Biotechnology. Unit 2: Microbiological Techniques. Student Materials [HIGHER] Margot McKerrell. abc. Learning and Teaching Scotland

Biotechnology Unit 2: Microbiological Techniques Student Materials [HIGHER] Margot McKerrell abc

The Scottish Qualifications Authority regularly reviews the arrangements for National Qualifications. Users of all NQ support materials, whether published by LT Scotland or others, are reminded that it is their responsibility to check that the support materials correspond to the requirements of the current arrangements. Acknowledgement Learning and Teaching Scotland gratefully acknowledge this contribution to the National Qualifications support programme for Biotechnology. The advice of Jim Stafford is acknowledged with thanks. First published 2004 2004 This publication may be reproduced in whole or in part for educational purposes by educational establishments in Scotland provided that no profit accrues at any stage. ISBN 1 84399 058 X

CONTENTS Introduction 5 Section 1: Growth limitation and sterilisation techniques 7 Section 2: Culturing techniques 15 Section 3: Identification of micro-organisms 37 Bibliography 41 Appendix: Advice for Outcome 2 45 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 3

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INTRODUCTION This unit introduces you to some of the techniques that are used to study micro-organisms. A micro-organism is any small organism that cannot be clearly seen without the help of a microscope. The microorganisms that you will use to carry out the study of microbiological techniques include bacteria, fungi and viruses. Many biotechnology processes rely on the use of micro-organisms and so it is important that you know how to work safely with them. That is why this unit is included in Higher Biotechnology. These student notes will provide you with the knowledge and understanding that you will need to carry out microbiological techniques. UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 5

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SECTION 1 GROWTH LIMITATION AND STERILISATION TECHNIQUES Growth limitation and sterilisation techniques Some micro-organisms can be harmful to humans as they cause disease. For this reason, techniques have been developed to control the unwanted growth and spread of micro-organisms. It is essential that you have an understanding of these techniques before you start to culture micro-organisms in the laboratory. This is to ensure that you safely culture and contain the micro-organism that you are interested in and do not contaminate yourself, others or the environment. Sterilisation and disinfection Sterilisation is a process that kills all micro-organisms, including endospores, within a material or object. (An endospore is a dormant structure formed from a bacterial cell that can survive extremely adverse conditions, including high temperatures. It can germinate into a bacterial cell if growth conditions become favourable.) Any physical or chemical agent that kills micro-organisms is said to be biocidal. There are several techniques used to sterilise materials and objects and these are discussed below. Autoclaving is a technique that kills micro-organisms using pressurised steam. Under normal atmospheric pressure the highest temperature that steam can reach is 100 C. While this temperature will kill many micro-organisms, it is too low a temperature to kill some endospores. In order to increase the temperature of steam above 100 C, it is pressurised in a closed container called an autoclave. Micro-organisms (including their endospores) are killed at these high temperatures because their enzymes and proteins are denatured, so they are no longer viable. In a laboratory, items are sterilised in autoclaves at 121 C for 15 minutes or at 126 C for 10 minutes. The times mentioned here are average sterilisation times. The time that objects and materials are autoclaved depends on their size as large objects take longer to sterilise. Also, the autoclave should be loosely packed so that the steam can circulate around the objects and so heat them to the correct temperature. If the UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 7

GROWTH LIMITATION AND STERILISATION TECHNIQUES correct temperature or time is not observed, then the items inside the autoclave cannot be guaranteed to be sterile. To ensure that the correct autoclaving procedure has been carried out, autoclave monitors such as Browne s tubes and test strips, are added to the autoclave with the items to be sterilised and then checked at the end of the procedure. In general, these monitors change colour to show that sterilisation has taken place; for example, Browne s tubes change from red to black when they have been sterilised correctly. The autoclave is used to sterilise objects and materials that are heatresistant such as glassware, cloth, rubber, metallic instruments, liquids, paper and heat-resistant plastic. In the laboratory in your school or college, you may use an autoclave to sterilise culture media, scalpels and glassware such as test tubes and conical flasks. Another technique that is used to sterilise materials and objects is the use of dry heat. Dry heat sterilisation takes place in a dry oven that has electrical coils that radiate heat. Dry heat kills micro-organisms by dehydrating them and denaturing their proteins. Higher temperatures are needed when using dry heat compared to using steam. In general, items are sterilised at 160 C for two hours. Dry heat is used to sterilise items that may be adversely affected by steam, such as powders, oils, glass pipettes and metal instruments that may corrode if exposed to moisture. Dry heat is not suitable for plastic, cotton, paper or for solutions that would boil and dry out, such as culture media. Some liquids cannot be sterilised by autoclaving or in dry-heat ovens because the temperatures used in these techniques cause the components in the liquids to denature. Heat-sensitive liquids can be sterilised using filtration. One type of filtration uses a filter that is composed of a mass of fibres. When liquid is passed through this type of filter, any micro-organisms present in the liquid adsorb (stick) to the fibres. Another type of filtration uses a filter that has pores (holes) large enough for liquid to pass through but too small to let micro-organisms through, so they become trapped on the filter. These filters are known as membrane filters. Filtration is used to sterilise solutions containing antibiotics, enzymes and glucose. It is also used to sterilise air, for example before it is pumped into a fermenter. 8 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

GROWTH LIMITATION AND STERILISATION TECHNIQUES Another technique used for sterilisation is gamma irradiation. Items to be sterilised in this way are placed in a machine that emits gamma rays and these items are exposed to the gamma rays for a specific time. Gamma rays kill micro-organisms by causing lethal mutations in the DNA that cannot be repaired. Gamma irradiation is used to sterilise plastics such as Petri dishes and syringes, and surgical gloves. Disinfection is when a chemical agent (disinfectant) is used to destroy micro-organisms but not endospores. Disinfectants do not normally achieve sterility because they do not always kill all micro-organisms present. However, they do reduce the number of micro-organisms to safer levels. In general, disinfectants are used on non-living objects because they are toxic to living tissues. Disinfectants that can be applied to living tissue are known as antiseptics. Some disinfectants are biocidal (look back to page 7 to remind yourself what this term means). Other disinfectants are biostatic. This means that micro-organisms are temporarily prevented from reproducing but they are not killed. When this type of disinfectant is removed, the microorganism can start to increase in numbers again. When using disinfectants, it is important to ensure that they are used at the correct concentration and that they are left to work for the correct length of time. In general, the higher the concentration of a disinfectant, then the faster the micro-organisms are killed. This is shown in the graph below: Figure 1 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 9

GROWTH LIMITATION AND STERILISATION TECHNIQUES Although high concentrations of disinfectant may be more effective at destroying larger numbers of micro-organisms more quickly, high concentrations of disinfectant tend to be more toxic and expensive. For these reasons disinfectants are generally used at the minimum concentration that effectively destroys micro-organisms. Apart from concentration, another factor that affects disinfectants is time. Most disinfectants require time to act on the micro-organisms. Also, the more micro-organisms that are present, then the longer the time needed to destroy them. Many laboratory disinfectants need to be prepared before they can be used. Some liquid disinfectants are diluted by putting a small volume of the disinfectant into a large volume of solvent. Powdered disinfectants are weighed out, then dissolved in an appropriate volume of solvent. In general the manufacturer of the disinfectant provides clear guidelines regarding the most appropriate concentration that should be used and how long it will remain active. In the laboratory, disinfectants are used for a variety of purposes such as swabbing a bench before and after use, for the sterilisation of surfaces, and for the disposal of used instruments such as Pasteur pipettes. There are many disinfectants available and some of the more common ones are discussed below: Chlorine Chlorine kills bacteria, fungi, viruses and endospores. It is an oxidising agent and works by denaturing proteins. It is used in the treatment of drinking water. Chlorine-based disinfectants are used in the home and laboratory for disinfecting surfaces. Phenolics Phenolics kill bacteria, fungi and some viruses. They work by dissolving cell membranes and denaturing proteins. They are found in carbolic soaps and some laboratory disinfectants. Alcohol Alcohol kills bacteria, fungi and some viruses (if left long enough). It works by dissolving cell membranes. It is used in the laboratory for swabbing benches and disinfecting instruments such as scalpels. 10 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

GROWTH LIMITATION AND STERILISATION TECHNIQUES Multi-oxidising detergents These destroy a wide variety of micro-organisms. They work by denaturing proteins. They are used in the laboratory for a variety of purposes such as swabbing benches and in disposal containers for used glass pipettes. Risk assessment and coping with spillages When working with micro-organisms, it is extremely important that you are aware of the possible hazards and risks associated with them. A hazard is the danger or harm that a micro-organism may cause to you. A risk is the probability or likelihood that you will be harmed by the micro-organism. Therefore, before working with micro-organisms, a risk assessment is carried out. To do this, you look at the procedures that you are intending to carry out using a micro-organism and then assess the potential risks associated with these procedures. Risk assessments generally fall into one of three main categories: simple, generic and novel risk assessments. Simple risk assessments involve the use of micro-organisms and procedures that pose a familiar hazard and there are well-known control measures available to minimise the risks associated with them. Generic risk assessments involve the use of an authoritative source of advice or code of practice for the safe handling of a micro-organism when using a particular procedure. Novel risk assessment is the procedure adopted when you come across a hazard that is unfamiliar to you and is not covered by the generic code of practice. In this case, you must research the potential risks from first principles. After you have identified the hazards and potential risks associated with working with micro-organisms, it is important to use appropriate control measures to reduce the risks to a minimum. Some control measures that are taken are outlined below: Choice of micro-organism. The most appropriate micro-organism with the fewest hazards associated with it is chosen. Micro-organisms should be obtained from known, reputable sources to ensure that you are working with a pure culture. The micro-organism should be UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 11

GROWTH LIMITATION AND STERILISATION TECHNIQUES stored in the correct conditions and be properly labelled with the name of the micro-organism and the date that it was obtained. A log book/record with details such as culture number and when it has been subcultured should be kept. Media selection. Media that are most suitable for the micro-organism should be used, for example selective media rather than generalpurpose media should be used. This helps to prevent the growth of unwanted micro-organisms. Media likely to encourage the growth of pathogenic (disease-causing) micro-organisms should be avoided. (You will find out more about different types of media later on in these notes.) Culture methods. The micro-organism should be grown in the correct conditions such as temperature, ph and with the correct oxygen requirements. For example, do not grow aerobic bacteria in anaerobic conditions as anaerobic pathogens may grow instead. Also, plastic culture vessels are preferable to glass as they are less likely to break if dropped. Choose appropriate handling procedures. For example, try to prevent aerosol formation. Aerosols are water droplets containing micro-organisms that are released into the atmosphere. They can remain in the air for long periods and be a source of contamination. There are many ways to prevent aerosol formation; for example, do not put a wire loop with lots of culture on it directly into the hottest part of the Bunsen burner. Instead, introduce the loop gradually into the flame. Also, aerosols are created when containers of liquid are opened, so take care when opening containers of micro-organisms. Other handling procedures that should be considered are the scale of the operation, the degree of containment and the likelihood of contamination. For all of these you need to look closely at your procedures and ensure that you have sufficient resources to cope with them. Protective equipment. A laboratory coat worn correctly protects your everyday clothes from contamination. Lab coats should be removed before leaving the lab to prevent taking contamination outside of the laboratory. Eye protection should also be worn to protect your eyes from air-borne contamination and chemicals. Gloves can be worn to protect your hands when handling microorganisms and can then be disposed of after use. If gloves are not worn (and they are not always needed!), then cuts should be covered with waterproof plasters. Hands should be washed thoroughly with anti-microbial soap before and after handling micro-organisms. 12 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

GROWTH LIMITATION AND STERILISATION TECHNIQUES Smoking, eating and drinking are not allowed in microbiology laboratories as your mouth and gastro-intestinal tract could be contaminated. Treatment of small-scale spillages of broth Accidents can happen and it is possible that you may spill a live culture of micro-organisms when you are working in the laboratory. If this happens, then it is important to prevent contamination of yourself, others in the laboratory and the laboratory itself. There are several steps that should be taken to treat a small-scale spillage. Firstly, the person disposing of the spill must wear a lab coat, disposable gloves and eye protection. The spill is covered with paper towels and disinfectant is poured around and over the towels to prevent aerosol formation. This is left for at least 10 minutes to ensure that the disinfectant has had enough time to work; then the paper towels are carefully put into a disposal bag and autoclaved. The gloves should be autoclaved too as they may be a source of contamination. If a culture vessel was broken in the accident, it should be put into a solid container or put into two disposal bags and autoclaved. Containment of large-scale spillages Large-scale spillages may happen in industrial processes using large fermenters. There are a number of precautions that are taken to contain spillages from fermenters, so preventing contamination of the environment. In rooms where there are large fermenters, drains do not directly lead to the main sewerage system. Instead they lead to sumps (large containers) where spillages can be decontaminated before being introduced into the main sewerage system. Floors and walls are sealed to ensure that they are waterproof. Doorways are positioned above ground level so that spillages do not leak out into the environment. You have now finished the first part of this unit and you should be familiar with the procedures used to prevent the unwanted growth of micro-organisms. You should be familiar with methods of sterilisation and disinfection; know the difference between a hazard and a risk; be aware of control procedures that you can carry out to minimise the risks of working with micro-organisms; know how to deal with smallscale spillages; and how large-scale spillages of broth are contained. You are now ready to be introduced to some of the techniques that are used to grow micro-organisms. UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 13

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SECTION 2 CULTURING TECHNIQUES Culturing techniques This section of the unit introduces you to the theory behind the techniques that are used to culture micro-organisms. However, you should remember that this unit is a hands-on practical unit and you will be given the opportunity to carry out many of the techniques in the laboratory so that you will become competent in the safe handling of micro-organisms. Your tutor will provide you with the protocols and methods that you need to carry out the techniques safely. While working with living micro-organisms you must develop and use good working/laboratory practices. These practices are important for several reasons: to ensure that you do not contaminate yourself or others in the laboratory, or contaminate the laboratory itself to avoid contaminating the cultures with which you are working to prevent accidentally taking micro-organisms out of the laboratory. Aseptic technique is the name given to all the procedures that are used when working with micro-organisms to prevent contamination. Some of the procedures that are used and the reasons for them are described below. Firstly, it is essential that you prepare yourself in readiness for carrying out practical work with micro-organisms. Long hair must be tied back, hands must be washed with soap and water, cuts covered with waterproof plasters (alternatively plastic gloves can be used) and personal protective equipment such as a lab coat and eye protection must be worn. You must ensure that the sleeves of your lab coat are rolled down and that it is buttoned up to protect your normal clothes from accidental spillages. Next, you must prepare your work space. Work benches used for microbiology must be smooth and non-absorbent so that they do not become contaminated if there is a spillage. If necessary, benches can be made suitable for microbiology work by covering them with nonabsorbent material such as benchcote. Before starting work, bench surfaces are always disinfected to reduce the possibility of contaminating the cultures with which you are working. It is good practice to have a UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 15

CULTURING TECHNIQUES container of disinfectant close to your work space so that apparatus, such as pipettes, can be disposed of quickly and easily without contaminating the environment. A Bunsen burner is placed on the work space. When lit, it provides an updraught that carries air away from the work space, so reducing contamination. It is also used to flame-sterilise equipment such as wire loops and the necks of test tubes and flasks before and after the transfer of micro-organisms. At the completion of work, your work space must be disinfected. Unwanted cultures and apparatus contaminated with micro-organisms must be disposed of properly. Unwanted cultures are autoclaved and then disposed of in the bin, in the case of used agar plates in Petri dishes, or down the sink in the case of liquid broth cultures. Contaminated items of apparatus are first put into disposal containers of disinfectant, then autoclaved. Media preparation and sterilisation Micro-organisms are grown in culture media that contain a range of nutrients necessary for growth. In a laboratory, culture media is contained within Petri dishes, test tubes, bottles and flasks. Prior to being used, all containers are labelled with the type of culture media that they contain and with the date when the culture media was prepared. They may also be labelled with the initials of the person who prepared them. Remember, a Petri dish is labelled on the bottom plate, never on the lid. Culture media is sterilised in an autoclave for the appropriate length of time, depending on the volume of media that is to be sterilised. When sterilising media in an autoclave, it is important that the containers are not filled to capacity, otherwise they may boil over in the autoclave or the container may crack or break. Sterile culture media is poured from one container to another using the appropriate aseptic technique. This includes flaming the neck of the container before and after the transfer of culture media from one container to another. 16 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

CULTURING TECHNIQUES The preparation of plates Culture media can be solidified by the addition of agar. Culture media containing agar becomes molten when it is autoclaved, then as it cools down to 42 C, the agar solidifies. Solid culture media in a Petri dish is known as an agar plate. Agar plates are prepared by adding water to the appropriate quantity of powdered culture media (follow the manufacturer s instructions), autoclaving, cooling to about 50 C, and then pouring aseptically into Petri dishes. When the agar has solidified to a flat, smooth surface, the agar plates are inverted and allowed to dry. This prevents condensation forming on the surface of the agar. The preparation of slopes Solid culture media can also be contained within a universal or McCartney bottle (this is a bottle with a flat bottom and a screw cap that holds about 20cm 3 liquid). These bottles are often used to prepare agar slopes. The bottle is filled with about 15cm 3 molten, sterilised agar media and kept in a tilted position while the agar solidifies, thus forming a slope of agar. The preparation of broths Liquid culture media is known as a broth and is contained within test tubes or conical flasks. These containers are always loosely stoppered to prevent contaminating micro-organisms from gaining entry, but allowing air to enter. Suitability of media for inoculation After culture media has been prepared, it is always examined for its suitability for inoculation. Culture media is examined to ensure that it is free from contamination with no visible signs of growth on solid or in liquid media. Agar plates should be dry, flat and smooth. Slopes should be flat and smooth. Types of media Culture media contains the nutrients in the correct proportions needed by micro-organisms for growth. UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 17

CULTURING TECHNIQUES The nutrients needed by micro-organisms for growth, why the nutrients are needed and the sources of these nutrients in culture media are shown in the following table: Nutrient Why the nutrient is Source of the nutrient needed by the in culture media micro-organism Nitrogen To make proteins Amino acids and and nucleic acids proteins Ammonium ions Nitrate ions Carbon To make carbohydrate, Sugars and amino proteins, nucleic acids acids and lipids As a source of energy Sugars Phosphorus To make nucleic acid Phosphate ions and certain types of lipids Sulphur To make proteins Sulphate ions In addition to nutrients, culture media contain buffers that ensure the ph of the culture media stays the same. This is important because microorganisms grow at optimum ph values and any change in ph may affect the growth of the micro-organism. Some micro-organisms produce acid as they grow which could change the ph of the culture media and so inhibit growth. Buffers help to prevent the acid produced from changing the ph of the media. As mentioned previously, some culture media is solidified by the addition of agar, a polysaccharide obtained from marine algae. It does not add nutrients to the media as few micro-organisms produce enzymes to metabolise it. Agar is molten at high temperatures and solidifies as it approaches 42 C. There are many different types of culture media, but they all fall into two main types, synthetic and complex media. 18 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

CULTURING TECHNIQUES Complex media are those in which the nutrients obtained from the ingredients are not present in defined (known) quantities. For example, some complex media contain peptone, which is a mix of proteins that have been partially hydrolysed (broken down). Peptone is a source of carbon, nitrogen and sulphur but the actual quantities may vary from batch to batch. Other complex media contain yeast or beef extract, which are sources of amino acids, sugars and vitamins. Synthetic media are those in which pure chemical components are added in known quantities. An example of a synthetic medium is Czapek Dox that is used to culture fungi. The quantity of ingredients used to make a volume of 1000cm 3 is shown below: Sodium nitrate 20.0g Potassium chloride 0.5g Magnesium glycerophosphate 0.5g Iron sulphate 0.01g Sucrose 0.35g Agar 12.0g General-purpose media are complex media that are designed to grow a broad spectrum of micro-organisms. Nutrient broth and nutrient agar are examples of general-purpose media that are used commonly in laboratories for the culture of bacteria. Selective media contain ingredients that allow the growth of some micro-organisms in a mixture but inhibit the growth of other bacteria in the same mixture. An example of selective media is mannitol salt agar that is used to select for Staphylococci bacteria. Differential media make it easy to identify colonies of one bacterium from colonies of another bacterium on the same agar plate. An example of differential media is MacConkey agar, which is used to identify human intestinal micro-organisms as a test for faecal contamination (just to confuse you, MacConkey agar is also an example of selective media! This is explained on pages 23 and 24). Isolating and culturing micro-organisms To culture a micro-organism, a sample (called the inoculum) is introduced into a culture medium that provides an environment in which the micro-organism can multiply. The observable growth that appears in the medium is known as a culture. UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 19

CULTURING TECHNIQUES A pure culture is one that contains a single known species or type of micro-organism. This type of culture is most frequently used for the study of micro-organisms in the laboratory. A mixed culture is one that has two or more known, easily differentiated species of micro-organisms. A contaminated culture is one that was once pure or mixed but has since had contaminants (unwanted and unknown micro-organisms) introduced into it. When culturing micro-organisms in the laboratory, it is important to use aseptic techniques at all times to reduce the risk of producing a contaminated culture. Micro-organisms are transferred from one culture medium to another, a process known as sub-culturing, before incubation. The following table shows the four sub-culturing techniques that can be carried out, the culture medium that the micro-organisms are taken from, and the culture medium to which the micro-organisms are transferred: Sub-culturing Culture medium Culture medium that technique that micro-organisms the micro-organisms are taken from are transferred to Solid to solid Agar plate Agar plate or or Agar slope Agar slope Solid to liquid Liquid to solid Agar plate or Agar slope Liquid broth Liquid broth Agar plate or Agar slope Liquid to liquid Liquid broth Liquid broth The inoculum is transferred using either a loop or pipette. A loop can be used in all four sub-culture techniques. A pipette can be used to transfer liquid to solid and liquid to liquid. 20 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

CULTURING TECHNIQUES When transferring micro-organisms from one medium to another, strict aseptic technique must be followed such as having a Bunsen burner lit on the work space, flaming the loop before and after each transfer, using sterile pipettes, and flaming the neck of containers with liquid broth before and after transfer of micro-organisms. One method for transferring micro-organisms onto an agar plate is called the streak plate technique. Micro-organisms can be transferred from liquid broth, from a slope or from an agar plate onto an agar plate by this method. Figure 2 shows the steps involved in streak plating. Figure 2 Firstly a loop-full of micro-organism is smeared across the edge of an agar plate (1). The loop is sterilised by flaming, and cooled. The loop is used to make several streaks (usually three) through the first set of streaks (2). Again the loop is sterilised and cooled and the process repeated a further twice (3) and (4). The number of micro-organisms decreases with each set of streaks, so that by the final set of streaks, individual colonies are produced (4) after the agar plate has been incubated. Growth of micro-organisms Following transfer into a growth medium, micro-organisms will reproduce and increase in number to produce a culture, providing they are given the correct growth conditions. While the culture medium provides the nutrients necessary for growth, micro-organisms also require the correct temperature, ph, level of oxygen and salt for maximum growth. UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 21

CULTURING TECHNIQUES All micro-organisms have their own optimum temperature at which they grow best. Some micro-organisms, such as those isolated from soil, have an optimum ph in the range between 0 C and 25 C. Other microorganisms, such as those that cause disease in mammals, have an optimum temperature in the range from 20 C to 40 C. There are some micro-organisms, which are found naturally in compost, that grow best in the temperature range from 45 C to 60 C. Micro-organisms also have an optimum ph for maximum growth. In general, fungi are acid tolerant and grow best in the ph range 5 to 6. Bacteria prefer to be grown at ph 6.5 to ph 7.5. Different micro-organisms have different requirements for oxygen. Obligate aerobes must be grown in the presence of oxygen whereas obligate anaerobes must be grown in the absence of oxygen. Facultative anaerobes can grow in the presence and absence of oxygen. Micro-organisms that live naturally in a salt environment, such as those that live in salt lakes and seas, must be provided with culture media that is supplemented with salt. Some micro-organisms will not grow unless they are provided with a minimum of 9% salt (that is 9 grams of salt per 100cm 3 medium). Assuming that the micro-organisms are provided with the correct nutrients and growth conditions, growth of the micro-organism in the culture media will occur. Growth is observed in liquid cultures when the broth turns cloudy or turbid. Growth is observed on agar plates by the appearance of colonies. It is difficult to be sure that a liquid culture contains a pure culture of micro-organisms and that it has not been contaminated. It is easier to observe if an agar plate contains a pure, mixed or contaminated culture. (Look back to the beginning of this part of the unit to remind yourself of the differences between a pure, mixed and contaminated culture). If a culture is pure, all the colonies on the plate are identical in colour, shape and size. In a mixed culture there are two or more (depending on the number of different species present) types of colonies with different colour, size and shape. 22 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

CULTURING TECHNIQUES Pure cultures In laboratories, pure cultures are normally used and there are many reasons for this: When studying the characteristics of a micro-organism, other microorganisms should not be present. Otherwise you will not know for certain that it is the micro-organism being studied that is carrying out the reaction. Contaminating micro-organisms use up nutrients in the medium, so there is less nutrient and so less growth of the actual micro-organism being studied. Contaminating micro-organisms may produce substances that prevent the growth of the micro-organism being studied. Pure cultures are also used in industrial fermentation where a commercial product is being made. Contamination in a fermentation process may lead to reduced product being formed (if there is less growth of the micro-organism carrying out the fermentation), or the product may be impure because of the presence of substances produced by the contaminating micro-organism. There are a number of ways that pure cultures can be obtained from a mixed culture, for example by using selective and differential media and by exploiting discrete growth characteristics of the micro-organisms. A selective medium contains one or more ingredients that prevent the growth of certain micro-organisms but not others. These media are important in the isolation of one type of micro-organism from samples containing lots of types of micro-organisms such as those found in faeces, saliva, water and soil. Mannitol salt agar is a selective medium that contains high concentrations of salt. This prevents the growth of most human pathogens (bacteria that cause disease) but the bacterium Staphylococcus grows in this medium, so it is used to select for this micro-organism. MacConkey agar is a selective medium that contains bile salts. This medium can be used to select for bacteria (such as E. coli) that normally grow in the human intestines as they can grow in the presence of bile salts. UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 23

CULTURING TECHNIQUES A differential medium is designed to show visible differences between micro-organisms such as different bacteria producing different colours of colonies. These differences arise from the ingredients of the media and the way that different micro-organisms react to them. Some differential media contain dyes that change colour if the ph changes. MacConkey agar is an example of a differential medium that contains a dye that is yellow if the ph is neutral and red if the ph is acidic. E.coli bacteria produce acid when grown on this agar and so E.coli colonies are red. Salmonella bacteria do not produce acid on this agar, so its colonies are a natural, off-white colour. In addition to using selective and differential media, discrete growth characteristics (such as temperature optimum, ph optimum, and oxygen requirements) of individual micro-organisms can be exploited to obtain pure cultures. For example, if a mixed culture contains aerobic and anaerobic bacteria, then anaerobic bacteria can be isolated from the aerobic bacteria by growing them in the absence of oxygen. Enumerating micro-organisms Enumerating micro-organisms means counting the number of microorganisms in a given sample. Counting micro-organisms is not always easy because of the large number of micro-organisms that can be present, even in a relatively small sample. For example, a small drop of liquid culture of bacteria could contain as many as several million bacterial cells. It is impossible to give the exact number of microorganisms that are present in a sample, so an estimate of the number of micro-organisms is given. A sample with many micro-organisms is diluted so that a smaller number of micro-organisms is present, which is easier to count. The number of micro-organisms in the diluted sample is then multiplied by the dilution factor to give an estimate of the number of micro-organisms in the original, undiluted sample. For example, if a sample is diluted by 10 5 (1 in 100,000) and the number of micro-organisms in the diluted sample is 25, then the original sample has 25 100,000 = 2.5 10 6 microorganisms in it. 24 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

CULTURING TECHNIQUES Generally, dilutions are carried out in a series of sequential steps known as a serial dilution. This is shown in Figure 3: Figure 3 When a 1 cm 3 sample is diluted in 9 cm 3, this is a 1 in 10 dilution. This is shown in Figure 3 where 1 cm 3 is taken from 1 and put into 2. When 1 cm 3 is taken from 2 and diluted a further 1 in 10, the resulting dilution in 3 is a 1 in 100 dilution (1 in 10 1 in 10). A 1 in 100 dilution is also known as a 10 2 dilution. When a 0.1 cm 3 sample is diluted in 9.9 cm 3, this is a 1 in 100 dilution. This is shown in Figure 3 when 0.1 cm 3 from 3 is put into 4. This is a 1 in 100 dilution of a sample already diluted 1 in 100. The sample in 4 has now been diluted 1 in 10,000 (1 in 100 1 in 100). A 1 in 10,000 dilution is also known as a 10 4 dilution. When 0.1 cm 3 is taken from 4 and put into 5, this is a 1 in 100 dilution of a sample already diluted 1 in 10,000. The sample in 5 has now been diluted 1 in 1,000,000 (1 in 100 1 in 10,000). A 1 in 1,000,000 dilution is also known as a 10 6 dilution. When enumerating micro-organisms, there are two counts that can be made: a total count and a viable count. A total count is a count of all living and dead micro-organisms in the sample. A viable count is a count of the living micro-organisms only. UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 25

CULTURING TECHNIQUES Direct methods of enumeration involve counting the number of microorganisms directly, for example by counting the number of colonies on an agar plate or by using a microscope to count the number of cells observed. Indirect counts can be made by growing micro-organisms in liquid broth. As micro-organisms grow, the broth becomes cloudy or turbid. The turbidity is measured using a device such as a colorimeter or a spectrophotometer. Direct counting of micro-organisms using a haemocytometer A haemocytometer is a counting chamber as shown in Figure 4. Figure 4 It is a thick glass slide containing a well in the central section. On the bottom of the well a grid is etched containing squares of known area. Each square is 0.04mm 2. A coverslip is placed over this well forming a chamber of known depth (0.1mm). Thus, the volume of each square is 26 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

CULTURING TECHNIQUES known (0.04 0.1= 0.004mm 3 ). The sample to be counted is placed in the well by placing a drop of the sample at the edge of the coverslip so that it runs into the well and over the grid. The haemocytometer is then viewed using a microscope. The number of micro-organisms in several squares is counted and an average number of micro-organisms is obtained. This number of micro-organisms is then used to calculate the number of micro-organisms in the original sample. Figure 5 shows bacterial cells observed on one large square of the grid. Figure 5 From this diagram, you can see that micro-organisms sometimes lie between one large square and the next. It is standard practice to count micro-organisms on the top and right-hand side of the square, but to ignore the bottom and left-hand side. From Figure 5: 5 micro-organisms are counted in one large square, so there are 5 micro-organisms in 0.004mm 3 sample so, in 1mm 3 there are 5 1 = 1250 micro-organisms per mm 3 0.004 so, in 1cm 3, there are 1250 1000 = 1.25 10 6 microorganisms UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 27

CULTURING TECHNIQUES If the original sample was diluted, the dilution factor must be taken into account, for example if the original sample was diluted 1 in 1000 (10 3 ), then the original sample would contain 1.25 10 6 1000 = 1.25 10 9 micro-organisms per cm 3. Indirect counting of micro-organisms using a colorimeter or spectrophotometer The total number of micro-organisms can also be estimated by indirect counting using a colorimeter or spectrophotometer. The number of micro-organisms in a suspension can be estimated from a turbidity reading on a colorimeter or spectrophotometer. These instruments measure the cloudiness (turbidity) of a suspension of micro-organisms. The more micro-organisms there are in the suspension, the higher the turbidity, so the higher the optical density reading from the instruments. The total number of micro-organisms in a suspension can be worked out from the turbidity reading if a standard curve is available. A standard curve is generated by obtaining the optical density of micro-organisms of known number in a colorimeter or spectrophotometer. This data is plotted to form a standard curve. An example of a standard curve is shown in the graph on the next page (Figure 6). 28 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

CULTURING TECHNIQUES Figure 6 The number of micro-organisms in an unknown sample can be estimated if its optical density is obtained. It is then a simple matter of reading the total count from the standard curve. From Figure 6, it can be seen that if the optical density is 0.5, then the total count is 2.5 10 6 microorganisms per cm 3. Viable count of micro-organisms in a sample A viable count is the number of living micro-organisms in a sample. The commonest method of doing this is to use a plate count. Firstly, a serial dilution is carried out on the sample. The diluted samples are then plated onto an agar plate. After incubation, colonies of micro-organisms appear on the agar plates. Plates with between 20 and 200 colonies are generally counted. The number of colonies observed on suitable plates is then multiplied by the dilution factor to estimate the actual number of colonies in the sample. UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 29

CULTURING TECHNIQUES Figure 7 shows the dilutions that were carried out on a sample of bacteria and the number of colonies observed on the plates at each dilution: Figure 7 Plates B and C would be counted as they contain between 20 and 200 colonies. For Plate B, 180 colonies grew using 0.1cm 3 of the 10 2 dilution. So, there are 1.8 10 5 colonies in 1cm 3 sample. (180 100 10) For Plate C 22 colonies grew using 0.1cm 3 of the 10 3 dilution. So, there are 2.2 10 5 colonies in 1cm 3 sample. (22 1000 10) The numbers obtained from Plates B and C are averaged to give an estimate of the number of micro-organisms in the original sample, and this works out to be 2.0 10 5 micro-organisms in 1cm 3 sample. Generally, more than one plate of each dilution is cultured and counted to increase the reliability of the estimate. 30 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

CULTURING TECHNIQUES Plaque assay to enumerate bacteriophage numbers This technique is used to estimate the number of bacteriophage in a sample. Bacteriophage grow only when they have infected bacterial cells. After growing within a cell, bacteriophage break open (lyse) the bacterial cell. If bacteriophage-infected bacterial cells are grown on agar plates, bacterial cells that have been lysed are observed on the plate because the agar is exposed as a clearing (known as a plaque) on the plate. This is shown in Figure 8: Figure 8 A plaque assay is carried out by doing a serial dilution of the bacteriophage and mixing each dilution with bacteria. A known volume of bacteriophage and bacteria is mixed with molten agar, and poured onto an agar plate. This is then incubated at the required temperature. Plaques (areas of clearing) are counted. Each plaque is assumed to have arisen from a single bacteriophage and so the original number of bacteriophage can be estimated in the same way as described for counting colonies on a plate (page 30). Preparing a bacterial lawn When a large number of bacteria are inoculated onto an agar plate, they do not grow as separate colonies. Instead, the colonies grow into each other and the entire surface of the plate becomes uniformly covered with bacteria. This is known as a bacterial lawn. UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 31

CULTURING TECHNIQUES There are two ways of preparing a bacterial lawn: by spreading a small volume (0.1cm 3 ) of a dense culture of bacteria over the surface of the agar plate by mixing a larger volume (1 cm 3 ) of bacterial culture with molten agar, then pouring the mixture into a Petri dish. Bacterial lawns are used when testing the sensitivity of a bacteria against a range of antibiotics. After the agar has been inoculated, discs impregnated with antibiotic are placed on top of the agar and the agar plate is incubated at the correct temperature for an appropriate length of time. Bacteria that are sensitive to an antibiotic do not grow around the disc and a clear area is observed. Bacteria that are resistant to an antibiotic can grow around the disc. An example of this is shown in Figure 9: Figure 9 This figure shows that this bacteria is sensitive to penicillin but resistant to tetracycline. 32 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

CULTURING TECHNIQUES Tissue culture It is possible to culture isolated plant tissue in the laboratory using techniques similar to those for culturing micro-organisms. When culturing plant tissues, strict aseptic technique is needed to maintain sterile conditions and to prevent contamination of the plant cells by micro-organisms. Plant tissue culture can be used to produce thousands of identical plants. It allows commercial plant growers to produce clones of plants such as pineapple, rose, orchid and palm oil in a relatively short period of time. Another advantage of producing plants in this way is that they are pathogen-free (this means that they are not diseased). One method by which plants are micropropagated is by isolating a piece of plant tissue, called an explant, and culturing it under sterile conditions, either in agar or liquid medium. If the explant is given the correct nutrients, vitamins and plant growth substances, the cells of the explant first divide into a mass of undifferentiated cells, called the callus, then the cells differentiate and finally they develop into a plant. Plants can also be micropropagated from the apical meristem of a plant. The apical meristem is the tissue found at the tip of the shoots. The cells in this tissue are actively dividing and produce new growth of stems. These cells are normally free from pathogenic (disease causing) microorganisms, so plants regenerated from them are pathogen-free. The meristem is removed from the plant and sterilised with disinfectant. It is then placed on agar containing nutrients until a shoot develops. The shoot is placed in agar containing plant growth substances that induce the development of roots. When roots have developed, the plantlets are planted in sterile compost. The nutrient medium used in micropropagation contains a variety of substances essential for growth. Some of the substances found in the nutrient medium are described below: It contains a source of carbon. A callus does not have chlorophyll, the pigment which is involved in photosynthesis (the process that produces sugar in a plant). Thus, the nutrient medium contains a source of carbon, usually in the form of a sugar. The sugar is used by the callus in aerobic respiration, the process that provides the plant with energy in the form of ATP. UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 33

CULTURING TECHNIQUES It contains vitamins because they are essential to make enzymes work correctly. If enzymes do not work, then the cells are unable to survive. It contains plant growth substances. These are critical for the successful growth and differentiation of the callus because they regulate growth and development. Two important plant growth substances are auxins and cytokinins. The following diagrams show the effect of different concentrations of auxin and cytokinin on the differentiation of the callus: (a) Agar containing nutrients and 0.2 mgdm 3 cytokinin The explant does not grow (b) Agar containing nutrients and 2mgdm 3 auxin The cells of the explant grow bigger (c) Agar containing nutrients and 2mgdm 3 auxin and 0.2 mgdm 3 cytokinin A mass of undifferentiated cells is produced 34 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

CULTURING TECHNIQUES (d) Agar containing nutrients and 2mgdm 3 auxin and 0.02 mgdm 3 cytokinin Roots form but shoots do not (e) Agar containing nutrients and 0.02mgdm 3 auxin and 1.0 mgdm 3 cytokinin Shoots form but roots do not These diagrams show that different concentrations of plant growth substances greatly influence the growth of explants. The correct concentrations are needed for shoot and root regrowth. UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 35

36 UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY)

SECTION 3 IDENTIFICATION OF MICRO-ORGANISMS Identification of micro-organisms One of the most fundamental tasks that someone working with microorganisms must perform is to identify micro-organisms. There are a number of ways that this can be done. Firstly, the micro-organism can be stained and viewed under the microscope. This gives information regarding the shape (morphology) of the micro-organism. Another way of identifying micro-organisms is to find out the reactions that the micro-organism carries out. This is known as biochemical testing. Once the morphology and biochemistry of the micro-organism is known, identification keys and tables can be consulted to work out the identity of the micro-organism being investigated. Use of the microscope in identifying micro-organisms Micro-organisms are often difficult to observe using a microscope and so they are generally stained before being viewed. A stain that is commonly used in the initial identification of a bacterium is the Gram stain. It identifies bacteria as being gram positive or gram negative, depending on the type of cell wall present in the bacterium. Gram-positive bacteria appear purple when viewed under the microscope whereas gram-negative bacteria appear red. (The gram stain is also discussed in Section 1 of the Student Materials for Unit 1: Microbiology see page 8.) When viewed under a microscope, bacteria are observed to have a definite shape (morphology). Three shapes are commonly seen round, rods and spirals. Figure 11 Round bacteria are called cocci: UNIT 2: MICROBIOLOGICAL TECHNIQUES (HIGHER BIOTECHNOLOGY) 37