Why are dilutions important?

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Check out another time lapse video explaining why dilutions are so important.

April 6, 2021

By Michael Beavitt

Accurate dilutions are one of the best used tools in microbiology for determining the number of microbes in something. No matter how many bacteria are in your initial sample, you can always dilute it down to a countable level.

In the timelapse video shown, a high concentration of E.coli was suspended in agar on the top left plate. From left to right, each plate contains a 1 in 10 dilution of that high concentration solution, then a 1 in 10 dilution of that, and so on and so forth.

Footage by Michael Beavitt

Plate one (top left) could be an example of a food sample tested in our lab with a very high bacterial load. After taking 10g of food and homogenising it with diluent, in the case of food tests, one millilitre of this solution would be added to agar, and incubated.

Each blue-green dot you see in the agar is representative of a single bacterium, which has grown into a colony. At a dilution of 1/10, there are far too many bacteria to be counted. They form a confluent lawn of growth across the entire medium. 1/100, and there’s still far too many.

The sample needs to be diluted all the way down to 1/1000000 from the original 10g suspension to allow countable colonies. Then, all it takes is some quick arithmetic to work out how many were in the 10g of food! The bottom right plate, at a dilution of 1/1000000, had 44 colonies. This means that 44000000 bacteria were present in 10g of food.

It’s very rare that anything will have this much E.coli growing on it – it was just used as an example in this case, because it’s easy to grow, and brightly coloured on the agar used. Ordinarily only a 1/10 dilution will suffice for food products! The same principles apply for other enumerations, however – an aerobic colony count will often require 1/1000 or 1/10000 for raw meat and other high risk food types.

If you’re interested, the video was shot for 24 hours, and the E.coli was incubated at 37ºC on TBX agar. Normally they’re incubated at 41.5ºC, but unfortunately my camera is not rated for those kinds of temperatures! The reason that the top 3 plates begin to crack and shrink is because all the lids were left off for ease of filming, allowing the water in the jelly to evaporate. The prolific bacterial growth also sped up the process.

Salmonella enterica Time-lapse

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There are many different types of Salmonella and they don’t all look the same. This is a short and sweet insight into what our microbiologists do each day in the form of a time-lapse. Enjoy!

March 2, 2021

By Michael Beavitt

Salmonella enterica subsp. enterica is one of the best-known food-poisoning culprits. While many associate it with undercooked chicken meat, in fact a large number of cases stem from the consumption of contaminated meat in general, as well as eggs and egg-containing products, and even sometimes fresh vegetables and fruit.

While a number of pathogenic varieties of Salmonella enterica exist, the most common are the Typhimurium and Enteritidis serovars. They account for the vast majority of food poisoning cases in the UK and can cause some pretty nasty symptoms.

Once they get into the body, they can trick the macrophage immune cells into “eating” them, before escaping the deadly digestive enzymes and hitching a ride to a multitude of organs and infectable sites. They can also trigger debilitating immune over-reactions, as well as producing large quantities of toxins.

Shown is a 48-hour time lapse of Salmonella enterica growing on XLD agar, which is used in our lab in the early confirmation stages of salmonella detection. One change that’s obvious is that the agar turns from orange/red to pink– this is due to the fermentation of xylose sugar and the decarboxylation of lysine (the “X” and “L” in XLD), turning the previously pH neutral surroundings alkaline. The pink colour comes from the indicator phenol red, which changes colour depending on the pH of the surrounding media. 

It’s important to note that not all Salmonella species look like this – some ferment other sugars to produce acid, turning the agar a bright yellow colour.

The other change of note is the formation of black spots in the centres of the colonies – Salmonella enterica species commonly produce H2S, a black precipitate, from sources of iron and sulphur. This allows you to quickly tell typical Salmonella species apart from other unwanted background flora that might still grow on the media.

Due to the various ways different Salmonella serovars grow on the XLD (and other selective agars used) it requires experienced microbiologists to interpret the plates as having or not having Salmonella present, and thus verifying if a food product is safe or not safe to eat.

Oysters: Dealing with Norovirus

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“If seven in 10 oysters have norovirus, should we still be eating them?”

February 23, 2021

By Jennifer Newton

It’s not often that I get enough time to ponder the specifics of food poisoning from molluscs. But, in the midst of feeling unwell after eating oysters, I found myself doing just that: wondering about the main contributors to food poisoning from oysters (in between trips to the bathroom, that is). A quick google search came up with “Nearly 200 ill in UK after eating oysters”, February 2020. But this is February 2021, so did I also fall ill to the same pathogen?

In the aforementioned article, the culprit was identified as the norovirus. The norovirus symptoms include:

  • Nausea.
  • Vomiting.
  • Stomach pain or cramps.
  • Watery or loose diarrhoea.
  • Feeling ill.
  • Low-grade fever.
  • Muscle pain.

Yep, I had all of them. So, looking pretty likely that I was being affected by the same pathogen that brought 200 people to their knees only a year previous. How did I get there? Well, I ate some raw oysters for Valentine’s day, and just over a 24 hours later I was feeling very poorly, which is the typical incubation period of the sickness (one to two days). And I felt unwell for just over a day, which is also in line with its classic diagnosis of lasting 1 to 3 days. The good news is; according to the CDC, people have temporary immunity from re-infection for up to 2 to 3 years.

Based on electron microscopic (EM) imagery, this three-dimensional (3D) illustration provides a graphical representation of a single norovirus virion, set against a beige background. The different colours represent different regions of the organism’s outer protein shell, or capsid.
Image by CDC

This led me to another line of questioning: I ate the oysters raw, but would I have been safe if I cooked them? Quick steaming oysters will not kill norovirus and other pathogens, according to the U.S. Centers for Disease and Prevention. To be safe, seafood must be cooked to an internal temperature of at least 145 degrees Fahrenheit (63°C). Other common sources of norovirus besides oysters include:

  • contaminated foods
  • shellfish
  • ready-to-eat foods, such as salads, ice, cookies, fruit, and sandwiches, that a worker with a norovirus infection has handled
  • any food that contains particles of the faeces or vomit of a person with norovirus

Norovirus is thought to be the most common cause of acute gastroenteritis (diarrhoea and vomiting illness) around the world. It spreads easily through food and drink and can have a big impact on people’s health. It was originally called the Norwalk virus, after the town of Norwalk, OH, where the first confirmed outbreak happened in 1972.

Can we test for norovirus?

Several methods have been developed to extract and test for total norovirus contamination (infectious and non-infectious virus particles) in foods; however, there are no internationally recognized standard methods to date. Despite improvements in our ability to extract viruses from foods, the analysis of rinses and extracts leaves much to be desired. Additionally, the method used to detect norovirus is based on PCR (Polymerase Chain Reaction) which can be costly and not normally a viable option to producers as a screening test.

“If seven in 10 oysters have norovirus, should we still be eating them?” Of course, there is a risk from eating oysters – they are harvested from the wild, after all. But the oyster itself is not the culprit, rather the water in which it is raised. All the oysters sold in the UK are purified for 42 hours, which largely nullifies any danger. But, to be completely safe, you can always cook oysters – or easier still, just zap them in a microwave, which would kill any residual traces of the virus.

So, should we still be eating oysters? Of course: they are good for you, and tasty, too!

Nutrition Facts: The Science Behind the Label

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All about nutritional values and how they are produced.

February 12, 2021

By Francisca Navarro Fuentes

Have you ever read the nutrition facts on your Friday nights double pepperoni frozen pizza? Did you ever wonder what those values mean, or think about how they are produced? Feed your curiosity with this post and find out about the science behind the label!

Nutrition facts are a powerful tool to help you understand the composition of a product, so that the consumer of the product is informed. Nowadays, the variety of products to choose from is endless, but knowing what is in your food can help you to pick what to buy.

By law, it is mandatory that nutrition facts are included in labels. This is controlled by Regulation (EU) No 1169/2011 [1] on the provision of food information to consumers. Therefore, nutrition declaration must include energy (KJ/Kcal), total fat and saturated fat (g), carbohydrates and sugars (g), proteins (g) and salt (g). Besides, it is optional to include values such as mono-unsaturates and polyunsaturates, polyols, starch, fibers and vitamins or minerals. The values are reported per 100g or 100mL of product, and often per portion too.

But do all products have to have a nutrition label? It may surprise you to learn that the answer is: no. There are products  exempt from this rule, such as waters, spices, salt, sweeteners, tea, food additives, gelatine, yeast and chewing gums, among others.

Most people don’t pay attention to the nutritional information and one of the reasons could be a lack of knowledge on how to interpret them. In order to help the consumer, the NHS has published a list of guidelines, summarized in Table 1.

So how are nutritional values produced?

Nutritional values are produced in analytical chemistry laboratories. A representative portion of the product arrives to the laboratory, this is then blended and homogenized into a paste (like your morning vegetable smoothy, ew!). This paste is stored in an airtight bag, ready for analysis.

Chromatography, flame photometry, spectrophotometry, high temperatures (up to 600 ºC!) and high pressures are techniques and conditions used to determine nutritional values. As you would imagine, in order to provide this information, trained chemistry analysts are required. I know, these techniques sound scary but, believe me, chemists love them! Although these machines are subject to break downs, chemists like me thrive on a broken machine’s challenge.

Now, how can you (or I), as a consumer, trust the values in the nutrition facts table? For that, a governmental body, known as United Kingdom Accreditation Service (UKAS), assesses and accredits analytical chemistry laboratories ensuring that the values obtained in laboratory A would be the same as the ones in laboratory B across the UK.

In conclusion, labels are educational as it helps to understand calories and nutrients. They regulated by Regulation (EU) No 1169/2011 and the values are generated in UKAS accredited analytical chemistry laboratories, ensuring the customer rights and accuracy of the data.

References

[1] EUR-Lex Website: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:02011R1169-20180101 Last visited 6/2/21

[2] NHS Website: https://www.nhs.uk/live-well/eat-well/how-to-read-food-labels/#:~:text=Nutrition%20labels%20are%20often%20displayed,certain%20nutrients%2C%20such%20as%20fibre. Last visited 6/2/21