ASTM: D5916: Clostridium perfringens from Water and Extracted Sediments

Summary
Analytes
Revision
Data and Sites

Official Method Name

Standard Test Method for Detection and Enumeration of Clostridium perfringens from Water and Extracted Sediments by Membrane Filtration (MF)

Current Revision

Current edition approved Feb. 10, 1996.

Media

WATER

Instrumentation

Membrane Filtration

Method Subcategory

Microbiological

Method Source

ASTM

Citation

Annual Book of ASTM Standards, Section 11, Water and Environmental Technology, Volume 11.02, Water (I)

Brief Method Summary

Appropriate volumes of water are passed through membrane filters (MF) that retain the bacteria present in the sample. The MFs are placed on mCP agar modified by Armon and Payment (1) from the medium of Bisson and Cabelli (2) and are incubated anaerobically at 44.5^oC for 24 h. The yellow, straw-colored C. perfringens colonies which turn dark pink to magenta on exposure to ammonium hydroxide are counted and reported C. perfringens colony forming units (CFU) per 100 mL. Because of the selectivity of the mCP medium, presumptive counts are normally reported for routine monitoring purposes. If verification is desired, colonies are confirmed by anaerobic growth in thioglycollate, a positive gram stain reaction and stormy fermentation of iron milk, and mCP counts adjusted based on the percent confirmation.
For sediment analyses, 1 to 10 g of wet sediment is weighed, water added, and mixed by vortex and sonication. After settling, the water layer is analyzed as described above.
Verification of counts is not required. However, if verification is desired, colonies can be confirmed by anaerobic growth in thioglycollate, a positive gram stain reaction and stormy fermentation of iron milk. The mCP counts may be adjusted based on the percent confirmation.

Scope and Application

This test method can enumerate Clostridium perfringens spores and vegetative cells from marine water, sediment, wastewater, ambient water, and drinking water. Since C. perfringens spores are present in large numbers in human and animal wastes and are resistant to wastewater treatment practices, extremes in temperature, and environmental stress, they are an indicator of present fecal contamination as well as a conservative tracer of past fecal contamination.

Applicable Concentration Range

1 and greater CFU/100 mL

Interferences

Waters containing sediment, large quantities of colloidal or suspended materials such as iron, manganese, alum floc, or algae can clog the filter pores and prevent filtrations or cause the development of spreading bacterial colonies which may mask target colonies and prevent accurate counting.
When bacterial densities are high, a smaller sample volume or sample dilution can be filtered to minimize the interference of turbidity or high background (nontarget) bacterial densities. Replicates of smaller sample volumes or dilutions of sample may be filtered and the results combined. However, the membrane filter techniques may not be applicable to highly turbid waters with low Clostridium densities.
Toxic materials such as metals, phenols, acids, caustics, chloramines, and other disinfection by-products may also adversely affect recovery of Clostridium vegetative cells on the MF.

Quality Control Requirements

Check and record temperatures in incubators daily to ensure operation within stated limits.
Check thermometers and record the results at least annually against a National Institute of Standards and Technology (NIST) certified thermometer or one traceable to NIST. Record results. Check mercury columns for separation.
As a quality control over anaerobic conditions, temperature, and media, spot test a separate mCP agar plate with a pure culture of C. perfringens and include in each test run. Examine for the appropriate response.
For general quality control recommendations, see" Quality Assurance for Microbiological Analyses of Water" in ASTM Special Technical Testing Publication 867.

Sample Handling

Collect water samples in sterile, nontoxic glass or plastic containers with leak-proof lids. Collect 10 to 20 g sediment samples and place in a sterile 4.5 oz plastic cup or other appropriate sterile container with leak-proof lid.
Use insulated containers to maintain water samples on ice or refrigerate at a temperature of 1 to 4^oC during transit to the laboratory. Take care that sample container tops and closures are not submerged in water during transit or storage. Refrigerate samples upon arrival in the laboratory and analyze as soon as possible after collection.
Although C. perfringens vegetative cells are sensitive to aerobic conditions and are not expected to survive well in storage, C. perfringens spores can survive for extended periods at 1 to 4^oC. However, if a correlation is planned with other indicator or pathogenic microorganisms, the holding time for C. perfringens should be limited to that of the other organisms.
Sampling procedures are described in Practices D 3370. Adherence to sampling procedures, preservation procedures, and holding time limits is critical to the production of valid data. Reject samples if appropriate sampling, preservation, and handling procedures have not been followed.

Maximum Holding Time

Relative Cost

Unknown

Sample Preparation Methods

This method has 1 analyte associated with it.

Analyte	Detection Level	Bias	Precision	Pct False Positive	Pct False Negative	Spiking Level
Clostridium perfringens(E-12204) C. perfringens C. perfringens Clostridium perfringens Clostridium perfringens Clostridium welchii Gangrene bacillus	1.000 CFU/100 mL	86% Rec (ML)	41.00			78.00 CFU/100 mL

Precision Descriptor Notes:	Sixteen analysts from nine laboratories analyzed a sediment, an unchlorinated wastewater, and three spiked waters (marine water, lake water, and a finished drinking water) as unknowns. Analysts were provided range values to reduce the number of dilutions necessary for the analyses. Of the 16 data sets, three sets were rejected out of hand because the labs either did not follow method instructions for the study or did not correctly report results. Other data sets for some samples were rejected because the laboratories used dilutions which produced results falling outside the desired counting range of approximately 15 to 80 CFU/agar plate. The triplicate plate counts were averaged and the average counts used to calculate the reported colony forming units (CFU).
Detection Level Note:	None.

Precision Descriptor Notes:

Sixteen analysts from nine laboratories analyzed a sediment, an unchlorinated wastewater, and three spiked waters (marine water, lake water, and a finished drinking water) as unknowns. Analysts were provided range values to reduce the number of dilutions necessary for the analyses. Of the 16 data sets, three sets were rejected out of hand because the labs either did not follow method instructions for the study or did not correctly report results. Other data sets for some samples were rejected because the laboratories used dilutions which produced results falling outside the desired counting range of approximately 15 to 80 CFU/agar plate. The triplicate plate counts were averaged and the average counts used to calculate the reported colony forming units (CFU).

Detection Level Note:

None.

Revision	PDF/Link
Current edition approved Feb. 10, 1996.

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ASTM: D5916: Clostridium perfringens from Water and Extracted Sediments

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