9215 HETEROTROPHIC PLATE COUNT*

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

measuring changes during water treatment and distribution or

in swimming pools. 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 the developing colonies. Choose the

procedure and medium that comply with the application of the

information. To compare data, use the same procedure and

medium. Four different methods and five different media are

described.

2. Selection of Method

a. Pour plate method: The pour plate method (9215B) is

simple to perform and can accommodate volumes of sample or

diluted sample ranging from 0.1 to 2.0 mL. The colonies produced are relatively small and compact, showing less tendency to

encroach on each other than those produced by surface growth.

On the other hand, submerged colonies often are slower growing

and are difficult to transfer. A thermostatically controlled water

bath is essential for tempering the agar. Heat shock to bacteria

from the transient exposure of the sample to 45 to 46°C agar may

occur.

b. Spread plate method: The spread plate method (9215C)

causes no heat shock and all colonies are on the agar surface

where they can be distinguished readily from particles and

bubbles. Colonies can be transferred quickly, and colony

morphology easily can be discerned and compared to published descriptions. However, this method is limited by the

small volume of sample or diluted sample that can be absorbed by the agar: 0.1 to 0.5 mL, depending on the degree to

which the prepoured plates have been dried. To use this

procedure, maintain a supply of suitable predried, absorbent

agar plates.

c. Membrane filter method: The membrane filter method

(9215D) permits testing of large volumes of low-turbidity

water and is the method of choice for low-count waters (1 to

10 CFU/mL). This method produces no heat shock but adds the

expense of the membrane filter. Further disadvantages include

the smaller display area, the need to detect colonies by reflected

light against a white background if colored filters or contrast

stains are not used, possible damage to cells by excessive filtration pressures, and possible variations in membrane filter quality

(see Section 9020B.4k).

d. Enzyme substrate method for heterotrophic bacteria: The

enzyme substrate method (9215E) can be used with samples

having a wide range of bacterial concentrations. The method†

uses a substrate-based medium in which the substrates are

hydrolyzed by microbial enzymes causing the release of

4-methylumbelliferone maximally after 48 h of incubation at

35°C. 4-Methylumbelliferone fluoresces when exposed to

long-wavelength (365-nm) ultraviolet light. The number of

fluorescing wells corresponds to a most probable number

(MPN) of bacteria in the original sample. The test may be

used for the analysis of drinking water and source water

samples. Individual colonies cannot be directly recovered for

subsequent analysis. This method produces no heat shock to

the bacteria in the sample. The enzyme substrate test is

comparable to the pour plate method.

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 table and bench tops having nonporous surfaces and

disinfect before any analysis is made.

4. Samples

Collect water as directed in Section 9060A. Initiate analysis as

soon as possible after collection to minimize changes in bacterial

population. The recommended maximum elapsed time between

collection and analysis of sample is 8 h (maximum transit time

6 h, maximum processing time 2 h). When analysis cannot begin

within 8 h, maintain sample at a temperature below 4°C but do

not freeze. Maximum elapsed time between collection and analysis must not exceed 24 h.

5. Sample Preparation

Mark each plate with sample number, dilution, date, and any

other necessary information before examination. Prepare at least

two replicate plates for each volume of sample or dilution

examined. For the pour or spread plate methods use sterile glass

(65 cm2

) or presterilized disposable plastic (57 cm2) petri dishes.

Thoroughly mix all samples or dilutions by rapidly making

about 25 complete up-and-down (or back-and-forth) movements.

Optionally, use a mechanical shaker to shake samples or dilutions for 15 s.

6. Media

Compare new lots of media with current lot in use according

to Section 9020B.5j.

* Approved by Standard Methods Committee, 2004.

Joint Task Group: R. Wayne Jackson (chair), Gil Dichter, Stephen C. Edberg, Ellen

P. Flanagan, Mark W. LeChevallier, Donald J. Reasoner, Peggy A. Roefer. † SimPlate® for HPC, Idexx Laboratories, Westbrook, ME.

1
a. Plate count agar (tryptone glucose yeast agar): Use for

pour and spread plate methods. This high-nutrient agar, widely

used in the past, may give lower counts than R2A or NWRI agar.

Tryptone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.0 g

Yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g

Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.0 g

Agar ............................................ 15.0 g

Reagent-grade water ................................1 L

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

b. m-HPC agar:‡ Use this high-nutrient medium only for the

membrane filter method

Peptone ........................................ 20.0 g

Gelatin ........................................ 25.0 g

Glycerol ....................................... 10.0 mL

Agar .......................................... 15.0 g

Reagent-grade water...............................1 L

For medium produced from basic ingredients, adjust to pH 7.2

with 1N NaOH. Heat slowly to dissolve thoroughly, add glycerol

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 7.1 0.2.

c. R2A agar: Use for pour plate, spread plate, and membrane

filter methods. This low-nutrient agar gives higher counts than

high-nutrient formulations.

Yeast extract ..................................... .0.5 g

Proteose peptone No. 3 or polypeptone . . . . . . . . . . . . . . . . 0.5 g

Casamino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g

Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g

Soluble starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g

Dipotassium hydrogen phosphate, K2HPO4. . . . . . . . . . . . . . . . . 0.3 g

Magnesium sulfate heptahydrate, MgSO4  7H2O....... 0.05 g

Sodium pyruvate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 g

Agar ........................................... 15.0 g

Reagent-grade water ...............................1 L

For medium produced from basic ingredients, adjust to pH 7.2

with solid K2HPO4 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. It is not

currently available in dehydrated form and requires preparation

from the basic ingredients; this makes its usage less desirable.

Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.0 g

Soluble casein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g

K2HPO4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g

MgSO4 ..................................................... 0.05 g

FeCl3 ....................................................... 0.001 g

Agar .......................................... 15.0 g

Reagent-grade water ..............................1 L

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

e. Enzyme substrate medium: See 9215E.3.

7. Incubation

For compliance monitoring purposes under U.S. EPA’s Surface Water Treatment Rule (40 CFR 141.74) provision on heterotrophic bacteria, incubate pour plates at 35°C for 48 h. Otherwise, select from among recommended times and temperatures

for monitoring changes in water quality. The highest counts

typically will be obtained from 5- to 7-d incubation at a temperature of 20 to 28°C.

During incubation, maintain humidity within the incubator

so the agar plates will have no moisture weight loss greater

than 15%. This is especially important if prolonged incubation is used. A pan of water placed at the bottom of the

incubator may be sufficient, but note that to prevent rusting or

oxidation of the incubator, the inside walls and shelving

should be of high-grade stainless steel or anodized aluminum.

For long incubation in nonhumidified incubators, seal plates

in plastic bags.

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 at 5 to 10°C for no more than 24 h, but

avoid this as routine practice. Record results of sterility controls

on the report for each lot of samples.

For compliance samples, count colonies manually using a

dark-field colony counter, such as a Quebec colony counter. If

such equipment is not available, for samples not for compliance

purposes, use other equipment, provided such equipment gives

equivalent magnification. Automatic plate counting instruments

are available. These generally use a television scanner coupled to

a magnifying lens and an electronics package. Their use is

acceptable if evaluation in parallel with manual counting gives

comparable results.

In preparing plates, pipet sample volumes that will yield from 30

to 300 colonies/plate. The aim is to have at least one dilution giving

colony counts between these limits, except as provided below.

Ordinarily, do not pipet more than 2.0 mL of sample; however,

when the total number of colonies developing from 2.0 mL is

less than 30, disregard this rule and record result observed. With

this exception, consider only plates having 30 to 300 colonies in

determining the plate count. Compute bacterial count per milliliter by the following equation:

CFU/mL  colonies counted

actual volume of sample plated, mL

If there is no plate with 30 to 300 colonies, and one or more

plates have more than 300 colonies, use the plate(s) having a

count nearest 300 colonies. Compute the count as above and

report as estimated CFU per milliliter.

If plates from all dilutions of any sample have no colonies,

report the count as less than one (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

less than 100 (100) CFU/mL.

If the number of colonies per plate exceeds 300, do not report

‡ Formerly called m-SPC agar. results as “too numerous to count” (TNTC). If there are fewer

HETEROTROPHIC PLATE COUNT (9215)/Introduction

2
than 10 colonies/cm2

, count colonies in 13 squares (of the

colony counter) having 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. (NOTE: The nominal diameter

of both disposable and nondisposable glass plates is 100 mm.

However, the internal diameter of disposable plates is nearer

to 85 mm and that of nondisposable plates is near to 90 mm.)

When bacterial counts on crowded plates are greater than 100

colonies/cm2

, report results as greater than () 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 colony-forming units per

milliliter.

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 because of an accumulation of

moisture 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 as individual colonies similar-appearing colonies growing in close proximity but not touching, provided that the distance between them is at least equal to the diameter of the

smallest colony. Count impinging colonies that differ in appearance, such as morphology or color, as individual colonies.

If plates have excessive spreader growth, report as “spreaders”

(Spr.). When plates are uncountable because of missed dilution,

accidental dropping, and contamination, or 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 filter

using a stereoscopic microscope at 10 to 15 magnification.

Preferably slant petri dish at a 45° angle on microscope stage and

adjust light source vertical to the colonies. Optimal colony

density per filter is 20 to 200. If colonies are small and there is

no crowding, 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 obtain an average count per square. For 10 to 20

colonies per square count 5 squares and obtain an average

count per square. Multiply average count per square by 100

and divide by the sample volume to give colonies per 100 mL.

If there are more than 20 colonies per square, record the count

as 2000 divided by the sample volume. Report averaged

counts as estimated colony-forming units. Make estimated

counts only when there are discrete, separated colonies without spreaders.

c. Enzyme substrate method: See 9215E.6.

9. Computing and Reporting Counts

The term “colony-forming unit(s)” (CFU) is descriptive of the

methods used; therefore, report all counts as colony-forming

units. Include in the 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 the

total number of colonies or average number (if duplicate plates

of the same dilution) per plate by the sample volume. For the

enzyme substrate method, use the MPN obtained from the MPN

tables, adjusted for sample 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 colony-forming units.

Avoid creating fictitious precision and accuracy when computing colony-forming units by recording only the first two

left-hand digits. Raise the second digit to the next higher number

when the third digit from the left is 5, 6, 7, 8, or 9; use zeros for

each successive digit toward the right from the second digit. For

example, report a count of 142 as 140 and a count of 155 as 160,

but report a count of 35 as 35.

10. Analytical Bias

Avoid inaccuracies in counting 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 the counts of other

analysts within 10% should discover the cause and correct

such disagreements.

HETEROTROPHIC PLATE COUNT (9215)/Introduction

3
9215 B. Pour Plate Method

1. Samples and Sample Preparation

See 9215A.4 and 5.

2. Sample Dilution

Prepare water used for dilution blanks as directed in Section

9050C.

a. Selecting dilutions: If possible (e.g., on the basis of historical information), select the dilutions so at least one plate in a

series will contain 30 to 300 CFU (Figure 9215:1). For example,

where a heterotrophic plate count as high as 3000 is suspected,

prepare plates with 102 dilution.

For most potable water samples, plates suitable for counting

will be obtained by plating 1 mL and 0.1 mL undiluted sample

and 1 mL of the 102 dilution.

b. Measuring sample portions: Use a sterile pipet for initial

and subsequent transfers from each container. If pipet becomes

contaminated before transfers are completed, replace with a

sterile pipet. Use a separate pipet for transfers from each different dilution. Do not prepare dilutions and pour plates in direct

sunlight. Use caution when removing sterile pipets from the

container; to avoid contamination, do not drag pipet tip across

exposed ends of pipets in the pipet container or across lips and

necks of dilution bottles. When removing sample, do not insert

pipets more than 2 to 3 cm below the surface of the same or

dilution.

c. Measuring dilutions: When discharging sample portions,

hold pipet at an angle of about 45° with tip touching bottom of

petri dish or inside neck of dilution bottle. Lift cover of petri dish

just high enough to insert pipet. Allow 2 to 4 s for liquid to drain

from 1-mL graduation mark to tip of pipet. If pipet is not a

blow-out type, touch tip of pipet once against a dry spot on petri

dish bottom. Less preferably, use a cotton-plugged blow-out type

pipet and a pipet bulb and gently blow out remaining volume of

sample dilution. When 0.1-mL quantities are measured, let diluted sample drain from chosen reference graduation until

0.1 mL has been delivered. Remove pipet without touching it to

dish. Pipet 1 mL, 0.1 mL, or other suitable volume into a sterile

petri dish before adding melted, tempered culture medium. Use

decimal dilutions in preparing sample volumes of less than

0.1 mL. In examining sewage or turbid water, do not measure a

0.1-mL inoculum of original sample, but prepare an appropriate

dilution. Prepare at least two replicate plates for each volume of

sample or dilution examined. After depositing test portions for

each series of plates, pour culture medium and mix carefully. Do

not let more than 20 min elapse between starting pipetting and

pouring plates.

3. Plating

a. Melting medium: Melt sterile solid agar medium in boiling

water or by exposure to flowing steam in a partially closed

container, but avoid prolonged exposure to unnecessarily high

temperatures during and after melting. Do not resterilize plating

medium. If the medium is melted in two or more batches, use all

of each batch in order of melting, provided that the contents

remain fully melted. Discard melted agar that contains precipitate.

Maintain melted medium in a water bath between 44 and 46°C

until used, preferably no longer than 3 h. In a separate container

place a thermometer in water or medium that has been exposed

to the same heating and cooling as the plating medium. Do not

depend on the sense of touch to indicate proper medium temperature when pouring agar.

Use plate count agar, R2A agar, or NWRI agar as specified in

9215A.6.

Figure 9215:1. Preparation of dilutions

HETEROTROPHIC PLATE COUNT (9215)/Pour Plate Method

4
b. Pouring plates: Limit the number of samples to be plated in

any one series so no more than 20 min (preferably 10 min) elapse

between dilution of the first sample and pouring of the last plate

in the series. Pour at least 10 to 12 mL liquefied medium

maintained at 44 to 46°C in each dish by gently lifting cover just

high enough to pour. Carefully avoid spilling medium on outside

of dish lid when pouring. When pouring agar from flasks or tubes

that have been held in a water bath, wipe with clean paper towel

and flame before pouring. As each plate is poured mix melted

medium thoroughly with test portions in petri dish, taking care

not to splash mixture over the edge, by rotating the dish first in

one direction and then in the opposite direction, or by rotating

and tilting. Let plates solidify (within 10 min) on a level surface.

After medium solidifies, invert plates and place in incubator.

c. Sterility controls: Check sterility of medium and dilution

water blanks by pouring control plates for each series of samples.

Prepare additional controls to determine contamination of plates,

pipets, and room air.

4. Incubation

See 9215A.7.

5. Counting, Recording, Computing, and Reporting

See 9215A.8 and 9.

6. Bibliography

BREED, R.S. & W.D. DOTTERER. 1916. The number of colonies allowable

on satisfactory agar plates. Tech. Bull. 53, New York Agricultural

Experimental Sta.

BUTTERFIELD, C.T. 1933. The selection of a dilution water for bacteriological examinations. J. Bacteriol. 23:355; Pub. Health Rep.

48:681.

ARCHAMBAULT, J., J. CUROT & M.H. MCCRADY. 1937. The need of

uniformity of conditions for counting plates (with suggestions for a

standard colony counter). Amer. J. Pub. Health 27:809.

RICHARDS, O.W. & P.C. HEIJN. 1945. An improved dark-field Quebec

colony counter. J. Milk Technol. 8:253.

BERRY, J.M., D.A. MCNEILL & L.D. WITTER. 1969. Effect of delays in

pour plating on bacterial counts. J. Dairy Sci. 52:1456.

GELDREICH, E.E., H.D. NASH, D.J. REASONER & R.H. TAYLOR. 1972. The

necessity of controlling bacterial populations in potable waters:

Community water supply. J. Amer. Water Works Assoc. 64:596.

GELDREICH, E.E. 1973. Is the total count necessary? Proc. 1st Annu.

AWWA Water Quality Technol. Conf., Dec. 3-4, 1973, Cincinnati,

Ohio, p. VII-1. American Water Works Assoc., Denver, Colo.

GINSBURG, W. 1973. Improved total count techniques. Proc. 1st Annu.

AWWA Water Quality Technol. Conf., Dec. 3-4, 1973, Cincinnati,

Ohio, p. VIII-1. American Water Works Assoc., Denver, Colo.

DUTKA, B.J., A.S.Y. CHAU & J. COBURN. 1974. Relationship of heterotrophic bacterial indicators of water pollution and fecal sterols.

Water Res. 8:1047.

KLEIN, D.A. & S. WU. 1974. Stress: a factor to be considered in

heterotrophic microorganism enumeration from aquatic environments. Appl. Microbiol. 37:429.

GELDREICH, E.E., H.D. NASH, D.J. REASONER & R.H. TAYLOR. 1975. The

necessity for controlling bacterial populations in potable waters:

Bottled water and emergency water supplies. J. Amer. Water Works

Assoc. 67:117.

BELL, C.R., M.A. HOLDER-FRANKLIN & M. FRANKLIN. 1980. Heterotrophic bacteria in two Canadian rivers. —I. Seasonal variation in

the predominant bacterial populations. Water Res. 14:449.

MEANS, E.G., L. HANAMI, G.F. RIDGEWAY & B.H. OLSON. 1981. Evaluating mediums and plating techniques for enumerating bacteria in

water distribution systems. J. Amer. Water Works Assoc. 73:585.

REASONER, D.J. & E.E. GELDREICH. 1985. A new medium for the enumeration and subculture of bacteria from potable water. Appl.

Environ. Microbiol. 49:1.

AMERICAN PUBLIC HEALTH ASSOCIATION. 1993. Standard Methods for the

Examination of Dairy Products, 16th ed. American Public Health

Assoc., Washington, D.C.

9215 C. Spread Plate Method

1. Samples and Sample Preparations

See Sections 9215A.4 and 5.

2. Laboratory Apparatus

a. Glass rods: Bend 4-mm-diam, fire-polished glass rods, 200

mm in length, 45° about 40 mm from one end. Sterilize before

using.

b. Pipet: 1.0-mL glass or plastic pipets.

c. Turntable (optional).*

d. Incubator or drying oven, set at 42°C, or laminar-flow hood.

3. Media

See 9215A.6a, c, and d. If R2A agar is used, best results are

obtained at 28°C with 5 to 7 d incubation; if NWRI agar is used,

incubate at 20°C for 7 d.

4. Preparation of Plates

Pour 15 mL of the desired medium into sterile 100- 

15-mm or 90-  15-mm petri dishes; let agar solidify. Predry

plates inverted so there is a 2- to 3-g water loss overnight with

lids on. See Figure 9215:2, Table 9215:I, or Figure 9215:3.

Use predried plates immediately after drying or store for 2

weeks in sealed plastic bags at 4°C. For predrying and using

plates the same day, pour 25 mL agar into petri dish and dry

in a laminar-flow hood at room temperature (24 to 26°C) with

the lid off to obtain the desired 2- to 3-g weight loss. See

Figure 9215:3. * Fisher Scientific, hand operated, No. 08-758 or Lab-Line, motor driven,

No. 1580, or equivalent.

HETEROTROPHIC PLATE COUNT (9215)/Spread Plate Method

5
5. Procedure

Prepare sample dilutions as directed in 9215B.2.

a. Glass rod: Pipet 0.1 or 0.5 mL sample onto surface of a

predried agar plate. Using a sterile bent glass rod, distribute

inoculum over surface of the medium by rotating plate by hand

or on a turntable. Let inoculum be absorbed completely into the

medium before incubating.

b. Pipet: Pipet desired sample volume (0.1, 0.5 mL) onto the

surface of the predried agar plate while plate is being rotated on

a turntable. Slowly release sample from pipet while making one

to-and-fro motion, starting at center of plate and stopping 0.5 cm

from plate edge before returning to center. Lightly touch the

pipet to plate surface. Let inoculum absorb completely into

medium before inverting and incubating.

6. Incubation

See 9215A.7.

7. Counting, Recording, Computing, and Reporting

See 9215A.8 and 9.

8. Bibliography

BUCK, J.D. & R.C. CLEVERDON. 1960. The spread plate as a method for

the enumeration of marine bacteria. Limnol. Oceanogr. 5:78.

CLARK, D.S. 1967. Comparison of pour and surface plate methods for

determination of bacterial counts. Can. J. Microbiol. 13:1409.

VAN SOESTBERGAN, A.A. & C.H. LEE. 1971. Pour plates or streak plates.

Appl. Microbiol. 18:1092.

CLARK, D.S. 1971. Studies on the surface plate method of counting

bacteria. Can. J. Microbiol. 17:943.

GILCHRIST, J.E., J.E. CAMPBELL, C.B. DONNELLY, J.T. PELLER & J.M.

DELANEY. 1973. Spiral plate method for bacterial determination.

Appl. Microbiol. 25:244.

PTAK, D.M. & W. GINSBURG. 1976. Pour plate vs. streak plate method.

Proc. 4th Annu. AWWA Water Quality Technology Conf., Dec.

Figure 9215:2. Drying weight loss of 15-mL agar plates stored separately, inverted with lids on. SOURCE: Unpublished data. Water Purification Lab., Chicago

Dep. Water.

TABLE 9215:I. EFFECT OF TEMPERATURE OF DRYING ON WEIGHT LOSS OF 15-ML AGAR PLATES STORED SEPARATELY*

Time for Plates to Lose 1 to 4 g of Water (Avg. for 5 Plates)

h

Temp. Plates Inverted with Lids on Plates Inverted with Lids Removed

°C 1g 2g 3g 4g 1g 2g 3g 4g

24 32 64 95 125 3.7 7.0 10.5 14.0

37 17 35 51 67 1.7 3.5 5.3 7.0

50 6 12 18 24 0.7 1.3 1.9 2.7

60 4 8 12 16 — — — —

* Referenced in Canada Centre for Inland Waters Manual, Burlington, Ont.

HETEROTROPHIC PLATE COUNT (9215)/Spread Plate Method

6
6-7, 1976, San Diego, Cal., p. 2B-5. American Water Works Assoc., Denver. Colo.

DUTKA, B.J., ed. 1978. Methods for Microbiological Analysis of

Waters, Wastewaters and Sediments. Inland Water Directorate,

Scientific Operation Div., Canada Centre for Inland Waters,

Burlington, Ont.

KAPER, J.B., A.L. MILLS & R.R. COLWELL. 1978. Evaluation of the accuracy and

precision of enumerating aerobic heterotrophs in water samples by the

spread plate method. Appl. Environ. Microbiol. 35:756.

YOUNG, M. 1979. A modified spread plate technique for the determination of concentrations of viable heterotrophic bacteria. STP

673:41-51, American Soc. Testing & Materials, Philadelphia, Pa.

GELDREICH. E.E. 1981. Current status of microbiological water quality

criteria. ASM News 47:23.

TAYLOR, R.H., M.J. ALLEN & E.E. GELDREICH. 1981. Standard plate count: A

comparison of pour plate and spread plate methods. Proc. 9th Annu.

AWWA Water Quality Technol. Conf., Dec. 6-9, 1981, Seattle, Wash.,

p. 223, American Water Works Assoc., Denver, Colo.

9215 D. Membrane Filter Method

1. Samples and Sample Preparation

See 9215A.4 and 5.

2. Laboratory Apparatus

See Section 9222B.1.

3. Media

See 9215A.6. Use m-HPC agar or, alternatively, R2A or

NWRI agar.

4. Preparation of Plates

Dispense 5-mL portions of sterile medium* into 50-  9-mm

petri dishes. Let solidify at room temperature. Prepared plates

may be stored inverted in a plastic box or tight container in a

refrigerator for no longer than 2 weeks.

* m-HPC agar may not be sterile.

Figure 9215:3. Weight loss of 25-mL agar plates (100  15 mm) dried separately in a laminar-flow hood at room temperature (24 to 26°C), relative

humidity (30 to 33%), and air velocity 0.6 m/s. SOURCE: Unpublished data. Alberta Environmental Centre, Vegreville, Alta.

HETEROTROPHIC PLATE COUNT (9215)/Membrane Filter Method

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5. Sample Size

The volume to be filtered will vary with the sample. Select a

maximum sample size to give 20 to 200 CFU per filter.

6. Procedure

Filter appropriate volume through a sterile 47-mm, 0.45-m-porediam, gridded membrane filter, under partial vacuum. Rinse funnel

with three 20- to 30-mL portions of sterile dilution water. Place filter on

agar in petri dish.

7. Incubation

Place dishes in close-fitting box or plastic bag containing moistened

paper towels. Incubate at 35 0.5°C for 48 h if using m-HPC agar, or

longer if using R2A medium, or at 20 to 28°C for 5 to 7 d if using

NWRI or R2A agar. Duplicate plates may be incubated for other time

and temperature conditions as desired.

8. Counting, Recording, Computing, and Reporting

See 9215A.8 and 9. Report as CFU/mL, membrane filter

method, time, and medium.

9. Bibliography

CLARK, H.F., E.E. GELDREICH, H.L. JETER & P.W. KABLER. 1951. The

membrane filter in sanitary bacteriology. Pub. Health Rep. 66:951.

STOPERT, E.M., W.T. SOKOSKI & J.T. NORTHAM. 1962. The factor of

temperature in the better recovery of bacteria from water by filtration. Can. J. Microbiol. 8:809.

TAYLOR, R.H. & E.E. GELDREICH. 1979. A new membrane filter procedure for bacterial counts in potable water and swimming pool

samples. J. Amer. Water Works Assoc. 71:402.

CLARK, J.A. 1980. The influence of increasing numbers of non-indicator organisms upon the detection of indicator organisms by the membrane filter and

presence-absence tests. Can. J. Microbiol. 20:827.

DUTKA, B.J., ed. 1981. Membrane Filtration, Applications, Techniques, and

Problems. Marcel Dekker, Inc., New York, N.Y. and Basel, Switzerland.

HOADLEY, A.W. 1981. Effect of injury on the recovery of bacteria on

membrane filters. In B.J. Dutka, ed. 1981. Membrane Filtration,

Applications, Techniques, and Problems, p. 413. Marcel Dekker,

Inc., New York, N.Y. and Basel, Switzerland.

9215 E. Enzyme Substrate Method

1. Samples and Sample Preparation

See Section 9215A.4 and 5.

2. Laboratory Apparatus

a. Pipets, 0.1-, 1.0-, and 10-mL, sterile, graduated, glass or

plastic.

b. Incubator set at 35 0.5°C.

c. Ultraviolet light source, long wavelength, 6-W, 365 nm.

d. Sample plates.*

3. Medium

The formulation is available commercially† in sterile medium vessels for 100-mL multi-dose procedures or is available in test tubes for

10-mL unit-dose procedures. Store medium between 2 and 25°C. The

product has a shelf life of up to 12 months from date of manufacture.

Test each lot for sterility by following the inoculation procedure (¶ 5

below), using 10 mL rehydrated medium without sample. Incubate for

48 h at 35°C. No wells should fluoresce after incubation.

4. Sample Diluent

Use either sterile deionized water, distilled water, buffered

water, or 0.1% peptone.

5. Procedure

Rehydrate medium by filling medium vessel to 100-mL mark with

sterile diluent, re-capping, and shaking until medium has dissolved.

Aseptically pipet 1.0 mL sample and 9 mL of rehydrated medium onto

the center of the sample plate. Alternatively, aseptically pipet 0.1 mL

sample and then 9.9 mL rehydrated medium onto center of the sample

plate. NOTE: Final volume of sample plus medium must be 10 

0.2 mL. Cover plate with lid and gently swirl to distribute mixture into

wells. Tip plate 90° to 120° to drain excess mixture into the absorbent

pad. Invert plate and incubate for 48 h (range of 45 to 72 h) at 35°C. If

a sample is suspected of having a count greater than 738 CFU/mL,

dilute by adding 1 mL sample to a sterile vessel containing 99 mL

sterile diluent. Make additional dilutions as required to keep counts

below 738 CFU/mL in final dilution.

6. Counting, Recording, Computing, and Reporting

After incubation, examine plate for fluorescent wells. Count number

of fluorescent wells by holding a 6-W, 365-nm UV light about 13 cm

above plate. Preferably use UV-filtering laboratory safety glasses during counting. Count number of wells exhibiting blue fluorescence.

Use MPN chart (provided by the medium 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.

7. Bibliography

STILLINGS, A., D. HERZOG & B. ROLL. 1998. Comparative assessment of the

newly developed SimPlate® method with the existing EPA-approved pour

plate method for the detection of heterotrophic plate count bacteria in

ozone-treated drinking water. Presented at International Ozone Assoc.

Conf., October 19-23, 1998, Vancouver, B.C., Canada.

JACKSON, R.W., K. OSBORNE, G. BARNES, C. JOLLIFF. D. ZAMANI, B. ROLL,

A. STILLINGS, D. HERZOG, S. CANNON & S. LOVELAND. 2000. Multiregional evaluation of the SimPlate® heterotrophic plate count

method compared to the standard plate count agar pour plate

method in water. Appl. Environ. Microbiol. 66:453.

* SimPlates®, IDEXX Laboratories, Westbrook, ME, or equivalent.

† IDEXX Laboratories, Westbrook, ME.

HETEROTROPHIC PLATE COUNT (9215)/Enzyme Substrate Method

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