Complete EMB Lab - lab PDF

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SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Microbiology Lab Notebook Report

Eosin Methylene Blue Agar - Exercise 4-3 March 24, 2020

Introduction Eosin methylene blue (EMB) agar is a complex (chemically undefined), selective, and differential medium. It contains digest of gelatin, lactose, and the dyes eosin Y and methylene blue. The gelatin provides nitrogen and organic carbon. Lactose is fermented to acid endproducts by coliforms such as Escherichia coli and Enterobacter aerogenes, whereas it is not by pathogens such as Proteus, Shigella, and Salmonella species.

Purpose EMB agar (Levine) is used for the isolation of fecal coliforms in food and dairy products as well as in environmental (especially water) samples. You will spot inoculate one EMB agar plate and one nutrient agar (NA) plate with four test organisms. The NA plate will serve as a comparison for growth quality on the EMB agar plate.

Materials • • • • • • • •

Microscope One eosin methylene blue agar (EMB) plate One nutrient agar (NA) plate Fresh broth cultures of these recommended organisms: Enterobacter aerogenes Escherichia coli Providencia stuartii Staphylococcus epidermidis

Procedure 1. Wear a lab coat, gloves, and chemical eye protection when performing this procedure.

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SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

2. Gently mix each culture according to your lab's standards. 3. Using a permanent marker, divide the bottom of each plate into four sectors. 4. Label the plates with the organisms' names, your name, and the date. Use the same positions for each specimen on both plates. 5. Spot inoculate the sectors on the EMB plate with the test organisms. 6. Repeat step 5 with the nutrient agar plate. Be sure to inoculate the specimens in the sectors that correspond to their position on the EMB plate. 7. Invert and incubate the plates at 35 ± 2 ℃ for 24 to 48 hours. 8. Save or dispose of the original cultures as directed by your instructor.

Results

Observations and Interpretations Results

Interperation

Presumptive ID

Poor growth or no growth

Organism is inhibited by eosin and/or methylene blue

Gram-positive

Good growth

Organism is not inhibited by eosin and/or methylene blue

Gram-negative

Growth is pink and mucoid

Organism ferments lactose with little acid production

Possible coliform

Growth is “dark” (purple to black and possible metallic green)

Organism ferments lactose with acid production

Probable coliform

Growth is “colorless” (not purple/ green/red/ or pink)

Organism does not ferment lactose

Noncoliform

ID #7330577

2

SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Conclusion This EMB plate was inoculated with a Gram-positive coccus (center) and two members of the Enterobacteriaceae, a coliform at the top and a noncoliform at the bottom. The Gram-positive coccus was inhibited by eosin and' methylene blue dyes but still grew enough to form a thin film on the agar. Vigorous lactose fermentation by the coliform gave its growth a shiny green appearance. The noncoliform was not inhibited by the dyes but also did not ferment the lactose, and so is a more natural color. EMB would normally be streaked for isolation, but you will spot inoculate pure cultures for the sole purpose of seeing what the different results look like.

Follow-Up Questions 1. -

Is EMB agar a defined or an undefined medium? Provide the reasoning behind your choice and explain why this formulation is desirable. It is undefined due to the gelatin in the medium. Gelatin is a family of related biochemicals, and the exact chemical formula of the gelatin used in making Levine EMB is not necessarily known. An undefined medium is desirable to support growth of a wide variety of the organisms being selected for.

2. In your own words, what are the roles of eosin Y and methylene blue in EMB agar? - Both act as selective agents against Gram positives. They also are responsible for the color changes produced by lactose fermenters. 3. In your own words, what is the role of lactose in EMB agar? - EMB agar is a differential medium based on the ability of an organism to ferment lactose to acid end products; lactose is the substrate of the fermentation. Coliforms perform this fermentation, whereas non-coliforms do not. -

4. Think about the results you recorded. a. Growth on the EMB agar and NA plates was recorded as "good growth," "poor growth," or "no growth." These are qualitative and, at least for the first two, subjective terms. What did you use to establish what constituted "good growth?" -The nutrient agar plate inoculated with the same organisms provided examples of what "good growth" for each organism looks like on a nonselective media. Without these for

ID #7330577

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SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

comparison, it wouldn't be possible to tell if sparse growth on EMB was due to inhibition or was just normal growth for that species.

b. Why wouldn't it be advisable to compare growth of the organisms on each plate to each other? There are at least two answers to this question! - On the NA plate, "good growth" for one organism may be very dense, whereas "good growth" for another might be fairly thin. In the context of this exercise you want to compare the amount of growth of each organism on the selective medium against its growth on NA to see if inhibition occurred. You wouldn't want to compare growth of the organisms on the EMB plate to each other because you wouldn't know which ones were inhibited and which were normal.

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