Know the Microbiology Laboratory Terms | Water Activity | Preservation and Storage of Cell Cultures | Real-Time PCR

Know the Pathogen:

Bacillus cereus | Clostridium botulinum | Clostridium perfringens | Coliforms | E.coli 0157 | Escherichia coli | Listeria | Salmonella
Foods implicated: milk, cheese, ice-cream, pate`s, coleslaw, meats and fermented sausages and salads. Found in animal faeces, soil, vegetation, water and sewage. Areas with excess moisture may provide a breeding ground for Listeria. Therefore drains, chillers and other areas with high moisture or condensation are most at risk. Optimum growth temperature is 37°C. Symptoms of L. monocytogenes poisoning include flu-like symptoms, diarrhoea, meningitis and spontaneous abortion.
E.coli 0157:H7:
This organism is regarded as the most deadly of the coliform group. Foods implicated in outbreaks include fresh and raw meats such as beef, poultry and dairy products. Onset of illness is usually within 4 days after ingestion with illness lasting for an average of 24 hours. Symptoms include onset of severe cramps and abdominal pain, bloody diarrhoea, vomiting, acute renal failure, fever, gastro-intestinal haemorrhage and sometimes death. Found in the gut of some ruminants. Contamination of meat usually occurs at the abattoirs. Optimum growth temperature is 37C. Water contaminated with faecal matter (sewage) will also cause infection.
Staphylococcus aureus:
Enters the food through human handling (poor hygiene), since it is naturally found in humans (nose, throat). Once present in the food, this bacterium grows and produces an enterotoxin, which causes intense vomiting 1 to 8 hours after ingesting the contaminated food. Generally causes problems in foods such as processed meat, hams, pastries, custards and sandwich spreads.
Salmonella species:
Causes gastroenteritis and has been known to cause death, but generally not fatal. Originates from the intestinal tracts of animals and mammals. Food poisoning in humans occurs through consumption of contaminated beef, poultry, eggs, water (sewage), and dairy products. Diarrhoea, fever, nausea, headaches and muscle aches are common symptoms. These symptoms could occur from 8 to 48 hours after consumption of contaminated foods.
Escherichia coli:
Commonly found in meat, raw milk, vegetables, and salads. Adequate cooking of meat, pasteurisation or sterilisation of milk and sanitising of vegetables (in chlorine bleach), should eliminate (kill) this bacterium. E.coli is widespread in nature therefore the risk of foods being contaminated is high. Food handlers must exercise good personal hygiene practices at all times, as they could be carriers of E. coli; which will result in food contamination and food poisoning by the faecal-oral route.
Clostridium botulinum:
Found in soil and the intestinal tracts of some animals. Spores of this bacterium are resistant to heat and if food is improperly processed, spores will germinate and the bacteria produce toxins. Gastrointestinal pain, headache, weakness and respiratory problems are experienced 18 to 24 hours after ingesting contaminated food. Foods implicated in this type of food poisoning are generally those that have undergone some form of preservation such as canning, pickling or smoking. Ingestion of such toxins may be fatal.
Clostridium perfringens:
These bacteria grow in re-warmed meat dishes and when ingested produce toxins. Grows best when there is little or no oxygen. Optimum growth temperature is between 43°C and 47°C. This organism is found in a wide variety of foodstuff including raw, dehydrated and cooked foods. C. perfringens is also found in soil, dust and vegetation. Symptom of C. perfringens food poisoning include watery diarrhoea and severe abdominal cramps and usually occur between 6 and 24 hours after ingestion.
Bacillus cereus:
Causes nausea and vomiting. The emetic illness is usually associated with boiled or fried rice and symptoms occur 1 to 6 hours after ingestion; the diarrhoea type illness is associated with a wider range of foods. Symptoms usually occur within 10 and 12 hours after ingestion. Uncooked food of plant origin is a common source of B. cereus. They produce spores which are heat stable. Optimum growth temperature is between 35°C and 40°C. Growth of this organism is inhibited by addition sorbic acid or potassium sorbate at low pH. High salt content also inhibits the growth of Bacillus cereus.
Coliforms include a wide range of organisms, such as E. coli, Klebsiella, Citrobacter and Enterobacter. Coliforms are defined as lactose fermenting gram-negative normal enteric (relating to the intestine) flora. Coliforms, other than E.coli, are regarded as mostly opportunistic pathogens in that they affect the young, the aged and immunocompromised persons.
An organism is said to be presumptive when the preliminary tests (screening tests) show all reasonable certainty that the organism is present. Confirmation tests are still required to determine a positive result for its presence.


What is:

petri dish:
A shallow dish (plastic or glass) with a lid, used to culture cells (bacteria/microorganisms).
Contains selective or non-selective nutrients which can be used in a semi-solid form (agar) or in a liquid (broth) form. Specifically used for the growth, storage or transport of bacteria/microorganisms.
A chemical compound/food (either liquid or solid) required by an organism in order to live, grow and multiply.
Liquid growth media containing nutrients which enable microorganisms/bacteria to live, grow and multiply.
In order to promote a particular reaction in a biochemical system, such as accelerated growth of microorgamnisms, specific temperature and humidty conditions need to be maintained.
Colony Forming Unit - a unit of measurement used to quantify (count) viable microorganisms. Example: number of visible individual colonies of bacteria counted that have grown (from a single bacterium) on nutrient agar in a petri dish.
A reproductive biological structure which is adapted for survival for extended periods of time, under unfavourable conditions. Spores form part of the lifecycles of many bactetria, plants, algae, fungi and some protozoans.
Are multicellular or unicellar biological structures and are found living in mainly soil or dead matter (as food). They are symbionts of plants, animals and mould.
Are a type of fungal organisms which can be readily seen by the naked eye. They include all species of microscopic fungi that grow in the form of multicellular filaments called hyphae, often having the appearance of "cottonwool".
Microscopic fungi that grow as single cells are called yeasts. Yeasts are spherical, ellipsoidal and sometimes cylindrical in shape, and are about 5 to 10 times larger in size than bacteria. They are unicellular, and have asexual reproduction via a process called budding (due to their appearance).
Agar, a gelling agent, is a solid complex polysaccharide at room temperature, but will melt at temperatures above 85°C. Once liquefied, agar does not re-solidify until it cools to about 42°C. It is used as a gell base to hold moisture and nutrients that enable bacteria to grow.
Is a disease causing agent to its host, such as animal or human. Examples are Staphylococcus aureus, Bacillus cereus and Clostridium perfringens.


Water Activity, what is it?

The Influence of Water Activity (Aw) and Moisture on the Shelf Life of Food Products

 Review article by our staff member, Nirvasha Ramparsad of Bio-Science Technologies

Many foods, including baked food such as biscuits, cakes, savory snacks and even nuts suffer in quality due to a gain in moisture (absorption) or loss of moisture (dehydration). This factor allows one to determine the choice of packaging to be used. Whether the growth of microorganisms occurs in food or not is an important factor determined by humidity (measured as water activity). Rates of moisture exchange through packaging and the change in the rate of water activity of food towards a critical limit will lead towards the determination of a product shelf life.

Generally there is an optimum water activity of most foods, which would yield a maximum shelf life. Thus, the shelf stability of products is formulated with respect to its water activity. A low water activity results in inhibition of mould growth. Water activity predicts how the migration of moisture affects the product. Note that the water activity of a food is not the same as its moisture (water) content. At a water activity of less than 0.6 units microbiological problems do not commonly occur.

The relationship between microbial spoilage and moisture is critical since microorganisms present a threat of foodborne illness, e.g. gastrointestinal infections caused by Salmonella spp; Klebsiella spp and Escherichia spp. Secretions caused by organisms such as Staphylococcus spp. and Bacillus spp. can cause food intoxications which can prove fatal to consumers. Allergic consequences, carcinogenicity, mutagenicity can be caused by mould, which also produce toxic substances. The water activity of foods influence the reproduction of these organisms.

Temperature changes are always occurring during handling, factory storage, distribution and in the shop; including the home of the consumer. The air in a package holds more moisture at elevated temperatures than at refrigerated temperature. Thus at a higher temperature the water activity of the air is reduced substantially, while at the lower temperature, the water activity of the air is increased.

Moisture, heat, freezing, oxygen and UV light are several factors, which affect shelf life. Moisture management means, that, when a package is exposed to varying environmental conditions, the products optimal water activity is maintained. This system allows the water activity in the package to remain constant by providing moisture to the product when needed, and removing it when necessary.

The packaging material controls the intake or escape of moisture. Preservation of the product in its optimal condition cannot occur indefinitely even if the packaging has excellent moisture barrier properties. A package may contain leaks because of cracking in the material due to flexing or flaws in the heat seal. The packaging itself, may also be, permeable to moisture. These factors affect the changing moisture levels of a food, impacting on the quality and shelf life of a food product.


How to Preserve and Store Cell Cultures:

 Review article by our staff member, Lineshree Pillay of Bio-Science Technologies

Cultures must be stored in such a way to eliminate genetic change, protect against contamination and retain viability. An organism may be kept viable by periodic transfer of cultures into fresh medium. However, at each cell division, there is a probability of mutations occurring, because repeated subculture involves many such divisions. There is a probability that strain degeneration would occur. Repeated subculture also carries with it the risk of contamination. To avoid all these problems, preservation techniques are as listed below:

Storage on agar slopes
Cultures grown on agar slopes may be stored in a refrigerator (4°C) or freezer (-20°C) and sub cultured at intervals of 6 months. If agar slopes are covered with sterile medicinal grade mineral oil, the time of subculture may be extended to 1 year.
Storage under liquid nitrogen
The metabolic activity of microorganisms can be reduced by storage at very low temperatures (-15 to -196°C) using liquid nitrogen. The culture is grown to the maximum stationary phase and the cells are re-suspended in a cryo-protective agent (such as 10% glycerol), and then frozen in sealed ampoules, before storage under liquid nitrogen.
The culture is grown to the maximum stationary phase and then stored in preservative agents such as milk, serum or sodium glutamate. A few drops of the suspension is transferred to an ampoule, which is then frozen and subjected to high vacuum until sublimation is complete, after which the ampoule is sealed and stored at refrigeration temperature; Lyophilisation stabilizes bacterial cultures for long-term storage, while minimizing the damage that may be caused by strictly drying the sample. This method is used in the biotechnology and biomedical industries to preserve vaccines, blood samples, purified proteins, and other biological material.


DNA testing in the Food Industry by Real Time PCR methodology

We can now offer quick results for Salmonella, Listeria, E coli and Campylobacter, using the latest Real-Time PCR technology!

This methodology for food safety testing is relatively new in South Africa and we at Bio-Science Technologies (Pty) Ltd are proud to be one of first independent testing laboratories to recently acquire this highly specialised instrumentation. Results using this technique are available within 24 hours compared to conventional methods which can take anything from 3 to 6 days. Product safety can be verified prior to dispatch from factory or warehouse, thus saving millions of rand due to product recalls, food poisoning and litigation costs.

Polymerase Chain Reaction, commonly referred to as PCR, is the amplification of DNA strands into millions of copies consisting of a specific DNA sequence. With conventional PCR, the results can be visualised on agarose gel only after the reaction has been completed but with Real Time PCR, results can be viewed as the reaction is taking place.

How does it work?

Samples are enriched in a nutrient medium for a couple hours. An aliquot of the sample is subjected to enzymes and a thermo cycler unit, in order to break the double stranded DNA and produce more DNA copies. Fluorescent labelled probes and dyes are added to the DNA copies in order for them to be detected. Every living organism has a different DNA sequence hence different probes and dyes have to be used. These dyes and probes bind to the specific DNA sequence causing it to fluoresce. The fluorescence is then measured to determine if the particular organism is present. Speed, sensitivity, specificity and excellent detection limit are just some of the advantages that Real Time PCR has over conventional microbiological test methods. The inclusion of certified positive and negative controls just adds more confidence to the results obtained!


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