DECLARATION 2 PUBLICATIONS
7. APPENDIX 1
7.6 Detection and enumeration of Staphylococcus
This method is the same as ISO. 6340:1995(E).
Specificity 100%
Selectivity 97.5%
Reporting Results
The results are reported as the presence or absence of Salmonella or Shigella in the sample volume.
Gram positive: Organism retaining the crystal violet-iodine stain of the differential Gram stain and taking up the safranin stain of the differential Gram stain.
Masking: Growth of non-target organisms which may interfere with the detection of target organisms.
Principles
Baird Parker medium contains lithium chloride and tellurite to inhibit the growth of accompanying microbial flora. It also contains pyruvate and glycine which selectively stimulate the growth of staphylococci. Staphylococci form black colonies on the medium surrounded by zones of clearing in the surrounding agar.
Health, Safety and Precautions
Interferences for this method are as follows:
Turbidity
Highly turbid samples can block membrane filters and therefore prevent proper analysis according to this method. If a sample stops going through the filter or takes an unusually long time to do so, further dilution of the sample before filtering should be considered or an MPN method should be used.
ToxicantsTocicants in the sample will interfere with the resuscitation of the organisms and therefore result in a low organisms count. This problem may occasionally be alleviated by diluting the sample.
Temperature
Too high a temperature reduces the survival of the target organisms, whilst too low a temperature permits the growth of other, non-target organisms.
Condensate
Inversion of Petri dishes for the duration of incubation prevents water droplets forming on the lid and dropping onto the surface of the medium with a resultant blurring of colonies.
Staphylococci can cause boils and other skin infections so should be handled with appropriate care.
Sample Handling Choice of Sample Size
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.
Potable water - 100 mL
River water - 0.2 and 0.01 mL Outfalls - 1 and 0.2 mL
Beaches - 5 and 1 mL
Pools - 100 mL
Apparatus and Equipment Bunsen burner
Autoclave
Biohazard cabinet Water purifier Automatic pipette Filter manifold and pump Forceps
Microwave Incubator Plate viewer
Reagents and Materials Baird-Parker Agar
API Staphylococcus test strips Brain-heart infusion broth (BHI)
Reagent Water
Water from the bacteriology water purifier which should have a conductivity of
<0.5mS/m.
Sterile water
Sterilise reagent water in autoclave at 1210C for 15 minutes.
Cool.
Store at <100C.
Sterile gridded 0.45 µm pore size, 47 mm diameter membrane filters. Individually packed membranes from acceptable supplier.
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 dispenser must have passed QC check within the last week.
Automatic pipettes must have passed QC check within the last week.
Medium batch must have passed QC check.
Laminar flow cabinets must have passed QC check within the last week.
Autoclave run must have passed QC check.
For each batch of media prepared, inoculate one Petri dish with positive and negative controls, namely E. coli and Enterococcus faecalis.
The E. coli colonies should be purple/blue and the Enterococcus faecalis. should be minimal and grey/white in colour.
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
Negative control E. coli
Procedure
Filter required volume of sample through a membrane filter.
Roll membrane onto surface of plate and incubate inverted for 45-48h at 35C.
Select plates containing 20-200 colonies for counting .
Colonies of S. aureus are circular, smooth, convex, moist, 2-3 mm in diameter on uncrowded plates, gray to jet-black, frequently with light-coloured (off-white) margin, surrounded by opaque zone and frequently with an outer clear zone; colonies have buttery to gummy consistency when touched with inoculating needle.
If confirmation is necessary, transfer suspect S. aureus colonies into small tubes containing 0.2-0.3 mL BHI broth and emulsify thoroughly.
Inoculate agar slant of suitable maintenance medium, e.g., TSA or nutrient, with a loopful of BHI suspension.
Incubate BHI culture suspension and slants 18-24 h at 35°C.
Retain slant cultures at room temperature for ancillary or repeat tests in case coagulase test results are questionable.
Add 0.5 mL reconstituted coagulase plasma with EDTA to the BHI culture and mix thoroughly.
Incubate at 35°C and examine periodically over 6h period for clot formation. Only a firm and complete clot that stays in place when the tube is tilted or inverted is considered positive for S. aureus. Partial clotting, formerly 2+ and 3+ coagulase reactions, must be tested further.
Test known positive and negative cultures simultaneously with suspect cultures of unknown coagulase activity.
Stain all suspect cultures with Gram reagent and observe microscopically. A latex agglutination test may be substituted for the coagulase test if a more rapid procedure is desired.
Incubation
Incubate the prepared membranes in an inverted position at 35 - 37oC for 24 hours.
Incubate samples within 30 minutes of filtering.
Enumeration
Count gray to jet black colonies using an illuminated plate counter.
Disposal
Collect all counted Petri dishes and place in autoclave bag.
Autoclave at 1210C for 15 minutes to sterilise.
Place sterilised cultures in hazardous waste disposal container for disposal.
Calculation
If a dilution has been performed, it is necessary to multiply the number of colonies counted by the inverse of the dilution factor to obtain number of colonies per 100 mL.
Reporting Results
All 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.