteriophages — Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis

ISO 10705-4 pdf download – Water quality – Detection and enumerationof bacteriophages — Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis.
7.7 Counting apparatus. with indirect, oblique light.
7.8 Deep freezer, thermostatically maintained at (—20 + 5) C.
7.9 Deep freezer, thermostatically maintained at (—70 + 10) ‘C or liquid nitrogen storage vessel.
7.10 Spectropholometer. equipped witti a tiller for the range of 500 nm to 650 nm with a maximum bandwidth of
+ 10 nm, capable of holding cuvettes (7.21) having an optical path length oIl cm or Hungate glass tubes (7.20) with
butyl rubber Stopper and screw cap or screw-capped glass culture tubes.
7.11 Anaerobic cabinet, or jars or bags, as well as anaeroblosls generators and anaerobdosls indicators.
7.12 RefrIgerator, temperature set at (5 ± 3) C.
7.13 Petrl dishes, having a diameter of 9cm, and vented.
7.14 Graduated pipettes. having a capacity of 0,1 ml. I ml, 5 ml and 10 ml and Pasteur pipettes.
7.15 Glass bottles, of suitable volumes.
7.16 Screw-capped glass bottles, of suitable volumes.
7.17 Culture tubes, with caps or suitable alternatives.
7.18 Screw-capped glass culture tubes
7.19 MeasurIng cyhnders, of suitable capacity.
7.20 Hungate glass tubes, with butyl rubber stopper and screw cap or screw-capped glass culture tubes which can fit in the spectrophotometer (see Figure I).
7.21 Cuvettes. having an optical patti length of 1 cm.
7.22 Membrane filter units, for decontamination, having a pore size of 0,2 m, preferably low protein-binding menranes. as for example, those composed of polyvinylidene difluoride.
7.23 PlastIcs vials, lidded. having a capacity of 3 ml.
7.24 Glass vials, screw-capped, having a capacity of 3 ml,
7.25 SyrInges and needles
7.26 Cotton swabs
8 Microbiological reference cultures
The recommended host strain is Bacte,oi’des fragrlis RVC2056 (ATCC 700786).’
Use bacteriophage B56-3 (ATCC 700786-Bi) infecting Bacferoides Iragilis RVC2056 for the preparation of reference materials (11.4).
NOTE The ATCC strains are available Irom American Type Culture Collection (ATCCI, 10901 Universaly Boulevard. Manassas, VA 20110.2209, USA. This information given lot the convenienoe of users ol this part 04 ISO 10705 and does not constitue an endorsement by ISO of the product named. Equivalent products may be used it they can be shown to lead to the same results,
Add BPRMB (BI) to a tube for anaerobic cultures (7.18) and warm to at least room temperature (laster growth will occur if the broth is prewarmed to (36 + 2) ‘C1. Transfer an aliquot of the above-mentioned culture, without shaking the tube and taking the aliquot from the middle part of the tube, to the tube containing prewarrned BPRMB in a ratio of culture-to-BPRM of 1,5:10 (volume). Be sure that the inoculated tube is completely filled (see 10.1.1). lnciiate the culture at (36 2) C to reach proximately 2 x 10 cfuIml.
Mix working culture and cryoproleclor (B.6) in a ratio of 11 (volume) avding bubble formation. Distribute Into screwcapped. preferably glass. vials (7.24) in alictuots of approximately 1,5 ml and store at (70 + 10) “C for a maximum of 12 months.
Ensure that the culture does not reach the stationary phase before mixing it with th€ cryoprotector. Absorbance stabilization will indicate the end of the log phase, which may fast 5 h to 8 h.
10.2 Calibration of absorbance measurements for counts of viable host bacteria
Remove a vial of working culture (10i 3) from the deep freeze (7.9) and allow the vial to equilibrate to room temperature (i.e. 15 C to 30 CC). Add BPRMB (8.1) to a tube for anaerobic cultures (7.iB) and warm to at least room temperature (faster growth will occur it the broth is prewarmed to (36 ± 2) CI. Before inoculation, adjust the spectrophotometer to zero. Transfer the working culture into BPRM8 (8.1) in a ratio of respectively 1:10 (volume), completely tilling the tie. Tubes for anaerobic cultures may be inoculated/sampled by puncture with sterile syrriges and needles (7.25), Incubate the cultwe at (36 ± 2) C. Every 30mm. measure the absorbance (using 7.10) and withdraw, by puncture, a 0,3 ml sample for viable cell counts. Ensure that the tube is out of the incubator for as short a time as possible.