Microbiology of the Respiratory Tract:

HAEMOPHILUS, CORYNEBACTERIUM, AND BORDETELLA

The genus Haemophilus contains a number of species of fastidious, gram-negative bacilli. Most of these
are found as normal flora of the upper respiratory tract. Haemophilus species can cause infections in a variety
of sites in the upper respiratory tract and elsewhere in the body.
Laboratory diagnosis is made by identifying these organisms in clinical specimens appropriately
representing the area of infection (throat swab, sinus drainage, sputum, conjunctival swab, spinal fluid, blood, or
other). A direct smear of the specimen may be useful, particularly for spinal fluid or an exudate from the eye, in
providing rapid, presumptive information. (Smears of material from the upper respiratory tract, with its mixed
flora, may have little value unless the organisms are present in large numbers.) Latex antibody tests can also be
performed directly with certain patient body fluids to detect Haemophilus antigen.
Until an effective vaccine came into widespread use in the early 1990s, most serious Haemophilus
disease was caused by H. influenzae serogroup b (H. influenzae strains are divided into serogroups af on the
basis of their antigenic polysaccharide capsule). This organism is seldom isolated in the clinical laboratory today,
but other Haemophilus species and H. influenzae serogroups other than serogroup b are occasionally
encountered.
The fastidious Haemophilus organisms require specially enriched culture media and microaerophilic
incubation conditions. Chocolate agar is commonly used for primary isolation of Haemophilus from clinical
specimens. This medium contains hemoglobin derived from bovine red blood cells as well as other enrichment
growth factors. Because the hemoglobin is dark brown, the agar in the plate has the appearance of chocolate.
Two special growth factors, called X and V, are required by some Haemophilus species. Some require one but
not the other. The X factor is hemin, a heat-stable derivative of hemoglobin (supplied in chocolate agar). The V
factor is a heat-labile coenzyme (nicotinamide adenine dinucleotide, or NAD), essential in the metabolism of
some species that lack it. Yeast extracts contain V factor and are one of the most convenient supplements of
chocolate agar or other media used for Haemophilus. Organisms other than yeasts elaborate V factor.
Staphylococci, for example, when growing on an agar plate secrete NAD into the surrounding medium.
Haemophilus species that need V factor may grow in the zone immediately around the staphylococci but not
elsewhere on the plate. This growth of the dependent organism is described as satellitism. X and V factors can
also be incorporated directly into agar media that do not contain these factors, or alternatively, they can be
impregnated in filter-paper disks that are pressed on the surface of X and V factordeficient media. In the latter
case, the growth factors diffuse into the agar in a manner similar to diffusion from disks impregnated with
antimicrobial agents.
The genus Corynebacterium is comprised of many species, but Corynebacterium diphtheriae has the
most important pathogenic properties. C. diphtheriae is the agent of diphtheria, a serious throat infection and a
systemic, toxic disease. If they have an opportunity to colonize in the throat, virulent strains of this organism
not only damage the local tissue (causing formation of a pseudomembrane), but they produce a powerful
exotoxin that disseminates through the body from the site of its production in the upper respiratory tract.
When this toxin reaches the cells of the myocardium, adrenal cortex, or other vital organs, it has very damaging
effects. The systemic effect of toxin is the primary cause of death in those patients with diphtheria who are not
promptly recognized and treated. In rare cases, the skin rather than throat is affected, but all toxic disease
manifestations are the same. The disease is controlled by maintaining active immunization with diphtheria
toxoid (purified toxin treated so that it is no longer toxic but remains immunogenic). Early clinical and
laboratory recognition of diphtheria infection developing in the throat is critical because prompt treatment with
antitoxin (antibody that neutralizes the toxin) and an appropriate antimicrobial agent are required for patient
recovery.
In the laboratory, the microbiologist must distinguish C. diphtheriae from other corynebacteria that are
harmless members of the normal flora but usually present in throat specimens. Identification must be made as
rapidly as possible, for the laboratory report is essential for clinical decisions. In patients with decreased
immune function (referred to as immunocompromised patients), corynebacteria other than C. diphtheriae may
cause disease by invading the weakened host to produce bacteraemia and pneumonia.
Corynebacteria are gram-positive, nonmotile, non-sporing bacilli that, like staphylococci, are widely
distributed on our bodies. Non-pathogenic species are often called diphtheroids because their microscopic
morphology resembles that of C. diphtheriae. These rods often contain granules that stain irregularly (they are
said to be metachromatic) and give the organisms a beaded or clubbed appearance. Pairs or small groups
characteristically fall into patterns that look like Chinese letters, or like Vs and Ys. Usually, C. diphtheriae is
longer, thinner, and more beaded in appearance than diphtheroids, which are generally short and thick by
comparison. This differentiation can be very difficult to make in examining a stained throat smear and cannot
be relied on for accurate diagnosis.
In culture, corynebacteria are not highly fastidious. They grow well aerobically on nutrient media. When
diphtheria is suspected, the primary isolation media used for throat swabs include those that are selective and
differential for C. diphtheria and also blood agar. Loefflers serum medium is commonly used for direct

inoculation and transport of the swab to the laboratory. This is a firm coagulated serum medium containing
nutrient broth, prepared as a tubed slant. Many of the normal throat flora organisms do not grow on Loefflers
medium, so it is somewhat selective. In addition, when C. diphtheriae grows on this medium its microscopic
morphology is characteristic. A methylene-blue-stained smear reveals thin, club-shaped bacilli and reddishpurple metachromatic granules. This appearance can lead to a rapid presumptive diagnosis of diphtheria. Blood
agar to which potassium tellurite has been added constitutes a good selective and differential medium for
primary isolation of C. diphtheriae. The tellurite not only suppresses many other throat flora, but it is
metabolized by C. diphtheriae with resulting blackening of its colonial growth. Thus the organism is
differentiated from others that can grow on the agar medium. The use of blood agar in the initial battery assures
the recovery of corynebacteria, as well as other pathogenic bacteria species that might be present, and
differentiates those that are hemolytic. The biochemical differentiation of C. diphtheriae from other
corynebacteria is based on carbohydrate fermentations.
Demonstration of toxin production is essential in reporting identification of a strain of C. diphtheriae, for
not all strains are toxigenic. Tests for virulence, that is, toxigenicity, are made either in experimental animals
(rabbits or guinea pigs) or by an in vitro method (Elek test). In the Elek test, antitoxin strips are placed on agar
plates to detect toxin produced by strains of C. diphtheria growing on the medium.
Bordetella pertussis is the etiologic agent of whooping cough. This very fastidious organism grows best
on special media. The two most common are Bordet-Gengou (BG) agar, which is enriched with glycerin, potato,
and 30% defibrinated sheep blood, and Regan-Lowe (RL) agar, which consists of charcoal agar, defibrinated
horse blood, and an antimicrobial agent to inhibit growth of normal respiratory flora. The charcoal is present to
adsorb toxic substances that might be present in the agar. Visible colonies are produced only after three to five
days incubation in a microaerophilic atmosphere. On BG medium, the colonies are raised rounded, and
glistening (resembling mercury droplets or a bisected pearl), and usually have a hazy zone of hemolysis. On RL
medium, the colonies are round, domed, shiny, and may run together slightly. B. pertussis is a gram-negative
bacillus resembling Haemophilus species, with which it was once classified. When whooping cough is suspected,
the best specimen for laboratory diagnosis is a nasopharyngeal swab, but throat swabs may be used in addition.

Laboratory Exercise:
Haemophilus, Corynebacterium and Bordetella
A. Haemophilus
Purpose:
To identify Haemophilus species in culture
Materials:
Blood agar plates, chocolate agar plates, nutrient agar plates, X and V disks,
alcohol lamps, inoculating loops, forceps
Broth/ chocolate agar cultures of Haemophilus influenzae and Haemophilus parainfluenzae
Procedures:
1. Make a Gram stain of each species of Haemophilus.
2. Divide a BAP and a chocolate agar plate in half with your marking pen or pencil. Label one half H. influenzae
and the other half H. parainfluenzae.
3. Inoculate H. influenzae on the appropriate side of each plate and streak for isolation within this half. Repeat
with H. parainfluenzae on the other half of each plate.
4. Incubate these plates in a candle jar or CO2 incubator at 35C for 24 hours.
5. Repeat step 2 using the NA plate, but inoculate each strain heavily and streak for confluent growth within its
half of the plate.
6. Now, using heated, cooled forceps, place an X and a V disk on the agar surface streaked with H. influenzae
and repeat on the H. parainfluenzae side. The two disks on each side should be placed not more than 1 inch
apart, and centered in the area streaked.

7. Incubate this plate in a candle jar or a CO2 incubator at 35C for 24 hours.
8. Describe your observations and indicate your interpretation of the appearance of the blood and nutrient
agar plates.

B. Corynebacterium
Purpose:
To identify Corynebacterium in smears and culture
Materials:
Blood agar plates, blood tellurite plate, tubed phenol red glucose broth, tubed phenol red
maltose broth, tubed phenol red sucrose broth,
Prepared Gram- and methylene-blue-stained smears of C. diphtheria,
Loefflers slant cultures of C. xerosis and C. pseudodiphtheriticum, NA slant culture of E. coli
Procedures:
1. Prepare a Gram stain and a methylene blue stain from either one of the Corynebacterium cultures. Read and
compare these with the Gram- and methylene-blue-stained smear of C. diphtheriae.
2. Inoculate a BAP with either one of the Corynebacterium cultures. Streak for isolation.
3. Divide the blood tellurite plate into two parts with your marker. Inoculate one side of the plate with a
Corynebacterium species, the other side with E. coli.
4. Inoculate the Corynebacterium culture into each of the three carbohydrate broths.
5. Incubate all plate and tube cultures at 35C for 24 hours.
6. Examine your cultures and record your observations.
C. Bordetella
Purpose:
To observe Bordetella pertussis in demonstration and to examine a throat culture on BordetGengou (BG) and Regan-Lowe (RL) media
Materials:
Prepared Gram stains of B. pertussis, Bordet-Gengou and Regan-Lowe agar plates
Procedures:
1. Examine the prepared Gram stains and record your observations.
2. Observe colonial morphology as demonstrated.
3. Collect a throat specimen and inoculate the Bordet-Gengou and Regan-Lowe plates.
4. Incubate the plates at 35C in a candle jar or CO2 incubator for 24 hours.
Study Questions (Answer on a separate sheet of bond paper):
1. What is chocolate agar?
2. Define X and V factors.
3. Name three species of Haemophilus and indicate the types of infection with which each may be associated.
4. What is the satellite phenomenon?
5. Why is a direct smear of spinal fluid essential when bacterial meningitis is suspected?
6. Name the etiologic agent of diphtheria and describe the media used to isolate it from a clinical specimen.
7. What is a virulence test and how is it performed?
8. Can diphtheria be transmitted directly via the respiratory route? If so, how?
9. How is diphtheria prevented?
10. Why is early laboratory diagnosis of diphtheria important?
11. How can transmission of respiratory infections be prevented?