Microorganisms through a Microscope

Question 1

Differentiate b/w an acidic dye and a basic dye, and give some examples of the commonly used dyes

(Why do basic dyes stain bacterial cells? Why don’t acidic dyes stain bacterial cells?)

Answer 1

1. Acidic Dye: Positive ions – Includes eosin, acid fuchsin, & nigrosin.
   - - Not commonly used because bacteria are slightly negatively charged at pH 7, and acidic dyes are not attracted to most types of bacteria
   - - The stain colors the background instead
2. Basic Dye: Negative ions – Includes crystal violet, methylene blue, malachite green & safranin

Question 2

Name the 3 kinds of staining techniques

Answer 2

1. Simple
2. Differential
3. Special

Question 3

Explain the simple staining technique. What’s the primary purpose of simple stain?

Answer 3

1. Simple staining technique uses an aqueous or alcohol solution of a single basic dye
2. The primary purpose: to highlight the entire microorganism so that cellular shapes and basic structures are visible

Question 4

What is the chemical called when added to intensify a staining solution?

Answer 4

Mordant

Question 5

What are the two common differential stain techniques?

Answer 5

1. Gram Stain

2. Acid-fast Stain

Question 6

Explain the Gram Staining procedures in 4 major steps

Answer 6

1. Application of crystal violet (purple dye)
   - - After a short time, wash off the purple dye with water
2. Application of iodine (mordant)
   - - After a short time, wash off the iodine with water
3. Alcohol or alcohol-acetone solution wash (decolorization)
   - - After a short time, rinse off the alcohol
4. Application of safranin (counterstain, red dye)
   - - After a short time, wash off safranin with water and blot dry before examination

Question 7

What color does Gram (+) bacteria appear? And Gram (-)?

Answer 7

Gram (+): Blue/Purple

Gram (-): Red/Pink

Question 8

Why can Gram (+) bacteria trap crystal violet stain, but not Gram (-)? Hint: cell wall structure / composition

Answer 8

1. Gram (+) bacteria: Thicker Peptidoglycan cell wall inhibits the Crystal violet-Iodine (CV-I) complex to be washed off by alcohol
   - - Peptidoglycan: disaccharides + aa’s
2. Gram (-) bacteria: Thin Peptidoglycan and they contain a layer of lipopolysaccharide (LPS), which is disrupted by alcohol wash, so the CV-I complex is washed out through the thin layer of peptidoglycan, appearing colorless until counterstained with safranin.

Question 9

Are Gram (+) or Gram (-) bacteria easily killed by Penicillin and Cephalosporin? Why?

Answer 9

1. Gram (+) bacteria are killed easily by PCN and Cephalosporin
2. Gram (-) bacteria are more resistant to these antibiotics because they can’t penetrate the LPS layer.

Question 10

Which genus of bacteria usually use the acid-fast staining technique? And give examples of the pathogens.

Answer 10

1. Mycobacterium, including Mycobacterium tuberculosis and Mycobacterium leprae.
2. Nocardia

Question 11

Explain the acid-fast staining procedures in 3 major steps

Answer 11

1. Application of red dye carbolfuchsin, and heat for several mins
   – Cool and Wash off stain with water
2. Application of acid-alcohol (decolorization)
   Note: red stain is removed is NOT acid-fast (colorless)
3. Application of methylene blue (counterstain)
   – Appear blue if not acid-fast

Question 12

How does acid-fast stain work?

Answer 12

Acid-fast stain is more soluble in the cell wall lipids (waxy material) than in the acid-alcohol, therefore, retaining the red color

Question 13

When is the special staining used? Give some examples

Answer 13

To color and isolate specific parts of microorganisms, such as Endospores, Flagella, and Capsules.

Question 14

Explain the Capsule Stain procedure in 2 major steps (capsule stain is also called negative stain)

Answer 14

1. Mix the bacteria in color solution: India ink or nigrosin
   - - Providing a dark background
2. Stain the bacteria with a Simple Stain, ex. safranin
   - - The Capsule doesn’t accept the dye, but the bacterial cell does. Thus, unstained halos appear microscopically

Question 15

Explain the Endo(spore) Staining procedure in 3 major steps. What color do we see under the microscope after the staining?

Answer 15

Schaeffer-Fulton endospore stain:

1. Application of Malachite green and steam for 5 mins
   - - Heat helps the stain penetrate the endospore wall
2. Wash with water for 30 secs
3. Application of safranin (counterstain)
   - - Stain bacterial cells other than the endospores.

Note: the endorspores appear green w/i red or pink cells

Question 16

Explain the Flagella Staining and why is it needed?

Answer 16

1. Application of a mordant and carbolfuchsin
   - - To build up the diameters of the flagella until they become visible
2. Bacterial flagella are generally too small to be see without staining

Question 17

How is total magnification of an object calculated?

Answer 17

Magnification of the objective lens X Magnification of the ocular lens

Question 18

When/why is immersion oil used?

Answer 18

It is used with the oil immersion lens to reduce light loss b/w the slide and the lens

Question 19

An electron microscope differs from a light microscope in that ___ focused by ___ is used instead of light, and the image is viewed not through the ocular lenses but on ___.

Answer 19

1. Objects smaller than about 0.2 microns
2. A beam of electrons (travel in waves)
3. Electromagnetic condenser lenses
   (Note: images are always black and white)

Question 20

The max. magnification of a compound microscope is ___; that of an electron microscope, ___.
The max. resolution of a compound microscope is ___; that of an electron microscope, ___.
One advantage of a scanning electron microscope over a transmission electron microscope is ___.

Answer 20

1. 2000X, 10,000X

Question 21

Endospores can be seen as refractile structures in unstained cells and as colorless areas in Gram-stained cells. Why is it necessary to do an endospore stain to verify the presence of endospores?

Answer 21

Because they can’t be differentiated from inclusions of stored material w/o a special stain