Bacteria That Vigorously Ferment Lactose Appear Metallic Green on Eosin Methylene Blue Agar

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Micro Lab Practical

Question	Answer
What is the purpose of Eosin Methylene Blue Agar?	It is used for isolation of fecal coliforms.
How is Eosin Methylene Blue (EMB) Agar put on a plate?	EMB agar can be streaked for isolation or used in the Membrane Filter Technique.
What are the ingredients of EMB agar?	Pepton, lactose, sucrose, and the dyes eosin Y and methylene blue.
What do the various ingredients in EMB agar do?	The sugars provide fermentable substrates to encourage growth of fecal coliforms. The dyes inhibit growht of Gram-positive organisms, and also produce a purple complex usually accompanied by a green metallic sheen in acidic conditions.
What does the green sheen in EMB agar mean?	This green metallic sheen serves as an indicator of the vigorous lactose and/or sucrose fermentation typical fo fecal coliforms.
What do small amounts of fermentation in EMB agar produce?	Smaller amounts of acid production result in a pink coloration of the growth.
What happens to nonfermenters in EMB agar?	Nonfermenters remain their normal color or take on the coloration of the medium.
What is the purpose of MacConkey agar?	It is used to isolate and differentiate members of the Enterobacteriaceae based on the ability to ferment lactose.
What is MacConkey agar made of?	It contains lactose, bile salts, neutral red dye, and crystal violet dye.
What do the various ingredients in MacConkey agar do?	Bile salts and crystal violet inhibit growth of Gram-positive bacteria. Neutral red dye is a pH indicator the is colorless above a pH of 6.8 and red at a pH below 6.8. Acid accumulating from lactose fermentation turns the dye red.
What happens for lactose fermenters and nonfermenters in MacConkey agar?	Lactose fermenters turn a shade of red on MacConkey agar whereas lactose nonfermenters remain their normal color or the color of the medium.
What is the purpose of a Gram stain?	It is used to distiniguish between Gram-negative and a Gram-positive cells. It also allows determiination of cell morphology, size, and arrangement.
What are the 2 stains used in Gram staining?	The primary stain is crystal violet and the secondary stain is Iodine.
What are the results of Gram staining?	Gram-negative cells are decolorized by the solution whereas Gram-positive cells are not. Gram-positive cells will appear purple and Gram-negative cells will appear reddish-pink.
Why do Gram-negative cells become decolorzied during Gram staining?	They have a higher lipid content and a thinner peptidoglycan layer. The alcohol in the decolorizer extracts the lipid, making the Gram-negative wall more porous and incapable of retaining the crystal violet-iodine complex, thereby decolorizing it.
What is the purpose of a catalase test?	This test is used to identify organisms that produce the enzyme catalse. It is most commonly used to differentiate members of the catalase-positive Micrococcaceae from the catalse-negative Streptococcaceae.
What happens in a catalse test?	Bacteria that produce catalase are detected easily using typical store-grade hydrogen peroxide. When hydrogen peroxide is added to a catalase-postive culture, oxygen gas bubbles form immediately. If no bubbles appear, the organism is catalase-negative.
What is the purpose of a DNase test?	It is mainly used to distinguish staphylococcus aureus from other staphylococcus species.
What is in a DNase test?	Peptides derived from soybean and casein, sodium chloride for osmotic balance, and DNA as the substrate.
What does a DNase test do?	DNase test helps determine the presence of the enzyme Deoxyribonuclease or DNase in the cell.
What is the purpose of the Oxidase test?	It is used to identify bacteria containing the respiratory enzyme cytochrome c oxidase.

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