BIOL 3150 Lab2 - ,youshould 1 2

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32 LABORATORY 2 – BASIC TECHNIQUES II Pre lab questions (to be handed in prior to the start of the Lab) [2 Marks] 1.How would you dilute a NaCl solution that has an initial concentration of 15% (w/v) to a 50mlsolution at 0.1%? 2.You have 5ml of an overnight culture (2x108cells per ml). How would you prepare a series of 1 or10ml dilutions (maximum dilution 1:100) to obtain a final 200ml culture at 1x103cell/ml? Showyour work for each step. 3.If 0.1ml (from a 1 ml sample )of a 105dilution of pond water was plated and yielded 52 colonies,how many bacteria were present per ml in the original water sample? Show your work. 4.If 0.1 ml of a urine culture from a 107dilution yielded 37 colonies, how many bacteria werepresent per ml in the original sample? Show your work. 1. Understand the Gram staining procedure and how to use Gram staining to distinguish Gram positive from Gram negative organisms. 2. Know how to perform differential staining to identify cellular structures such as endospores. 3. Perform serial dilutions and perform Spectronic 20 measurements. Reading: Madigan et al (2015): Ch 2: pp 26 29 and 32 33; Ch 5: pp 155 158 and Ch 6: pp 156 157.
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33 which distributes almost all bacteria into one of two groups. Other differential stains are used for organisms not distinguishable by the Gram stain or for those possessing important cellular characteristics such as acid fastness (a property of Mycobacterium spp. ), a capsule, endospores or flagella. Gram Stain The Gram stain is a differential stain in which a decolourization step occurs between the application of two basic stains. The primary stain is Crystal Violet while Iodine is added as a mordant to enhance Crystal Violet staining by forming a Crystal Violet iodine complex. Decolourization by alcohol follows and is the most critical step in the procedure. The cells decolorized by the alcohol are termed Gram negative while the other cells are Gram positive . Gram negative cells are then colorized by the counterstain , Safranin, to improve their visibility. Thus, after the staining procedure, Gram positive cells appear purple while Gram negative cells are pink. Analysis of Gram positive and Gram negative organisms has revealed that the
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