1. Examination of throat
and mouth specimens
Lecture 2

2. Possible pathogens





Gram positive
Streptococcus
pyogenes
Corynebacterium
diphtheriae
Corynebacterium
ulcerans

Gram negative
Vincent’s organisms

3. Note: Pathogens in the upper respiratory tract
such as Bordetella pertussis, Streptococcus
pneumoniae, and Neisseria meningitidis, are
usually more successfully isolated from nasopharyngeal secretions collected by aspiration

4. Notes on pathogens





S. pyogenes, Lancefield Group A beta-haemolytic
Streptococcus is the commonest cause of bacterial
pharyngitis (sore throat), especially in young
children. Its association with rheumatic heart
The term scarlet fever is used when streptococcal
pharyngitis is accompanied by a characteristic skin
rash
Sore throat
Fever
Bright red tongue with a "strawberry" appearance

5. Notes on pathogens
● C. diphtheriae produces a powerful and often fatal exotoxin and
therefore when diphtheria is suspected, the patient is treated
immediately with antitoxin. The role of the laboratory is to confirm
the clinical diagnosis.
● Infection with Vincent’s organisms (Borrelia vincenti in
association with Gram negative anaerobic fusiformbacilli) causes
Vincent’s angina (Vincent’s gingivitis), an ulcerative tonsilitis with
tissue necrosis.

6. Commensals
Gram positive
Viridans streptococci
Non-haemolytic streptococci
Gram negative
Moraxella catarrhalis
Neisseria pharyngitidis
Staphylococcus epidermidis
Streptococcus pneumoniae
Micrococci
Fusobacteria
Coliforms
Bacteroides species
Lactobacilli
Diphtheroids
Haemophilus influenzae
Also various spirochaetes, actinomycetes, aerobic
and anaerobic spore forming.

7. COLLECTION AND TRANSPORT OF
THROAT AND MOUTH SWABS
In a hospital with a microbiology laboratory
1 In a good light and using the handle of a spoon to
depress the tongue, examine the inside of the
mouth. Look for inflammation, and the presence
of any membrane, exudate, or pus.
– With diphtheria, a greyish-yellow membrane
(later becoming greyish green-black and smelly)
can often be seen extending forwards over the
soft palate and backwards onto the pharyngeal
wall.
– With a streptococcal sore throat, the tonsils are
inflamed and often covered in yellow spots.
– With Vincent’s angina, there is ulceration of the
mouth, throat, or lips.

8. COLLECTION AND TRANSPORT OF
THROAT AND MOUTH SWABS
2 Swab the affected area using a sterile cotton wool
swab. Taking care not to contaminate the swab with
saliva, return it to its sterile container.
Important: For 8 hours before swabbing, the patient must
not be treated with antibiotics or antiseptic mouthwashes (gargles).
Caution: It can be dangerous to swab the throat of a child
with acute haemophilus epiglottitis because this may
cause a spasm that can obstruct the child’s airway.
Blood for culture should be collected instead.
3 Within two hours of collection, deliver the swab
with a completed request form to the laboratory.

9. In a health centre for dispatch to a
microbiology laboratory
1 Using a sterile swab (supplied in a tube of
silica gel by the microbiology
laboratory), collect a specimen from the
infected area as described under the hospital
collection of throat swabs.
2 Taking care not to contaminate the
swab, return it to its tube. Seal with adhesive
tape and label the tube.

10. Culture the specimen
Blood agar
– Inoculate the swab on a plate of blood agar Use the loop to
make also a few stabs in the agar (well area). Colonies of S.
pyogenes growing below the surface will show more distinct
zones of haemolysis because of the anaerobic conditions
provided.
– When a swab is received in silica gel (e.g. from a health
centre), moisten it first with sterile nutrient broth and then
inoculate the plate.
– Add a 0.05 unit bacitracin disc (Reagent No. 15) to the plate.
This will help in the identification of S. pyogenes (see subunit
7.18.2). Some workers also add a co-trimoxazole disc (as used
for
susceptibility testing) which prevents the growth of other
bacteria, making it easier to see betahaemolytic S. pyogenes
colonies.
– Incubate the plate preferably anaerobically or, when this is not
possible, in a carbon dioxide enriched atmosphere overnight at
35–37 C. Candle jar incubation will detect

11. Culture : Blood agar: S. pyogenes produces betahaemolytic
 colonies, i.e. the colonies are surrounded by a zone of complete
haemolysis with decolorization of the haemoglobin. Colonies are
usually small (0.5–1 mm), colourless, dry, shiny or mucoid.
Haemolysis is more marked under anaerobic conditions as seen in
colonies growing below the agar surface (following stabs made in the
culture medium.
Choice of blood
 To isolate beta-haemolytic streptococci, use sheep blood (1st
choice), horse, rabbit or goat blood to prepare blood agar plates. Do
not use human blood because this may contain unwanted substances
such as citrate (e.g. donor blood), antibiotics, or antibodies such as
ASO or anti-M protein that could interfere with the growth or
haemolytic activity of S. pyogenes.
 Note: Other beta-haemolytic streptococci belonging to other
Lancefield groups) also produce colonies similar to S. pyogenes.
Betahaemolysis may also be seen with some strains of S.
aureus, Haemophilus (particularly from throat
swabs), Corynebacterium and Moraxella. It is therefore important to
examine a Gram stained smear of the culture.
 A catalase test can be used to differentiate streptococci (negative)
from staphylococci (positive).

12. Group B: Streptococcus

13. Immunological detection of S.pyogenes.

Direct detection of antigen A from throat swab extracts
Several tests have been developed to detect antigen A directly
extracted from throat swabs without the need to culture the
specimen, thus providing an early presumptive diagnosis of
streptococcal sore throat.

Direct antigen A detection tests are highly specific but sensitivity
varies between manufacturers.

Test is also influenced by the quality of the specimen. Culturing is
required when infection with S. pyogenes is suspected and a direct
antigen test is negative

14. PYR (pyrrolidonyl) test:


This detects pyrrolidonyl peptidase enzyme
activity. Besides S. pyogenes, Enterococcus
species and occasionally streptococci
belonging to groups C and G are also PYR
positive.
The test can be rapidly and simply performed
using PYR impregnated strips

15. Culture of specimen when diphtheria
is suspected
When diphtheria is suspected and culture is
specifically requested, inoculate the swab on
Tinsdale medium or tellurite blood agar Incubate the
plate aerobically at 35–37 C for up to 48
hours, examining for growth after overnight
incubation.
Tellurite blood agar: This medium is widely
used as a primary medium for isolating C.diphtheriae
from throat and nasopharyngeal swabs. C. diphtheriae
reduces tellurite and produces grey or grey-black
colonies measuring 0.5–2 mm in diameter after 24–
48 h incubation

16. Corynebacterium diphtheriae cultivated on
Tinsdale agar.Cultivation 72 hours, 37 °C in an
aerobic atmosphere

17. Tinsdale medium
Tinsdale medium: After 24–28 h incubation,
C. diphtheriae colonies are grey-black, raised,
and surrounded by a dark brown area. The brown color
is due to the hydrogen sulphide produced from the
cystine interacting with the tellurite.
 Occasionally commensal diphtheroids and other
respiratory tract commensals may grow on Tinsdale’s
medium but the colonies are not surrounded by a
brown halo like those of C. diphtheriae.


18. Bordetella pertussis




Specimens: Preferably nasopharyngeal secretions collected
by aspiration or a correctly taken pernasal swab
Cultur: Bordetella species are strict aerobes. Specimens for
the isolation of B. pertussis must be cultured as soon as
possible after they are collected. A selective and enrichment
medium such as charcoal cephalexin blood agar is
recommended for the primary isolation of B. pertussis.
Charcoal cephalexin blood agar: When incubated for 2–6
days at 35–37 ºC in a moist aerobic atmosphere, B. pertussis
produces small pearly-grey, shiny (mercury-like), usually
mucoid colonies.
B. parapertussis grows more rapidly and forms larger colonies
than B. pertussis. It produces a pigment in the medium and is
able to grow aerobically on blood agar and nutrient agar

19. Examine the specimen microscopically
Gram smear
spread smear of the specimen on a slide. Allow the smear to
air-dry in a safe place. Fix, and stain by the Gram technique .
Use dilute carbol fuchsin (1 in 10 dilution) as the counter stain
in preference to safranin or neutral red (stains Vincent’s
organisms better). Examine the smear for pus cells and
Vincent’s organisms:Vincent’s organisms: These are seen as
Gram negative spirochaetes (B. vincenti) and Gram negative
fusiform rods
Other bacteria: No try should be made to report routinely
other bacteria in a Gram stained smear from a throat swab
because the throat contains a wide variety of commensals
that cannot be distinguished morphologically from pathogens.

20. Pathogenesis

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Vincent angina is caused by Borrelia vincenti.
Borrelia vincenti is anaerobic spirochaete which lies
commensal in healthy human mouth.
Under certain conditions which cause injury of the
mucous membrane, Borrelia vincenti together with
anaerobic cigar shaped fusiform bacilli (fusobacterium)
multiply in the lesions causing ulcers.
These ulcers may become covered by
pseudomembrane containing pus cells and necrotic
tissue.

21. Laboratory diagnosis

Specimen: smears prepared from the
pseudomembrane.
Direct microscopic examination stained with
Gram:
Gram negative borrelia in large numbers +
Gram negative cigar shaped fusiform bacilli
+ pus cells.


23. Albert stain
Albert stained smear when diphtheria is suspected, we can see
pleomorphic rods containing dark-staining volutin granules . The
pleomorphic rods tend to join together at angles giving the appearance
of Chinese letters.
 Pleomorphism and granule formation are best seen in smears from a
Loeffler serum or Dorset egg medium culture.

Smears directly from specimens may not show these features.
Volutin granules

It is also possible for commensal diphtheroids to contain volutin
granules but the commensals are not pleomorphic like C. diphtheriae.
 When C. diphtheriae is cultured on tellurite blood agar and modified
Tinsdale medium, granule formation is usually limited.
 Note: In Gram stained smears, C. diphtheriae stains variably and
weakly Gram positive, whereas commensal diphtheroids appear
strongly Gram positive.


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