Identification of bacteria Aims Correctly follows written instructions to carry out experimental techniques or procedures. Carries out techniques or procedures methodically, in sequence and in combination, identifying practical issues and making adjustments when necessary. Obtains accurate, precise & sufficient data for experimental & investigative procedures & records this methodically using appropriate units and conventions. 1.23 The use of differential staining techniques 1.20 The classification and characteristics (size of cell, type of cell, presence of organelles) of microorganisms. It can be important to identify the species and even strain of bacteria in medicine. For example, some bacteria are immune to certain antibiotics and knowing that someone is infected by one species/strain rather than another can mean the difference between effective treatment and the death of the patient. There are a number of methods that can be used to identify bacteria. They include the use of microscopes, differential staining, colony morphology, gene analysis and mass spectrometry. Today we are going to look at two of these – colony morphology and differential staining. Equipment Equipment Microscope Why have you used this piece of equipment? Slide and cover slip Crystal violet Safranin Agar plates Wire loop Bunsen burner Plastic spreader Risk Assessment Equipment Hazard Potential risk How will you minimise this risk? Gram stain The Gram stain was first used by Hans Christian Gram in 1884 to visualise bacteria in samples of lung tissue. It involves two stains – crystal violet (purple) and safranin (red) – which are added sequentially along with Gram’s iodine. 1. Make a slide of cell sample to be stained. Heat fix the sample to the slide by carefully passing the slide with a drop or small piece of sample on it through a Bunsen burner three times. 2. Add the crystal violet to the sample/slide and leave for 1 minute. Rinse slide with a gentle stream of water for a maximum of 5 seconds to remove unbound crystal violet. 3. Add Gram's iodine for 1 minute. Again, rinse slide with a gentle stream of water for a maximum of 5 seconds to remove iodine 4. Rinse sample/slide with acetone, proponine or alcohol for ~3 seconds and rinse with a gentle stream of water (careful, if the alcohol remains on the sample for too long, it may decolorize Gram positive cells). 5. Add the safranin, to the slide and leave or 1 minute. Wash with a gentle stream of water for a maximum of 5 seconds. 6. If the bacteria is Gram positive, it will retain the primary stain (crystal violet) and not take the secondary stain (safranin), causing it to look violet/purple under a microscope. 7. If the bacteria is Gram negative, it will lose the primary stain and take the secondary stain, causing it to appear red when viewed under a microscope. You have some different samples of bacteria to study today. You need to make slides and stain each sample and view them under the microscope using a 100X OIL lens in order to be able to judge if they are Gram positive or Gram negative. Results When you have viewed each slide, take a photograph of the results and save them for later. You can add them to a document and print it off to include with this worksheet to show evidence of your skills. Complete the table below for all the bacteria samples you have examined. Name of bacteria (Use correct binomial nomenclature) Colour and shape of cells under a microscope following Gram stain Result – is the bacteria Gram positive or Gram negative? Complete a biological drawing on a blank piece of paper. Include your diagram when you hand in this sheet for marking Answer the questions 1. How does the Gram stain method work? i.e. How does it determine if a cell is Gram positive or Gram negative? 2. What structural differences exist between Gram positive and Gram negative bacteria? 3. What are the possible medical implications linked to a bacterial infection if it is Gram positive or Gram negative? Aseptic techniques, environmental swabs and colony morphology Another way to identify bacteria is through colony morphology. This requires the bacteria to be inoculated onto agar in a petri dish and incubated for a time before analysis. The shape, opacity and colour of the colonies are distinct for different species of bacteria and further differentiation can be achieved by using different media (i.e. Blood Agar or restricted agar). For this practical, we are going to practise some aseptic inoculation methods (how you get the bacteria from the liquid culture onto the plate without contamination) onto basic nutrient agar and also perform some environmental swabs. The agar plates from these will be incubated at 30oC for 24 – 72 hours (depending on growth) and then stored at 4oC until we can analyse them in a future lesson using colony morphology. Labelling plates Agar plates need to be stored ‘upside down’. This means you should label the base of the petri dish rather than the lid. Labelling should be done around the edge of the dish so as not to obscure the middle where you will find the colonies growing. Each dish should be labelled with: - Your initials The date you used the plate The method used (Pour, spread, streak, environmental swab) The species of bacteria (if known) or the location the sample came from. Aseptic techniques Aseptic technique comprises a number of methods used to ensure sterile practise in microbiology. It makes use of Bunsen burners, alcohol and other methods to ensure tools and solutions are as sterile as possible. Before you carry out any of these methods, watch the demonstration of the techniques performed by your tutor, paying attention to the aseptic methods. Note the methods used down in the tables below. Pour plates The Pour plate method involves adding a bacterial inoculum to liquid agar. One use is a method to determine viable cell counts using serial dilution of the original sample and counting the colonies. What aseptic techniques were used here? How do they assure sterility? Spread plates/Bacterial Lawn What aseptic techniques were used here? How do they assure sterility? Streak plates The streak plate method is used to isolate single colonies of a bacteria for further analysis by other methods. It involves performing a series of streaks on the agar with a wire loop, sterilising between each stroke. What aseptic techniques were used here? How do they assure sterility? Environmental swabs This is a method used to sample things in the environment. It can be a way to establish the source of an infection and can even be used on human tissues (i.e. an open wound). You can sample anywhere in the lab, including the people in it (with consent). However, for health and safety reasons you are not allowed to sample: - Inside a human body (i.e. the mouth, nose, wounds, anus etc.) Toilets or other areas exposed to faeces or other bodily waste Foodstuffs, especially meat. To sample, follow this method: 1. 2. 3. 4. 5. 6. 7. 8. 9. Divide your plate into two sections. Label them with the areas you are going to sample. Open the bottle of sterile water, flame the lip of the bottle. Dip the cotton bud into the bottle. Flame the lip of the bottle and replace the lid. Rub the cotton bud over the area you are sampling, using firm pressure. Carefully remove the lid of the petri dish as shown in the demonstration. Gently rub the cotton bud over the agar where you have marked for that area, be careful not to damage it. Repeat for your other environmental area. Replace the lid of the petri dish, turn the plate over and seal with two pieces of tape on opposite sides of the petri dish. 10. Put in the tray for incubation. It requires at least 12 – 24 hours of incubation before colonies will start to appear. To analyse the colonies you will need a hand lens and a reference for what the different colony shapes and colours mean. Practical Competencies: How do you rate in the following skills? Selfassessment (RAG) Correctly follows written instructions to carry out experimental techniques or procedures. Carries out techniques or procedures methodically, in sequence and in combination, identifying practical issues and making adjustments when necessary. Obtains accurate, precise & sufficient data for experimental & investigative procedures & records this methodically using appropriate units and conventions. 1.23 The use of differential staining techniques 1.20 The classification and characteristics (size of cell, type of cell, presence of organelles) of microorganisms. Tutor assessment (RAG) Results When the plates are analysed in the next lesson, complete the tables. If you need any more room, create a new table on a blank sheet of paper. Also, take a photograph of the plate. Area 1: Description of colony Species of bacteria What conclusions can you draw from the presence of this bacteria here? Species of bacteria What conclusions can you draw from the presence of this bacteria here? Area 2: Description of colony
