Lab test for exercise 12, 14, 24, 25, 45 and 46

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Lab final for microbiology
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1

Mannitol salt agar

- Selective and differential medium
- NaCl selective for Staphylococcus
Some can tolerate high NaCl concentrations
- Differential
- Mannitol fermenters
-agar turns yellow due to acid production
- Mannitol non-fermenters
- no color change of agar

2

Eosin Methylene Blue (EMB)

- Selective and differential
- Used for isolation of fecal coliforms
- Dyes (eosin and methylene blue) in agar inhibit Gram+ bacteria
- Under acidic conditions, eosin and methylene blue, will form complex --- metallic green

3

MacConkey Agar

- Selective and differential
- Isolates and differentiates between members of Enterobacteriaceae
- Used to differentiate between lactose fermenters (pink colonies) and non-fermentors
- Bile and crystal violet make it selective

4

Carbohydrate Fermentation Media

- Broths (liquid media)
Uninoculated color: red
- Contain
- One type of sugar
- Peptone
- Durham tube (inverted tube)
- Phenol red (pH indicator)
- Yellow (< 6.8)
- Red (pH 7.0)
- Pink/magenta (> 7.4)

5

MRVP Media (2 tests in one)

- Broths (liquid media)
- Uninoculated color: colorless
- 2 Tests in same media: Methyl red test and Voges-Proskauer Test
- Tests for
- mixed acid fermentation (MR Test)
- acetoin production (VP Test)
- Contains
- Peptone
- Glucose
- Phosphate buffer (resists pH change)

6

Methyl Red Test

- Mixed Acid Fermentation
- Used to detect bacteria that produce large amounts of mixed acids
- Acids are stable and overcome buffer
Cause pH change
- After incubation, methyl red is added
- Methyl red is a pH indicator
- Red at pH < 4.4 (*only true indication of a positive result)
- Yellow at pH 6.2
- Orange (between 4.4-6.2)
- Orange is negative or inconclusive

7

Voges-Proskauer Test

- Fermentation of glucose to acetoin
- Addition of reagents:
- V-P reagent 1
- α-naphthol
-V-P reagent 2
- Potassium hydroxide
- Catalyst
- Red color = positive
- Copper color = negative

8

MR test steps

- 5 drops of methyl red
- Red color indicates positive methyl red test

9

V-P test steps

- 12 drops of V-P reagent I
- 2-3 drops of V-P reagent II
- Pinkish-red will indicate positive V-P test

10

Use-dilution test

1) Dip metal rings in bacterial culture
2) Allow metal rings to air dry
3) Dip metal rings in chemical agent for 10 min
4) Place metal rings in nutrient broth

11

Kirby-Bauer disk diffusion method (steps)

- Swap culture in three directions, 45 degree angles
- Let stand 5 min
- Place impregnated disk on surface
- gently tap disk into agar

12

Bacteria of the skin (First period)

- place swap in saline
- swap skin
- swap 1/3 of Mannitol agar plate
- use streak plate method with loop

13

Bacteria of the skin (second period)

- Perform a gram stain of colonies with yellow halos
- take catalase test on desired colony
- with toothpick and add H2O2
- subculture a catalase-positive, gram-positive onto another mannitol plate

14

Bacteria of the skin (third period) test for coagulase

- place loop full of rehydrated coagulase plasma on slide
- add loop full of water
- add heavy suspension of bacteria

- observe for clumping
- clumping = positive
- no clumping = negative

15

Bacteria of the respiratory tract

- swab throat
- swab 1/2 of blood agar
- streak remainder of plate
-INCUBATE-
- observe for hemolysis

16

Optochin inhibition

use a optochin disk to identify pathogen streptococcus pneumoniae