MBIO 1010 / Lab 3

How do you smear for a gram stain?

1. Get a loopful of water on the slide.

2. Get a needleful of culture on the slide. Tap five times.

3. Mix the culture and water into nickel with loop. If cloudy, tens of thousands of cells.

4. Air dry.

5. Heat fix with 3–4 passes over pilot flame.

How do you stain for a gram stain?

1. Perform gram smear.

2. Add crystal violet. Let stay for 1 minute.

3. Wash off with gentle water. Blot dry with soft pressure.

4. Add iodine. Let stay for 1 minute.

5. Wash off with gentle water. Do not blot dry.

6. Hold slide at angle, and slowly drip 5–10 drops (6th is enough) directly on top of the smear and watch colour bleed out of the smear.

7. Wash off with gentle water. Blot dry with soft pressure.

8. Add safranin. Let stay for 1 minute.

9. Wash off with gentle water on both bottom and top side of slide. Blot dry with soft pressure.

How do you do a gram string test?

1. Place a loopful of culture on slide. For plate cultures, collect 3–4 colonies with loop. For slant cultures, scrape 3–4 colonies with loop.

2. Add a drop of KOH.

3. Mix bacteria with loop for 30 seconds.

4. When “retrieving” your loop, slimy reaction occurs with gram negative bacteria. The mixture is DNA.

The lab manual instructions say to "re-streak your pet bacterium onto a new T-soy plate." To to do this, you should…

Begin by touching a single well isolated colony with your loop, and then streak that onto the first section to begin the streak plate.

When counting spread plates with a lot of colonies, it's a good idea to…

Turn the plate upside down, and touch the back of the plate with a permanent marker to record each colony that has been counted.

By convention, plate counts from liquid cultures are always reported in scientific notation accurate to…

One decimal place.

To get just the right amount of water on your slide to make a nice smear for microscopy it is best to…

Hold your loop under a dripping tap, and catch a loopful of water that can then be placed on the microscope slide.

Which type of bacterial cell do you expect will be broken open and turn 'stringy' after being treated with a drop of 3% potassium hydroxide?

Gram negative cells, because they have thinner peptidoglycan.

When performing the KOH string test, you should use how much bacteria?

Use a visible amount of bacteria (which may be several colonies) because the more bacteria you have, the easier it will be to determine the results.