Microbiology lab

Question 1

Difference between selective medium, differential medium, and enrichment medium

Answer 1

Selective medium = permits growth of a PARTICULAR organism (MacConkey grows only Gram -)

Differential medium = uses biochemical property to differentiate from one organism to another (MacConkey shows what ferments lactose!)

Enrichment medium = provides growth advantage for one type of bacteria over another (MacConkey - probs gram - lactose fermenters!)

Question 2

Is MacConkey Agar selective, differential, or both? What are its general features?

Answer 2

Selective and differential medium: grows only gram (-) bacteria and will differentiate between lactose and non-lactose fermenters.

Bile salts and crystal violet in the medium prevent growth of gram (+) and some -(-)

(fermenters=red/pink colonies, non-fermenters=colorless)

Question 3

Is Hektoen Enteric Agar selective, differential, or both? What are its general features?

Answer 3

Highly selective and also differential: primarily to isolate
salmonella and shigella; inhibits growth of gram (+) and somewhat inhibitory for common enteric bacteria, like e.coli

Differential via fermentation of carbohydrates in medium (+ fermentation=orange, non-fermenters=green-blue.

Salmonella and shigella = non-fermenters, appear as greenish-blue translucent colonies

H2S production=black = salmonella

Question 4

Is Sheep Blood Agar selective, differential, or both? What are its general features?

Answer 4

Only differential based on degree of blood hemolysis

beta-hemolysis: complete clearing of red cells from a zone surrounding the colony (Group A strep, staph aureus)

alpha-hemolysis: incomplete hemolysis and appears as a green color (Strep. viridans and pneumonia)

gamma-hemolysis: lack of any change around the colony
(staph epidermidis, nisseria)

Question 5

Is Pseudomonas Isolation Agar selective, differential, or both? What are its general features?

Answer 5

Highly selective for pseudomonas and also somewhat differential. Enhances the growth of P. aeruginosa, which form = blue colonies

Question 6

How to test for beta-lactamase production?

Answer 6

Use a chromogenic cephalosporin that when hydrolyzed by the enzyme changes color.

Beta-lactamase (+) organisms wil turn disk with substrate from yellow to pink.

Question 7

Test most commonly used to identify gram (+) cocci as streptococcus or staphylococcus?

Answer 7

Catalase test (enzyme converts hydrogen peroxide to water and oxygen), bubbles on slide = (+)

Streptococcus = catalase (-)
Staphylococcus = catalase (+)

Question 8

How does the oxidase test work and what are typical (+) and (-) organisms?

Answer 8

Assays the c component of the cytochrome-oxidase complex, turns test solution colorless to pink to black if (+)

Pseudemonas and Nisseria = (+)

E.coli and salmonella = (–)

Question 9

How does serotyping Salmonella species work?

Answer 9

Specific antibodies for the LPS O-anitgens are added to slurry of bacterial cells. Clumps will form if antiserum binds to the right O-antigens.

Question 10

What are steps in a gram stain? Also explain logic for why this works.

Answer 10

1. Crystal violet solution for 1 min
2. Gram iodine solution for 1 min
3. Decolorize for 15 sec
4. Counterstain with safranin for 1 min

The crystal violet penetrates both gram (+) and (-) and forms a complex with the iodine. The decolorizing step washes out the complex from gram (-), but in gram (+) the complex is stuck in the thick PG layer, looks purple. The counterstain stains the now colorless gram (-) pink.