Microbiology Questions and Answers

Microbiology Questions and Answers Aseptic Free from contamination by microorganisms Autoclave Container using steam, produced under high pressure and temperature, to sterilise equipment - items can be kept dry by wrapping tightly in tinfoil. Name 5 Aseptic techniques 1. Working in a sterile bench-space 2. Accessing a glass container 3. Accessing a Petri dish 4. Flaming to sterilise instruments 5. Securing a petri dish Aseptic techniques; 1. Working in a sterile bench-space - Work surfaces must be disinfected, before and after use. - Hands must be washed with antibacterial soap, before and after work. - Any transfer or microorganisms must be done close (say within 20cm) to a Bunsen burner where warm air currents draw any airborne microorganisms upwards. Aseptic techniques; 2. Accessing a glass container - On opening a flask, bottle or test tube containing agar or microbial culture, its neck must be held over a Bunsen flame to kill any surface microbes, having removed the plug with the little finger curled towards the palm. Aseptic techniques; 3. Accessing a Petri dish - An agar plate is prepared by pouring liquid sterile nutrient agar (sterilised by autoclaving) into a sterile Petri dish, having lifted its lid only at one side to allow access. Aseptic techniques; 4. Flaming to sterilise instruments - A wire loop, sterilised by heating within a Bunsen flame, is used to transfer a microbial culture (from an existing culture on an agar slope or in a nutrient broth) to a nutrient agar plate and sterilised afterwards. - Forceps, used in the transfer of antimicrobial discs, are also sterilised in a Bunsen flame (before and after use) - L-shaped glass spreaders are sterilised by dipping in ethanol then flamed (before and after use). Aseptic techniques; 5. Securing a petri dish - The inoculated plate must be secured by fixing two or four short strips of adhesive tape at opposite ends of the dish. Commercial sterilisation Petri dishes, dropping (Pasteur) pipettes and spreaders are commercially available already sterilised (by irradiation). Using aseptic techniques to transfer bacteria from a broth culture to a Petri dish In what can bacterial cultures be obtained? Bacterial cultures may be obtained in a liquid broth, or on an agar slope in a McCartney bottle. Effects of antibiotics on the growth of bacteria Effects of antibiotics on the growth of bacteria Culture A microbial culture is a nutrient medium (broth or agar surface) in which a specific microorganism can reproduce and multiply. Inoculum A sample amount of material containing microorganisms used to start a new culture. Investigating the effectiveness of different antibiotics; Preparation - A sterile Petri dish containing nutrient agar is used. - A sample is removed from a broth culture (only bacteria from the 'safe microorganisms' list provided by the Society for General Microbiology must be used) using a sterile dropper pipette, and a few drops are placed onto the surface of the agar in the Petri dish. - A sterile spreader is then used to spread the inoculum over the entire surface of the agar. - The inoculum is allowed to dry. Investigating the effectiveness of different antibiotics; Carrying out the experiment - Sterile discs of filter paper (commercially available) are soaked in various antibiotics. - Using sterile forceps, a disc is then placed onto the agar plate. - For example, if comparing four different antibiotics, each would be placed centrally within separate quadrants of the dish. - Alternatively, a Mast ring (a ring of paper with several 'arms' each treated with a different antibiotic) can be used. - Label the underside of the plate with initials, date, name of microorganism and antibiotic used (or coded A, B, C etc.). - The dish is taped and placed in an incubator at 20-25ºC for 2-3 days. Investigating the effectiveness of different antibiotics; After incubation - After incubation, the plate will look opaque where bacteria have grown, but where the antibiotics have inhibited growth, clear zones called inhibition zones will be seen. - The diameter of the inhibition zones may be measured in mm, or the area of these zones may be determined by placing mm-squared paper below the dish. - This information may be used to decide which antibiotic is most effective at inhibiting the growth of the bacterium. - The diagram shows the results for a particular Mast ring. Investigating the effectiveness of different antibiotics; Exam tip Be aware that an antibiotic found to be more effective against one species or strain of bacterium will not necessarily be the most effective against other strains or species. Investigating the effective concentration of an antibiotic; MIC - If a low concentration of an antibiotic inhibits growth then it can be regarded as effective in dealing with a bacterial infection. - The lowest concentration of an antibiotic that inhibits the growth of a bacterial strain is called its minimal inhibitory concentration (MIC). Investigating the effective concentration of an antibiotic; Determining the precise concentration - The precise concentration of antibiotic required to inhibit growth can be determined by preparing an inoculated agar plate (as in the previous investigation), adding an e-strip and incubating the culture. - An e-strip has an exponential gradient of antibiotic concentrations on a paper strip. - The result for a particular e-strip, exhibiting an elliptical zone of inhibition is shown in the diagram. Investigating the effective concentration of an antibiotic; Example - The diagram shows the antibiotic's MIC for the inoculated bacterium. - For patients with a specific infection, an antibiotic with a low MIC score will be administered. - The dosage used might be several times the MIC score to ensure effective treatment and also prevent the evolution of antibiotic-resistance microbial strains. Preparing a streak plate to isolate single colonies Preparing a streak plate to isolate single colonies Method - A sterile wire loop is used to remove a sample from the bacterial culture. - The inoculum is smeared backwards and forwards across the surface of the agar (region A in the diagram). - Flame the loop, cool and, having turned the Petri dish 90º, streak the inoculum from A across the surface of the agar in parallel lines (B). - This process is repeated twice more as shown in C and D. - After a suitable period of inoculation, colonies of bacteria will have grown on the agar. Typical results - In areas A, B and C (from the previous diagram) they will probably form a continuous mat. - In area D, however, you should find small colonies formed by the division of a single bacterial cell. - The diagram shows the result of a streak plating after inoculation of the agar plate. Investigating the antimicrobial properties of plants Investigating the antimicrobial properties of plants Antimicrobial properties of plants - Some plants contain chemicals with antimicrobial properties, allowing them to resist infection from, for example, bacteria and fungi. - Plants reputably with antibacterial or antifungal properties include those with culinary uses such as herbs and spices (eg garlic, thyme, cinnamon) or those producing essential oils (eg oil of cloves, lavender). Method for investigating antibacterial activity - Inoculated agar plates are produced, and sterile filter paper discs containing plant extracts are placed on the agar surface. - To make a plant extract, 3g of the plant material is crushed in 10cm³ ethanol. - The paper discs are soaked in the extract and allowed to dry (within a sterile Petri dish) before using. - The agar plates should be incubated for 2-3 days and measurements made of any inhibition zones. Method for investigating anti fungal properties - A sterile agar plate is inoculated with a mould using a sterile wire loop, which is also used for spreading the mould across the surface. - Fungi grow best at lower temperatures than bacteria, and so incubating at room temperature should suffice. Possible Questions Possible Questions Reasons for aseptic techniques - Prevent contamination while working with microorganisms - Prevent contamination of microbe culture and of individuals working with the microbes What is MIC? Minimal inhibitory concentration - The lowest concentration that inhibits the growth of a bacterial strain. Why is MIC important? It ensures effective treatment and prevents the evolution of antibiotic-resistant microbial strains. Describe how you would transfer bacteria from tube A to petri dish B so that contamination is avoided - Sterilise loop/use sterilised disposable loop - Open culture bottle (tube A) and hold at angle/ keep lid off bench - Use loop to scrape the bacteria from surface of slop in culture bottle - Once loop is removed, replace lid of culture bottle immediately/flame lid - Partially open Petri dish and spread bacteria over agar - Replace lid immediately and tape closed - Sterilise loop again or safely dispose. After bacteria has been transferred, a Petri dish was incubated at 25ºC; Why was it not incubated at a higher temperature? To avoid culturing pathogens that thrive in higher temperatures