9215 HETEROTROPHIC PLATE COUNT (2017)

Abstract:9215 A. Introduction

1. Applications

The heterotrophic plate count (HPC), formerly known as the standard plate count, is a
procedure for estimating the number of live, culturable heterotrophic bacteria in water and
for measuring changes in swimming pools or during water treatment and distribution.
Colonies may arise from pairs, chains, clusters, or single cells—all of which are included in
the term colony-forming units (CFU). The final count also depends on interaction among
developing colonies. Choose the procedure and medium that best suit how the resulting
information will be used. Only compare data generated using the same procedure and
medium. Four methods and five media are described.

2. Selection of Method

a. Pour plate method: The pour plate method (9215B) is simple to perform and can
accommodate sample or diluted-sample volumes ranging from 0.1 to 2.0 mL. It produces
subsurface colonies that are relatively small, compact, and less apt to encroach on each
other than surface colonies do. However, submerged colonies often are slower growing
and difficult to transfer. A thermostatically controlled water bath is essential for tempering
the agar.

b. Spread plate method: The spread plate method (9215C) causes no heat shock. All
colonies are on the agar surface, where analysts typically can easily distinguish them from
particles and bubbles, and discern different colony morphologies. If not, they can transfer
colonies quickly and easily discern colony morphology via comparisons to published
descriptions. However, the agar in this method can only absorb a small volume of sample
or diluted sample (0.1 to 0.5 mL, depending on the degree to which prepoured plates have
been dried). Also, spreading colonies could be interferences. To use this procedure,
maintain a supply of suitable pre-dried, absorbent agar plates (see 9215C.4).

c. Membrane filter method: The membrane filter method (9215D) can be used to analyze
large volumes of low-turbidity water and is the method of choice for waters with low
numbers of heterotrophic organisms (<1 to 10 CFU/mL). It produces no heat shock.
However, there is the expense of the membrane filter, the smaller area for colony
development, possible damage to cells by excessive filtration pressures, possible variations
in membrane-filter quality (see Section 9020B.5i), and the need to detect colonies via
reflected light against a white background if colored filters or contrast stains are not used.

d. Enzyme substrate method: The enzyme substrate method, SimPlate® (9215E), can be
used to analyze drinking- and source-water samples with a wide range of bacterial
concentrations. It produces no heat shock. Comparable to the pour plate method, this
method† uses a medium in which substrates are hydrolyzed by multiple microbial enzymes,
causing the release of 4-methylumbelliferone after 48 h of incubation at 35 ± 5°C. The 4-
methylumbelliferone fluoresces when exposed to long-wavelength (365–366 nm) ultraviolet
(UV) light, and the number of blue fluorescing wells corresponds to a most probable
number (MPN) of bacteria in the sample. Unfortunately, individual colonies cannot be
directly recovered for subsequent analysis

3. Work Area

Provide a level table or bench top with ample area in a clean, draft-free, well-lighted room
or within a horizontal-flow laminar hood. Use tables or bench tops with nonporous surfaces,
and disinfect before any analysis is made (Sections 9020B.3cand e).
4. Samples

Collect water as directed in Section 9060A. Initiate analysis as soon as possible after
collection to minimize changes in bacterial populations, and do not exceed a holding time of
24 h (or, if the sample was collected for regulatory purposes, the maximum holding time
specified by the regulation). If the sample cannot be tested within 30 min after collection,
maintain it at <10°C but do not allow it to freeze during transit.

5. Sample Preparation

Mark each plate with sample number, dilution, date, and any other necessary information
before examination. For pour- or spread-plate methods, use sterile glass (65 cm2) or
presterilized disposable plastic (57 cm2) Petri dishes. For pour-plate, spread-plate, or
membrane-filtration methods, prepare at least two replicate plates for each volume of
sample or dilution examined for compliance testing. Duplicates also are recommended for
non-compliance testing.

Thoroughly mix all samples or dilutions by shaking vigorously for 7 s by hand

(approximately 25 times with a 1-ft movement) or via a mechanical shaker for 15 s at low
speed. Analytical results depend on adequate sample mixing; if the sample is not
adequately shaken before aliquots are removed, the actual bacterial density could be
underestimated.

6. Media

Users should ensure that the formulations of purchased media match those described
below. Compare new lots of media with current lot in use according to Sections
9020B.5j and 9b.

a. Plate count agar (also called tryptone glucose yeast agar): This medium can be used to
examine heterotrophic bacteria in a wide variety of matrices. It can be used for both pour-
and spread-plate methods. This high-nutrient agar, widely used in the past, may give lower
counts than R2A or NWRI agar. Commerically available dehydrated medium should be
used when available.

The pH should be 7.0 ± 0.2 after autoclaving at 121°C for 15 min.

b. m-HPC agar (formerly called SPC agar): This can be used to count heterotrophic
bacteria in waters with low levels of heterotrophs, such as treated potable water. This high-
nutrient medium is used only with the membrane-filter method. Commerically available
dehydrated medium should be used when available.
For medium produced from individual ingredients, adjust to pH 7.2 with 1 N NaOH. Heat
slowly to dissolve thoroughly, add glycerol, and sterilize at 121°C for 5 min. Commercially
prepared medium should not require post-sterilization pH adjustment; see Section
9020B.5j1). Final pH is 7.1 ± 0.2.

c. R2A agar: Use for pour-plate, spread-plate, and membrane-filter methods. This low-
nutrient agar was developed for use with potable treated water to monitor trends in potable-
water production; this agar and a longer incubation period can improve the recovery of
stressed and chlorine-tolerant bacteria. This medium may yield higher counts than high-
nutrient formulations.

For medium produced from individual ingredients, adjust to pH 7.2 with solid K 2HPO4 or
KH2HPO4 before adding agar. Heat to dissolve agar and sterilize at 121°C for 15 min.
Commercially-prepared medium should not require post-sterilization pH adjustment; see
Section 9020B.5j1). Final pH is 7.2 ± 0.2.

d. NWRI agar (HPCA): Use for pour-plate, spread-plate, and membrane-filter methods.
This low-nutrient medium is likely to produce higher colony counts than high-nutrient media.
This agar typically is not available in dehydrated form; it must be prepared from individual
ingredients, making its use less desirable.

Final pH is 7.2 ± 0.2 after autoclaving at 121°C for 15 min.

e. Enzyme substrate medium: SimPlate® medium is available commercially‡ in sterile

vessels for 100-mL multi-dose procedures or in test tubes for 10-mL unit-dose procedures.
Store medium between 2 and 25°C and use before expiration date.

7. Incubation

The media described in 9215A.6 should be incubated as follows:

a. Plate count agar (PCA): Incubate at 35°C for 48 ± 3 h.

b. m-HPC agar: Incubate at 35°C for 48 ± 3 h.

c. R2A agar: Incubate at 20–28°C for 5 to 7 d.

d. NWRI agar: Incubate at 20°C for 7 d.

e. SimPlate® medium: Incubate at 35 ± 0.5°C for 48 h (range of 45 to 72 h).

During incubation, maintain humidity so agar plates will not lose >15% moisture weight; this
is especially important during prolonged incubation. A pan of water placed in the bottom of
the incubator may be sufficient so long as the incubator's interior walls and shelving are
made of high-grade stainless steel or anodized aluminum, which will not rust or oxidize. If
the incubator is not humidified, seal plates in plastic bags, removing as much air as
possible before sealing bag.

8. Counting and Recording

a. Pour and spread plates: Count all colonies on selected plates promptly after incubation.
If counting must be delayed temporarily, store plates refrigerated for ≤24 h, but avoid this
as routine practice. Record results of sterility controls on the report for each lot of samples.

Count colonies manually using a dark-field colony counter, such as a Quebec colony
counter or equivalent. If such equipment is unavailable, then other counters can be used on
non-compliance samples so long as they provide equivalent magnification. Automatic plate-
counting instruments are available; they generally use a computer program and scanner
and give computed results. Their use is acceptable if, when evaluated by being run in
parallel with a manual method, counting results are comparable.

When preparing plates, pipet sample volumes that will yield between 30 and 300
colonies/plate. The aim is to have at least one dilution yield colony counts within these
limits, except as provided below.

Ordinarily, do not pipet >2.0 mL of sample; however, if this sample volume yields <30
colonies, record the result observed. Otherwise, use only plates containing 30 to 300
colonies when determining plate count. Compute bacterial count per milliliter as follows:

If no plate contains 30 to 300 colonies, and one or more plates have >300 colonies, use the
plate(s) whose count is nearest 300 colonies. Compute the count as above and report as
estimated CFU/mL.

If plates from all dilutions of a sample have no colonies, report the count as <1 divided by
the corresponding largest sample volume used. For example, if no colonies develop from
the 0.01-mL sample volume, report the count as <100 CFU/mL.

If the number of colonies per plate exceeds 300, do not report results as “too numerous to
count” (TNTC). If there are <10 colonies/cm2, count colonies in 13 squares (of the colony
counter) with representative colony distribution. If possible, select seven consecutive
squares horizontally across the plate and six consecutive squares vertically, being careful
not to count a square more than once. Compute estimated colonies per plate as follows:
When the plate is 65 cm2 (the typical area of a glass plate), multiply the sum of the number
of colonies in 13 representative square centimeters by 5; when the plate is 57 cm2 (the
typical area of a plastic plate), multiply the sum of the number of colonies in 19
representative square centimeters by 3. When there are >10 colonies/cm 2, count four
representative squares, take the average count per cm2, and multiply by the appropriate
factor (57 for disposable plastic plates and 65 for glass plates) to estimate colonies/plate.
(NOTE: The nominal diameter of both disposable plastic and nondisposable glass plates is
100 mm. However, the internal diameter of disposable plates is closer to 85 mm and that of
nondisposable plates is closer to 90 mm.) When bacterial counts on crowded plates are
>100 colonies/cm2, report results as >6500 divided by the smallest sample volume plated
for glass plates or >5700 divided by the smallest sample volume plated for plastic plates.
Report as estimated CFU/mL.

If spreading colonies (spreaders) are encountered on the plate(s) selected, count colonies
on representative portions only when colonies are well distributed in spreader-free areas
and the area covered by the spreader(s) does not exceed one-half the plate area.

When spreading colonies must be counted, count each of the following types as one: a
chain of colonies that appears to be caused by disintegration of a bacterial clump as agar
and sample were mixed; a spreader that develops as a film or growth between the agar
and bottom of the Petri dish; and a colony that forms in a film of water at the edge or over
the agar surface. The last two types largely develop due to moisture accumulation at the
point from which the spreader originates. They frequently cover more than half the plate
and interfere with obtaining a reliable plate count.

Count similar, adjacent colonies as individual colonies so long as they do not touch and the
distance between them is at least equal to the diameter of the smallest colony. Count
impinging colonies that differ in appearance (e.g., morphology or color) as individual
colonies.

If plates have excessive spreader growth, report as “spreaders” (Spr.). When plates are
uncountable due to missed dilution, accidental dropping, or contamination, or else the
control plates indicate that the medium or other material was contaminated, report as
“laboratory accident” (LA).

b. Membrane filter method: Count colonies on membrane filters using a stereoscopic

microscope at 10 to 15× magnification. Preferably slant Petri dish at a 45° angle on a
microscope stage and adjust light source vertical to the colonies. Optimal colony density
per filter is 20 to 200. If colonies are small and uncrowded, a higher limit is acceptable.

Count all colonies on the membrane when there are ≤2 colonies per square. For 3 to 10
colonies per square, count 10 squares and determine an average count per square. For 10
to 20 colonies per square, count 5 squares and determine an average count per square.
Multiply average count per square by 100 and divide by the sample volume to give colonies
per mL. If there are >20 colonies per square, record the count as >2000 divided by the
sample volume. Report averaged counts as estimated CFU. Make estimated counts only
when there are discrete, separated colonies without spreaders.

c. Enzyme substrate method: Count the number of blue fluorescent wells by holding a 6-W,
365–366 nm UV light about 5 in. above plate, preferably with UV-filtering safety glasses or
in a UV cabinet. Alternatively, count fluorescent wells through the back of the inverted plate
following the procedure indicated above.

Use the appropriate MPN Table (multi-dose or unit dose) provided by the manufacturer to
calculate MPN/mL. Adjust MPN to reflect sample volume and/or dilution made to yield a
corrected MPN value. Record as MPN/mL.

9. Computing and Reporting Counts

The term colony-forming unit(s) (CFU) is descriptive of the methods used; therefore, report
all counts as CFUs. Also report the method used, the incubation temperature and time, and
the medium. For example: CFU/mL, pour plate method, 35°C/48 h, plate count agar.

To compute the heterotrophic plate count for pour plate, spread plate, and membrane filter
methods (CFU/mL), divide either the total or average number of colonies per plate by the
sample volume. (Use the average number of colonies if duplicate plates of the same
dilution.) Record sample volumes used and number of colonies on each plate counted or
estimated.

When colonies on duplicate plates and/or consecutive dilutions are counted and results are
averaged before being recorded, round off counts to two significant figures only when
converting to CFUs.

Avoid creating fictitious precision and accuracy when computing CFUs by recording only
the first two digits. Round up the second digit when the third digit is 5 through 9 and round
down when the third digit is 1 through 4 (e.g., report a count of 142 as 140, 155 as 160, and
35 as 35).
For the enzyme substrate method, record the MPN/mL obtained from the MPN tables
provided by the manufacturer, adjusted for sample dilution if required.

10. Analytical Bias

Avoid counting inaccuracies due to carelessness, damaged or dirty optics that impair
vision, or failure to recognize colonies. Laboratory workers who cannot duplicate their own
counts on the same plate within 5% and other analysts' counts within 10% should discover
the cause and correct such disagreements.