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Chapter 17 : Reagents, Stains, and Media: Bacteriology

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Abstract:

A number of classical and rapid tests are used for the identification of medically important bacteria. -acetyl-L-cysteine-sodium hydroxide (NALC (mucolytic agent)-NaOH), Cetylpuridium chloride-sodium chloride (CPC-NaCl), oxalic acid are used for decontamination agents. A variety of dyes and indicators are used to detect specific reactions such as pH and oxygen production. A variety of stains can then be used to help visualize and differentiate bacteria from the specimen. Gram staining is the differential staining procedure most commonly used for microscopic examination of bacteria. Based upon the staining reaction, bacteria are classified as gram-positive organisms, which retain the primary crystal violet dye and appear deep blue or purple, and gram-negative organisms, which can be decolorized, thereby losing the primary stain and subsequently taking up the counterstain safranin and appearing red or pink. Louis Pasteur in 1860 was the first to use culture media for growing bacteria in the laboratory. Almost immediately, additional media began to be developed by Robert Koch and his colleagues, who used animal and plant tissues as sources of nutrients to support bacterial growth. Using solid media permits the isolation of pure cultures of bacteria. Agar is the most common solidifying agent used in microbiological media. Many media contain selective components that inhibit the growth of nontarget bacteria. Some pathogens are anaerobic, and factors such as thioglycolate are included in some media to reduce the availability of molecular oxygen so that anaerobes may be cultured. Bacteria that reduce nitrate to nitrite turn the reagents red or pink.

Citation: Atlas R, Snyder J. 2011. Reagents, Stains, and Media: Bacteriology , p 272-303. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch17

Key Concept Ranking

Chemicals
0.6406133
Gram-Negative Bacteria
0.53705984
Antimicrobial Susceptibility Testing
0.5162868
Bile Esculin Azide Agar
0.4597504
Gram-Positive Bacteria
0.4532345
Lipids
0.42831436
0.6406133
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References

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Tables

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TABLE 1

Nitrite and nitrate reductase activities

Courtesy of Georges Wauters and Mario Vaneechoutte.

Addition of Zn is necessary only when both broths remain colorless. It can then differentiate between situations 3 and 4.

In case of normal nitrite reductase positivity (p), as in situation 2, nitrite is still present. In the case of very strong positive nitrite reductase activity (pp), as in situation 3, all nitrite is further reduced quickly, resulting in a false-negative nitrate reductase reaction upon addition of Griess reagents to the nitrate broth. In that case, addition of Zn is needed to confirm that nitrate is no longer present.

Citation: Atlas R, Snyder J. 2011. Reagents, Stains, and Media: Bacteriology , p 272-303. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch17
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TABLE 2

Dyes and pH indicators

Citation: Atlas R, Snyder J. 2011. Reagents, Stains, and Media: Bacteriology , p 272-303. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch17
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TABLE 3

McFarland standards protocol

Citation: Atlas R, Snyder J. 2011. Reagents, Stains, and Media: Bacteriology , p 272-303. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch17
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TABLE 4

Culture media for enrichment, isolation, and cultivation of pathogenic bacteria

Citation: Atlas R, Snyder J. 2011. Reagents, Stains, and Media: Bacteriology , p 272-303. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch17
Generic image for table
TABLE 5

Commercial sources of chromogenic agar media for bacteria

Citation: Atlas R, Snyder J. 2011. Reagents, Stains, and Media: Bacteriology , p 272-303. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch17

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