Microbiology

• Microbiology Lesson 04 involves checking out water samples, performing serial dilutions, spreading plates, and pouring plates.
• Lesson 05 involves enumeration of colony morphologies, streak plate method, and broth inoculation.
• Lesson 06 involves simple staining and gram staining for preliminary identification.
• Deliverables for Lesson 04 include a Lab Record Sheet to be submitted by the end of Lesson 6.
• Pour plate, water sample, team 1A/B, and date should be labelled.
• Materials required for labelling include 9ml saline, sterile 0.9% saline, pipette aid, and a sterile pipette.
• The pipette aid should be used to aid in the opening and closing of the serological pipette.
• The serological pipette should be pressed to dispense and aspirated to aspirate.
• The serological pipette should be placed back into the sleeve before discarding into the biohazard bin.
• Diluent should be aliquoted to all the tubes before performing serial dilutions.
• Aseptic zone includes a flame, which cannot be flamed.
• Performing a dilution involves picking up a new pipette tip, picking up a tube, flaming, opening the tube, dispensing, closing the tube, and replacing the tube back to the rack.
• For serial dilution, a new pipette tip should be used and the process should be repeated from Step 3.
• The basic microbiological techniques important for media preparation and bacterial culturing include sterile technique, aseptic technique, and proper use of equipment.
• Time for Lesson 04 includes a recap of Lesson 3, a lab safety awareness briefing, a lecture on quantitative analysis of microbial population in water samples, and an activity on preparation of LB agar, autoclave, and pouring of agar plate.
• The purpose of media used during bacterial culturing is to provide a suitable environment for the growth of bacteria.
• The different components used in media preparation and bacterial culturing include agar, saline, and bacterial media.
• The procedure to prepare microbiological growth media involves the selection of appropriate ingredients, their mixing, and sterilization.
• The equipment used in media preparation and bacterial culturing include sterile bottles, flasks, and Petri dishes.
• Bacterial culture should be free from contaminants as they can interfere with the growth of bacteria.
• The preparation and pouring of molten agar involves heating agar to a specific temperature, adding bacteria, and pouring the mixture into a Petri dish.
• Aseptic techniques are important in the preparation of growth media and bacterial culturing as they help to prevent the introduction of contaminants.
• A sterile work environment can be achieved by the use of a Bunsen Burner.
• The implications of poor aseptic techniques include contamination of growth media and bacterial culturing, inaccurate results, and potential health risks.
• Lesson 05 involves a lecture on the importance of serial dilutions, calculations for serial dilution, and a hands-on activity to perform serial dilution of water sample and positive control.
• Water cooler – Team 1 (Pair/trio A & B)
• Poolside shower – Team 2 (Pair/trio A & B)
• Jacuzzi – Team 3 (Pair/trio A & B)
• Tap in toilet serving swimming pool – Team 4 (Pair/trio A & B)
• Shower in toilet serving swimming pool – Team 5 (Pair/trio A & B)
• Are these water samples contaminated with E. coli as well?
• Serial dilution, Spread/Pour plates, Water sample, Possible results, Enumeration, Identification, Considerations: Nutritional Requirements of Bacteria (recall L02) and Media Preparation, Aseptic Techniques, Experimental Controls.
• Negative control, Positive control, The importance of experimental controls, Positive and negative controls are required to validate the experimental procedure.
• Hands on Activity to Perform: Serial dilution of water sample and positive control, Practicing basic aseptic techniques, Aseptic Techniques: a set of routine measures taken to prevent cross contamination of cultures, sterile media stocks and other solution by foreign microorganisms inherent in the environment and to prevent infection of the handler and others who may be exposed to the microbial samples.
• Labelling of agar plates: <Sample/+ve control> <Dilution Factor> <Team> <Date>lid base
• Labelling of tubes: Dilution Factor cap tube
• Full labelling: Label 6 tubes: <water sample>, 101, <team 1A/B> and <date>
• Positive, 101, <team 1A/B> and <date>
• Negative, neat, <team 1A/B> and <date>
• Lesson 06 involves a lecture on experimental plan and controls, spread plate method, pour plate method, and cleaning up and review of lesson/PQ discussion.