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Microbiology for Allied Health Students: Lab Manual

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    This lab manual was created to support a microbiology course for allied health students. The first section of the manual was adapted from the OpenStax Microbiology textbook, of which a remixed version, Microbiology for Allied Health Students, is used as the text for the course. The next section, staining methods, encompasses three essential staining procedures used in any microbiology lab. The manual concludes with descriptions of the major biochemical tests students must perform in order to identify an unknown microorganism. While many traditional lab manuals are lengthy and comprehensive, descriptions of the labs in this manual are kept minimal to encourage students to further research the procedures and results on their own.

    • 2.1: Gram Stain Procedure
      The Gram stain procedure is a differential staining procedure that involves multiple steps. It was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques.
    • 2.2: Endospore Stain Procedure
      Endospore Staining is a technique used in bacteriology to identify the presence of endospores in a bacterial sample, which can be useful for classifying bacteria.
    • 2.3: Acid-fast Stain Procedure
      Acid-fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids. These acids resist staining by ordinary methods such as a Gram stain. It can also be used to stain a few other bacteria, such as Nocardia.
    • 3.1: Carbohydrate Fermentation
      During fermentation most bacteria convert carbohydrates into organic acids, with or without the production of gas. One can test for this by adding a pH indicator and an inverted tube (a Durham tube) to the culture medium. We will use phenol red as the pH indicator. If acid is produced the phenol red will turn yellow (pH below 6.8). Any gas produced will form a bubble in the inverted tube.
    • 3.2: Starch Agar
      Amylase is an exoenzyme (an enzyme released from the bacteria into its surroundings) that breaks starch by cleaving large starch molecules into monosaccharide and disaccharide units that can then enter the cell and be metabolized.
    • 3.3: Gelatin Agar
      Gelatinase is an exoenzyme that digests the protein gelatin into amino acids and shortchain peptides. There are two ways to test for the production of gelatinase. One method, the gelatin liquefaction test, examines the ability of gelatinase to liquefy nutrient gelatin. However, the tubes need to be incubated for up to 7 days. However, the test that we will use takes advantage of the ability of strong acids to denature proteins and form a visible precipitate.
    • 3.4: Catalase
      Catalase is an enzyme that breaks hydrogen peroxide into water and oxygen. Hydrogen peroxide is a common byproduct of metabolic reactions occurring in an environment where water and oxygen are present, but it is toxic to cells. Therefore, most organisms that survive in the presence of oxygen contain enzymes to degrade the hydrogen peroxide.
    • 3.5: Motility Agar
      Because the flagellar staining procedure often produces poor results in the hands of novices, other tests for motility (and the presence of flagella) have been developed. One type of test involves using a semi-solid medium that allows motile bacteria to penetrate.
    • 3.6: SIM Agar
      SIM (sulfide, indole, motility) medium is an example of a multi-test medium, that is, it tests more than one aspect of the bacterium’s metabolism at a time. In this case, the production of hydrogen sulfide, the formation of indole, and motility.
    • 3.7: Tryptone Broth
      Bacterial tryptophanase coverts the amino acid tryptophan into pyruvate, ammonia, and indole. The medium used to test for this enzyme is 1% tryptone in water.
    • 3.8: Methyl Red - Voges-Proskauer (MR-VP) Broth
      When glucose is fermented, a variety of products are possible. This is a test used to determine some of those products. Most organisms differentiated by this test convert the glucose to acids. Some bacteria continue to produce more acid in ‘mixed acid fermentation.’ Some genera use the glucose but produce end products with a more neutral pH like those that result from ‘butanediol fermentation.’
    • 3.9: Simmons Citrate Agar
      Simmons Citrate Agar is a defined medium that tests for an organism's ability to use citrate as its carbon source and ammonia as its nitrogen source. Only bacteria that can transport citrate into the cell from the medium grow well in Citrate Agar.
    • 3.10: Urea Broth
      Some bacteria are able to break urea into carbon dioxide and ammonia by means of the exoenzyme urease.
    • 3.11: Phenylalanine Agar
      Bacteria that produce phenylalanine deaminase are able to remove the amino group from the amino acid phenylalanine yielding ammonia and phenylpyruvic acid
    • 3.12: Triple-Sugar Iron Agar
      Triple-sugar iron agar (TSI agar) is another example of a multi-test agar. It tests for the fermentation, with or without gas production, of glucose, lactose, and sucrose. It also tests for the production of hydrogen sulfide from amino acids. Phenol red is the pH indicator used in this test medium.
    • 3.13: Levine EMB Agar
      Levine EMB (eosin methylene blue) agar is an example of a selective and differential medium. This means that only some bacteria will grow on this agar and that the appearance of those that do grow will be different. In particular, EMB agar inhibits the growth of Gram-positive bacteria and helps differentiate some of the Gram-negative rods.
    • 4: Microbiology Laboratory Safety Contract
      Microbiology is a hands-on laboratory class. You will be doing many laboratory activities which require the use of potentially hazardous bacteria and chemicals. Safety in the microbiology laboratory is #1 priority of the instructor and the student. To ensure a safe microbiology laboratory, a list of rules has been developed and provided to you in this student safety contract. These rules must be followed at all times.

    This page titled Microbiology for Allied Health Students: Lab Manual is shared under a CC BY license and was authored, remixed, and/or curated by Molly Smith and Sara Selby (GALILEO Open Learning Materials) .

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