plate counts and bactiquant - mycometer · mycometer recommends: when comparing bactiquant analysis...

HETEROTROPHIC BACTERIA:

Heterotrophic bacteria are broadly defined as bacteriathat require organic carbon for growth. These bacteria are traditionally quantified usingheterotrophic plate counts (HPC).

HPC METHODS:

There is no universal “HPC measurement.”Althoughstandardized methods have been formalized, HPCtest methods involve a wide variety of test conditionsthat lead to a wide range of quantitative and q u a l i t a t i v e results.Temperatures employed typically range from around20°C to 40°C, incubation times from a few hours toseven days or a few weeks, and nutrient conditions from low to high. The HPC methods do not indicate the specific heterotrophic bacteria present or their sources.Instead, HPC testing indicates the culturable organisms present, which could be as low as 0,1 -1% of the total bacteria present in a water sample. The result will differ significantly according to whichmethod is used. The actual organisms recovered in HPC testing canalso vary widely between locations, between seasons and between consecutive samples at asingle location (WHO, Heterotrophic Plate Countsand Drinking Water Safety, Editors; J. Bartram etal., 2003)

THE CONSEQUENCE IS THAT:

1 Plate counts are not a quantitative measure oftotal bacteria in a water sample

2 Results on identical samples using different HPCmethods are often not correlated

3 The HPC method measures the presence ofbacteria that can grow under a specific set ofgrowth conditions; temperature, incubation time, pH, substrate media etc.

HPC TEST VARIABLES

SPREAD PLATING:

The advantage of the spread plating technique isthat the bacterial colonies rapidly become visible. They grow on the surface and not into the agar.The disadvantage is that it selects for motile andrapid growing bacteria. Another drawback is that the spread plating is notcompatible with long incubation times as it becomesdifficult to differentiate between individual coloniesafter more than three days of incubation. The consequence is that slow growing bacteria arenot detected due to substrate competition, inhibitionand over growth.

POUR PLATING:

The advantage of the pour plating technique is thatcolony interaction is significantly reduced. This makes it possible to differentiate colonies evenafter long incubations times (2-3 weeks). The pour plate technique allows for detection ofslow growing bacteria as well as rapid growing bac-teria. The pour plate technique in combination withlonger incubation times provides a more true repre-s e n t a t i o n of the bacterial presence in water samplesas compared to spread plating. The disadvantage is that it is more elaborate andtime consuming protocol.

PLATE COUNT AGAR (PCA):

Contains high concentrations of yeast extract andtryptone or peptone. The colonies will often becomevery big due to the high substrate load. Small colonies will rapidly become overgrown by larger colonies which make it difficult to count thesmaller colonies.

PLATE COUNTS AND BACTIQUANT

Bactiquant®water - Heterotrophic Plate Counts (HPC); Recommendations for protocols when comparing with Bactiquant®-water analysis resultsCopyright © Mycometer A/S, March 2016, first edition, version 2016.3 • www.mycometer.com

Technical paper 01-032016Morten Miller, PhD., and Jan Nielsen, MSc., Mycometer A/S:4

R2A AGAR:

Contains low concentrations of yeast extract and tryptone or peptone as well as pyruvate. The colonies will be relatively small and will not o v e r g r o w other colonies. The pyruvate ensures abetter survival for stressed/damaged cells.

INCUBATION TIME:

The most common incubation time for HPC methodsis in the range 48 hours to 72 hours. The incubationtime is closely related to the number of plate countsobserved. By prolonging incubation time from 72hours to 7 days, plate counts will typically, increaseby a factor of 5 to 100 times. Many bacteria inwater 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 predominantlyoccur in flakes and in conglomerates of varying consistency. The specific physical characteristicsvary with water type and location. The particles may be bound together by organic orinorganic constituents, including filamentous- andslime producing bacteria. Also a significant proportion of the total bacteria ina water sample can be particle associated bacteria(PAB). In the laboratory the occurrence of PAB andbacteria in various conglomerates, is a significantconsideration, because one colony forming unit(CFU) can originate from a large flake, debris, aswell as individual organisms. It is thus not possibleto distinguish between flakes and debris, whichmay harbor a high number of bacteria, and individual organisms.

The consequence is a gross underestimation ofbacteria associated with various conglomerates andparticles when using plate count methods.

PLATE COUNTS IN CHLORINATED

(SODIUM HYPOCHLORITE) WATER:

The efficiency of inactivation of bacteria by chlorineis affected by a number of factors including;pH, contact time, reactions of the chlorine with inorganic and organic constituents in the water,occurrence of particles and bioflims that can protectbacteria from the biocidal effects of chlorine and concentration of chlorine. Also it is important to notethat the disinfection action of chlorine is not instantaneous. Exposure time is required to kill bacteria and the time for different bacteria to become inactivated varies widely.

Plate counts are often used to document the biocidal effect of chlorine on heterotrophic bacteriain water samples. In most cases incubation times of48 hours are used. However, caution has to beexercised when using plate counts for verification ofchlorine treatments. This is readily observed in datafrom chlorination of water sampled from a medicalequipment in a health care facility in Germany(Table 1, Appendix). The plate counts throughout the observation period-5 to 26 days (commencement of chlorination) showed zero plate counts after 48 hours of incubation. A prolongation of the incubation periodto 7 days showed high plate counts up to above >100.000 CFU/ml after 7 days. In this case a 48 hour incubation did not give a truerepresentation of chlorination efficiency.



Technical paper 01-032016Morten Miller, PhD., and Jan Nielsen, MSc., Mycometer A/SPage 1:4

MYCOMETER RECOMMENDS:

When comparing Bactiquant analysis results andplate counts to demonstrate a correlation with indi-genous planktonic bacteria Mycometer recom-mends using the standard method DS/EN ISO 6222with the following modifications:

1. Use the pour plate method2. Use R2A agar when sampling potable water3. Apply incubations times of minimum 7 day4. Make a coarse filtration of the sample prior to

plate count and Bactiquant analysis to eliminateparticle associated bacteria. Coffee filters, cheesecloth or commercially available filterswith comparable pore sizes, 10-15 µm, can beemployed.

Figure 1 (appendix) shows data from plate countanalyses of a non-chlorinated tap water sample inDenmark. Water samples were analyzed accordingto the recommendations above.For comparing Bactiquant analysis results with

plate counts on monocultures of bacteria, it isimportant to take into consideration, that some culture media can interact with the Bactiquant analysis.

Mycometer recommends the use of the following cultivation media: Bacteriological peptone from the company Oxoid: Max. 500 mg/l

Glucose or fructose or sucrose or starch:Max. 500 mg/l

NaCl: Max. 9 g/l

For pH adjustment use: NaOH or HCL. Do not useH3PO4 based buffers. All other buffers can be usedat max. 500 mg/l.




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

APPENDIX PLATE COUNTS AND BACTIQUANT



Table 1. Total Viable Count (TVC), Tryptic Soy Agar, 22oC and 37oC, spread plating. Chlorination varied 0,8 – 2 mg/l.

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

plate counts and bactiquant - mycometer · mycometer recommends: when comparing bactiquant analysis...

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