RESPONSE OF MICROORGANISMS USED IN ASSAY
In the study of microorganisms, microbes can also be used as reagents to evaluate or determine quantitatively, the presence or absence of certain chemical compounds in other products, foods or materials. This field of microbiology which uses microorganisms as reagents for the quantitative determination of certain chemical compounds or substances is generally known as analytical microbiology.
The response or reaction of microorganisms when used in assays is an important aspect of analytical microbiology. Microorganisms react or respond differently when used or introduced into certain environments or medium. Measurements of microbial response in an assay can be used to determine the presence or absence of a given microbe and/or harmful chemical substances in the product being assayed or evaluated. Microbes are metabolizing entities, and they produce various responses in their environment. These responses coupled with the metabolic profiles of the microbes can be used to identify or inform of their presence, absence or activity in any environment.
Microbes used as reagents in microbiological assays may be called microbial reagents. The selection of microorganisms that can be used as a reagent is important to the success of any microbiological assay. Using the wrong microorganism, especially those that cannot be used as a reagent will result in the failure of the assay. Not every microorganism can be used for microbiological assay. Microorganisms used as reagents for microbiological assays must meet certain criteria before they can be qualified to be used for microbiological assay or examination. Microbes used as microbial reagents in assaying techniques include bacteria, fungi (yeast), protozoa and viruses. However, bacteria are mostly used because of its fast growth and amenability to various conditions.
Ideal test microbes used as microbial reagents in microbiological assays must possess some unique features. And these features are outlined as follows:
- The microbe must be easily cultivated.
- It must be sensitive to the substance being assayed.
- The microbe should have or produce some metabolic function or response that can be measured.
- It must not be easily susceptible to variation in sensitivity or phase(s) of the assaying technique.
- The microbe should have specificity, if possible.
- It should not be pathogenic, as much as possible. A non-pathogenic microbe is often preferable in assaying technique. However, when assaying for the efficacy of antimicrobial agents, a pathogenic microbe can be most preferable in such an assay. Pathogenic microbes may put the lives and safety of the scientists, researchers or technicians (carrying out the assay) at risk.
TYPES OF MICROBIAL RESPONSE
The type of microbial response measured in an assay or during an assay is usually determined by the type of substance or product being assayed for. In principle, the substance or product being assayed should be able to bring about a unique chemical reaction or biochemical effect on the metabolism of the microbe(s) used in the assay. Therefore, the type of response measured in the assay is also dependent on the type of biochemical effect that the substance being assayed causes on the microbe’s metabolism. There are basically two (2) types of microbial responses exhibited by microbes used in assays: 1. Growth response 2. Metabolic response
Growth response: Microbes are living organisms, and they can grow either in size or cell mass. They respond in different ways when introduced into certain environments. If the organism grows, it is said to be viable. But those that fail to grow may be regarded as dead cells. A handful of microbiological procedures used for analysis depend on the growth response of the microbe. The growth response can either be positive or negative. The growth response is positive if the microbes respond to the substance being assayed with increased growth. On the other hand, the growth response is negative if the microbes fail to grow in the presence of a particular substance or material being assayed. Microbial growth rate or lack of growth can be measured by: simple microbial counts (e.g., viable counts, total counts), use of haemocytometer, turbidity measurement or turbidimetric method, bioluminescence, use of spectrophotometer (to measure optical density), and by the use of microscopy and other growth rate measuring techniques.
Metabolic response: Metabolic response is another type of microbial response aside the growth response that is measured in microbiological assay. Microbes react differently to certain materials or substances, and their ability to do so can be used in evaluating their response in an assay. Microbes produce metabolic products that can be measured. They can also have a change in their function, which is also measurable. The metabolic response of microbes can either be positive or negative. Examples of measurable metabolic responses in microbes include: pigment production, haemolysis of red blood cells (RBCs), acid production, oxygen uptake, carbondioxide production, antiluminescent reaction, nitrate reduction or reduction of nitrates, sterilizing or curing of the growing hyphae of fungal mycelium, and inhibition of spore formation or germination.
METHODS USED FOR MICROBIOLOGICAL ASSAYS
The methods used for microbiological assays include:
- Diffusion methods: This method is used to determine the onward dispersal or flow of a chemical reactant or substance from one point to another. This can be seen in antibiotic susceptibility disks used for antibiogram – in which there is diffusion of antimicrobial compounds from the disk into the surrounding culture environment where the microbe or bacteria is growing.
- Turbidimetric methods: This method is used to measure or determine microbial cell concentration or growth – which is usually seen as turbidity. It is best for measuring microbial response in broth cultures, solutions or suspensions. The more bacterial cells there are in a suspension, the greater will be its turbidity or cloudiness. However, the less bacterial cells there are in a suspension, the less will be its turbidity. Turbidometry is the technique that is used to measure the degree of turbidity. Instruments used for measuring turbidity include: spectrophotometer, nephelometer and calorimeter.
- Metabolic response methods: In metabolic response methods such as bioluminescence, the light-emitting capability of certain insects (e.g., fire flies) and some luminous bacteria (i.e., bacteria engineered to emit light) are used.
- Gravimetric methods: The gravimetric methods are techniques through which the amount of an analyte (the ion being analyzed) can be determined through the measurement of mass. It depends on comparing the masses of two compounds containing the analyte.
DETERMINATION OF MICROBIOLOGY QUALITY OF FOOD, BEVERAGES AND OTHER INDUSTRIAL PRODUCTS
Food, beverages and other industrial products such as antibiotics and other pharmaceutical agents are intended for internal or in vivo usage. These products are mainly consumed by humans or animals as either food or medication. And it is vital that such substances entering the body are of good hygiene, quality and safe for consumption. Food products and other materials or substances intended for human or animal usage especially in vivo should be free from contaminants and pathogenic organisms or substances capable of destabilizing the normal physiological and metabolic functions of the human or animal host consuming it. This is why the determination of the microbiology quality of food, beverages and other industrial products such as antibiotics and water is important for virtually all industrial processes involved in their production. The presence of contaminants and pathogens in these products impact consumer acceptability of taste, texture, aroma, and other perceptions associated with any of the products.
Importance of dilution: When the number of microorganisms in a sample (food, beverage, pharmaceutical) is large or the sample is suspected to be heavily contaminated, it is important to perform serial dilution in order to enumerate fully the number of bacteria present in the sample. To fully enumerate the number of microorganisms in a food sample, it is necessary to prepare serial dilutions of the food samples or food homogenate to be analyzed. And this also applies to other industrial products including antibiotics, beverages, and other pharmaceuticals to be examined for microbial load. Serial dilutions are usually done using some specific solvents or diluents that have little or no chemical effect on the sample to be analyzed. Examples of diluents to be used for serial dilution include normal saline, peptone saline solution or distilled water. Generally, serial dilutions are prepared from the sample homogenate or whole sample (as the case may be) by adding 1 mL of sample homogenate to 9 mL of diluent, and this process can be repeated several times until the required endpoint is reached.
General microbiology techniques used for enumerating or determining the microbiology quality of food, beverages, and other industrial products such as antibiotics and other pharmaceuticals
- Membrane filtration technique: This technique is suitable for enumerating microbes including bacteria in water, beverages, and other liquid food products. Membrane filtration technique can be used to analyze large sample sizes. It uses membrane filtration equipment that contains membrane filters with specific pore sizes. The samples are usually passed through the membrane filtration equipment already containing a membrane filter. After passing the sample or diluted sample through the membrane filtration equipment, the membrane filter is aseptically removed using sterilized forceps, and then it is placed on selective culture media and incubated at the required temperature for bacteria enumeration. Serial dilution of samples can be performed prior to membrane filtration technique – if the sample(s) is likely to contain large numbers of microorganisms.
- Culture technique: Culture technique include dip slide culture, pour plate,
- Dip slide culture technique: This technique is used for estimating the numbers of bacteria in liquid food products. It can also be used to estimate bacteria numbers in food homogenates (i.e., solid food that have undergone some levels of dilution using normal saline or peptone saline solution).
- Pour plate technique: Pour plate technique is most suitable for liquid food samples or food homogenates. Samples may be diluted depending on its extent of contamination. In this technique, an aliquot of the sample or dilution (1 mL) is aseptically placed on a Petri dish, and a specific volume of the molten clear culture medium (e.g., 15 mL) is also aseptically added to the same Petri dish. The culture media plates (containing both the sample and molten agar medium) are mixed well by rotating the plate in a vertical, clockwise, horizontal and anticlockwise direction. All plates are incubated based on the temperature condition of the suspected organism. The culture medium used for pour plate technique should be as clear as possible in order to allow for the counting of bacteria colonies that have grown below the surface of the medium.
- Spiral plate technique: Spiral plate technique is suitable for food homogenates or food products. This technique uses a spiral plater which dispenses a specific volume of the diluted or liquid food sample on the surface of a rotating culture media plate. Unlike the pour plate technique, all food particles in the liquid food samples are allowed to settle before carrying out the spiral plate test. This is necessary to avoid food particles blocking the stylus portion of the spiral plater. The stylus is the portion of the spiral plater from where the liquid is dispersed from, and onto the culture media plate. The stylus should be properly disinfected using 2-5 % sodium hypochlorite solution or bleach, and then rinsed in sterile distilled water before using the spiral plater. Culture plates are incubated as appropriate for the organisms sought for; and bacterial count is performed later to enumerate the microbes.
- Surface drop technique: Surface drop technique is also suitable for food products or food homogenates. This technique is performed by starting with the highest dilution of the sample. The samples should be mixed well using a vortexer or vortex mixer. A given volume of the liquid should be drawn using an automatic pipettor and sterile tip, and the aspirated liquid should be dispensed as a drop onto the sector of two culture agar plate. The process is then repeated for other plates to produce more sectored plates. All plates should be incubated as appropriate for the organism being sought for. And the colonies of bacteria on all sectors containing 30 or fewer colonies per drop should be counted and recorded.
- Surface spread plate technique: Surface spread plate technique is also used for food products and food homogenates. Two plates should be prepared at least for each dilution of the food sample to be analyzed. An aliquot of about 0.1-0.5 mL of the test dilution sample should be transferred to the surface of a culture media plate using automatic pipettor and sterile pipette tips. The inoculum should be spread across the entire surface of the plate using a sterile spreader without touching the sides of the culture plate. The plates should be incubated as appropriate for the organisms being sought.
- Most probable number (MPN): The MPN technique can also be called multiple tubes method. MPN is suitable for food products or food homogenates. MPN is usually based on the probability of finding bacterial growth after the culture of successive dilutions of the food sample in a liquid culture medium. There are three stages of the MPN test, and they are:
- The presumptive test
- The confirmed test
- The completed test
MPN cannot be used for total microbial or bacterial count on a sample including food, water and beverages. They are used for the enumeration of specific microorganisms or groups of microorganisms including Escherichia coli and coliforms or some members of the Enterobacteriaceae family.
Other methods used for enumerating or determining the microbiology quality of food, beverages, and other industrial products
- Surface contact methods: In surface contact methods, the surface of a sterile agar is used to make contact with the test surface. The surface contact methods are available as dip slide method, agar sausage, and surface contact plates. These plates usually come in commercially available forms.
- Surface swab methods: It is used for surface swabbing of a known area.
- Membrane slide culture methods: Membrane slide culture technique is a surface testing technique.
- Rinse methods: This method is used for enumerating fruits and vegetables.
- Bottle rinse and plate count methods: This method is used to determine the effectiveness of milk bottle washing.
Microorganisms are ubiquitous and they are found in food and food products, especially when aseptic measures are not properly imbibed during food processing, storage and distribution. Contaminants including spoilage organisms and pathogens can also be found in beverages and medications if proper aseptic measures are not imbibed during their production. Food safety and the safety of other products meant for human consumption especially, is an important issue around the world particularly in the developing countries – where persistent hygienic practices may not be imbibed by some food handlers, food producers and industries involved in food production. It is important to enumerate and determine the occurrence of food spoiling microbes as well as food borne pathogens in food or food products so that the quality and safety of food can be guaranteed for the consumers. As an analytical microbiologist, you can work in government establishments where your role may be, not just to determine the safety of food and food products, but to help the government in regulating and enforcing legislation on food safety and food hygiene in your country or elsewhere.
ENUMERATION OF MICROORGANISMS IN FOOD, BEVERAGES AND OTHER INDUSTRIAL PRODUCTS
There are several methods available for the enumeration of microorganisms in food or food products. However, the type of enumeration method(s) to be used is usually dependent on several factors including:
- Type of food sample (solid, liquid or semi-solid).
- Characteristics, including the physiological state, of specific microbe sought.
- Lower limit of enumeration required.
- Purpose of the enumeration or examination.
- Time available for the analysis.
In summary, the enumeration of microorganisms in food or food products, beverages and other industrial products is mainly geared towards achieving the following important points.
- Estimation of the viable (colony) count (colony forming units, cfu/ml).
- Estimation of the number of Escherichia coli and other members of the Enterobacteriaceae This is because the presence or absence of E. coli and/or members of the Enterobacteriaceae family in the food or food product is indicative of the standard of hygiene of the food, especially during its processing or preparation. E. coli and members of the Enterobacteriaceae family are generally known as indicator organisms. Indicator organisms are organisms that may indicate or signify the evidence of contamination or pollution of a food product or water, usually by faecal origin. E. coli and coliform bacilli or members of the Enterobacteriaceae family are typical examples of indicator organisms.
- Detection of food spoilage organisms. Detecting food spoiling organisms in food will help to determine the shelf life of the food.
- Detection of pathogenic microorganisms. Presence of pathogens in food can cause food borne diseases or infection. And this is why food spoiling organisms and pathogens should be detected in order to contain and assuage their possible implication in disease outbreak or spread.
HAZARD ANALYSIS CRITICAL CONTROL POINT (HACCP)
Hazard analysis critical control point (HACCP) is an internationally recognized food safety system that is employed in the production line of food and food products in food processing industries to ensure food safety, food hygiene and good food quality. It is a quality assurance technique or system that is used especially in food processing and production industries to evaluate the possible hazards or risks that are eminent in the entire manufacturing process of food and food products so that sustainable measures could be put in place to contain and minimize them. HACCP is mainly made up of seven (7) steps or principles that set the foundation for ensuring hygiene conditions and requirements by food companies in the production lines of food (Figure 1). HACCP is a detailed assessment of the production process and control of potential hazards at certain critical points in the production line of a given product (e.g. food).
HACCP technique identifies risks in food production, evaluates these risks and hazards and controls all the possible hazards that generally affect food safety and hygiene. In HACCP, the personnel’s involved in the production, testing techniques of end products, quality control of the production process and the maintenance of a hygienic production environment are all covered. The HACCP technique is a key part of the GMPs of food industries because it provides the basic operating and environmental conditions necessary for the production of hygienic, suitable and safe food. Most of the microbiological tests carried out in food production plants are mainly designed to detect the presence or contamination of food by some non-specific organisms; and majority of these tests look out for gas production amongst other factors that are key indicators for the presence of contaminating microorganisms in the food under test.
The “hazard” in this case generally refers to factors which would adversely affect the entire process as well as the end product if not avoided at an early stage. This is only possible after carrying out a detailed assessment of known hazards of the production process including those associated with the personnel and raw materials used for the production. After this, the identification of critical control points (CCPs) is another step to be taken in the production process. The CCPs generally refers to specific stages of the production process at which interventions can be taken or implemented in order to minimize or avoid completely the negative effects of particular risks. At the CCPs, the production process is routinely and carefully monitored; and this is mainly aimed at ensuring that the entire process proceeds in such a manner that will avoid contamination and ensure the production of quality products aside other important factors.
Figure 1: Illustration of the processes involved in HACCP.
Nester E.W, Anderson D.G, Roberts C.E and Nester M.T (2009). Microbiology: A Human Perspective. Sixth edition. McGraw-Hill Companies, Inc, New York, USA.
Pelczar M.J Jr, Chan E.C.S, Krieg N.R (1993). Microbiology: Concepts and Applications. McGraw-Hill, USA.
Prescott L.M., Harley J.P and Klein D.A (2005). Microbiology. 6th ed. McGraw Hill Publishers, USA.
Talaro, Kathleen P (2005). Foundations in Microbiology. 5th edition. McGraw-Hill Companies Inc., New York, USA.
Thakur I.S (2010). Industrial Biotechnology: Problems and Remedies. First edition. I.K. International Pvt. Ltd. New Delhi, India.
Dictionary of Microbiology and Molecular Biology, 3rd Edition. Paul Singleton and Diana Sainsbury. 2006, John Wiley & Sons Ltd. Canada.
Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of microorganisms. 12th edition. Pearson Benjamin Cummings Publishers. USA. Pp.795-796.