Cognitive Apprenticeship (L06)

Question 1

Difference between Gram-positive and Gram-negative bacteria

Answer 1

Gram-positive:
- 2 Layers (Cell membrane & thick peptidoglycan cell wall)

Gram-negative:
- 3 layers (Cell membrane, outer membrane & thin peptidoglycan cell wall)

Question 2

What is the role of bacterial cell wall?

Answer 2

Determines the shape of the bacteria
Provide structural support to prevent bacterium from lysing due to osmotic pressure
- In isotonic solution, no net movement of water
- In hypotonic solution, water moves into the cell and cause the cell to burst

Question 3

Characteristics of Gram-positive cell wall

Answer 3

Thick peptidoglycan cell wall embedded with polyalcohol

Overall negative charge due to the presence of phosphodiester bonds between teichoic acid monomers

Question 4

Characteristics of Gram-negative cell wall

Answer 4

Thin peptidoglycan cell wall with lipopolysaccharides and lipoproteins
Overall negative charge due to the negatively charged lipopolysaccharides

Question 5

How to stain bacteria to ease visualization?

Answer 5

Before staining of bacteria, thin film of bacteria culture needs to be fixed onto a glass slide

Question 6

What are the steps to prepare smear for staining?

Answer 6

Liquid culture:
1. Label the glass slide with the name of the microorganism on the frosted surface with a wax pencil

2. Draw a circle at the bottom of the glass slides to mark the region for smear
3. Smear 3-4 loopfuls of the cell suspension onto the microscope slide in a circular fashion within the marked region for smear
4. Air-dry the smear
5. Pass the air-dried smear twice briefly over a flame to heat fix the smear

Solid media:
1. Label the slides with the name of the microorganism on the frosted surface with a wax pencil

2. Draw a circle at the bottom of the glass slide to mark the region for smear
3. Place a loopful of RO water on the slide
4. Transfer a small amount of a well isolated colony into the water droplet on the slide
   Spread in a circular fashion within the marked region for smear to obtain a homologous suspension
5. Air-dry the smear
6. Pass the air-dried smear twice briefly over the flame to heat fix the smear

Question 7

What happens when too much water is added (step 3 if solid media), when too many bacteria is used or when smear is too thin?

Answer 7

When too much water is added, it will take a longer time for the smear to air-dry.
When too many bacteria are being used, the smear will be too thick and it would be difficult to observe individual bacterial cells under the microscope.
When the smear is too thin, it would not be ideal for staining as it would be difficult to observe bacterial cells under the microscope.

Question 8

Why do we need to heat fix the smear?

Answer 8

The proteins on the surface of the bacteria will denature
Hence causing adhesion to the slide
Which prevents the smear from being washed off during staining

Question 9

Why do we need to air-dry the smear before carrying out heat fixation?

Answer 9

When the smear is not completely air-dried prior to heat fixation, brief heat fixing would not result in adhesion of bacterial cells to the slides
Longer heat fixation would then cause alteration to the shape and size of the bacteria

Question 10

Why do we need to carry out staining?

Answer 10

With the lack of contrast of unstained bacteria, it would be hard to visualize under a light microscope
The color dyes would increase the contrast and aid in visualization of bacterial cells

Question 11

What is the difference between simple staining and differential staining?

Answer 11

Simple staining is non-differential while differential staining allows differentiation of bacteria by cell properties

Question 12

Why is Gram-positive and Gram-negative bacteria differentiated during decolorization?

Answer 12

Gram-positive bacteria:
Thick peptidoglycan layer traps the crystal violet-iodine complex
Retaining the purple color

Gram-negative bacteria:
Lipids in the outer membrane dissolved, leading to the removal of crystal violet-iodine complex
Colorless cell wall will pick up the red color from safranin

Question 13

What would the results look like in Gram-positive and Gram-negative?

Answer 13

Gram-positive: purple/blue color

Gram-negative: red/pink color

Question 14

How to ensure that staining is done properly?

Answer 14

Perform Gram stain using bacteria of known Gram control

Positive control:
Use known Gram-positive bacteria
Should appear purple

Negative control:
Use know Gram-negative bacteria
Should appear red

Question 15

What are the steps for Gram staining?

Answer 15

1. Place the fixed slides on the staining rack
2. Gently cover smears with crystal violet and let it stand for 1 minute
3. Pour off the stain and wash smears with Gram’s iodine solution
   Cover the smear with Gram’s iodine for 1 minute
4. Pour off the iodine and wash the smears with 95% ethanol
   Stop washing when ethanol running off the slide is faintly violet, to avoid decolorization
5. Immediately wash the slides gently with RO water using the Pasteur pipette
6. Cover the smears with safranin for 30 seconds
7. Pour off the safranin
8. Blot gently with C-fold paper towel

Question 16

What are the steps we need to take when using a microscope?

Answer 16

1. Clean all the objective lens with 70% ethanol using Kim wipes
2. Adjust the stage to the lowest position possible using coarse adjustment knob
3. Turn the revolving nosepiece so that the lowest objective is above the specimen
4. Place the sample slide on the stage and secure it with a stage clip
5. Adjust the coarse adjustment knob to achieve focus for 4x objective
6. Switch to the next objective
7. Adjust fine adjustment knob to achieve focus for 10x objective
8. Switch to the next objective
9. Adjust fine adjustment knob to achieve focus for 40x objective
10. Shift the objectives such that the slide sits between 40x and 100x objective
11. Place a drop of immersion oil onto the specimen and switch to the oil immersion objective
12. Adjust fine adjustment knob to achieve focus for 100x objective
13. Wipe the oil off 100x objective using Kim wipes with 70% ethanol

Question 17

Why do we need to use immersion oil?

Answer 17

Prevents refractive loss and increase the resolution of the bacteria sample