Modified Track plating Method for quantification of Enterobacteriaceae in Faecal samples SOP version 1 Author: N
Fawcett April 2015, Nuffield Department of Medicine, University of Oxford
Equipment required:
1)
Peptone water /(Normal Saline) in 3ml aliquots.
2)
Sterile 96-well plate
3)
1ml Pipette, 200ul pipette and Multichannel Pipettes – 200ul and 10ul
4)
Large-bore 1ml tips, 200ul tips and 10ul tips with filters (you may need to chop the ends off filtered narrow-bore
1ml tips with filters with sterilised scissors or risk having your pipetteman contaminated with faecal sample…)
5)
Agar plates – e.g. Oxoid UTI Brilliance Chrom-agar pre-poured plates.
Method:
Purpose: Protocol for solid faecal sample. Note: for diarrhoeal samples it may be appropriate not to dilute the sample prior
to addition to 96-well plate. Recommended that no more than 6-8 samples are processed at once, as the entire procedure
should be performed within 15 minutes of diluting the first faecal sample.
1)
Prepare 96-well plate. Add 180ul peptone water to each well in rows 2-6, with 1 column per sample. Leave row 1
empty.
2)
Label agar plates and inspect for excess moisture and bubbles on the surface, which may interfere with track
plating. Remove excess water (which may drip and interfere) from plate lids.
3)
Homogenise faecal sample with sterile spatula as best able. Fume hood may reduce smell.
4)
Aliquot out approximately 250mg of faecal sample into 7ml bijou and weigh*
5)
Dilute with peptone water/normal saline at ratio of 1:9 (around 2250ul) (Dilution 10-1 ).
6)
Vortex sample for 10 seconds.
7)
Using 1ml large-bore tips, pipette 333ul of faecal slurry into 3ml peptone water bottle. (Dilution 10-2 )
8)
Vortex for 10 seconds.
9)
Take 200ul from the peptone water bottle and pipette into row 1 of the 96-well plate. Repeat with other faecal
samples if required.
10) Using the 200ul multichannel pipette, take 20ul from each well in row 1, and add to row 2. Try not to touch the
contents of the well with the pipette tip. Discard pipette tips.
11) Using fresh tips, pipette up and down in row 2 to mix the sample 5 time. Withdraw 20ul and repeat to row 3-6,
creating 10-1 serial dilutions (10-2 - 10-7 )
12) Using the 10ul multichannel pipette take 10ul from each well in Column A, rows 1-6.
13) Hold Agar plate at 45’ angle from horizontal with other hand facing towards the pipette.
14) Eject 10ul carefully so a 10ul bead of fluid forms at the pipette tip.
15) Touch the fluid bead onto the agar plate, and rotate the plate to vertical, allowing the fluid to form tracks down the
agar plate.
16) Before the fluid reaches the edges of the plate, place horizontally on the bench, and allow to dry for 1 minute.
(Sufficient for UK lab and Oxoid UTI Brillance Chrom-agar, may differ depending on humidity of lab and agar).
17) Repeat process with other faecal samples in other columns if required.
18) When dry, plates can be incubated upside down.
19) Consider appropriate control plates.
20) Following appropriate incubation period, colonies can be counted and CFU/ml calculated.
*This protocol makes the assumption that 1g of faeces has a volume of 1cm3. Depending on water content this may vary. A commonly
quoted particle density for soil is 2.65g/cm3 with a bulk density of 1-1.6g/cm3, thus this assumption may mean that the first 1/10 dilution
may be up to 1/30 and result in an under-estimation of the CFU/ml by a factor of 3. The protocol is designed to quantify CFU to the nearest
power of 10, and use inexpensive equipment. Alternative methods of volume measurement may be appropriate
Colony counting method used in abstract:
1)
Count the track with the highest number of discrete colonies between 10-50
2)
If there is no track with this number, choose the track with between 50-100 colonies. If in this track there are more
than 2 instances where multiple colonies are growing so close that you are having to guess, choose the track with
<10 colonies.
Note: depending on colony morphology and size, criteria for choosing best dilution may differ. Using a microscope and
lighting conditions may also optimise counting ability.
Calculation:
CFU/ml = colony count / dilution of track x 102
( colony count/dilution = CFU in 10ul)
e.g. 25 colonies counted in row 2 (10-3) final CFU/ml = 25 x 105 CFU/ml
Further steps:
1)
2)
3)
4)
For screening for resistance, selective agar can be used in parallel, such as ESBL agar and kanamycin-containing
agar.
MALDI-TOF identification of unique colony morphologies performed
Formal testing for resistance profile according to British Society of Antimicrobial Chemotherapy standards for all
unique colony morphologies
For identification/resistotyping, always select colony from the highest dilution track where colony morphology is
visible.
Note: Method design to resistotype the dominant strain. Faecal sample may contain many morphologically identical strains
with different resistance profiles and assessment of all of these phenotypes is not feasible. Performing culture in parallel with
screening selective agar (e.g. ESBL agar) can increase sensitivity to minority resistant strains, whilst still giving phenotype
of numerically dominant culturable strain.
Health and Safety:
A full risk assessment taking into account local guidance for the safe handling of faecal samples should be performed.
Specific hazards:
1)
2)
Vortexing faecal sample may create aerosols. It may be recommended to open all vortexed samples in a fume hood
or similar to reduce the very low risk of transmission of pathogens through aerosol.
Risk of culture and handling of high-risk organisms (eg. Salmonella Typhi) should be assessed based on local
policy and proposed faecal sample identity.