1. Equipment and materials

   1. Spiral plater

   2. Spiral colony counter with special grid for relating deposited sample volumes to specific portions of petri dishes

   3. Vacuum trap for disposal of liquids (2-4 liter vacuum bottle to act as vacuum reservoir and vacuum source of 50-60 cm Hg)

   4. Disposable micro beakers, 5 ml

   5. Petri dishes, plastic or glass, 150 x 15 mm or 100 x 15 mm

   6. Plate count agar (standard methods) (M124)

   7. Calculator (optional), inexpensive electronic hand calculator is recommended

   8. Polyethylene bags for storing prepared plates

   9. Commercial sodium hypochlorite solution, about 5% NaOCl (bleach)

   10. Sterile dilution water

   11. Syringe, with Luer tip for obstructions in stylus; capacity not critical

   12. Work area, storage space, refrigerator, thermometers, tally, incubator, as described for Conventional Plate Count Method, above.

   13. Sodium hypochlorite solution (5.25%). Available commercially.

    

2. Preparation of agar plates.

   Automatic dispenser with sterile delivery system is recommended to prepare agar plates. Agar volume dispensed into plates is reproducible and contamination rate is low compared to hand-pouring of agar in open laboratory. When possible, use laminar air flow hood along with automated dispenser. Pour same quantity of agar into all plates so that same height of agar will be presented to spiral plater stylus tip to maintain contact angle. Agar plates should be level during cooling.

   The following method is suggested for prepouring agar plates: Use automatic dispenser or pour constant amount (about 15 ml/100 mm plate; 50 ml/150 mm plate) of sterile agar at 60-70°C into each petri dish. Let agar solidify on level surface with poured plates stacked no higher than 10 dishes. Place solidified agar plates in polyethylene bags, close with ties or heat-sealer, and store inverted at 0-4.4°C. Bring prepoured plates to room temperature before inoculation.

    

3. Preparation of samples.

   As described in Chapter 1, select that part of sample with smallest amount of connective tissues or fat globules.

    

4. Description of spiral plater.

   Spiral plater inoculates surface of prepared agar plate to permit enumeration of microorganisms in solutions containing between 500 and 500,000 microorganisms per ml. Operator with minimum training can inoculate 50 plates per h. Within range stated, dilution bottles or pipets and other auxiliary equipment are not required. Required bench space is minimal, and time to check instrument alignment is less than 2 min. Plater deposits decreasing amount of sample in Archimedean spiral on surface of prepoured agar plate. Volume of sample on any portion of plate is known. After incubation, colonies appear along line of spiral. If colonies on a portion of plate are sufficiently spaced from each other, count them on special grid which associates a calibrated volume with each area. Estimate number of microorganisms in sample by dividing number of colonies in a defined area by volume contained in same area. Studies have shown the method to be proficient not only with milk (4) but also with other foods (7,10).

    

5. Plating procedure

   Check stylus tip angle daily and adjust if necessary. (Use vacuum to hold microscope cover slip against face of stylus tip; if cover slip plane is parallel at about l mm from surface of platform, tip is properly oriented). Liquids are moved through system by vacuum. Clean stylus tip by rinsing for 1 s with sodium hypochlorite solution followed by sterile dilution water for 1 s before sample introduction. This rinse procedure between processing of each sample minimizes cross-contamination. After rinsing, draw sample into tip of Teflon tubing by vacuum applied to 2-way valve. When tubing and syringe are filled with sample, close valve attached to syringe. Place agar plate on platform, place stylus tip on agar surface, and start motor. During inoculation, label petri plate lid. After agar has been inoculated, stylus lifts from agar surface and spiral plater automatically stops. Remove inoculated plate from platform and cover it. Move stylus back to starting position. Vacuum-rinse system with hypochlorite and water, and then introduce new sample. Invert plates and promptly place them in incubator for 48 ± 3 h at 35 ± 1°C.

    

6. Sterility controls

   Check sterility of spiral plater for each series of samples by plating sterile dilution water. CAUTION: Prepoured plates should not be contaminated by a surface colony or be below room temperature (water can well-up from agar). They should not be excessively dry, as indicated by large wrinkles or glazed appearance. They should not have water droplets on surface of agar or differences greater than 2 mm in agar depth, and they should not be stored at 0-4.4°C for longer than l month. Reduced flow rate through tubing indicates obstructions or material in system. To clear obstructions, remove valve from syringe, insert hand-held syringe with Luer fitting containing water, and apply pressure. Use alcohol rinse to remove residual material adhering to walls of system. Dissolve accumulated residue with chromic acid. Rinse well after cleaning.

    

7. Counting grid

   1. Description. Use same counting grid for both 100 and 150 mm petri dishes. A mask is supplied for use with 100 mm dishes. Counting grid is divided into 8 equal wedges; each wedge is divided by 4 arcs labeled l, 2, 3, and 4 from outside grid edge. Other lines within these arcs are added for ease of counting. A segment is the area between 2 arc lines within a wedge. Number of areas counted (e.g., 3) means number of segments counted within a wedge. Spiral plater deposits sample on agar plate in the same way each time. The grid relates colonies on spiral plate to the volume in which they were contained. When colonies are counted with grid, sample volume becomes greater as counting starts at outside edge of plate and proceeds toward center of plate.

   2. Calibration. The volume of sample represented by various parts of the counting grid is shown in operator's manual that accompanies spiral plater. Grid area constants have been checked by the manufacturer and are accurate. To verify these values, prepare 11 bacterial concentrations in range of 10⁶-10³ cells/ml by making 1:1 dilutions of bacterial suspension (use a nonspreader). Plate all Incubate both sets of plates for 48 ± 3 h at 35 ± 1°C. Calculate concentrations for each dilution. Count spiral plates over grid surface, using counting rule of 20 (described in H, below), and record number of colonies counted and grid area over which they were counted. Each spiral colony count for a particular grid area, divided by aerobic count/ml for corresponding spirally plated bacterial concentrations, indicates volume deposited on that particular grid area. Use the following formula:

   
   

    

   To check total volume dispensed by spiral plater, weigh amount dispensed from stylus tip. Collect in tared 5 ml plastic beaker and weigh on analytical balance (± 0.2 mg).

                      

   Figure 1. 10 cm plate, area (3b)

   

8. Examination and reporting of spiral plate counts.

   Counting rule of 20. After incubation, center spiral plate over grid by adjusting holding arms on viewer. Choose any wedge and begin counting colonies from outer edge of first segment toward center until 20 colonies have been counted. Complete by counting remaining colonies in segment where 20th colony occurs. In this counting procedure, numbers such as 3b, 4c (Fig. l) refer to area segments from outer edge of wedge to designated arc line. Any count irregularities in sample composition are controlled by counting the same segments in the opposite wedge and recording results. Example of spirally inoculated plate (Fig. l) demonstrates method for determining microbial count. Two segments of each wedge were counted on opposite sides of plate with 31 and 30 colonies, respectively. The sample volume contained in the darkened segments is 0.0015 ml. To estimate number of microorganisms, divide count by volume contained in all segments counted. See example under Fig. l.

   If 20 CFU are not within the 4 segments of the wedge, count CFU on entire plate. If the number of colonies exceeds 75 in second, third, or fourth segment, which also contains the 20th colony, the estimated number of microorganisms will generally be low because of coincidence error associated with crowding of colonies. In this case, count each circumferentially adjacent segment in all 8 wedges, counting at least 50 colonies, e.g., if the first 2 segments of a wedge contain 19 colonies and the third segment contains the 20th and 76th (or more), count colonies in all circumferentially adjacent first and second segments in all 8 wedges. Calculate contained volume in counted segments of wedges and divide into number of colonies.

   When fewer than 20 colonies are counted on the total plate, report results as "less than 500 estimated SPLC per ml." If colony count exceeds 75 in first segment of wedge, report results as "greater than 500,000 estimated SPLC per ml." Do not count spiral plates with irregular distribution of colonies caused by dispensing errors. Report results of such plates as laboratory accident (LA). If spreader covers entire plate, discard plate. If spreader covers half of plate area, count only those colonies that are well distributed in spreader-free areas.

   Compute SPLC unless restricted by detection of inhibitory substances in sample, excessive spreader growth, or laboratory accidents. Round off counts as described in I-D, above. Report counts as SPLC or estimated SPLC per ml.

References

1. American Public Health Association. 1984. Compendium of Methods for the Microbiological Examination of Foods, 2nd ed. APHA, Washington, DC

2. American Public Health Association. 1993. Standard Methods for the Examination of Dairy Products, 16th ed. APHA, Washington, DC.

3. Association of Official Analytical Chemists. 1990. Official Methods of Analysis, 15th ed. AOAC, Arlington, VA.

4. Donnelly, C.B., J.E. Gilchrist, J.T. Peeler, and J.E. Campbell. 1976. Spiral plate count method for the examination of raw and pasteurized milk. Appl. Environ. Microbiol. 32:21-27.

5. Gilchrist, J.E., C.B. Donnelly, J.T. Peeler, and J.E. Campbell. 1977. Collaborative study comparing the spiral plate and aerobic plate count methods. J. Assoc. Off. Anal. Chem. 60:807-812.

6. International Dairy Federation. 1987. Milk and Milk Products: Enumeration of Microorganisms--Colony Count at 3°C. Provisional IDF Standard 100A. IDF, Brussels, Belgium.

7. Jarvis, B., V.H. Lach, and J.M. Wood. 1977. Evaluation of the spiral plate maker for the enumeration of microorganisms in foods. J. Appl. Bacteriol. 43:149-157.

8. Niemela, S. 1983. Statistical evaluation of Results from Quantitative Microbiological Examinations. Report No. 1, 2nd ed. Nordic Committee in Food Analysis, Uppsala, Sweden.

9. Tomasiewicz, D.M., D.K. Hotchkiss, G.W. Reinbold, R.B. Read, Jr., and P.A. Hartman. 1980. The most suitable number of colonies on plates for counting. J. Food Prot. 43:282-286.

10. Zipkes, M.R., J.E. Gilchrist, and J.T. Peeler. 1981. Comparison of yeast and mold counts by spiral, pour, and streak plate methods. J. Assoc. Off. Anal. Chem. 64:1465-1469.

Hypertext Source: Bacteriological Analytical Manual, Edition 8, Revision A, 1998. Chapter 3.
*Authors: Larry Maturin and James T. Peeler