BIOL 3150 Lab 2 and 3.pdf

BIOL 3150 Lab 2 and 3.pdf - LABORATORY 2 – BASIC...

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34 LABORATORY 2 BASIC TECHNIQUES II Please note: You must bring graph paper and loose leaf paper to complete the in-lab assignments. EXERCISE 1: Gram Staining Introduction: Differential Staining Last week, you learned to use simple stains to examine the gross morphology of various bacterial species. This week, you will practice using a number of differential stains . Differential stains allow a microbiologist to detect differences between organisms or differences between parts of the same organism. In practice, differential stains are used much more frequently than simple stains because they allow determination of cell size, shape and arrangement (as with a simple stain), but provide information about other features as well. The most commonly used differential stain in bacteriology is the Gram stain 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 difference in Gram staining is due primarily to differences in cell wall structure in the two groups of organisms. Gram-positive cell walls possess a thicker layer of peptidoglycan which traps the Crystal Violet- iodine complexes and prevents their removal during decolourization. Objectives for Week 2 - After completing these exercises, you should: 1. Understand the Gram staining procedure and how to use Gram staining to distinguish Gram positive from Gram negative organisms. 2. Perform serial dilutions and perform Spectronic 20 measurements. 3. Student pairs should work together in this lab, not each one to prepare his own experiments. Reading: Madigan et al (2018): Ch 1: p. 11-16; Ch 2: p. 53-56; Ch 5: p. 149-152
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35 While some organisms give Gram-variable results, most variable results are due to poor technique (most often in the decolourization step). It is possible to “over - decolorize” by leaving the alcohol on too long and get pink Gram-positive cells. It is also possible to “under -decolo rize” and produce purple Gram-negative cells. For this reason, you must be very careful.
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  • Winter '13
  • MichaelGadson
  • Bacterial growth

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