CMS SOM App. H, Tag V257

7.2.3 Heterotrophic plate count

Samples that cannot be cultured within 1 to 2 hours can be refrigerated for up to 24 hours.

Use of a calibrated loop to apply the sample to the agar plate is NOT permitted.

Interpretive Guidance § 494.40(a)

ANSI/AAMI RD52:2004

7.2.3 Heterotrophic plate count

The reference method for culturing is the membrane filtration technique. With this method, a

known volume of sample or diluted sample is filtered through a 0.45 µm membrane filter and the

membrane filter is aseptically transferred to the surface of an agar plate. Trypticase soy agar

(TSA, a soybean casein digest agar) is the medium of choice for culturing water and dialysate;

other acceptable media include standard methods agar and plate count agar (also known as

TGYE). Blood and chocolate agars are not appropriate for this test. The spread plate technique

may also be used. With this method, an inoculum of at least 0.5 mL of sample is spread equally

over the surface of the agar plate.

AAMI Rationale for the Development and Provision of This Recommended Practice

A.7 Strategies for bacterial control

A.7.2.3 Heterotrophic plate count

Sensitive culturing methods must be used to measure the low total viable microbial counts

permitted for water used for hemodialysis applications under the provisions of ANSI/AAMI

RD62:2001 and recommended for dialysate in this recommended practice. The membrane filter

technique is particularly suited for this application because it permits large volumes of water to

be assayed. Because the membrane filter technique may not be readily available in clinical

laboratories, the spread plate assay can be used as an alternative.

However, if the spread plate assay is used, a calibrated loop shall not be used to apply sample to

the plate. The sensitivity of an assay performed with a calibrated loop is low. A standard

calibrated loop transfers 0.001 mL of sample to the culture medium, so that the minimum

sensitivity of the assay is 1,000 CFU/mL. This sensitivity is unacceptable when the maximum

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allowable limit for microorganisms is 200 CFU/mL. Therefore, when the spread plate method is

used, a pipette should be used to place 0.1 mL to 0.5 mL of water on the culture medium.

Additional Guidance:

Responsible staff must be knowledgeable of the culture methods in use and must inform any

outside labs that the samples are water and dialysate and that use of a calibrated loop to

inoculate the culture plate is not acceptable.