ABOUT THE Heterotrophic Plate Count Test
The value of HPC testing
Back in 1883 Robert Koch published "About Detection Methods for Microorganisms in Water", a historic scientific paper that marked the introduction of microbial indicators for monitoring water hygiene. In his paper, Koch described HPC measurement for the first time and showed its value as a measure of water treatment performance.
Source : W.H.O. : Heterotrophic Plate Counts and Drinking-water Safety - The Significance of HPCs for Water Quality and Human Health
HPC TESTING AND OTHER ENUMERATION METHODS
ATP AND PROTEIN CONCETRATION TESTING
Some widely used devices estimate microorganism concentration by measuring ATP or protein concentration, both substances found in microorganisms. However with these techniques, because we are measuring the substance rather than the number of organisms, results may have only a loose correlation with the number of organisms present. This can be for two main reasons firstly, not all microorganisms contain the same amount of ATP or protein – 10 bacteria will give a very different result from 10 yeast – and secondly because ATP or protein present from other sources (not microbes) might be present in the sample and that can skew the result.
However, in general, this kind of test is accurate enough for some applications such as verifying surfaces after cleaning, and these tests are helpful sometimes because they give real-time results.
In samples of up to 0.1ml it is often easiest to enumerate organisms by spreading the sample over a large solid medium, such as a 90mm agar-filled petri-dish.
"nomad Testers are based on the culture method, the most established and well-documented microbial method"
A BIT OF HISTORY...
"You can go a long way with very simple tools"
- Only microorganisms that are able to grow appear on the culture.
Other techniques such as PCR do not easily differentiate between living and dead microorganisms, which is less indicative of the future population and therefore of the risk.
- Culture techniques have been widely used for over a century
For this reason, the majority of current reference methods are culture methods, having been grandfathered into modern quality assurance systems because they are well understood, in spite of their occasional weaknesses.
- They are sensitive
Even the most advanced instrumental technique requires a minimum number of molecules for analysis, and individual bacteria do not contain much of any molecule, so it can be very hard to record them at low concentrations without amplification. Conversely, culture methods can create a colony from a single individual – some microorganisms can multiple to generate 1 million individuals from a single cell in just 24 hours – rendering them visible to the naked eye! How about that for inexpensive simple and powerful amplification!
- Testing requirements are minimal
Nutrients, water and correct temperature for incubation are all readily available.
CONTAMINATION RANGE WHERE NOMAD IS SUITABLE
Counting colonies on a test-device is typically considered acceptably reliable between 1 and 300 cfu / tester. Above 300 cfu / tester, colonies interact too much with each other to count the number reliably (overlap, competition for nutrients and so on).
If your sample already falls within this range, perfect!
If your sample is more contaminated than 300cfu/ml, you may be able to dilute it simply in a buffer solution to reduce the concentration before testing. After you obtain results, multiply the results by the dilution factor to obtain the CFU/ml, although it is better if this can be avoided as it adds further possibility of sample contamination and handling errors.
If your sample contains less than 1 CFU per ml, you can concentrate it by filtering it through a 0.45µm membrane filter disk in a filtration funnel, followed by transferring the membrane (keeping it uncontaminated) onto culture media or onto micro-colony counting equipment.
For direct testing without dilution or concentration, the most practical method for different expected microorganisms is indicated on the chart below.
HPC TESTING MEDIA
nomad Testers make testing for total counts easy!
Total Counts are a good indicator of the performance of your processes. Sanitation and hygiene problems often result in increased microbial growth, which result in higher-than-usual Total Counts and may indicate the presence of a pathogen.
There are many Total Count media for enumerating oxygen-tolerant microorganisms including:
- Aerobic Mesophillic Count (AMC)
- Aerobic Colony Count (ACC)
- Standard Plate Count (SPC)
- Aerobic Plate Count (APC)
- Heterotrophic Plate Count (HPC)
- Comptage mésophilique aérobie (AMC)
All these media have similar characteristics when incubated at room temperature. For more information, see the comparative study between nomad HPC and a reference method using PCA-filled Petri-Dishes and the funnel filtration method.
Most microorganisms present in working environments are capable of growth at room temperature. When using Total Count media, incubations between 20 and 25˚C will essentially result in similar counts. If Total Count Testers are incubated at temperatures lower than 20˚C, check that counts are stable after day 3 (no new colonies are appearing). Once counts are stable they can be assumed to be similar to the counts on day 3, incubated at 20–25˚C
For additional information about HPC media, W.H.O: Heterotrophic Plate Counts and Drinking-water safety – The significance of HPCs for Water Quality and Human Health.
"In most cases, HPC Testers can be incubated at room temperature, with no need for special equipment"
SPECIATION AND POPULATIONS
Knowing how many microorganisms are present in a sample is an important data-point when running microbial surveillance.
Knowing what type of microorganisms are present gives us a complete picture:
- Among populations observed, are there any potentially troublesome ones?
- Is there a new population here, that I had not seen before?
- Where do these populations normally belong (what is their natural habitat)?
- Is this new population in sample A, one that I have already observed elsewhere, in which case I have a clue as to how it go here?
- Non-selective media, i.e. total count media provide all the nutrients necessary for the largest variety of populations to grow.
Example: HPC media contained in the red nomad Testers
- Selective media are used to allow only selected microorganisms to grow.
This media most frequently includes a component that is toxic for the microorganisms not relevant for the analysis.
Example: Yeast and Mould media contained in the yellow nomad Testers
- Differential media distinguish one microorganism type from another growing on the same medium.
This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients to visibly indicate the defining characteristics of a microorganism.
Example : Coliform media contained in the blue nomad.In this case, the media is also selective and so is the incubation temperature (35°C)
- Chromogenic media are culture medium used to isolate, identify and differentiate specific microorganisms from a diverse population. They contain a chromogenic substrate which is utilised by the microorganism to give a colour that is specific for a microorganism type and accurately differentiates it from others.
Example : CHROMagarTM media
"Our 3 nomad flavors cover many environmental, hygiene and process monitoring needs"
RECOGNIZING MICROORGANISMS ON HPC TESTERS
To obtain precise microorganism profiles, molecular identification can be used. As with human genetic profiling, DNA techniques can be used to compare 1 strain to another with extreme precision and identify the organism provided the type you are observing exists in the database you have available.
These techniques require appropriate facilities, specific and often expensive equipment and trained operators. They are available from laboratory service providers. Should you want to confirm the identity of a colony observed on a nomad Tester, your service provider will know what to do if you provide them with the Tester in question marked with the colony to identify.
"Get to know your common HPC populations. It will help you map microbial populations in your facility, and understand how they move around"
CULTURE METHODS vs RAPID METHODS
"Rapid can be good in some cases... but thorough methods are the right choice in most circumstances "