Gram-Postive Bacilli

Question 1

A Gram-stained smear from growth on an anaerobic blood agar plate shoes small gram-positive bacilli that are pleomorphic and club shaped. They are arranged parallel to each other and in V and Y formation. To which genus do these organisms most likely belong?

Answer 1

Corynebacterium.

Question 2

Name a rapid test to differentiate non-hemolytic streptococci from diphtheroids (nonpathogenic corynebacteria).

Answer 2

Catalase. Diphtheroids are catalase-positive; streptococci are catalase-negative.

Question 3

Name the organism that causes diphtheria.

Answer 3

Corynebacterium diphtheria.

Question 4

What media are used to isolate C. diphtheriae?

Answer 4

Cystine tellurite or modified Tinsdale is used for development of characteristic colonial morphology. C. diphtheria produces black colonies with dark brown halos on modified Tinsdale agar, and black or gray colonies on Cystine tellurite. Loeffler medium is used for development of characteristic microscopic morphology. It stimulates production of metachromatic granules.

Question 5

Which test is required for definitive identification C. diphtheriae?

Answer 5

Toxigenicity testing to separate the toxin-producing pathogen, C. diphtheria, from the nonpathogenic corynebacteria. Toxigenicity testing is performed in reference labs.

Question 6

What is the clinical significance of Corynebacterium jeikeium (CDC Group JK)?

Answer 6

It is part of normal skin flora and can cause infections in immunocompromised patients and in patients with catheters & prosthetic devices. It is the most common cause of diptheroid prosthetic valve endocarditis in adults and it is very resistant to antibiotics.

Question 7

Which gram-positive sporeforming rod is contracted form handling contaminated wool or hides, looks like bamboo on a Gram stain, and produces large, nonhemolytic “Medusa head” colonies on blood agar?

Answer 7

Bacillus anthracis.

Question 8

Which organism causes anthrax?

Answer 8

Bacillus anthracis. Anthrax is primarily a disease of herbivores. It has been rare in the U.S., with only occasional cases due to occupational exposure of workers who handle hides, wool, goat or camel hair, or cashmere. Anthrax is of renewed concern with the advent of bioterrorism.

Question 9

Describe the Gram stain morphology of B. anthracis.

Answer 9

Broad, gram-positive rods 1-1.5 x 3-5 micrometer. Oval, central to subterminal spores, 1 x 1.5 micrometer with no significant swell of the cell. (Spores are usually not seen in clinical specimens.)

Question 10

Describe B. anthracis in direct smears from clinical specimens.

Answer 10

Encapsulated, broad, gram-positive rods in short chains of 2-4 cells. An India ink stain should be used to visualize the capsule. Spores are usually not present in clinical specimens.

Question 11

Describe the colonial characteristics of B. anthracis.

Answer 11

Blood agar:
Colonies 2-5 mm in diameter
Gray-white with ground glass appearance
Flat or slightly convex
Irregularly round with edges that slightly undulate, often with comma-shaped protrusions from the edge (Medusa head colonies)
Tenacious consistency (When teased with a loop, the growth will stand up like beaten egg white.)
Non-hemolytic

MacConkey agar:
no growth

Question 12

What is the role of the level A community hospital microbiology laboratory in the identification of B. anthracis?

Answer 12

It should be able to rule out B. anthracis based on colonial morphology, Gram stain, and motility. When B. anthracis cant be ruled out based on these criteria, the isolate should be referred to a higher level lab (public health laboratory) for identification. B. anthracis can be safely handled in a Class II biological safety cabinet.

Question 13

What constitutes a presumptive identification of B. anthracis in a Level A laboratory and what should be done?

Answer 13

Large, aerobic gram-positive rods
Non-hemolytic
Non-motile
The isolate should be referred to the State Public Health Laboratory

Question 14

A level A laboratory receives a culture swab from a lesion suspected of being cutaneous anthrax. The direct Gram stain shows large gram-positive rods. The specimen is plated onto blood agar and MacConkey agar. Flat, non-hemolytic colonies grow on the blood agar. There is no growth on MacConkey. A motility test is performed and the isolate is motile. Could this isolate be B. anthracis?

Answer 14

No. The positive motility rules our B. anthracis.

Question 15

Describe Listeria’s microscopic appearance.

Answer 15

Pleomorphic, non-sporeforming gram-positive coccobacilli in diphtheroid arrangement.

Question 16

What diseases does Listeria cause?

Answer 16

Meningitis and septicemia of the newborn and debilitated, and food poisoning.

Question 17

Discuss characteristics of Listeria that are helpful in tis identification.

Answer 17

Listeria produces tiny colonies with narrow zones of indistinct beta-hemolysis. It is catalase-positive. It exhibits tumbling motility on wet mount and umbrella growth in motility agar at room temperature. It grows from 0.5-45C.

Question 18

How can Listeria be differentiated from streptococci?

Answer 18

By catalase test. Listeria is positive; streptococci are negative.

Question 19

How can Listeria be differentiated from diphtheroids?

Answer 19

By motility. Listeria is positive; diphtheroids are negative.

Question 20

Pus from a draining sinus is submitted fro Gram stain and culture. Granules are detected in the specimen which, when crushed and Gram stained, reveal purple branching filaments. What should be suspected and what other stain should be performed?

Answer 20

Nocardia. A modified acid fast stain should be performed. Nocardia is partially acid-fast.

Question 21

CSF from a baby in the neonatal intensive care unit is cultured and tiny, translucent, gray colonies with narrow zones of indistinct beta-hemolysis are observed on blood agar and CNA. The Gram stain shows gram-positive coccoid organisms. The technologist sets up a hippurate hydrolysis and CAMP test. The organism hydrolyzes hippurate and is CAMP-positive. What is the most likely organism and what additional tests would be helpful in confirming the identification?

Answer 21

Both Listeria monocytogenes and group B streptococcus (GBS) should be considered. A bile-esculin test and a catalase test would distinguish between the two. Listeria is positive for bile-esculin and catalase. GBS is negative for both. Motility at room temperature is another important distinguishing characteristic of Listeria.

Question 22

A saline wet prep of vaginal discharge shows many epithelial cells covered with pleomorphic rods. The long, slender rods usually seen in vaginal wet preps are absent. These findings are suggestive of what condition?

Answer 22

Bacterial vaginosis due to Gardnerella vaginalis. The cells observed are clue cells.

Question 23

Which organism is the predominant vaginal flora during reproductive years?

Answer 23

Lactobacillus. It is a highly pleomorphic gram-positive rod that forms chains in thioglycolate broth and is catalase-negative.