Clinical Microbiology Made Ridiculously Simple 6th Edition / Клинична Микробиология направена изключително лесна: CHAPTER 1. BACTERIAL TAXONOMY

All organisms have a name consisting of two parts: the genus followed by the species (i.e., Homo sapiens). Bac­ teria have been grouped and named primarily on their morphological and biochemicaVmetabolic differences. However, bacteria are now also being classified accord­ ing to their immunologic and genetic characteristics. This chapter focuses on the Gram stain, bacterial morphology, and metabolic characteristics, all of which enable the clinician to rapidly determine the organism causing a patient's infection.

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GRAM STAIN

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Because bacteria are colorless and usually invisible to

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light microscopy, colorful stains have been developed to visualize them. The most useful is the Gram stain, which separates organisms into 2 groups: gram-positive bugs and gram-negative bugs. This stain also allows the clinician to determine whether the organism is round or rod-shaped.

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For any stain you must first smear the substance to be stained (sputum, pus, etc.) onto a slide and then heat it to fix the bacteria on the slide.

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There are 4 steps to the Gram stain:

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1)

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Pour on crystal violet stain (a blue dye) and wait 60 seconds.

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2)

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Wash off with water and flood with iodine solu­

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tion. Wait 60 seconds.

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3)

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Wash off with water and then "decolorize" with

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95% alcohol.

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4)

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Finally, counter-stain with safranin (a red dye).

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Wait 30 seconds and wash off with water.

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When the slide is studied microscopically, cells that absorb the crystal violet and hold onto it will appear blue. These are called gram-positive organisms. How­ ever, if the crystal violet is washed off by the alcohol, these cells will absorb the safranin and appear red. These are called gram-negative organisms.

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Gram-positive = BLUE

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I'm positively BLUE over you!!

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Gram-negative = RED

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No (negative) RED commies!!

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The different stains are the result of differences in the

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cell walls of gram-positive and gram-negative bacteria.

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Figure 1-1

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Both gram-positive and gram-negative organisms have more than 1 layer protecting their cytoplasm and nucleus from the extracellular environment, unlike animal cells, which have only a single cytoplasmic membrane composed of a phospholipid bilayer. The layer just outside the bacterial cytoplasmic membrane is the peptidoglycan layer or cell wall. It is present in both gram-positive and gram-negative organisms.

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Fig. 1-1. The peptidoglycan layer or cell wall is composed ofrepeating disaccharides with 4 amino acids in a side chain extending from each disaccharide.

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Fig. 1-2. The amino-acid chains of the peptidoglycan covalently bind to other amino acids from neighboring chains. This results in a stable cross-linked structure. The enzyme that catalyzes the formation of this linkage is called transpeptidase and is located in the inner cytoplasmic membrane. The antibiotic penicillin binds to and inhibits this enzyme. For this reason the enzyme is also called penicillin binding protein (see page 160)

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Figure 1-3

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Fig. 1-3. The gram-positive cell wall is very thick and

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has extensive cross-linking of the amino-acid side chains. In contrast, the gram-negative cell wall is very thin with a fairly simple cross-linking pattern.

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Fig. 1-4. The gram-positive cell envelope has an outer cell wall composed of complex cross-linked peptidoglycan, teichoic acid, polysaccharides, and other proteins. The inner surface of the cell wall touches the cytoplasmic membrane. The cytoplasmic membrane contains proteins

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GRAM-POSITIVE CELL ENVELOPE

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Figure 1-4

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that span the lipid bilayer. The bacterial cytoplasmic membrane (unlike that of animals) has no cholesterol or other sterols.

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An important polysaccharide present in the gram­ positive cell wall is teichoic acid. It acts as an antigenic determinant, so it is important for serologic identifica­ tion of many gram-positive species.

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Fig. 1-5. The gram-negative cell envelope has 3 layers, not including the periplasmic space. Like gram-positive bacteria, it has 1) a cytoplasmic membrane surrounded by

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a peptidoglycan layer. 3) In addition, a gram-negative

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2)

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cell has a unique outer cell membrane.

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The inner cytoplasmic membrane (as in gram­ positive bacteria) contains a phospholipid bilayer with embedded proteins. Gram-negative bacteria have a periplasmic space between the cytoplasmic membrane and an extremely thin peptidoglycan layer. This periplasmic space is filled with a gel that contains pro­ teins and enzymes. The thin peptidoglycan layer does not contain teichoic acid, although it does have a small helical lipoprotein called murein lipoprotein. This lipoprotein is important because it originates from the peptidoglycan layer and extends outward to bind the unique third outer membrane. This last membrane is similar to other cell membranes in that it is composed of two layers of phospholipid (bilayer) with hydrophobic tails in the center. What makes it unique is that the outermost portion of the bilayer contains lipopolysac­ charide (LPS).

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Fig. 1-6. Lipopolysaccharide (LPS) is composed of 3

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covalently linked components:

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1)

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Outer carbohydrate chains of 1-50 oligosaccharide

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units that extend into the surrounding media. These differ from one organism

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nants. This part is called the 0-specific side chain or the 0-antigen. Think of 0 for Outer to help remember this.

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2)

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The center part is a water soluble core polysac­

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charide.

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