BIOL 351 Lab 10

What is the purpose of MacConkey agar?

To isolate and differentiate members of Enterobacteriaceae based on lactose fermentation.

Is MacConkey agar selective, differential, or both?

Both selective and differential.

What are the four key components of MacConkey agar?

Lactose, bile salts, neutral red, and crystal violet.

What makes MacConkey agar selective?

Bile salts and crystal violet inhibit Gram-positive bacteria.

Which bacteria are selected for on MacConkey agar?

Gram-negative bacteria.

What makes MacConkey agar differential?

Lactose and the neutral red pH indicator differentiate lactose fermenters.

What is the pH indicator in MacConkey agar?

Neutral red.

What color are colonies on MacConkey agar above pH 6.8?

Colorless.

What color are colonies on MacConkey agar below pH 6.8?

Pink or red.

Why do lactose fermenters turn pink on MacConkey agar?

Lactose fermentation produces acid, lowering the pH and changing the indicator color.

What does pink growth on MacConkey agar indicate?

Growth of bacteria that ferment lactose.

What does colorless growth on MacConkey agar indicate?

Growth of bacteria that do not ferment lactose.

What does no growth on MacConkey agar indicate?

The organism is inhibited by the selective medium.

What are the three MacConkey agar outcomes shown in the lecture?

No growth; growth without lactose fermentation; growth with lactose fermentation.

Where are biochemical tests listed on the enteric identification chart?

Along the left side.

Where are organism names listed on the enteric identification chart?

Across the top.

What do numbers on the enteric chart represent?

The percentage of 100 tested samples giving a positive result.

What percentage is considered positive on the enteric chart?

Greater than 50%.

Which tests are considered more reliable on the enteric chart?

Biochemical tests from indole through malonate.

Which tests should be used first to narrow down an unknown enteric organism?

The more reliable biochemical tests from indole through malonate.

Why should carbohydrate tests be considered after the other biochemical tests?

The lecture identifies carbohydrate tests as less reliable for initial identification.

How are genera with multiple species displayed on the enteric chart?

The genus is written horizontally above the species names.

How is an organism with only one listed species displayed on the chart?

Its full name is written vertically.

How many unknown organisms are assigned in the identification exercise?

Two.

What are the supplies for unknown identification Day 1?

Two unknown slants, two TSA plates, and two MacConkey plates.

What must be recorded before streaking on Day 1?

Observations of growth on the original unknown slants.

What streaking method is used on Day 1?

Quadrant streaking on TSA and MacConkey plates.

At what temperature are the Day 1 plates incubated?

37°C.

What information must be written on each plate?

Initials, section number, date, unknown number, and medium type.

What should happen to the original unknown slants?

Return them to the TA for safekeeping.

Where are unknown identification results recorded?

On lab manual worksheets submitted to Canvas.

Which biochemical test results are reviewed from the previous lab?

MSA, BEA, citrate, malonate, MIO, and nitrate reduction.

What does MIO stand for?

Motility, indole, and ornithine.

Which MIO results must be read before adding Kovacs reagent?

Motility and ornithine.

How much Kovacs reagent is added to an MIO tube?

3–5 drops.

Where should the Kovacs reagent color change be observed?

In the reagent layer on top of the agar.

How much nitrate reagent A and B is added?

Eight drops of each reagent.

What should be done if no color change occurs after adding nitrate reagents A and B?

Add a small amount of zinc and observe for a color change.

What are the main procedures on unknown identification Day 2?

Record plate observations; perform catalase, oxidase, and Gram stain tests; examine morphology and arrangement.

Which plate supplies the isolated colony for the Gram stain?

The TSA plate.

What three characteristics are examined under the microscope?

Gram reaction, cellular morphology, and cellular arrangement.

Which Gram stain reagents are listed in the Day 2 supplies?

Crystal violet, iodine, and safranin.

Where should Gram stain decolorizing waste be collected?

In the provided ethanol waste container.

Where are used glass slides discarded after the catalase test?

In the glass disposal box.

At what temperature are the plates returned after Day 2?

25°C.

How is the unknown organism group number determined?

Using the lab manual after completing the required tests and verifying results with the TA.