Lecture 1 - Intro to Bacteria & Gram Staining

Question 1

What are the 5 categories of microbiology?

Answer 1

1. Bacteriology [bacteria]
2. Virology [viruses]
3. Mycology [study of fungi and yeast]
4. Parasitology [study of protozoa, ameboa & worms]
5. Immunology [immunity and resistance]

Question 2

What is the arrangement of organisms based on their size? [smallest to largest]

Answer 2

smallest: prions & viruses [30-300nm/ 0.01 - 0.1 microns] (need a electron microscope to look at them)
intermediate: Bacteria & Fungi [1-10 microns] (need a microscope to look at them)
largest: parasites/worms & Mammals [>1000 microns / >1mm]

Question 3

How do we name bacteria?

Answer 3

based on relatedness of genetic information (rRNA)

genus & species names i.e. Escherichia Coli is [E.coli]

Question 4

3 examples of good bacteria in our bodies?

Answer 4

1. bacteria in our gut that help break down glycans [sugars from milk] into usable sugars
2. bacteria help produce vitamin B12 (for our RBC) and vitamin K (for blood clotting)
3. Bacteria provide protection against diseases [i.e. Salmonella]

Question 5

What are virulence factors?

Answer 5

toxins that are released by pathogenic bacteria that cause damage to tissue and cells

Question 6

What are the 3 bacteria shapes that can be found?

Answer 6

1. Cocci [round]
2. Bacilli [rod shaped]
3. Spiral [corkscrew shape]

Question 7

3 Types of cocci bacteria?

Answer 7

1. staphylococci [clumped together]
2. streptococci [joined end to end]
3. diplococci [two cells joined together]

Question 8

3 types of bacilli bacteria?

Answer 8

1. Coccobacilli [small, oval shape]
2. diplobacilli [paired bacilli joined end to end]
3. streptobacilli [chains of bacilli joined together]

Question 9

3 types of spiral bacteria?

Answer 9

1. vibriospiral [short, looks like a lima bean]
2. spirillaspiral [wavy shape, rigid] (looks like a worm)
3. Spirochaetespiral [helical, tightly coiled looking but flexible]

Question 10

What are two types of Bacterial Staining procedures? and example

Answer 10

1. Simple staining –> only used to detect if bacteria is present; does not give us enough information about diagnosis [i.e. methylene blue]
2. Differential Staining –> tells us what kind of bacteria is present; gives us useful information for treatment and diagnosis [i.e. gram staining & acid fast/ZN stain]

Question 11

What is the acid fast stain used for?

Answer 11

• differential stain [useful for diagnosis]

- used for bacteria that have a waxy outer coating on their cell walls [i.e. Mycobacteria Tuberculosis TB]

Question 12

What is the gram stain used for?

Answer 12

separates bacteria into gram positive or gram negative characteristics based on how they absorb a dye
- it is a diagnostic test used to identify disease causing bacteria

Question 13

What are 5 steps of gram staining?

Answer 13

1. Fix Specimen on the slide & then flood the slide with methanol or pass it through a flame to sterilize
2. add crystal violet on the surface of the slide [this is the primary stain]
3. add iodine on the slide to wash off the excess crystal violet [this is called a mordant and it helps the stain stay only on the cells]
4. apply decolorizer like acetone/alcohol [this is the differentiation step]
5. Add Safranin to the slide [this is a counterstaining test]

Question 14

What is the purpose of step 2 in the gram staining procedure?

Answer 14

addition of crystal violet is used because its a dye that penetrates into the peptidoglycan layer of all cells on the slide

Question 15

What is the purpose of step 3 in the gram staining procedure?

Answer 15

Iodine helps the stain stay in the cells by combining with the crystal violet and forming a CV-I complex. This complex cant leave the cell because its entrapped in the peptidoglycan layer

Question 16

What is the purpose of step 4 in the gram staining procedure?

Answer 16

Alcohol/Acetone rinses away the remains of CV

• Acetone dissolves some lipids in the peptidoglycan layer
• alcohol can dehydrate the peptidoglycan and make it have holes
• this is the step where gram + cells will remain purple coloured while gram - cells lose their color because their peptidoglycan layer is thin

Question 17

What is the purpose of step 5 in the gram staining procedure?

Answer 17

This is the counterstaining step

• Safranin is added to make the gram - cells turn pink and show up after they became colourless in step 4
• Gram + cells will remain purple colour

Question 18

What colour should a gram + bacteria be after a gram stain procedure?

Answer 18

should be purple/blue colour

Question 19

What colour should a gram - bacteria be after a gram stain procedure?

Answer 19

should be a pink colour

Question 20

What is peptidoglycan?

Answer 20

its a mesh layer on the outside of the plasma membrane of bacteria; it makes up the bacteria’s cell wall

Question 21

What is the difference between the peptidoglycan layer of gram + vs. gram - bacteria?

Answer 21

gram + bacteria have very thick peptidoglycan layer [this is why it does not leak or dry out much after the addition of acetone/alcohol in staining procedure]
gram - bacteria has a thinner layer of peptidoglycan, and this is why it needs to be counterstained during the gram staining procedure because the addition of alcohol and acetone makes it leaky and lets out the CV dye