Bacteriological Media

Question 1

What are enriched media?

Answer 1

Media with supplements added to support the growth of fastidious organisms. Blood agar and chocolate agar are examples of enriched media.

Question 2

What are selective media?

Answer 2

Media with substances such as bile salts, dyes, or antibiotics added to inhibit certain bacteria. Examples of selective media are CNA, PEA, EMB, MacConkey, XLD, Hektoen Enteric, Modified Thayer Martin, and Martin Lewis.

Question 3

What are differential media?

Answer 3

Media formulated to provide distinct colonial appearance based on certain biochemical reactions, such as lactose fermentation or hydrogen sulfide production. MacConkey, EMB, XLD, and Hektoen Enteric are differential media.

Question 4

What might cause blood agar plates to appear brown?

Answer 4

Lysis of the RBCs. This can result from exposure to temperature extremes or from age of the medium.

Question 5

What can be added to media to make it selective for gram-negatives?

Answer 5

Crystal violet, bile salts, eosin, or methylene blue.

Question 6

What can be added to media to make it selective for gram-positives?

Answer 6

Colistin, nalidixix acid, or phenyethyl alcohol.

Question 7

What type of bacteria grow on CNA and PEA?

Answer 7

Gram-positives.

Question 8

When growth is observed on blood agar and MacConkey agar but not on NA or PEA, what type of organism has been isolated?

Answer 8

A gram-negative rod.

Question 9

Why is chocolate agar used?

Answer 9

To grow fastidious organisms such as Neisseria and Haemophilus. (Most other organisms also grow on chocolate agar.)

Question 10

When is xylose lysine deoxycholate agar (XLD) used?

Answer 10

For the recovery of Salmonella and Shigella from stool cultures. Hektoen enteric agar or SS agar may be used instead.

Question 11

A pure growth of colorless colonies is observed on a MacConkey plate. What type of colonies would be expected on the XLD plate set up from the same specimen?

Answer 11

Colorless or red colonies which may have black centers if the organism produces H2S.

Question 12

Name four media specifically used fro the isolation of anaerobes.

Answer 12

CDC anaerobic blood agar, Bacteroides bile esculin agar (BBE), laked kanamycin-vancomycin blood agar (LKV), phenylethyl alcohol sheep blood agar (PEA), thioglycolate broth, and chopped meat broth.

Question 13

Will anything other than anaerobes grow in thioglycolate broth?

Answer 13

Yes. Most aerobes grow as well. The location of the growth indicates the organism’s oxygen requirements. Aerobic organisms grow at the top of the tube, strict anaerobes at the bottom, and facultative anaerobes throughout.

Question 14

In the Gulf states, where one might expect to isolate Vibrio, what medium should be added to those routinely used for stool cultures?

Answer 14

Thiosulfate citrate bile salts sucrose (TCBS). Alkaline peptone water may be used as an enrichment broth.

Question 15

Which medium is best for recovery of Legionella pneumophila from clinical specimens?

Answer 15

Buffered charcoal yeast extract agar (BCYE).

Question 16

Which medium should be used to recover Bordetella pertussis from a nasopharyngeal specimen?

Answer 16

Bordet Gengou, Regan Lowe, or Jones Kendrick charcoal agar.

Question 17

What media are used to isolate mycobacteria?

Answer 17

Lowenstein Jensen, Middlebrook 7H10 and 7H11, and broth media.

Question 18

When are Thayer Martin or Martin Lewis agar used?

Answer 18

To isolate pathogenic Neisseria from sites with normal flora. Antibiotics is these media suppress the normal flora.

Question 19

E. coli was isolated on the Thayer Martin plates from several different patients in the same day. What might account for this unexpected finding?

Answer 19

The antibiotics were left out of the medium or had deteriorated. This illustrates the importance of performing quality control on media.

Question 20

Which media are used for fecal/rectal cultures?

Answer 20

A supportive medium (blood agar), a medium selective for gram-negative rods (MacConkey or EMB), a medium selective for Salmonella and Shigella (XLD or Hektoen enteric), and Campylobacter blood agar. Some labs also use an enrichment broth for SAlmonella and Shigella (GN, tetrathionate, or selenite broth). Many include MaConkey sorbital agar (SMAC) for E. coli O157:H7. In certain geographic areas, CIN agar for Yersinia enterocolitica and/or TCBS for Vibrio may be included.

Question 21

In some laboratories, MacConkey plates inoculated with stool specimens are incubated at room temperature when which fecal pathogen is suspected?

Answer 21

Yersinia enterocolitica. The optimum temperature for Y. enterolitica is 25-30C.

Question 22

When is bismuth sulfite agar used?

Answer 22

When Salmonella is suspected. S. typhi produces black colonies surrounded by a metallic sheen.

Question 23

Name a medium that is selective for Clostridium difficile.

Answer 23

Cycloserine sefoxitin fructose agar (CCFA). C. difficile produces yellow ground glass colonies.

Question 24

What media are inoculated for urine cultures?

Answer 24

Blood agar and MacConkey or EMB.

Question 25

What is added to holding media to absorb fatty acids present in the specimen that could kill fragile organism such as Neisseria gonorrhaeae?

Answer 25

Charcoal.

Question 26

Which cultures require incubation in increased CO2?

Answer 26

Blood, CSF, genital, Respiratory, wound, and cultures for AFB and Campylobacter.

Question 27

Which plates are routinely incubated in increased Co2 at 37C?

Answer 27

Chocolate, modified Thayer Martin or Martin Lewis, Human Blood Tween (HBT) and Vaginalis (V) agar.

Question 28

Which plates are routinely incubated in increased Co2 at 42C?

Answer 28

Campylobacter blood agar plates.

Question 29

Why is media heated before autoclaving?

Answer 29

To dissolve the agar.

Question 30

How is most media sterilized?

Answer 30

By autoclaving for 15 minutes at 15 pounds of pressure (121C).

Question 31

How should urea broth be sterilized and why?

Answer 31

By filtration. Urea cannot withstand high temperatures.

Question 32

How should most agar plates be stored?

Answer 32

Plates are stored upside down to prevent condensation water form falling on the surface of the agar. Most media is stored under refrigeration.

Question 33

What quality control is required on media?

Answer 33

It should be checked with organisms expected to grow or to give positive results and with those expected not to grow or to give negative results. Five percent of each batch should be checked for sterility by incubation for 48 hours. Plated should be checked daily for moisture, sterility, color, and hemolysis.