DECLARATION 2 PUBLICATIONS
7. APPENDIX 1
7.3 Detection and enumeration of Enterococcus on Enterococcus selective agar
Reporting Results
All total coliform and E. coli results are entered into LIMS where the count per 100 mL is automatically calculated. Specification limits have been set for each water type and any results outside these limits will appear on the LIMS screen in red. These results must be further investigated according to the procedure for the handling of non-conforming results.
Definitions
Enterococcus: This group is a subgroup of the faecal streptococci and includes E.
faecalis, S. gallinarum and S. avium. All give a positive reaction with Lancefield’s Group D antisera and have been isolated from the faeces of warm blooded animals. For this method, enterococci are those bacteria which produce black colonies on Enterococcus selective agar and are catalase negative and Gram positive.
Principles
Sodium azide is a selective agent which suppresses the growth of Gram positive organisms.
Esculin hydrolysis and bile tolerance are regarded as being reliable, constant characteristics of faecal streptococcii. The microorganisms hydrolyse the glycoside esculin to dextrose and esculin which forms an olive green to black complex with iron (III) ions.
Health, Safety and Precautions
Temperature Incubation temperature is critical. The incubator must hold the 37±1oC temperature throughout the chamber over a 48 hour period.
Turbidity Membranes are unsuitable for use with waters of high turbidity as the membrane will block before sufficient water can be filtered. In such a case either smaller volumes of the sample could be used or a pre-filter may be fitted ahead of the membrane filter to trap the non-microbial suspended material.
Toxicants Toxic metals
Toxic organics such as phenols.
Bacteria Staphylococcus aureus may cause a false positive result.
Sample Handling
Keep samples chilled from the time they are sampled till delivery to the Laboratory.
Use insulated boxes and freezer bricks to keep the samples cold.
Analyse samples as soon as possible.
Do not hold samples for more than eight (8) hours before analysis.
Use volumes yielding 20 to 50 colonies per membrane where possible.
When the bacterial density is unknown, filter several decimal volumes to establish the density.
Estimate the volume expected to yield a suitable membrane colony count and select two additional volumes representing approximately one tenth and ten times this volume respectively.
The bacterial load of different types of water varies and this is compensated for by using different volumes of water for analysis. The following volumes have been found to be generally appropriate.
Matrix Volume (mL)
Potable water - 100
River water - 0.2
0.01
Outfalls 1
0.2
Beaches 5
1
Pools 100
Apparatus and Equipment Electronic colony counter.
Automatic pipettes of suitable volume.
Membrane filtration unit and manifold.
Line vacuum, electric pump and filter flask.
Flask for safety trap containing silica gel.
Forceps with smooth tips to handle filters.
Bunsen burner for sterilisation.
Petri dishes, sterile plastic.
Tubes, glass for dilutions.
Membrane filters, white, gridded, 47 mm diameter.
Incubator.
Reagents and Materials Enterococcus Selective Agar
Suspend 55g Enterococcus Selective Agar in 1 lire reagent water. Boil whilst stirring until completely dissolved. Autoclave at 121°C for 15 minutes. Cool to 45 - 50°C. Mix well and pour into plates.
pH 7.1 ± 0.2 pH units.
Composition g/L
Esculin 1.0
Ferric Ammonium Citrate 0.5
Ox Bile 10.0
Peptone 3.0
Sodium Azide 0.5
Sodium Citrate 1.0
Tryptone 17.0
Yeast Extract 5.0
Agar 12.0
Calibration
Balance must have passed QC check within the last week.
pH meter must have passed QC check within the last week.
Incubator must show a steady temperature on the Laboratory Temperature Logger.
Automatic pipettes must have passed QC check within the last week.
Media batches must have passed QC check.
Laminar flow cabinets must have passed QC check within the last week.
Autoclave run must have passed QC check.
If any of the above calibrations has failed, the method should not be deemed fit for use before remedial action has been shown to be successful.
Quality Control
Perform positive and negative controls on each batch of media.
Positive control is Enterococcus faecalis Negative control is Escherichia coli.
Procedure Technique
Take out of the fridge sufficient Petri dishes of Enterococcus medium for the expected number of samples and allow to warm to room temperature.
Where a 100 mL volume is used, filter directly onto the filter membrane.
Where dilutions are required, pipette 5 mL, 2 mL, 1mL, 0.2 mL or 0.01 mL volumes as necessary out of the well mixed sample and place in approximately 30 mL of sterile reagent water.
Filter the sample or aliquot under vacuum through a 0.45µm pore size membrane filter in a sterile filter assembly.
Open the filter assembly and remove the membrane using sterile technique.
Roll it onto the surface of the agar in a Petri dish.
For each batch of 10 samples or less, one sample must be run in duplicate. The selected sample must have a replicate entered against it in LIMS.
Incubation
Invert the Petri dishes.
Incubate the prepared membranes at 37±1oC for 48 hours.
Incubate samples within 30 minutes of filtering.
Enumeration
Enterococci form dark brown to black colonies with esculin production
Count colonies with a colony counter employing illumination and magnification.
Verification
Pick selected colonies and streak for purity onto brain heart infusion agar.
Incubate at 37±1oC for 24-48 hours.
Transfer a loop full to each of two microscope slides.
Add a few drops of fresh 3% hydrogen peroxide to one slide.
If no bubbles appear, the culture is catalase negative.
Make a Gram stain of the other slide.
Enterococci are catalase negative, Gram-positive, ovoid cells, 0.5 - 1.0µm in diameter, mostly in pairs or short chains.
OR API Strept.
Calculation
Compute the density from the sample quantities which produce counts within the desired range of 20 - 50 colonies.
Enterococcus / 100 ml = Enterococcus x 100 mL of sample filtered
Method Performance Assessment
This is the standard method for enumeration of faecal streptococci and enterococci – Standard Methods no 9230 C and is deemed suitable for use in water analysis.
Reporting Results
Results are expressed as colony forming units per 100 mL.