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2420-Lab 2- Bacterial Staining Techniques-I Directions: Answer following questions after reading the information and watching the video from the link below. Use color RED or BLUE for your answers. Submit the completed document on eCampus for grading. Refer to Refer to the textbook Openstax Microbiology Chapter 2 Link: Bacterial Staining Techniques-I, Virtual Edge Experiment-2 Another optional but helpful link Simple Staining This document has 17 questions Students MUST use scientific terminology while answering all questions For each wrong answer 0.5 points will be deducted Aseptic transfer of the specimen with necessary safety precautions You will watch videos that show transferring specimens from a tube to a slide OR from a plate to a tube or slide. While you watch this video understand a few things listed below that are not explained in the video. Always start by spreading the CDC recommended disinfectant evenly throughout the work area with a disinfectant using clean disposable paper towels and let it dry by allowing the disinfectant to evaporate before you begin work. This removes micro-organisms that settled down through air or left over from previous work. Wash hands with antimicrobial soap after wiping the area and disposing off the wipes. This is to prevent introducing micro-organisms from your hands. Once you begin work, you should your hands should not touch anything else (body parts, personal items like phone, keys until you complete the experiment). This way, you will not get infected with bacterial samples and bacterial samples will not get contaminated with bacteria from one’s body or personal items. you should avoid unnecessary talking to avoid introducing microbes from your mouth in the work area. you should not walk around if work does not require to avoid creating air draft intruding germs A lab notebook and a lab-pen is all that can stay in the working area away from the working material including bacterial cultures and burner. It’s a good idea to keep a dedicated notebook and a pen/pencil which stays in the lab. If at all some drops of bacterial culture splashes on these items, bacteria will not leave the lab. Bunsen burner used for two reasons Direct heating to sterilize equipment Create an updraft of air taking particles, unwanted microorganisms away from the work area keeping some area around the burner sterile. Observe the pictures below. Picture-1: shows updraft of hot air Picture-2: shows how a current is created leaving some are sterile Picture-3: shows how close to the burner you need to work so the specimens will not be contaminated The circular “sterile” area shown in the pictures A and B below is the only area where you could expose bacteria. Any area beyond that can lead to contamination of bacterial cultures. Inoculation loop: Used to transfer bacterial specimen. Also called a “Nichrome loop”. Made of an alloy of Nickle and Chromium. Main properties useful in aseptic transfer of bacterial specimens: Heats and cools quickly Doesn’t get oxidized Deteriorate with repeated heating and cooling Never touch the loop to check whether it’s cooled down. Touching can introduce contamination into the bacterial culture or it could give you an infection. If the loop is too hot, fingers could get burnt. Once the loop is red hot, it’s sterile. Hold the sterile loop for a few seconds in the sterile area around the burner to cool down to avoid contamination. Preparation of a smear: (Watch the video-2A) Why do you mark both sides of a slide before adding the specimen to the slide? Why do you pass the mouth and the lid of the water bottle through the flame? What is the need to air-dry the smear? What is the need to heat-fix the smear? Direct Stain or Simple Stain: How many stains are used in this process? Why are basic dyes used in simple stains? Name the organisms seen on the slide: List the advantages and disadvantages of simple staining. Using Oil Immersion lens: Watch How to use oil immersion lens What is the purpose of oil while using 100X objective lens? Identify the bacterial shape in the following picture: Negative Stains: (Go to the experiment- 2C) Which stains- acidic or basic- bind to bacteria directly and why? What is negative staining? List the advantages and disadvantages of negative staining. Which stain and color was picked up by bacteria in negative staining? In the following picture, point out (or describe) the bacterial cells and air bubbles: Hanging-Drop Method to view Motility of Bacteria: Bacteria use various ways that helps them move. Some use flagella, some secrete polysaccharide as in biofilm and glide. Many bacteria show chemotaxis (movement towards and away from chemicals). Bacterial phototaxis (movement towards and away from light) and magnetotaxis (movement towards and away from magnetic field) has also been shown. Many bacteria show a random pattern called Brownian movement. Some examples are shown below: Bacterial movement in a petri dish: Bacterial movement in a tube with agar Tube A Tube B Tube C In the above picture identify which tube has freely motile bacteria? Bacterial movement on a slide: Hanging drop One of the easy techniques in undergraduate microbiology class called a “hanging drop” method is used to view bacterial motility. Usually the slide is prepared with one motile and one non-motile bacterial species with different shapes. Most commonly used combination is Staphylococcus aureus as non-motile cocci and Proteus vulgaris as motile rod-shaped. No smearing involved instead a drop is added on a coverslip, not on the slide. Petroleum jelly/Vaseline is added on the corners of the coverslip so the coverslip can adhere to the slide. The glass slide used has depression at the center so, it’s called a depression slide. Heat fixing step is eliminated as we need live moving bacteria. The setup is shown below: You can see the drop that is hanging from the coverslip carries bacterial sample is not touching the slide for effective bacterial movement. What is the “hanging drop” method used for?