Should You Chill or Freeze Your Retention Samples?

A question often arises at FCS regarding how retention samples should be kept. Should you chill (refrigerate) or freeze your retention samples?

Before we can answer this question, we need to understand how bacteria grow and survive as well as understand why one is required to keep retention samples.

What is a retention sample?

It is important here to distinguish the difference between what is required from a food manufacturer of products and a food service business (catering, hotel, restaurant). Although the retention sample may be kept for the same reason, ie. The purpose of keeping retention samples is to support or verify the food products’ shelf-life period, quality, microbiological, physical, and chemical attributes.  

Retention samples may also be used as part of complaint investigations. It is the time period that makes the difference.

In manufacturing the retention sample is kept for the duration of the use-by/before date in the same environmental conditions in which the product is sold. In other words, the same temperature requirements at which the product is sold at the supermarket, either chilled or frozen. This could be a period of 5 days up to 1 year, depending on the product.

In foodservice, the food is consumed either immediately or within a day or two. In the obvious case of hot foods, the retention sample cannot be kept hot for a period of 2 days. Thus, reviewing the definition of a retention sample, it is then finding a system that maintains the product in a condition that is as close as possible to the final product that was served to the customer.

It is also important to distinguish the time period for how long the sample should be kept. It is reasonable to assume that if the food product is consumed with a day or two of preparation and allowing sufficient time for any potential food poisoning symptoms to present themselves and allow time for reporting such an incident. A period of 3 to 7 days for the retention of such samples is reasonable.

We may intuitively know that in order to keep food in a ‘safe state’, we would need to chill or freeze such a sample.

Bacterial growth and temperature ranges.

There are a number of factors that affect the growth of bacteria. Chief among them are temperature and time. Bacteria can be divided into separate groups defined by the temperature at which they grow at their best.

Psychrophiles or cryophiles are extremophilic organisms that are capable of growth and reproduction in low temperatures, ranging from −20 °C to +10 °C.

mesophile is an organism that grows best in moderate temperature, neither too hot nor too cold, with an optimum growth range from 20 to 45 °C. The term is mainly applied to microorganisms. Organisms that prefer extreme environments are known as extremophiles.

Thermophiles (literally heat lovers) are organisms that grow at temperatures above those (25–40°C) that sustain most life forms. Typically, a thermophile shows maximum growth rates at temperatures above 45°C.

Almost all pathogens, the bacteria involved with food poisoning are mesophiles, growing at their best with the critical danger zone, 20 – 45 °C. The exception is Listeria monocytogenes. L.monocytogenes is a bacteria that is notorious for growing well, between 0 – 10 °C.

This effectively means that one cannot keep retention samples at ambient or room temperature. Doing so would encourage the growth of bacteria at an exponential rate, causing the product to expire at the fastest possible rate.

Realizing this, barring the L.monocytogenes exception, means samples must be kept outside the optimal growth range in order to maintain the integrity of the sample, (by which we mean, keeping the microbiological environment in the same state) as at the point of consumption.

Realistically, the means chilling or freezing. It is also important to understand what effect this would have on the bacteria.

How does temperature affect optimal growth?

Reviewing the graph, below, we can observe a basic explanation of the temperature growth factor. We now know that bacteria grow their best at the optimal temperature range, in the case of food poisoning bacteria, between 20 – 45 °C. And do not grow at their best at their minimum or maximum temperature range.

bacteria growth curve graph

However, this may not mean that the bacteria die off or are killed at these ranges. But specifically, do not grow at their best, but do still grow to some extent. 

Why do we chill foods?

Because the refrigeration temperature range falls into the minimum range of a mesophiles growth range, bacteria grow much slower. This means that when we chill foods, the shelf-life of the product can be extended as compared to leaving foods out at room temperature.

The difference between the two is a matter of hours as compared to a matter of days if correctly chilled.

However, we cannot keep food indefinitely in a fridge or freezer, bacteria do eventually grow to cause foods to become unsafe for consumption. The image below further explains this principle.

bacteria growth curve graph

The rate of growth is much slower as compared to the optimal temperature range.

Chilling food does not kill bacteria, but it does stop them from growing to harmful levels.  Because of this, it is a legal requirement that perishable foods should be kept refrigerated at 7ºC or below.

Why do we freeze food?

We already understand that keeping foods frozen further extends the shelf-life of a product beyond that of what we can achieve if we were to chill foods. In this instance, the shelf-life is a matter of weeks and months compared to days.

What happens when you freeze foods?

Freezing delays spoilage and keeps foods safe by preventing bacteria from growing and by slowing down the enzyme activity that causes food to spoil. As the water in the food freezes into ice crystals, it becomes unavailable to those bacteria that need it for growth.

This is great news for keeping foods for longer periods of time.

What happens to bacteria when you freeze foods?

The ice crystals that develop as a result of the freezing process can have an adverse effect on the bacterial cells. Very low temperatures affect cells in many ways. Membranes lose their fluidity and are damaged by ice crystal formation.

Chemical reactions and diffusion slow considerably. Proteins become too rigid to catalyze reactions and may undergo denaturation.

In such a case of causing environmental stress such as freezing. Some bacteria are able to survive, some die off and others undergo sublethal or metabolic injury. These are due to in part ‘cold shock’, the lethal concentration of nutrients to the bacteria, and internal ice formations.

What is of particular interest is that the injured bacteria cannot be detected in the laboratory using standard detection methods. Such bacterial cells need to be resuscitated prior to these methods being used.

Despite this, the bacteria are still present in the frozen food and are often revived in their environment (the food product) during the thawing or defrosting process.  Many such cases exist of food poisoning during incorrect defrosting procedures, particularly for Clostridium perfringens bacteria.

It is these sorts of instances that yield what is called a false-negative result.

A false negative error, or false negative, is a test result that wrongly indicates that a condition does not hold. For example, when a pregnancy test indicates a woman is not pregnant, but she is, or when a pathogen is not detected but is known to be present these are false negatives.

The results can have an adverse effect when for example, the food product is served and consumed by the public and causes a food poisoning incident. Because the results showed a negative result, the food was served. However, if the result is a false negative, those individuals will get sick.

What does this mean for retention samples / what is the intention?

When considering the intention of keeping retention samples, the idea is to accurately identify whether a food product had a pathogen present that could have caused food poisoning. Such results are important in identifying the cause of a problem. If a pathogen is present, this becomes the starting point in the process of investigating the cause in resolving any potential cause of the contamination.

If the result produces a negative result, meaning no pathogens are detected, no further investigations are needed, as the product is deemed safe. If the results are indeed a false negative, further cases of food poisoning could happen with no cause being identified.

This certainly defeats the point of the retention sample, and may indeed cause more harm than what had originally occurred.

How should you take and keep retention samples?

Our recommendation is that samples be kept in well-sealed and correctly labeled containers or sample bags, placed in a dedicated area of a fridge or cold room that is operating at a temperature of between 0 – 5°C. Samples should be kept for a period of between 3 – 7 days.

But freezing microorganisms is a way of preserving them

This is true, most microorganisms, including bacteria, are frozen as a way to preserve for transport or for later use. This is done by many medical and research laboratories. However, these are specially prepared using the appropriate media to ensure survival and are frozen at temperatures of -70°C and colder.

These are done by individual bacterial species and not a collective community of bacteria that may be present in food. Both the special media and temperatures are well beyond the means of a kitchen or food production facility.

Most kitchen freezers do not operate beyond -18°C and certainly, in a working kitchen struggle to maintain -10°C. Research shows that temperatures of -1°C to -5°C seem to be more ‘dangerous’ for common pathogens such as E.coli. More so, that temperatures of -20°C and colder. This means one cannot realistically ‘preserve’ the bacteria in a kitchen freezer.

The speed at which the freezing occurs also seems to play a role in the bacteria’s survival. Slowing freezing, over a period of a few hours to a day, as would happen in a kitchen freezer, causes the bacterial cells to develop ice crystals which will either kill them or metabolically injure them.

Fantastic for preserving foods and extending the shelf-life of a product. But, less useful for the intended purpose of a retention sample.

Rapid freezing, as would happen during cryogenic freezing has been shown to be less lethal to the cells.

Similar results could be achieved through the use of a blast freezer, being able to completely freeze the product to -18°C with 2 – 4 hours. The problem here is that different foods freeze at different rates. There are several factors that determine this. Density, water content, and fat content all affect the time required to achieve temperature as well as weight loadings and the thickness of the product.

But, if we keep the samples in a fridge, the bacteria will grow

This is true, to an extent. It is important to remember the intention of keeping the samples, we want to detect whether there are pathogens present in the food or not.

Keeping foods in the fridge does not cause pathogens to grow, without the pathogen is present in the first place. So, without a pathogen is present, no pathogens can grow and none will be detected.

What does happen, is that food spoilage bacteria will grow slowly (remember how temperature affects bacterial growth) eventually causing the food to spoil or go off. This, however, makes no difference in terms of the food poisoning investigation.

In terms of microbiology, significant growth would be exponential or logarithmic growth, meaning 1000 CFU/ml to 10 000 CFU/ml, or in the case of spoilage to >1 million CFU/ml where food may begin to expire. 

But, won’t the pathogens if present, grow too?

Yes, that is true. This is where the understanding of pathogens is important. The presence of a pathogen indicates a food safety concern and should not be present in the first place.

Some pathogens may require very low numbers of cells to cause illness.

Meaning 10 to 100 cfu/g can be dangerous for us to consume. Whether there are 10 cfu/g present or 500 cfu/g presents, makes no difference in being able to detect them, nor to the fact that they are present and could have caused food poisoning.

Our SANAS accredited testing methods are very sensitive, meaning we are able to detect even 1 cfu/g within a food product. Pathogens are unlikely to grow to significant levels in a fridge.

Should we chill or freeze our retention samples?

In conclusion, we would highly recommend that retention samples be kept in a correctly operating fridge (0 – 5°C), where these samples are chilled and not frozen in order to ensure that the sample is as close as possible to the product that was served to the customer.

Basic retention sampling procedure for clients holding high-risk foods:

  • Samples should be placed into new, sealable containers.
  • Preferably “Zip seal” bags or cleaned and sanitised containers i.e. Ziplock bags, or small sauce containers with lids.
  • Samples should be placed into the containers using a clean tablespoon that has been soaked in a correctly diluted sanitiser solution for 5 minutes.
  • For the above, chlorine sanitiser would be the best for this application. Alternatively, the bowl of the clean spoon could be left in boiling water for 10 minutes before being used. In the case of the latter please be careful when using the newly boiled spoon.
  • Write a description of the sample, including the service period and date, on the sample container before sampling the item. i.e. Potatoes/Dinner/2019/05/02
  • 100g of the sample should be placed into the container to ensure all tests can be performed.
  • Place the samples into the cold room or under-counter fridge.
  • NOTE: the samples should be kept for a 7-day rotation to allow for delays in incident reporting and for ease of sample rotation.
  • It is recommended that one closable container for each day of the week is placed into the storing fridge. Interlocking or portioned boxes might be best.
  •  The foods would then be discarded and retained in rotation based on the day.
  •  On day 8, the second Monday for example, the foods from day 1, the first Monday, would be discarded and replaced with day 8’s foods. BUT keeping all the other foods from the previous 6 days (day 2-7/Tuesday- Sunday). The rotation would then follow this trend as the week goes on.

Should there be an incident reported please contact FCS to arrange the collection or transport of the food retention samples for testing.

Guide to identifying high-risk foods:

  • Uncooked vegetables for consumption (salads/salad base mixes)
  • Multi-component bulk foods (sauces/soups/stews/curry)
  • Bulk held cooked and chilled foods (rice/mashes/cooked vegetable mix/pre-cooked pasta)
  • Multi-component Ready To Eat Foods (sandwiches/all kinds of salads)
  • Meals containing dairy (cream cakes/sauces/pasta sauces/cut soft cheese)
  • Most foods held in hot and cold holding Bain maries/warmers/displays
  • Processed supplied products (sausages(cooked)/cold meats/processed meats)

Food Consulting Services is a private South African-owned Food Testing Laboratory and Food Safety Consultancy, that tests water and food to ensure compliance with South African National Standards. We also perform hygiene survey audits to the hospitality and food manufacturing industry. If you’d like to learn more about Food Consulting Services, visit our website or feel free to enquire with us. We look forward to hearing from you and becoming your food safety partners. Get in touch with us for more information.

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