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Name: Meghna Kakkar Student ID: 21163972 Page 1 PRACTICAL REPORT TWO INFECTIOUS AGENTS Microscopic examination of bacteria INTRODUCTION In forensic science bacteria can play a role as:  a weapon (e.g. in biological warfare or as a toxic contaminant in food or other products),  the cause of violence (e.g. if infection with a bacteria causes a disease that alters a persons mental state and results in them committing a crime),  a tool to solve crime (e.g. to link a
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   Name: Meghna Kakkar Student ID: 21163972 Page 1 PRACTICAL REPORT TWO INFECTIOUS AGENTS Microscopic examination of bacteria INTRODUCTION  In forensic science bacteria can play a role as:   a weapon (e.g. in biological warfare or as a toxic contaminant in food or other products),   the cause of violence (e.g. if infection with a bacteria causes a disease that alters a persons mental state and results in them committing a crime),   a tool to solve crime (e.g. to link a suspect to a crime scene)   the subject of crime (e.g. the theft of bacteria from a laboratory, or the breaking of a legal patent). Bacteria are typically 1  –  5  m in size, and are therefore able to be visualised with a light microscope at 1000 X magnification. Preparations of bacteria are stained with a coloured dye to enhance the contrast between the cells and the background making them much easier to see. A simple stain  makes use of a single dye and reveals basic cell shapes and cell arrangements. A differential stain  makes use of two or more dyes and distinguishes between two kinds of organisms or between two different parts of an organism. The Gram stain, named after Dr Christian Gram, is the most important differential stain currently used for the classification and differentiation of bacteria. It divides bacterial cells into two major groups; Gram positive and Gram negative. The Gram stain requires the use of four chemical reagents that are applied sequentially to a heat-fixed bacterial smear. The first and second reagents are used to colour all of the cells in the smear. In order to establish a colour contrast, the third reagent is a decolourising agent. This will remove the first two reagents from certain cells (in accordance with cell wall structure). The final reagent has a contrasting colour to the first reagent and is absorbed by cells from which the first stain has been removed. Different bacteria have different cell wall structures which affects the absorption of reagents used in the Gram stain. The two main types of cell wall structures are termed Gram positive and Gram negative, and their composition are illustrated below. Forensic scientists often culture and grow bacteria found at crime scenes or extracted from remains. The assignment of an organism as Gram positive or negative, a description of the morphological features of the cell observed microscopically and the appearance of surface colonies on solid media are fundamental to the identification and classification of bacteria. This experiment uses the Gram stain to observe several different types of bacteria under the light microscope. peptidoglycan outer membrane periplasmic space cell membrane Gram positive cell wall Gram   negative cell   wall   Name: Meghna Kakkar Student ID: 21163972 Page 2 PROCEDURE You are provided with grown bacterial cultures on solid media. A bacterial smear needs to be made of each  sample on a separate  slide. Take care not to muddle them up during the practical. 1. Take a clean, grease-free slide from the alcohol using a pair of forceps. Replace the lid of the container to prevent accidental ignition of the alcohol. Carefully flame the slide in the Bunsen burner to remove the alcohol. 2. Flame a microbiological loop in a hot Bunsen flame until it is glowing red to sterilise it. Using the flamed loop, aseptically add one small drop  of sterile distilled water to the slide. 3. Flame the loop as above. 4. Aseptically transfer a small amount of a bacterial colony into the water on the slide and spread over the slide to form a thin film. 5. Flame the loop. 6. Leave the slide to air-dry. 7. When dry the slides must be heat-fixed . To heat-fix, pass the slides through a hot Bunsen flame 4-5 times using forceps to hold the slide. 8. Place the slide on the staining tray and flood the slide with Crystal Violet. Leave for 2 minutes. 9. Pour off the Crystal Violet into the staining tray and replace with Gram’s  Iodine. Leave for 1 minute. 10. Wash the Iodine off with Alcohol until it almost runs clear. 11. Wash with tap water. 12. Flood the slide with Safranin. Leave for 2 minutes. 13. Wash with tap water and carefully blot the slide dry. Do not rub the slide as you will remove the smear! 14. Examine under the light microscope at 1000X magnification. All tissues and gloves should be placed into the biohazard bag. Slides should be placed in the disinfectant pots provided.   Name: Meghna Kakkar Student ID: 21163972 Page 3 ANSWER THE FOLLOWING REPORTING THE EXPERIMENTAL RESULTS 1. Describe the appearance of the colonies produced by each organism with respect to their size, shape, colour and appearance using the following guidelines.  The Image below ( Image 1) shows the colonies that were produced by the organisms. The sample that was taken for this experiment was obtained by swabbing the floor near the door of the lab. The colony that was used for the microscopic observation has been indicated on Image 1 . The description of this colony is: SHAPE:  Irregular SIZE:  Approximately 10mm ELEVATION:  Flat SURFACE:  Roughened COLOUR:  Translucent CONSISTENCY:  Mucoid ( 2 marks ) 2. Draw your microscopy observations for each sample, illustrating the relative size, shape and grouping of the bacterial cells.   Image 2 shows the microscopic   observation; this image was obtained by taking a picture through the microscopic lens.  Image 1 Colony used for microscopic observation using Gram staining  process.  Image 2   Name: Meghna Kakkar Student ID: 21163972 Page 4 ( 3 marks )  
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