Wk 3. Bacterial Staining 1.pptx

QUIZ 1 TODAY, lets get settled in 1. Set your things down (do not place anything on the lab bench) 2. Disinfect your lab bench 3. Wash your hands 4. Take out a pencil or pen 5. Make sure your backpack is stowed away neatly under your bench or you may place it on the table in the right side of th...

QUIZ 1 TODAY, lets get settled in 1. Set your things down (do not place anything on the lab bench) 2. Disinfect your lab bench 3. Wash your hands 4. Take out a pencil or pen 5. Make sure your backpack is stowed away neatly under your bench or you may place it on the table in the right side of the back of the classroom. 6. Optional: Put on your gloves before or after the quiz B A  BACTERIAL STAINING TECHNIQUES 1 BIOL 2015 – Microbiology Allied Health Lab Karen Galvan  Bacterial Cultures Bacteria: unicellular microorganisms which lack a true nucleus, they have a nuclear region Culture: a medium that contains living microbes. Pure culture: a culture that contains a single species. Fig. 1. LB Broth and plated media agar. Fig. 2. Various cultures in petri dishes. Fig. 3. Bacterial cultures in agar slants During the experiment Today’s lab will be INDIVIDUAL & PAIRED. I want all of you to get hands-on experience. If your glove rips, get a new set. If your gloves get heavy stain on them, get a new set. If you want to take photos of your microscopy images, you can. But never ever use a gloved hand to touch your personal items including your phone and face/body/hair. AND DO NOT leave your phone on the lab bench. – Remove your gloves, take your phone out, and take the image, but do not touch the microscope or your lab bench – When you are done, put your phone away, put on new gloves, and resume ASEPTIC TECHNIQU E Aseptic transfer: the transfer of microbes from their pure culture to a sterile medium without contamination of yourself, others, the environment, the source culture, or the medium being inoculated. Keeping objects sterile, in a sterile field/environment Work within the sterile field  Inoculating Loops used for gliding on surface of agar Inoculating Needle/Wire  used for stabbing into agar Wait 20-30 seconds for the sterilized loop to cool. But don’t put it down  Bacterial Colonies Take a small chunk from an isolated colony. – Isolated colonies are descended from one or a few cells of the same species of bacterium, termed colony-forming units (CFUs). Isolated colonies are needed to make pure cultures. You do not need to take the entire colony! Why do you think we should take a small amount and not too much? PART 1: SIMPLE STAINING  MAKING A BACTERIAL SMEAR We will not be dipping the inoculation loop inside the water. It is really important to not grab TOO much culture, or the cells will appear crowded and not stain properly Do not open the gas valve all the way! Just slightly. You can adjust the height of the flame once you ignite it. BACTERIAL SMEAR You will prepare TWO different bacterial smears at the same time prior to staining so they can air dry together. After heat fixing, go ahead and turn off the Bunsen burner. – What do you think is the significance of heat fixing?  MAKING A BACTERIAL SMEAR 1. With a sharpie, label the underside of the slide by drawing a circle in the center and putting the name of the culture being used. Purpose: it is the underside so it will not fade away with subsequent washing. The circle is to locate the bacterial smear 2. Turn on the Bunsen burner and sterilize your inoculation loop. Let the loop cool down (do not waft it or set it down). Have your bacteria ready on the benchtop within your sterile field. Purpose: you use the Bunsen burner to create a sterile field and keep aseptic technique 3. Add a small drop of water on your inoculation loop and transfer it to your slide within the circle you drew. Purpose: the water is necessary to get a good dispersal of the bacteria 4.Sterilize the inoculation loop. Let the loop cool down (do not waft it or set it down). Pick up a small amount of bacteria and smear it/mix it with the water on the slide (within in the circle as well). Purpose: you do continuous sterilizations to keep aseptic technique. The bacteria is mixed with the water for better staining 5. LET IT AIR DRY. Once the bacterial smear is dry, heat fix the bacteria by passing it over the flame 2-3 times using a clothes pin. Purpose: Heat fixing kills bacteria and adheres them to the slide so that it will increase the permeability of the stain. If we do not do this step, the bacterial cells will be rinsed off.  SIMPLE STAINING What is the purpose of staining?  Bacteria is almost colorless. So, we want to increase the contrast between the organisms and the background. Start @ 6:58 We will not be doing the hot plate trick  blotting paper instead SIMPLE STAINING You must do TWO different stains on the TWO different organisms. – You will stain them both at the same time. After you are done staining, get the microscopes out! – You will be doing a 100X oil-immersion. – Be sure to not get oil elsewhere on the microscope, other than on the 100X objective lens. If so, clean it up before doing the second organism. ** If you stain your gloves with dye, DO NOT touch the microscope.  change gloves SIMPLE STAINING 1. Place it over the sink, bacteria face up 2. Place 1-2 drops on slide. Let it sit for 1 minute. Stains:  They are basic.  Crystal Violet  Methylene blue  Safranin 3. Tilt the slide, let the stain drip off and rinse with DISTILLED WATER. 4, Blot the slide between the blotting paper, make sure it is dry. When using the stains, always do OPEN-USE-CLOSE  What is the significance of the stains being basic? Lets review the microscope steps! When setting up your microscope: 1. Make sure the stage is all the way down 2. Start with your lowest objective 3. Mount your slide and make sure it is aligned on the stage and with the objective 4. Adjust the light to your preference 5. Adjust the course knob until you can make out an image  only at 4x and 10x 6. Adjust the fine knob until you can see the image clearly  40 x and 100 x – Be careful not to crack your slide once your objective lens is close to your slide! At this point, you should really only be using the fine knob. 7. You can adjust the stage left/ right or up/down depending on what you are going to magnify 8. When you decide what you are going to magnify, make sure it is in the center of your field of view. Why? Any questions? Lets get started on part 1 PART 2: NEGATIVE STAINING NEGATIVE STAINING The dye solution is acidic and thus carries a negative charge  the negative charge of the bacterial surface _REPELS so, the cells remain _UNSTAINED_____ compared to the background. You will prepare ONE bacterial smear IN PAIRS Turn the Bunsen burner back on to create a sterile field and inoculate the loop again. Give yourself space between the flame and the microscope. We will NOT be heat fixing after air drying this slide – What would this imply in terms of caution/ lab safety? NEGATIVE STAINING 1. Place a paper towel on the bench and then the glass slide on top. 2. Turn on the Bunsen burner to create a sterile field. 3. Place 1 drop of the negative stain on the side of the slide. You need to do this more towards one side of the slide, NOT centrally. 4. Sterilize the inoculation loop, let it cool down, get a dense colony of bacteria and place it within the negative stain on the slide. Mix it in small circles. 5. Sterilize the inoculation loop, let it cool down, and place on tabletop. 6. Get another slide, slide it over the stain about half-way away from you, and then slide it towards you. 7. Let the slide air-dry. 8. Dispose of the slider slide. 9. OVERALL: NO HEAT-FIXING. CONCLUSIONS Make sure all slides are properly disposed of Make sure the gas valves are shut properly! CLEAN YOUR MICROSCOPES – Make sure to get all the oil off – Before you put them away Clean up procedures – Clear off your benchtop – Disinfect your benchtop – Remove and dispose of your gloves – Wash your hands