Unit 1 lab Exam Flashcards Preview

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Flashcards in Unit 1 lab Exam Deck (29):
1

What's the purpose of an autoclave?

sterilizing

2

How do you prepare a smear?

FROM A SLANT
1. aseptically transfer (w/ loop) a drop of water to slide
2. transfer a small amount of bacteria to water (on slide) & spread to a thin film
3. air dry a thin layer of specimen
2. heat fix the slide (before staining)

FROM A BROTH
no need to add water
roll tube between hands to mix bacteria
transfer a loop full of broth to slide
air dry
heat fix

3

Why must slide be air dried before heat-fixing?

If slide is heat fixed when wet, the water surrounding the bacterial cells will boil, causing the cells to lyse (burst).

4

Purpose of heat-fixing?

1. Kills bacteria
2. Adheres them to the slide so no need for a coverslip.

5

simple staining procedure

After heat-fixing:
1. cover smear with crystal violet
2. gently rinse with water bottle
3. blot with bibulous paper

6

Basic dyes

crystal violet
safranin
methylene blue

surface of bacteria with neutral pH attract basic dyes

7

Acidic dyes

Congo red
Nigrosine

stain the background & leave microbe transparent

8

Gram stain: what does it do?

G(+) have thick cell wall so stain adheres to it; G(-) has thin cell wall so are decolorized when rinsed with acetone alcohol.

9

Gram stain - procedure

1. Primary stain - crystal violet
2. Rinse w/distilled water
3. Mordant - iodine
4. Decolorize - acetone alcohol
5. Counterstain - safranin
6. Rinse w/distilled water
7. Blot w/bibulous paper

10

Acid Fast Stain (Kinyoun method) - purpose

distinguishes acid-fast Mycobacterium from other bacteria due to a waxy substance in its cell wall.

11

Acid Fast stain process

1. Primary stain - carbol fuchsin
2. Rinse - distilled water
3. Decolorize - acid alcohol
4. Rinse - distilled water
5. Counterstain - Kinyoun brilliant green
6. Rinse - distilled water
7. Blot w.bibulous paper

12

Endospore stain - purpose

Endospores have a tough outer covering made of the protein keratin that resists staining. Heat (steaming) is used to drive the stain inside the endospore.

13

Endospore stain process

1. Cover smear w/small piece of bibulous paper; apply malachite green to paper
2. Steam slide
3. Decolorize - distilled water
4 Counterstain - safranin
5. Rinse - distilled water
6. Blot w/bibulous paper

14

Negative stain (capsule stain) - purpose

Used to identify bacteria with a thick glycocalyx capsule. Both G(+) and G(-) bacteria can form capsules. The negative stain (congo red) stains the background & the positive stain (crystal violet) stains the cell body. The capsule is left unstained.

15

Negative stain (capsule stain) process

1. Add one drop of Congo Red to slide
2. Aseptically transfer bacteria to stain, swirl loop around to mix the two
3. Place edge of "spreader slide" in dye & push dye across slide, making a thin smear
4. Dispose of spreader slide in bleach
5. Allow smear to completely air-dry (do not heat fix - can cause cells to shrink, creating a possible "halo effect" which looks like a capsule, but isn't)
6. Add drop of Crystal Violet
7. Rinse - distilled water
8. Air dry

16

Which species needs Acid Fast stain?

Mycobacterium smegmatis - G(+) - cell walls are thick and waxy

17

Which Acid Fast species are pathogenic?

Mycobacterium leprae - leprosy

Mycobacterium tuberculosis - tuberculosis

18

Bacillus subtilis

G(+) - purple/blue
bacilli
endospore former

19

Staphylococcus aureus

G(+) - purple/blue
cocci

20

Escherichia coli

G(-) - pink/red
bacilli
pathogenic - food poisoning

21

Streptococcus pyogenes

G(+) - purple/blue
cocci

22

Pathogenic endospore formers

Bacillus cereus - food poisoning
Bacillus anthracis - anthrax
Clostridium tetani - tetanus
Clostridium botulinum - botulism
Clostridium difficile - diarrhea
Clostridium perfringens - gas gangrene

23

Capsule formers

- use negative stain
- Klebsiella pneumoniae

24

Mycobacterium smegmatis

G(+)
bacilli
use acid fast stain (b/c it has a waxy cell wall)

25

Klebsiella pneumoniae

G(-)
bacilli
capsule former (use negative stain)

26

Proteus mirabilis

G(-)
bacilli
causes UTIs

27

Pathogenic bacteria

Proteus mirabilis - UTIs
Mycobacterium leprae - leprosy
Mycobacterium tuberculosis - tuberculosisBacillus cereus - food poisoning
Bacillus anthracis - anthrax
Clostridium tetani - tetanus
Clostridium botulinum - botulism
Clostridium difficile - diarrhea
Clostridium perfringens - gas gangrene

28

Pseudomonas aeruginosa

G(-)
bacilli
pathogenic - can cause pneumonia, UTI

29

Conditions of an autoclave

121 degrees
15-20 minutes
15 psi