ABOUT THE Heterotrophic Plate Count Test

nomad Testers use Heterotrophic plate count (HPC) testing, a way of estimating the number of bacteria in a given sample.

Discover why HPC testing is a powerful tool in understanding microbial and bacterial contamination in your industry.

In 1883 Robert Koch published an article entitled: About Detection Methods for Microorganisms in Water. In that historic paper that marked the introduction of the application of microbial indicators for surveillance of water hygiene, Koch described for the first time the methodology for HPC measurement in water, and showed its value as a measure of water treatment technology performance.

 

If you are not familiar with microbiology tools, in this section you will find background information on microbial enumeration techniques, Culture Media, ranges and detection, recognizing and identifying bacterial populations and when rapid methods are really useful.

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

In industry it is well understood that finished products are supposed to be free from microbial pathogens. Often this is checked with a release test, where a random sample of the product is taken and tested.

Most environmental organisms, even pathogens, do not represent a threat to product safety or processes unless their concentration is 'too high'. In some cases, there are industry guidelines and standards which define what 'too high' means in a given situation. In others, companies have internal guidelines which they create tailored to their specific process and product, and regular monitoring alerts them if the number of organisms increases which means they might have a problem.

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.


SMALL SAMPLES

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.


LARGER SAMPLES

For much larger sample volumes, 2 other enumeration techniques can be used:
- Most Probably Number (MPN) techniques and
- Membrane Filter (MF) techniques (the type used in nomad testers)

MPN is a technique used to estimate the number of microorganisms, which is particularly helpful when the sample cannot be filtered. MF was introduced in the late 1950s as an alternative to microbiological analysis of water samples. It has the advantage of creating individual colonies from each separate bacterium or microbe in the original sample, allowing easy counting.
You can learn more about MPN at Microbonline.

nomad Testers are based on the membrane filtration technique, during which microorganisms form isolated colonies (CFU or Colony Forming Unit) which makes it easy to count and identify individuals in the original sample.
Comparison of MPN and MF methods from WHO Guidelines for drinking water quality.

nomad Testers are used to count isolated colonies, which indicate the number of culturable individual microorganisms in the original sample.

"nomad Testers are based on the culture method, the most established and well-documented microbial method"

A BIT OF HISTORY...

When Robert Koch, considered the founder of modern microbiology, started his research on microorganisms some 150 years ago, he used potato slices and later liquid nutrients to which he added agar, the same product used today to set jams and deserts.

With those simple techniques he made key discoveries in public health and was awarded the Nobel Prize in 1905.

The microbes in our environment, their population diversity, size and location represent contamination risks for industrial processes and procedures.

nomad Testers are the result of combining established techniques in a new way, to help you understand the microorganisms around you.

"You can go a long way with very simple tools"

Why use culture methods?

Culture methods involve taking a sample containing microorganisms, and then encouraging those microorganisms to multiply to produce one or more colonies, visible to the naked eye.

Culture methods present several advantages over other methods for determining microbiological contamination:
  • 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

Normal microorganism concentrations can vary widely from none in a large volume (sterile product), to millions in a small volume (waste waters).
nomad Testers are suited for concentrations between 1 and 300cfu per tester.


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

Total Counts are frequently indicated in microbiological specifications to help in the assessment of Good Manufacturing Practices, Good Hygiene Practices and HACCP (Hazard Analysis Critical Control Point). Total Count means the total number of culturable microorganisms present in a sample.
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?

CULTURE MEDIA

The key ingredients for microbial growth: water, temperature, oxygen and nutrients are all quite easily available. The culture medium contains the nutrients necessary for the microorganisms we're testing for to grow... if they're there!

Media types include:
  • 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

To observe populations, initially we can study visual colony characteristics such as color, shape and size on a non-selective medium such as in the red nomad Tester.
Since each colony composed of a million individual cells, originating from the same mother cell, the genetic profile of microorganisms may lead to recognizable characteristics. Some types are easy to recognise, some are almost impossible and some can be recognized with observation and practice.


"Our 3 nomad flavors cover many environmental, hygiene and process monitoring needs"

RECOGNIZING MICROORGANISMS ON HPC TESTERS


Here are examples of environmental test results, explained by an expert. Recognizing distinct populations is a matter of looking closely. Identifying them is a question of experience.
Sample origin: cooling unit internal surface/refrigerated storage room

Blue Circle > Brownish bacteria colony. Could be a Micrococcus : from skin, dust or water, or Serratia with degraded pigmentation: ubiquitous, biofilm producer

Green Circle > Translucent yellowish: Most probably Pseudomonas or related : widespread in water and plant seeds. Motile and can be biofilm producer and fluorescent. Commensal Micrococcus (from the skin) : would exhibit a more pronounced yellow color generally. Commensal Staphylococcus aureus (from the skin) : would be opaque and more pronounced yellow

Red Circle > Fried egg morphology : bacillus, ubiquitous, spore forming (resistant to adverse environments). Unlikely Listeria: from soil, grows in cold environments. Unlikely Enterococcus: from animals, tolerant to cold

Purple Circle > Could be an Enterobacteria: from animals, found in soil or water or lactic bacteria : found in decomposing plants and milk

Yellow Circle > Mold: looks like Mucor. Mucor is ubiquitous, likes humidity and metal supports. Fluffy, cotton candy appearance


Sample origin: storage cold room surface, in contact with vegetables

Yellow Circle > Mold: looks like Mucor. Mucor : ubiquitous, likes humidity and metal supports.Fluffy, cotton candy appearance

Blue Circle > Flat with small dark spots: Pseudomonas related from water and plants (Xanthomonas: from plants, motile, Flavobacterium: from soil and water)


Sample origin : treated water, after carbon filtration

Green Circle > Big mucous colony: possible Klebsiella: ubiquitous, from skin and animals

Yellow Circle > Brown mold: possible Cladosporium: ubiquitous, from living or dead plant material. Spoils foods, damages materials e.g. browns bathroom seams. Hard to eliminate once present

Blue Circle > Pigmented yellow: probable Pseudomonas, widespread in water. Motile and can be biofilm producer and fluorescent

Red Circle > Small white colonies: probable Enterobacteria, common in water



The second approach to identify microorganisms is to use culture media specifically designed to select or differentiate specific populations.

Either use such a media for the test or pick the colonies of interest from a general purpose media and spread it on a more selective/differentiating media.This second option requires a microbiology laboratory, specific material and some training.

Take a look at CHROMagar chromogenic media for Food & Water QC

MOLECULAR IDENTIFICATION

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 Microbiology Methods (RMM) have been the holly grail for decades, and for the thirty last years we have been "almost there".

The recent article "Rapid microbiological methods. They are rapid! Are they fast?" (PDF) lists and reviews 17 of them, with some good results for specific use-cases.

Yet still a lot of people resort to culture methods. Why?

Putting aside the apparently universal trade-off between performance, speed and price, which in practical terms means that fast in microbiology either means resource intensive (expensive and complex equipment) or poorly quantitative (hygiene tests), speed is perhaps not always paramount.

When is 'fast' necessary?
Situation: Accepting a consignment of perishable raw material, or releasing an intermediate product.
Your decision to go forward depends on the result
You need a traffic light yes/no indicator and the sooner the better: in minutes !
Example : ATP swab tests

When is 'real-time' needed?
i.e. you obtain the result fast enough that you can still influence the outcome of what is being done.
Situation: Monitoring a day-long process
Your decision to carry on or change strategy depends on a value
Like a GPS warning of traffic ahead, you want a qualified indicator to decide what route to take before it is too late to change your mind!
Example: Several techniques may provide such information within hours, such as autofluorescence, flow cytometry, genotyping, solid phase cytometry, ..The names alone give you an idea of the resources required for implementation! Some of these techniques are very successful and can greatly contribute to process control and efficiency improvements

But if you want to understand...
Situations : assess a process or facility vulnerability to contamination, detect slow drifts in sanitation/decontamination performance, document environmental flora, decide where and when you should implement rapid or Real-Time techniques i.e. know how wide, steep, straight, well paved, etc. the road is you intend to drive.
Your decision to take the highway or the country road will commit you, and depends on as much qualitative and quantitative data you can get.
Like choosing your route on a road-trip, taking into account hotels, stops for gas, music festivals, tolls etc.
Example : quantitative and qualitative culture methods
Standard, inexpensive, versatile, informative, can be easy to implement with a wealth of public information available.

nomad Testers are good starting point! Find out how to use them in your business!

"Rapid can be good in some cases... but thorough methods are the right choice in most circumstances "

WANT TO KNOW MORE ABOUT IMPLEMENTING MICROBIAL TESTING IN YOUR BUSINESS?

We'd love to hear from you to answer any questions you might have!