Short-term Laboratory Training Report

Host Institution: French Research Institute for Exploitation

of the Sea (IFREMER), Brest (France)

Period: 15 – 22th of May 2010

Sarah Purnell and Daniel Ekane Nnane

(University of Brighton)

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Introduction

One of the main objectives of the AquaManche project is the promotion of cross-fertilisation
and cross-border mobility of research expertise through the exchange of both senior and
early-career scientists between partner institutions for training purposes. It was on the basis of
this objective that Sarah Purnell and Daniel Ekane Nnane visited Ifremer, Brest (France). The
visit was a follow up of an earlier exchange that saw Marie-Paule Caprais and Cecile Le
Mennec from IFREMER visiting University of Brighton to learn the phage-lysis of
Bacteroides technique, which is used to trace human faecal pollution in environmental
samples.

Aim of visit

To learn quantitative real time polymerase chain reaction (qRT-PCR) method for F-RNA
specific bacteriophages.

Monday, May 17th

Monday began with us discussing the timetable of activities for the week with Marie-Paul
Caprais. Following that meeting, we were introduced to some Ifremer colleagues and shown
round the laboratory. During the laboratory tour, we were shown the different rooms, and
also the equipment we were to use for the various analyses. The next activity was the
preparation of oyster samples for bacteriophages detection and enumeration by culture in the
afternoon.

Processing of oysters

Washing and opening:

1. First, oysters were scrubbed and washed under a running tap using scourers.
2. Oysters were then opened using an oyster knife (shucking knife) or ‘couteau à huitre.’
3. Once opened, the knife is used to carefully separate (shuck) the flesh from the shell.
4. The flesh and intravalvular fluid are then put into a Petri dish. Nine to ten oysters
(approximately 100g) were put into each Petri dish.

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The instruments used were all sterilised. Throughout the process of shucking and dissection,
the oysters were placed on ice (Figures 1 & 2).

Figure 1 Opening of oysters using ‘couteau à huitre’

Figure 2 An oyster knife

Dissection to remove the digestive gland

1. Before dissection, the weight of intravalvular fluid and flesh was taken and recorded.
2. The intravalvular fluid was then discarded.

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 3. Each oyster flesh was then dissected to separate out the digestive gland (Figure 3).

Figure 3 Oyster dissection

4. The separated glands were placed into sterile centrifuge tubes for further preparation.

Preparation of digestive gland samples for culture and PCR

The digestive glands were finely chopped and then weighed. 2 x 6g of the digestive gland
was kept for PCR analysis and the remainder was weighed and kept for phage analysis by
culture. The 2 x 6g of digestive gland for PCR was stored overnight at -80oC.

The diluent, EPS (1g of peptone and 8.5g of NaCl in 1 litre of distilled water), was added to
the digestive gland for culture. For every X g of digestive tissue, 2 X volume of diluent were
added (for example, for 3g of tissue, 6ml of diluent was added). This was then homogenised
using a hand held mixer (homogeniser) for approximately 2 x 20 seconds. The homogenised
samples were then centrifuged at 4oC for 15 minutes at 3500rpm. The supernatant was used
for the detection and enumeration of F-specific RNA bacteriophages.

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Detection and enumeration of F-specific RNA bacteriophages

Enumeration of F-specific RNA bacteriophages was accomplished by the ISO10705-1

standardized method (Figure 4).

Figure 4 Detection and enumeration of F-specific RNA bacteriophages

Briefly, the host strain, Salmonella typhimurium (WG49) was grown in tryptone-yeast
extract-glucose broth (TYGB) to a turbidity that corresponds to a cell density of
approximately 108cfu/ml. 1ml of WG49 was added to 2.5ml of TYG semi-solid agar, then 1
ml of sample was added and poured onto 90mm diameter Petri-dishes containing a bottom
layer of TYG agar. Once solidified the plates were inverted and incubated at 37oC ± 1oC for
18h ± 2h. The presence of plaque forming units (PFU) indicated the presence of phages
(Figure 5). For each water and oyster sample, 10ml and 5ml was analysed respectively. A
positive control of MS2 phage was used at the beginning and end of each analysis.

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 Figure 5 Plate showing plaque forming units (PFU)

Tuesday, 18th May

Most of Tuesday was spent picking phages and concentrating samples for genotyping on
Wednesday. Water samples were concentrated using Centricon Plus-70 (Millipore).

Concentration of water samples using Centricon Plus-70

The Centricon Plus-70 centrifugal filter is a disposable, single-use device designed for rapid
processing of aqueous biological solutions in volume ranging from 15 to 70 ml. Each filter
can contain 70 ml (maximum volume) of sample each time it is used and can concentrate the
70 ml down to 350 µl in about 25 minutes. The filters are rinsed before first use because the
filters contain trace amounts of glycerol which was used as a humectant (substance that
retains moisture). The glycerol is flushed out by centrifuging 70 ml of a buffer solution
through the filter for 5 minutes.

The procedure for concentrating the water samples is described below:

 Seventy ml of the sample is added to the filter cup and sealed with the cap provided.
The filter cup is placed on top of the filtrate collection cup.
 The Centricon Plus-70 complete assembly is then placed in the centrifuge and spun at
3,500 rpm for approximately 5 minutes. It takes progressively longer as residue builds
up of in the filter following successive filtrations.

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  Each time the device is centrifuged the sample moves through the filtration device to
the bottom compartment. This liquid is discarded and the next 70 ml of sample is
centrifuged until at least 500 ml has been filtered.
 Once the sample has been concentrated, the concentrate cup is turned upside down
and placed on top of the sample filter cup.
 5ml of eluent (Beef extract (1 g in 100 ml), Tween 80 (3 ml), 0.5M NaCl (2.9 g)) is
added to the device. The whole unit is then inverted and vortexed. Following
vortexing, the sample is centrifuged at 1,000 rpm for up to 2 minutes (Figure 6).
 Following the concentration of the water sample, 2 ml is put aside for culturing and 3
ml for PCR.

Figure 6 Centricon Plus-70 units inverted

Picking phages for genotyping

The plates from Monday were counted (PFU). Then, 24 plaques (phages) for genotyping
were picked from the 10 plates we had for each sample. The phages were picked with
pipettes and transferred to 2 ml centrifuge vials containing phosphate-buffered saline (PBS)
with 20% glycerol. The vials were labelled, vortexed and then frozen at -80oC.

Wednesday, 19th May

On Wednesday concentration of water samples was achieved by using Vivaflow 50/200

modules. Genotyping was completed in the afternoon.

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Concentration by ultrafiltration using Vivaflow 200

Ultrafiltration using Vivaflow 200 modules was another method we used to concentrate water
samples. These modules are able to filter larger volumes of sample in comparison to the
Centricon Plus-70. For each sample site 2 litres were concentrated.

 The system was set up as shown in Figure 7.

 Each module was rinsed with distilled water to remove glycerine and sodium azide
before use.
 The 2 litres of each water sample was pumped through and concentrated to the desired
volume of 35 ml.
 10 ml of glycine buffer was run through the filtration units and added to the 35 ml to
make a final volume of 45 ml. The glycine buffer is used because 10 ml remains in
the Vivaflow unit and this enables that last 10 ml to be extracted.
 Of the 45 ml, 5 ml was put aside for culturing whilst 40 ml for extraction of RNA
with Nuclisen method following further concentration with PEG during the night.

For fresh stream waters 1 litre was concentrated whilst for saline waters 2 litres was
concentrated for each 45ml volume.

Figure 7 Vivaflow 50/200 set up

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Genotyping: Methodology I

Prior culturing and recuperation of isolates was required. We used the phages picked the day
before. We utilised a quantitech (qiagen) kitII after a step of denaturation. QRT-PCR was
used to differentiate between two major genotypes.
 A plan for the placement of isolates in a 96 well plate was completed before
beginning. To this plan negative and positive controls were included.
 Isolates were placed into a 96 well polypropylene plate, which was then placed onto
an ice base (Figure 8).

Figure 8 Transferring the isolates into the 96 well polypropylene plates

 7.5µl of each sample isolate was put into the wells of the tray according to the plan.
This was done in duplicate for each isolate as they were to be tested for two major
genotypes.
 The tray was then centrifuged at about 1900rpm for six minutes in order to get all
samples to the bottom of the wells.
 After this the tray was placed in the thermo-cycler (DNA machine) at 95oC for 5
minutes.

Whilst the isolates were in the thermo-cycler we prepared the mix for PCR. Two mixes were
made, one for the GA genotype and one for the MS2 genotype. The composition of the mix
is shown below:

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  2rt (reverse transcription) Master Mix 12.5*53= 662.5
 Enzyme 0.25*53 =13.25 (10µL to economise, this is always added last)
 Primer F a 40µM 0.25*53 = 13.25
 Primer r a 40µM 0.25*531 = 13.25
 Probe 15 µM 0.25*53 = 13.25
 Water 4*53 = 212 (actually use 215.25 µL)
 To each of the wells containing 7.5 µL of isolate, 17.5 µL of the mix was added.
 The plate was centrifuged to make sure the liquid was at the bottom of the well.
 The tray then went onto the qRT-PCR machine (stratagene).

Thursday, 20th May

RNA extraction
The method used for the extraction of RNA was the ‘Extraction des acides nucléiques-PEG-
BM méthode eau’ (Water method BM-PEG-Nucleic acids extraction) (Figure 9).

Figure 8 Nucleic acids extraction

Genotyping: Method II

After the RNA extraction, one step qRT- PCR system (Invitrogen) kit for reverse
transcription and amplification was utilised.
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  Like the previous method described, a plan for the isolates placements in the 96 well
plates was completed first. Positive and negative controls were included.
 Isolates were placed into a 96 well polypropylene plate, which was then placed onto
an ice base.
 7.5µl of each sample isolate was put into the wells of the tray according to the plan.
 The mix used for RT-PCR one step is shown below:
Water 9.12 µl
5x reaction mix RNA ultrasens 5 µl
RT R primer (10 µM) 2.25 µl
F PCR primer (10 µM) 1.25 µl
Probe (10 µM) 0.625 µl
Rox reference dye (50x) 0.5 µl
Enzyme mix RNA ultrasens 1.25 µl
RNA 5 µl
 The one step RT-PCR programme is either run for 30 minutes at 55 °C or 5 minutes at 95
°C.

Friday, 21st May

On Friday we discussed the processes learnt throughout the week and their potential
application back at the Brighton University laboratory. The outcomes are stated below. To
finish the week, we gave a presentation on recent work being done at the University of
Brighton for the AquaManche project (Figure 10)

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 Figure 10 Oral presentation at Ifremer

Outcomes

 Exploring the detection and enumeration of phages by qPCR at Brighton University

 Pilot study at University of Brighton using Centricon concentration
 Phage conservation and analysis for method comparison
 Exchange of samples for analysis using methods used in our respective laboratories
 Possible method comparison publication

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