Bactiquant Tec paper Legionella by Pemann

Technical paper 01-032016 Morten Miller, PhD., and Jan Nielsen, MSc., BactiQuant Page 1:4

Plate counts and BactiQuant HETEROTROPHIC BACTERIA: Heterotrophic bacteria are broadly defined as bacte-

HPC TEST VARIABLES SPREAD PLATING:

ria that require organic carbon for growth.

The advantage of the spread plating technique is that

These bacteria are traditionally quantified using het-

the bacterial colonies rapidly become visible. They

erotrophic plate counts (HPC).

grow on the surface and not into the agar. The disadvantage is that it selects for motile and rapid growing

HPC METHODS:

bacteria.

There is no universal “HPC measurement.” Although

Another drawback is that the spread plating is not

standardized methods have been formalized, HPC

compatible with long incubation times as it becomes

test methods involve a wide variety of test conditions

difficult to differentiate between individual colonies

that lead to a wide range of quantitative and qualita-

after more than three days of incubation.

tive results.

The consequence is that slow growing bacteria are

Temperatures employed typically range from around

not detected due to substrate competition, inhibition

20°C to 40°C, incubation times from a few hours to

and over growth.

seven days or a few weeks, and nutrient conditions from low to high.

POUR PLATING:

The HPC methods do not indicate the specific het-

The advantage of the pour plating technique is that

erotrophic bacteria present or their sources. Instead,

colony interaction is significantly reduced.

HPC testing indicates the culturable organisms pre-

This makes it possible to differentiate colonies even

sent, which could be as low as

after long incubations times (2-3 weeks).

0,1-1% of the total bacteria present in a water sample.

The pour plate technique allows for detection of slow

The result will differ significantly according to which

growing bacteria as well as rapid growing bacteria.

method is used.

The pour plate technique in combination with longer

The actual organisms recovered in HPC testing can

incubation times provides a more true representation

also vary widely between locations, between seasons

of the bacterial presence in water samples as com-

and between consecutive samples at a single loca-

pared to spread plating.

tion (WHO, Heterotrophic Plate Counts and Drinking

The disadvantage is that it is more elaborate and

Water Safety, Editors; J. Bartram et al., 2003)

time consuming protocol.

THE CONSEQUENCE IS THAT:

PLATE COUNT AGAR (PCA):

1 Plate counts are not a quantitative measure of total

Contains high concentrations of yeast extract and

bacteria in a water sample 2 Results on identical samples using different HPC methods are often not correlated 3 The HPC method measures the presence of bacteria that can grow under a specific set of growth

tryptone or peptone. The colonies will often become very big due to the high substrate load. Small colonies will rapidly become overgrown by larger colonies which make it difficult to count the smaller colonies.

conditions; temperature, incubation time, pH, substrate media etc.

BactiQuant ®water - Heterotrophic Plate Counts (HPC); Recommendations for protocols when comparing with BactiQuant ®-water analysis results Copyright © BactiQuant, March 2016, first edition, version 2016.3 • www.bactiquant.com

Technical paper 01-032016 Morten Miller, PhD., and Jan Nielsen, MSc., BactiQuant Page 2:4

Plate counts and BactiQuant R2A AGAR: Contains low concentrations of yeast extract and

PLATE COUNTS IN CHLORINATED (SODIUM HYPOCHLORITE) WATER:

tryptone or peptone as well as pyruvate.

The efficiency of inactivation of bacteria by chlorine

The colonies will be relatively small and will not over

is affected by a number of factors including;

grow other colonies. The pyruvate ensures a better

pH, contact time, reactions of the chlorine with in-

survival for stressed/damaged cells.

organic and organic constituents in the water, oc-

INCUBATION TIME: The most common incubation time for HPC methods is in the range 48 hours to 72 hours. The incubation time is closely related to the number of plate counts

currence of particles and bioflims that can protect bacteria from the biocidal effects of chlorine and concentration of chlorine. Also it is important to note that the disinfection action of chlorine is not instan-

observed. By prolonging incubation time from 72

taneous. Exposure time is required to kill bacteria and

hours to 7 days, plate counts will typically, increase

the time for different bacteria to become inactivated

by a factor of 5 to 100 times. Many bacteria in water

varies widely.

samples are slow growing and require longer incubation times to produce visual colonies. PLATE COUNTS AND PARTICLE ASSOCIATED BACTERIA (PAB): It is well known that bacteria in water predominantly occur in flakes and in conglomerates of varying con-

Plate counts are often used to document the biocidal effect of chlorine on heterotrophic bacteria in water samples. In most cases incubation times of 48 hours are used. However, caution has to be exercised when using plate counts for verification of chlorine treat-

sistency. The specific physical characteristics vary

ments. This is readily observed in data from chlorina-

with water type and location.

tion of water sampled from a medical equipment in a

The particles may be bound together by organic or

health care facility in Germany

inorganic constituents, including filamentous- and

(Table 1, Appendix).

slime producing bacteria.

The plate counts throughout the observation pe-

Also a significant proportion of the total bacteria in

riod -5 to 26 days (commencement of chlorination)

a water sample can be particle associated bacteria

showed zero plate counts after 48 hours of incuba-

(PAB). In the laboratory the occurrence of PAB and

tion. A prolongation of the incubation period to 7 days

bacteria in various conglomerates, is a significant

showed high plate counts up to above > 100.000

consideration, because one colony forming unit (CFU) can originate from a large flake, debris, as well as individual organisms. It is thus not possible to distinguish between flakes and debris, which may harbor

CFU/ml after 7 days. In this case a 48 hour incubation did not give a true representation of chlorination efficiency.

a high number of bacteria, and individual organisms. The consequence is a gross underestimation of bacteria associated with various conglomerates and particles when using plate count methods.

Technical paper 01-032016 Morten Miller, PhD., and Jan Nielsen, MSc., BactiQuant Page 3:4

Plate counts and BactiQuant BACTIQUANT RECOMMENDS:

For comparing BactiQuant analysis results with

When comparing BactiQuant analysis results and

plate counts on monocultures of bacteria, it is impor-

plate counts to demonstrate a correlation with indig-

tant to take into consideration, that some culture me-

enous planktonic bacteria BactiQuant recommends

dia can interact with the BactiQuant analysis.

using the standard method DS/EN ISO 6222 with the following modifications:

BactiQuant recommends the use of the following cul-

tivation media:

Use the pour plate method

2. Use R2A agar when sampling potable water

Bacteriological peptone from the company

3. Apply incubations times of minimum 7 day

Oxoid: Max. 500 mg/l

4. Make a coarse filtration of the sample prior to plate count and BactiQuant analysis to elimi-

Glucose or fructose or sucrose or starch:

nate particle associated bacteria. Coffee filters,

Max. 500 mg/l

cheesecloth or commercially available filters with comparable pore sizes, 10-15 µm, can be

NaCl: Max. 9 g/l

employed. For pH adjustment use: NaOH or HCL. Do not use Figure 1 (appendix) shows data from plate count

H3PO4 based buffers. All other buffers can be used at

analyses of a non-chlorinated tap water sample in

max. 500 mg/l.

Denmark. Water samples were analyzed according to the recommendations above.

Technical paper 01-032016 Morten Miller, PhD., and Jan Nielsen, MSc., BactiQuant Page 4:4

APPENDIX Plate counts and BactiQuant Table 1. Total Viable Count (TVC), Tryptic Soy Agar, 22°C and 37°C, spread plating. Chlorination varied 0,8 – 2 mg/l. 22°C, CFU/ml Incubation days

Treatment

37°C, CFU/ml Incubation days

(Days)

-5 0 1 3 6 8 10 13 16 19 26

(Days) 7 > 100.000 0 3000 > 100.000 > 100.000 2460 3800 3200 400 320 4000

N.D. 0 0 0 0 0 7 0 0 M+ M+

7 N.D. 0 0 M+ 7 0 M+ 0 0 M+ M+

1580 460 1900 3000 2400 2720 7680 3200 160 320 3120

N.D.; no determination, M+; Micro-colonies observed – not counted.

Figure 4. CFU/ml and Bactiquant Value in tap water sample from Mycometer in Hoersholm, stored at 22 C.

Figure 1. CFU/ml and BactiQuant Value in tap water sample, stored at 22° C.

1.000.000

100.000

10.000

1.000 CFU/ml ( plates incubated 3 days)

1.000

100

CFU/ml ( plates incubated 7 days)

Bactiquant Value

10.000

CFU/ml

100.000

Bactiquant Value 100

Time (days)