Methods for controlling microbial contamination

Transcription

1 Unit 2: Industrial Microbiology. 23 Methods for controlling microbial contamination Why could contamination of culture media or equipment in industrial microbiological processes by other microorganisms be a problem? Before microbial cultures are set up, all equipment is sterilised, so that no contaminants remain. The equipment also has filters to prevent entry of contaminants during the fermentation process. This is because contaminants may: produce other metabolites that could be toxic or cause spoilage compete with the required microorganisms for nutrients or oxygen and reduce their growth and hence the product yield infect the required microorganisms, for instance, bacteriophage viruses infect and kill bacteria. If contamination occurs then considerable time and money is wasted while the fermentation process is halted, fermenters are emptied and all equipment is sterilised and then cooled to allow a new batch to be set up. On successful completion of this topic you will: understand the methods for the control of microbial contamination (LO3). To achieve a Pass in this unit you will need to show that you can: assess the effectiveness of physical and chemical methods of reducing microbial growth (3.1) assess the effectiveness of the types of chemicals used (3.2). 1

2 Table 2.3.1: Features of some physical methods of microbial control. 1 Physical and chemical methods for controlling microbial growth Physical methods In industrial microbiology heat and filtration are the most widely-used physical methods, although in some cases radiation may be used. Other methods such as cold temperatures, osmotic effect and desiccation are used in the food industry. Table summarises the features of some physical methods of microbial control. Method Mode of action Effectiveness Some examples of use dry heat direct flaming incineration hot air sterilisation oxidation reduces contaminants to ashes and carbon dioxide oxidation can use an oven at 170 C for 2 hours all give very effective sterilisation inoculating loops waste materials glassware and instruments moist heat autoclaving boiling of freeflowing steam tyndallisation (fractional sterilisation) denatures proteins uses steam under pressure to give a temperature of 121 C for minutes denatures proteins denatures proteins intermittent sterilisation, involves 3 x heatings at 100 C for 30 minutes, interspersed with a day at 30 C, to allow endospores to germinate so the next heat treatment can kill the resulting vegetative cells kills vegetative bacterial cells and endospores kills bacterial, viral and fungal contaminants within 10 minutes less effective for bacterial endospores this kills endospores, some of which can withstand temperatures of 100 C for 20 hours microbial nutrient media, dressings, overalls, equipment glass and metal equipment and pipes used in the food industry and may be used to sterilise nutrient media if no autoclave is available pasteurisation kills by denaturation involves heating to 63 C for 30 minutes or to 72 C for seconds kills only pathogenic bacteria used for milk, cream, milk products (such as cheese and yoghurt), wine and beer filtration membrane filters made of cellulose acetate or nitrocellulose have small pores to mechanically remove microorganisms pore sizes of µm exclude bacteria (and larger microorganisms such as protoctists and fungi) pore sizes of 0.01 µm exclude viruses inlets on fermenters air inlets of laminar flow cabinets drinking water may be filtered radiation ionising radiation, such as X-rays and gamma rays UV radiation damage or destroy DNA sterilise by producing heat and may also damage DNA effective and do not produce heat radiation is not very penetrating can be used for plastic and other thermolabile substances used to sterilise pharmaceuticals and medical and dental supplies and plastic Petri dishes UV light used inside laminar flow cabinets 2

3 Unit 2: Industrial Microbiology Key terms Sterilisation: Removal or destruction of all forms of microbial life, including endospores, on an object or material. Vegetative cells: Metabolically-active bacterial cells, not containing endospores. Endospores: Dormant state in some bacteria to overcome adverse conditions. The cell contents become metabolically inactive and are enclosed in a protective coat, inside the cell wall. These can survive high heat for long duration. Figure shows an industrial autoclave used to sterilise instruments and Figure shows milk processing. Milk is filtered to remove solid impurities and pasteurised to kill pathogenic microorganisms. Figure 2.3.1: Industrial autoclave used to sterilise instruments. Figure 2.3.2: Milk processing. Milk is filtered to remove solid impurities and pasteurised to kill pathogenic microorganisms. 3

4 Table 2.3.2: Features of some chemical methods of microbial control. Chemical methods Antiseptics and disinfectants are chemical substances used to prevent contamination. Several factors influence the efficacy of these chemicals, such as: concentration length of exposure of microorganisms to it environmental temperature at which they are used type of microorganism on which they are used spore-forming bacteria and bacteria with slime capsules are particularly resistant to chemicals (as they are to heat) surface on which the microorganism is living chemicals may combine with an organic matrix and their efficiency is then reduced. Table summarises the features of some chemical methods of microbial control. Method Mode of action Effectiveness Some examples of use gases ethylene oxide formaldehyde gas ozone denatures proteins inactivates proteins oxidation very effective sterilising agent, especially where heat cannot be used very effective sterilising agent, especially where heat cannot be used corrosive and irritating but kills viruses and bacteria in a short time and prevents regrowth of microorganisms used to sterilise spacecraft such as for moon landings fumigation disinfects waste water more effective than chlorination liquids alcohols aldehydes dyes halogens denature proteins and dissolve lipids of cell membranes inactivates proteins interfere with nucleic acids oxidising agents and denature proteins 70% ethanol is effective for killing bacteria and viruses but does not kill endospores effective sterilising agents effective at killing bacteria effective sterilising agents cleans skin before injections although most microbes are removed by the wiping action; cleans work surfaces often used in combination with another antiseptic formalin (solution of formaldehyde) used in embalming glutaraldehyde used to sterilise medical equipment crystal violet (aniline dye) flavines (acridine dyes) used in antiseptics iodine used as antiseptic for livestock and wounds; chlorine gas used to disinfect water; chlorine compounds (e.g. bleach) used to disinfect dairy equipment, eating utensils and glassware Continued on next page 4

5 Unit 2: Industrial Microbiology Method liquids continued phenolics, e.g. carbolic acid, cresol (Lysol ) hexachlorophene chlorohexidene thymol hydrogen peroxide surfactants soaps and detergents solids salts of heavy metals such as silver, zinc, copper and mercury Mode of action disrupt cell membranes and denature enzymes Effectiveness very effective microbicide disrupts cell membranes and denatures enzymes disrupts cell membranes disrupts cell membranes oxidation reduce surface tension, disrupt cell membranes, inactivate enzymes coagulate proteins Key term Fungicide: Agent that kills fungi. effective and persistent bactericide fungicide effective as disinfectant for surfaces but poor antiseptic as blood contains the enzyme catalase that breaks down hydrogen peroxide to water and oxygen bactericidal, virucidal, fungicidal, fast acting, non-corrosive and nontoxic effective against bacteria but limited effectiveness against fungi Some examples of use carbolic acid used by Lister to pioneer antiseptic surgery but causes skin irritation all chemical sterilising agents are compared to phenol to describe their effectiveness surgical scrubs mouthwashes disinfectant skin cleansing, sanitising equipment in food and dairy industries; antiseptics for skin, instruments and utensils and rubber goods used in wound dressings nanoparticles of silver in bandages are small enough to enter bacteria and are very effective bactericides Figure shows some pipes in a brewery being inspected. They carry liquids from the fermenting vats to the bottling line and must be sterilised between batches. Figure 2.3.3: These pipes in a brewery are being inspected. 5

6 Key term Phenol coefficient: A measure of how effectively a chemical compound destroys bacteria in relation to phenol. Take it further Although phenol is no longer widely used as a disinfectant it is still the standard used for comparison of the effectiveness of other disinfectants. Each chemical is given a rating called the phenol coefficient, which indicates its effectiveness. Three test organisms are used: Staphylococcus aureus (Gram positive), Salmonella typhi (Gram negative) and Pseudomonas aeruginosa (Gram negative and resistant to antimicrobials) are grown in broth cultures under standard conditions. They are then exposed to the test chemical for a specified time, before being plated onto nutrient culture media plates and incubated to see if there is any microbial growth. If the test chemical has to be used at a greater concentration or a longer time than phenol to get the same results, then the phenol coefficient is less than 1. It is less active than phenol. If the test chemical is effective at a lower concentration or needs to be used for a shorter time than phenol to kill the microbes, it has a phenol coefficient of greater than 1 and is more active than phenol. For example: Disinfectant Brand X at a dilution of 1:100 has the same effectiveness as phenol at a dilution of 1:50 for killing bacteria on a work surface. The phenol coefficient of Brand X is 100/50 = 2. It is twice as effective as phenol. Disinfectant Brand Y, diluted 1:20, is as effective as phenol diluted 1:50. Calculate the phenol coefficient of Brand Y. Which brand, X or Y, is best to use to disinfect this work surface? Activity: Flow chart Make a large flow chart to give an overview of the physical and chemical methods used to control the growth of microorganisms. Activity: Plan an investigation to compare heat treatment times and temperatures to kill three species of microorganism You have access to test tubes, water baths set at a range of temperatures, Petri dishes with nutrient agar, Bunsen burner, inoculating loop, incubator, colony counter and cultures of Escherichia coli (E. coli, G negative bacterium), Staphylococcus albus (G positive bacterium) and Saccharomyces cerevisiae (yeast). Plan an investigation to find out the temperature and length of duration of exposure that is needed to kill each of these organisms. List the equipment you will need. State your aim and a hypothesis to test. State the independent variables and the dependent variables. Describe the confounding variables and explain why and how you can control them. If possible, carry out your investigation and write a report of your findings. 6

7 Case study: University Researcher Anna Jansen carries out research into antibacterial properties of substances such as essential oils at a university in the Netherlands. Her findings are published in the International Journal of Food Microbiology. Her work is important in finding new antibacterial substances that can be safely added to food. It has been estimated that up to 30 per cent of people in developed countries suffer from a foodborne disease each year. With reductions of salt in processed foods and the preference of many people for not having additives in food, more green products that are considered acceptable may be needed to restrict bacterial growth in food products. Essential oils are aromatic liquids from various parts of plants, usually obtained by steam distillation. Of the 3000 essential oils known, about 300 are commercially important mainly for flavouring. However, many of them (as well as garlic and many spices, e.g. turmeric) have antibacterial properties. Some are used in dental root canal sealers, antiseptics and in feed supplements for lactating sows and weaned piglets. Some natural food preservatives are already marketed and contain oils of sage, rosemary and citrus. Extracts from coriander, mint, mustard, cinnamon, oregano, eucalyptus, cloves, sage, rosemary and thyme have antibacterial chemicals such as thymol, cymene, carvacrol, citral, geraniol and terpinene. Anna uses the disc diffusion method, agar well method and time-kill analysis to investigate antibacterial properties of essential oils that she extracts from plants such as tea tree. She tests the extracts on bacteria such as E. coli, Salmonella typhimurium, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. Once antibacterial action has been shown in vitro, the oil needs to be tested when in food, where a high fat content can reduce the antibacterial effect as the oils dissolve in the food fats and do not reach any bacteria present. Therefore the required dose in vivo may be greater than the in vitro dose. The storage conditions of the food can also affect effectiveness of the essential oils, so many variables are involved in this research. Essential oils may act by damaging cell membranes and their proteins, breaking down cell walls causing leakage of cell contents, and disabling the proton-motive force used by motile bacteria. Sometimes two oils can work synergistically and sometimes they may work antagonistically. What does working synergistically mean? Activity: Investigating antibacterial properties Plan and carry out an investigation to compare the effectiveness of bleach, hydrogen peroxide, a skin antiseptic, and a disinfectant at preventing the growth of three types of bacteria. Alternatively, extract one or more essential oils from plant matter and investigate their antibacterial properties. You can use the well method in solid nutrient agar or the disc diffusion method and measure zones of inhibition, or the time to kill exposure method, involving making serial dilutions of each disinfectant and exposing bacteria to them for 10 minutes, then plating them out, incubating and counting colonies to measure growth. Write your plan and include: clear statement of the aim of the investigation; equipment needed and justification for its use; clear method; risk assessment and precautions to be taken; how you will collect and analyse the data. 7

8 Checklist In this topic you should now be familiar with the following ideas: processes involving making products by utilising microorganisms provide suitable conditions for contaminant organisms to grow and spoil the product or pose a threat to the health of personnel or consumers of the product there are strict guidelines about contamination control (see Topic guide 2.5) there are physical and chemical methods to control microbial growth physical methods include moist and dry heat, pasteurisation, filtration and exposure to short wavelength ionising radiation chemical methods include gases such as ozone and formaldehyde; liquids such as ethanol, phenol, halogens, hydrogen peroxide and surfactants; and solids such as salts of heavy metals. Further reading Annets, F. (2010) BTEC Level 3 National Applied Science Student Book, London: Pearson Education. Cappuccino, J. and Sherman, N. (2013) Microbiology: A Laboratory Manual (10th edition), Benjamin Cummings. Case, C. Funke, B. and Tortora, G. (2012) Microbiology: An Introduction (11th edition), London: Pearson. Prescott, L., Harley, J. and Klein, D. (2004) Microbiology (6th edition), McGraw-Hill Higher Education. Taylor, J. (1990) Microorganisms and Biotechnology, Nelson Thorne. Acknowledgements The publisher would like to thank the following for their kind permission to reproduce their photographs: (Key: b-bottom; c-centre; l-left; r-right; t-top) Corbis: Photoquest Ltd / Science Photo Library 1; Michael Rosenfeld / Science Faction 3b; Shutterstock.com: Robert A Levey Photography LLC 3t; Science Photo Library Ltd: RIA Novosti 5 All other images Pearson Education In some instances we have been unable to trace the owners of copyright material, and we would appreciate any information that would enable us to do so. 8