IA. General microbiology | 4. Microscopic examination of bacteria. Staining methods used in bacteriology.

Question 1

I. Microscopic Examination
1. What are the 2 types of Microscopic Examination?

Answer 1

1. Simple Microscope Examination
2. Stained Preparations

Question 2

I. Microscopic Examination
2. What is Simple Microscope Examination?

Answer 2

Examination of microbes that are still alive

Question 3

I. Microscopic Examination
3. What are the 2 types of simple microscopic examination?

Answer 3

1. Native preparation
2. Vital staining

Question 4

I. Microscopic Examination - Simple Microscope Examination
4. What is native preparation?

Answer 4

preparation of flat drop/wet mount (glass slide and cover slip)

Question 5

I. Microscopic Examination - Simple Microscope Examination
5. What are 5 steps of vital staining?

Answer 5

1. Place drop of yeast (Candida Albicans) suspension with a pipette in center of slide
2. Place small drop of methylene-blue to the suspension wait 10mins, mix w. Loop
3. Wait 1min
4. Cover with a cover slip
5. Examine with dry lens

Question 6

I. Microscopic Examination - Stained Preparations
6. What are stained preparations?

Answer 6

Prepare fixed smear → killed microorganisms

Question 7

I. Microscopic Examination - Stained Preparations
7. What are the 2 types of stained preparations?

Answer 7

1. Simple stain
2. Complex stain

Question 8

I. Microscopic Examination - Stained Preparations
7A. What is simple stain?

Answer 8

• Only 1 stain ~ 1min (fuchsin, methylene blue, crystal violet etc)
• All organism are stained in the same colour in smear, dye just provides contrast

Question 9

I. Microscopic Examination - Stained Preparations
7B. What are examples of stains in simple stain?

Answer 9

fuchsin, methylene blue, crystal violet etc

Question 10

I. Microscopic Examination - Stained Preparations
7C. What are the 5 steps of simple stain?

Answer 10

1. Make fixed smear (see below)
2. Lay the slide with fixed film up across the rods over the sink
3. Flood with methylene-blue or fuchsin or other dyes
4. Rinse w. water after 2-5mins; allow the slide to dry by applying edge to a filter paper
5. Locate the smear under 10x (low) power and then examine it under oil
   immersion

Question 11

I. Microscopic Examination - Stained Preparations
7D. How to prepare fixed smear?

Answer 11

1. Degreasing the slide (5x fire)
2. Making bacterial suspension
   a. One drop of saline → mix w. a small loop of bacteria (decrease cells number = increase quality) → let air dry
3. Fix the smear with fire (3x or until dry)

Question 12

I. Microscopic Examination - Stained Preparations
8A. What is complex stain?

Answer 12

stain → washing/decolourization → counter stain

Question 13

I. Microscopic Examination - Stained Preparations
8B. What are the 4 types of complex stain?

Answer 13

1. Neisser Stain
2. Ziehl-Neelsen Stain (acid fast stain)
3. Capsule stain
4. Gram Stain

Question 14

I. Microscopic Examination - Stained Preparations
8C. What are the steps of Neisser Stain?

Answer 14

Neisser Stain – demonstrating polymetaphosphate granules
1. Cover slide with Neisser’s stain and allow to act for 3-5mins
2. Wash in water and blot dry
3. Cover slide with Neisser’s iodine and allow to act for 1mins
4. Wash and blot dry
*Granules appear bluish black or part of bacterium yellow or light green.

Question 15

I. Microscopic Examination - Stained Preparations
8D. What are the steps of Ziehl-Neelsen Stain (acid fast stain)?

Answer 15

1. Flood the slide with carbolfuchsin, heat to steaming (3x)
2. Cool slide and waster with water
3. Decolorize the slide with acid alcohol for 15s
4. Rinse w. water
5. Counterstaining w. Methylene-blue, leave on smears for 15-20s
6. Wash with water and let it dry
   *acid fast organism (red) other tissue elements (blue)

Question 16

I. Microscopic Examination - Stained Preparations
8E. What are the steps of Capsule stain?

Answer 16

1. Place a small drop of a negative stain (India Ink, Congo Red, Nigrosin, or Eosin) on the slide.
2. Smear the sample into the ink
3. smear the sample over the slide with another slide
4. Dry, heat fix
5. Simple counter stain could be applied
   ———-
6. Place a drop of broth culture of Klebsiella w. flamed loop into near the short edge of the slide. Add a drop of India ink. Mix w. the loop.
7. Using the short edge of a second slide, spread the drop over the entire slide. Allow to dry in air. Discard this second slide carefully as it is contaminated.
8. Heat-fix
9. Stain w. Fuchsin for 1min
10. Rinse w. water
11. Allow to dry in air. Observe with oil immersion
    *Capsules are hard to stain, we use “negative staining”: staining of the bacteria and the background. Capsule is seen as a unstained halo surrounding the cell

Question 17

I. Microscopic Examination - Stained Preparations
8F1. What are the steps of Gram stain?

Answer 17

1. Prepare smear (heat-fixed emulsion)
2. Cover the smear w crystal violet → 1min
3. Wash the slide with water
4. Cover the smear with Gram’s iodine → 1min
5. Wash the slide with water
6. Decolonize w. 96% ethanol → stop when the alcohol is no longer obviously purple
7. Wash the slide with water
8. Counterstain with safranin, keep → 1min
9. Use filter paper to gently dry the stained smear
10. Examine under 100x lens , w oil immersion

Question 18

I. Microscopic Examination - Stained Preparations
8F2. Explain the result of Gram stain?

Answer 18