Advanced Food Microbiology

Module 9: Course assessment

Key concepts and terminology

This lesson introduces you to some of the key concepts and terminology used in food microbiology. We will be discussing these concepts in more detail as the course progresses, but it is important to have a basic understanding of the underlying principles and concepts discussed here prior to continuing with the rest of this course.

In the list below, click on the arrow to reveal an explanation or more information. You can refer back to this module frequently as you progress through the course.

Bacteria
Photo by CDC on Unsplash

Bacteria are tiny, single-celled organisms that are ubiquitous in our environment (ie they can occur everywhere). Bacteria are too small to be seen individually without the aid of a microscope – however, they can multiply to form groups or colonies on a food source. When individual cells have multiplied a sufficient number of times, the resulting colony of bacteria can be seen with the naked eye.


Viewed with a microscope, different kinds of bacteria will have different shapes or forms and this is one way in which bacteria are grouped together. Many bacteria have either a spherical shape or an elongated rod shape. A spherical shaped bacterium (singular) is called a coccus, and a group of spherical-shaped bacteria (plural) are called cocci. A rod shaped bacterium is called a bacillus, and a group of rod-shaped bacteria are called bacilli. There are other types and shapes, such as the staphylococci, which form grape-like clusters.

Colony Forming Unit (CFU)

A colony forming unit is what is counted on an agar plate at the laboratory when your testing is undertaken using a quantitative method of analysis (ie providing a COUNT of the number of bacteria in a specified amount of sample).

An amount of your product is diluted and spread onto a specific medium (agar or other type of plate) before being placed in a temperature controlled environment. After a specified period of time, the plates are removed and the ‘dots’ are counted. Each dot represents a colony of bacteria (or yeast or mould) which grew from an initial single cell.

This is why on your certificate of analysis you will see the results presented in CFU (colony forming units) per gram or mililitre of product (grams for solid food and mililitres for liquids). The format will be either CFU/g or CFU/mL. Therefore, if 10 ‘dots’ were present on the plate, the results would be presented as 10CFU/g if 1gram of product was sampled.

It is more common for solid food to be diluted prior to spreading on the plate, so if 1g of product was diluted in 99g of water, the results would be presented as 10CFU/100g.

Often, you will see the results presented with a less than sign in front of the number, thus: <10CFU/100g. In this case, it means that no colonies were grown or counted on the plate, but that there is a margin or error implicit in the testing.

This may be expressed as the Limit of Detection (LOD), the Limit of Reporting (LOR) or the Limit of Quantitation (LOQ). Whichever is the case, the lab cannot say that there are no bacteria present in the sample with quantitative methods, so it is expressed as less than.

Death phase

During the death (or decline) phase of the Microbial growth curve, bacteria die off because conditions have become unfavourable to survival. It could be that all of the available nutrients have been consumed, or the temperature has become too high or low to support survival.

Extrinsic growth factors

Extrinsic factors are imposed from the environment in which the food product is present, such as temperature, relative humidity, gaseous environments, packaging or presence of competitor microorganisms. Minor fluctuations in these factors, either individual or multiple, can alter the stability of a food product and make them susceptible to the growth of spoilage or pathogenic microorganisms.

As not all microorganisms are equal, a combination of various factors are utilised to influence the separate behaviours of bacteria, yeasts, and moulds. A thorough understanding of how the intrinsic and extrinsic factors are used in a multicomponent approach is essential for ensuring food safety and quality, which is covered in detail in Module 5 of this course.

Food composition

A suitable supply of nutrients is the most important condition affecting growth of bacteria. Every living cell requires certain nutrients to multiply. These include solutions of sugars or other carbohydrates, proteins, and small amounts of other materials such as phosphates, chlorides and calcium. If the nutrient supply is removed, bacteria will not multiply.

Fungi

The fungi consist of two major groups of microbes: moulds and yeasts. Moulds are multicellular organisms. Yeasts are single-celled organisms. Moulds and yeasts tend to be significantly larger than bacteria. Both moulds and yeasts are widely distributed in nature, both in the soil and in dust carried by air.


Moulds have a branching filamentous structure, and can develop into colonies visible as a colorful, furry or downy coating on food or surfaces. They reproduce by producing small spores, which are not related to bacterial spores. Mould spores can be picked up and spread by air currents. If mould spores settle on suitable surfaces, they will begin to germinate and produce new mould growth.


Yeasts are usually egg-shaped, and tend to be smaller than moulds. Like moulds, yeasts can be spread via air currents. They reproduce by a process known as budding. Visible colonies of yeast are generally slimy in appearance and creamy white.

HACCP

HACCP stands for Hazard Analysis and Critical Control Points – it is a systematic approach to the identification, evaluation and control of food safety hazards. Hazards are grouped as biological, chemical, physical or radiological and the approach is proactive rather than reactive, as it seeks to prevent or reduce hazards to acceptable levels, rather than relying on end product testing for safety.

There are 7 HACCP principles:

  1. Conduct a hazard analysis
  2. Identify critical control points (CCPs)
  3. Establish critical limits for each CCP
  4. Establish CCP monitoring requirements
  5. Establish corrective actions
  6. Establish procedures for verifying that the HACCP system is working as intended
  7. Establish record keeping procedures

There are also an additional 5 preliminary steps to HACCP, as below:

  1. Build a HACCP team and assemble the resources
  2. Describe the product and its method of distribution
  3. Identify the intended use and consumers of the product
  4. Construct a process flow diagram
  5. Confirm the process flow diagram on site
Hazard vs risk

It is important to distinguish between a hazard and risk, in terms of food safety planning.

A hazard is a source of harm, whereas risk is the likelihood of being exposed to the hazard and the subsequent likelihood of being harmed by the hazard if exposed.

Therefore, a microbiological hazard could be the presence of Listeria monocytogenes in fresh, bagged salad. The risk would be the potential for that salad to make someone sick: low risk would be a bagged salad with a refrigerated shelf life of less than 5 days and high risk would be a refrigerated shelf life of more than 5 days.

Intrinsic growth factors

The food environment can support or reduce the ability of microorganisms to persist, establish and grow. Each one presents as a natural characteristic of a food ingredient or adjusted through manufactured processes. Intrinsic and extrinsic factors play very important roles to maintain a microbiologically safe food system.

Intrinsic factors include those that are internal to the food product itself, such as nutrient content, pH levels, water activity, redox potential, and other antimicrobial components acting as defense mechanisms against microbes.

Lag phase

In the microbial growth curve, the lag phase is the stage where bacteria are becoming established in a food product. They are adapting to the growth conditions and individual bacteria are not yet able to divide, The cells are not dormant, although they undergo very little change. The lag phase can last from 1 hour up to several days, depending on the growth conditions present.

Log
The image above demonstrates the correlation between log and percentage reduction from a starting point of 10 million microbes.

‘Log’ is short for logarithm, a mathematical term for a power to which a number can be raised. For example, if using 10 as a given number, a Log 3 increase can be shown as 103 or 10 x 10 x 10 = 1,000.

A log reduction takes the power in the opposite direction. For example, a log reduction of 1 is equivalent to a 10-fold reduction or, to put it another way, moving down one decimal place or a 90% reduction.

During product efficacy testing, the microbiology laboratories count the number of colony forming units (CFUs) of the given pathogen present at the start of the test.  They then apply the disinfection product being tested, alongside a control product and wait the required test time before recounting the number of CFUs present.

The result of the difference between the control and the test product is then expressed as a Log reduction. For example, if the number of CFUs in the control was found to be 1,000,000 (or 106) and the end result using the product was only 1,000 (103), that would be a Log reduction of 3 or a reduction of 99.9%.

As a basic rule of thumb, for every additional Log reduction number you add a 9 to the percentage reduction – so a log reduction of 3, as illustrated above, is a 99.9% reduction compared with a log reduction of 6 which is equivalent to a 99.9999% reduction.

Log phase

In the Microbial growth curve, the log phase (or exponential phase) is a period of constant growth as the cells reproduce by doubling (binary fission). Depending on the nature of the food medium and characteristics it contains, both daughter cells may survive and this leads to exponential microbial growth in the food.

Microbial growth curve

If favorable environmental conditions exist, bacterial growth occurs. For our purposes, we will use the term growth to refer to an increase in microbe numbers, not an increase in size of an organism. Bacteria reproduce by dividing, a process called binary fission. When a bacterial cell is ready to divide, the material within it gradually increases until the cell’s volume is almost doubled. The cocci shapes become oval while rod shapes stretch to nearly twice their length. The cell then constricts in the middle. This constriction deepens until the cell contents are held in two distinct compartments separated by a wall. These two compartments finally separate to form two new cells, which are duplicates of the former cell and each other.

Theoretical growth patterns can be represented by a graph of bacterial numbers over time and broken down into four different stages or phases.

The first phase is called the lag phase. The lag phase occurs when a bacterial population first enters a nutrient rich environment. The rate of growth is very slow because the bacterial cells are adjusting to their new environment. In a nutrient-rich environment, such as on a meat or poultry product, the lag phase is generally short; however, the length of the lag phase is the most variable of the four phases. For example, it will take a bacterium longer to adapt to temperatures below the optimum growth range for that bacterium. Therefore, good temperature control will prolong the lag phase. Other environmental factors, including pH, water activity (aw), and competition with other microbial species for nutrients, can also impact the length of the lag phase for a particular microbe.

After some hours or days, depending on environmental conditions and characteristics of the particular bacterial species, the bacterial cells begin to rapidly multiply. This phase is called the log phase because growth occurs exponentially and is depicted on a logarithmic scale on the vertical axis of the growth curve. A logarithmic scale basically allows a wide range of values to be displayed on a graph of manageable size and in a visually meaningful way. Such a scale is necessary because bacterial growth can occur at an exponential rate, i.e., 1 cell becomes 2 cells, the 2 cells become 4, then 8, then 16, then 32, then 64, etc. With each successive replication, the total number of cells doubles. The time it takes for the population of bacteria to double is referred to as doubling time or generation time. This doubling time can vary among species of bacteria, but for most is between 10 to 30 minutes under optimal conditions for growth. It is important to note that, while the starting bacterial count may not have an effect on doubling time it will have a tremendous effect on the numbers of bacteria after each doubling. For example, the numbers of bacteria will differ tremendously after 1 doubling time if the initial count is 200 cells (become 400 cells with the first doubling) vs. 2 cells (become 4 cells with the first doubling). Effective sanitation to reduce bacterial load will limit the number of cells available to contribute to proliferation during this phase.

The third phase is the stationary phase. In this phase the rate of bacterial growth is the same as the rate of bacterial death, because the population of bacteria has reached its maximum due to limitations in the availability of nutrients and an increase in bacterial waste products.

The fourth phase is the death phase. In this phase, more bacterial cells are dying than those that are dividing. There is a net loss in the number of viable bacterial cells in the environment. This is the result of increasingly hostile environmental conditions associated with decreasing availability of nutrients and increasing waste products. Initially the rate of death is exponential, but it may slow down after significant numbers of bacterial cells have died. Spore-forming bacteria may begin to sporulate and remain viable but dormant. Because the nutrient supply in meat and poultry products is almost unlimited, an exponential rate of death may not occur.

Oxygen availability

Similar to temperature, oxygen availability can determine which microbes will be active. Microbes that have an absolute requirement for oxygen are called obligate aerobes. Those that require the total absence of oxygen are called obligate anaerobes. Some microbes are called facultative anaerobes, because they can grow in the presence or absence of oxygen. Molds require oxygen for growth. Yeasts grow best under aerobic conditions, but some can grow slowly under anaerobic conditions. The kinds of bacteria that cause food spoilage tend to be aerobes, but those that cause foodborne illness are anaerobes or facultative anaerobes.

Parasites
Amoeboid protozoan: Credit: Melba Photo Agency/Alamy Stock Photo

Parasites are living organisms that derive nourishment and protection from other living organisms called hosts. These organisms live and reproduce within the tissues and organs of infected human and animal hosts. There are different types of parasites, and they range in size from single-celled protozoa to multi-cellular worms. Protozoan parasites are visible only through a microscope. Many adult parasitic worms are visible without a microscope; however, a microscope is necessary for detecting eggs and preadult forms of some worms. Identification of the adult forms of certain parasitic worms can also require microscopy.


The respective lifecycle of different parasites also varies. While some parasites use a permanent host, others go through a series of developmental phases using different animals or human hosts. They may be transmitted from host to host through consumption of contaminated food and water. Several parasites have emerged as significant causes of foodborne and waterborne illness.

pH

The degree of acidity or alkalinity of an aqueous solution is expressed on a scale between 0 and 14 referred to as the pH scale. As acidity increases, we move down on the pH scale (i.e., the pH is lower). As alkalinity increases, we move up on the pH scale (i.e., the pH is higher). The pH of pure water is 7.0, which is referred to as neutral pH.

The pH of a meat or poultry product can have a profound effect on the growth and viability of microbial cells. Each species of microbe grows within an optimal range of pH values. Most microbes thrive when the pH is near neutral or slightly acidic, but there are exceptions. Most bacteria will not grow at pH levels below 4.6 because the environment is too acidic. Many moulds and yeasts can grow at a lower pH than do bacteria. On the basis of pH, food products are often grouped as high-acid foods (pH below 4.6) and low-acid foods (pH 4.6 and above). The pH of fresh meat ranges between 5.3 and 6.4 (i.e., high pH or low-acid). Meat with a pH in the 6.0 to 6.4 range spoils faster than meat in the lower pH range of 5.3 to 5.7, because spoilage microbes are more active in the pH range of 6.0 to 6.4. We will discuss later how you can control pH to limit microbial growth.

Presence/Absence testing

A presence/absence test is what is also known as a qualitative test – the analysis is performed on a specified amount of product to test for the presence or absence of a specified microorganism.

This type of testing is used when compliance with strict specifications is required – for example, the Food Standards Code dictates that in certain high risk products, Listeria monocytogenes must be absent in 25g of product.

The results are presented as either Detected or Not Detected – for example, ND in 25g.

Presence/absence testing should not be used if wishing to quantify the number of microorganisms in a specified amount of product.

Quantitative analysis

A quantitative analysis of a food sample will provide a count of the number of colony forming units of a particular microorganism.

Quantitative test methods should be used when it is important or useful to know the number or organisms in a given sample.

For example, in the Food Standards Code, ready to eat food products that do not support the growth of Listeria monocytogenes have a limit of <100 cfu/g – in this instance a Presence/Absence test is not appropriate, so a quantitative method is used.

Quantitative methods are also useful for establishing the hygiene of a process. Organisms such as Enterobacteriaceae are used as ‘indicator’ of hygiene standards, as their presence indicates recent contamination with enteric bacteria. High numbers in ready to eat manufactured food products are undesirable and should entail further investigation.

Stationary phase

Following the log phase of the Microbial growth curve, the stationary phase occurs when the microbes are in stasis – that is, the growth rate and death rate are equal. This occurs when a growth limiting factor is present, such as the depletion of nutrients in the food.

Temperature

All bacteria, moulds, and yeasts have an optimum, maximum, and minimum temperature for growth. These temperatures can vary among different species of microbes. Therefore, environmental temperature not only impacts the rate of growth of microbes but can determine which microbial species thrive. A temperature difference of only a few degrees may favor the growth of an entirely different population of microbes. Below approximately 5°C proliferation of spoilage microbes is slow, and growth of most pathogenic microbes stops.

Listeria monocytogenes (Lm), a bacterial pathogen of concern in many ready-to-eat products, is a notable exception. While Lm grows optimally at temperatures in the range of 30 to 37°C, it is capable of growing at a temperature as low -0.4°C. Lm’s rate of growth at that temperature may be slow, but can still be significant as will be discussed in a later module. At temperatures above 60°C most microbes begin to die, although the time needed for cell destruction at a particular temperature will vary for different species of microbes and may depend on other environmental factors such as humidity. In food processing, the temperature range of 5 – 60°C is commonly referred to as the danger zone, because the optimum, maximum, and minimum temperature for growth of most microbes will fall somewhere within that range. However, it is important to note that time is a major factor associated with the rate of growth at a particular temperature. For example, depending on other factors, the rate of growth of many pathogens may be extremely slow in the 5 to 10°C temperature range.

Time

Permitting sufficient time for microbes to adapt to their environment (lag phase) is necessary before they can enter the rapid growth phase (log phase). The doubling time for most bacterial species is between 10-30 minutes under optimal conditions for growth. Generally doubling times in this range would only occur under ideal laboratory conditions. Bacteria would grow much more slowly in meat and poultry products, especially if those products are properly handled and stored. Allowing the temperature of meat and poultry products to remain in the danger zone for a sufficient period of time will promote significant proliferation of microbes and microbial toxins. In addition, time may be a factor involved in how well certain microbes adhere to the surface of a meat or poultry product. For example, it may be more difficult to eliminate bacteria from the surface of a carcass by washing or with antimicrobial sprays the longer the bacteria are allowed to remain on that carcass before washing or spraying.

Viruses

Viruses are much smaller than bacteria. They are too small to be seen with a standard light microscope. An electron microscope is necessary to see viruses. These microbes are not true living organisms. They are composed of genetic material—either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)—enclosed in a protein coat. A virus must invade a living host cell in order to replicate. Once inside the host cell, the viral genetic material directs the host cell’s “machinery” to make more virus particles, which interferes with normal host cell function and may result in destruction of the host cell.

Water Activity (Aw)

The availability of water in a food (referred to as water activity, or Aw) is an important factor for microbial growth. Nutrients for microbial growth must be in a soluble form for microbes to utilise them. Generally, bacteria have the highest Aw requirements, moulds have the lowest, and yeasts are intermediate. It is important to note that Aw is not necessarily equivalent to measures of moisture content (e.g., Moisture Protein Ratio or MPR) in a product. Most moist food products will have greater water availability to support microbial growth than dryer food products, but there are exceptions. For example, some processing methods might incorporate certain chemical ingredients (e.g., salt) that bind to free water and in sufficient concentrations significantly reduce Aw – limiting the growth of some microbes.

Microorganisms can grow only when conditions are favourable – with Aw, there are lower limits for growth for all bacteria, yeasts and moulds.

The table below is a guide to which microorganisms are able to grow at specific Aw levels and which foods are commonly associated with the specific Aw.