L17 - Medical Microbiology Laboratory (Corynebacterium and Listeria)

1. Medical Microbiology Laboratory
Gram Positive Bacilli (Rods)
(Corynebacterium and Listeria)
Hussein A. Abid
Medical Laboratory Scientist
Member at American Society of Microbiology
Chairman of Iraqi Medical Laboratory Association
Teacher at Middle Technical University

2. Non-spore formingSpore forming
• Corynebacterium spp.
• Lactobacillus spp.
• Mycobacterium spp.
• Clostridium spp.
• Bacillus spp.
2
GRAM POSITIVE RODS
 Bacterial spores are highly resistant, dormant
structures (i.e. no metabolic activity) formed in response
to adverse environmental conditions.
 They help in the survival of the organisms during
adverse environmental conditions; they do not have a
role in reproduction.

3. 3
TAXONOMY
Scientific nameRank
• CorynebacteriaceaeFamily
• CorynebacteriumGenus
• C. diphtheriae
• C. ulcerans
• C. pseudotuberculosis
• C. striatum
• C. urealyticum
• C. jeikeium
Species
(medically important spp.)

4. 4
Corynebacterium (pathogenic species)
 C. diphtheriae
 Toxigenic strains – diphtheria; respiratory, cutaneous
 Nontoxigenic strains – pharyngitis, endocarditis
 Human pathogen
 C. ulcerans
 Respiratory, diphtheria
 Veterinary pathogen
 C. jeikeium (group JK)
 Septicemia, endocarditis, wound infection, foreign body (catheter,
shunt, prosthesis) infection
 Human skin normal flora NF
 C. pseudotuberculosis
 Lymphadenitis, ulcerative lymphangitis, abscess formation
 Veterinary pathogen

5. 5
Corynebacterium (pathogenic species)
 C. ulcerans toxigenic strains produce disease similar to
diphtheria, but less severe
 C. jeikeium those with underlying disease; bacteremia,
meningitis, peritonitis, wound infection
 C. pseudotuberculosis those with exposure to animals (cattle,
sheep, horses, goats, deer); pneumonia, lymphadenitis
 Other species are normal flora of skin, may occasionally cause
disease, particularly immunocompromised patients.

6. 6
Corynebacterium diphtheriae
 Pleomorphic Gram positive rods
 often palisading & they arrange as “Chinese letters’’
 can be club-shaped
 On BAP may have a very small zone of β-hemolysis
 Catalase +ve
 Nitrate +ve
 Glucose +ve
 Maltose +ve
 Sucrose –ve
 Urease –ve

7. 7
Corynebacterium jeikeium
 Microscopic features are the same of C. diphtheriae.
 On BAP appears as non-hemolytic, opaque colonies.
 Catalase +ve
 Nitrate –ve
 Glucose +ve
 Maltose variable
 Sucrose –ve
 Urease –ve
 Key to identification: resistant to most antibiotics used to
treat Gram +ve infections. It is “S” to vancomycin
 Causes infections in immunocompromised patients
Negative Positive
Nitrate reduction test

8. 8
Diphtheroids
 Microscopic features are the same of C. diphtheriae & C.
jeikeium
 On BAP appear as C. jeikeium (opaque, γ-hemolysis
colonies)
 Catalase +ve
 Nitrate variable
 Glucose variable
 Maltose variable
 Sucrose variable
 Urease variable
 Considered normal skin flora or contaminant.
Ureasetest

9. 9
Corynebacterium diphtheriae
Specimen collection: (throat swab)
 Both tonsillar pillars and the oropharynx should be swabbed. Do
not allow the swab to touch the tongue.
1. The patient is instructed to tilt his/her head back and breath
deeply. The tongue is gently depressed with a tongue blade to
visualize the tonsillar fossa and posterior pharynx.
2. The swab is extended between the tonsillar pillars and behind the
vulva, care should b taken not to touch the lateral walls of the
buccal cavity or the tongue to minimize contamination with
commensal bacteria.
3. The posterior pharynx should be firmly rubbed with the swab.
4. After collection, the swab should be placed immediately into
sterile tube or other suitable container for transport to the
laboratory.

10. 10
THROAT SWAB
Who will collect the specimen?
 Physician, Medical technologist,
Microbiologist, experienced nurse.
Quantity of specimen:
 One or two swabs
Time relapse before processing the
sample:
 30 min.
Storage:
 Maintain specimen at room temperature

11. 11
C. diphtheriae (culture)
 Tinsdale medium: potassium
tellurite is the selective agent
that turns the media brown-
black as a result from the
reduction of potassium
tellurite to metallic tellurite.
 Blood agar plate (BAP): may
have a very small zone of
beta hemolysis. Incubate the
plate aerobically with enriched
CO2 atmosphere.

12. Black colonies with
gray periphery Large, gray colonies Small, dull, gray-black
12
C. diphtheriae (culture)
 Tellurite-Blood agar (TBA or CTBA): selective & differential
media.
 Selective for Corynebacterium, because the tellurite inhibits
many non-coryneform bacteria.
 Differential for tellurite reduction, so there are 3 biotypes of C.
diphtheriae depending on colony morphology as shown below:

13. 13
C. diphtheriae (Albert’s stain)
 Prepare the smear as described
previously. Fix with alcohol and stain by
the Albert staining technique (methylene
blue stain).
 Examine the smear for bacteria that
could be C. diphtheriae. Look for
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.

14. 14
C. diphtheriae
Toxigenicity testing: (other techniques)
1. In vivo test: by the culture into antitoxin-protected &
unprotected guinea pigs subcutaneously.
2. Tissue culture neutralization technique
3. In vitro test: immunodiffusion assay (Elek test)
4. Detection of toxin gen by PCR

15. 15
ELEK PLATE DIPHTHERIA TOXIN TEST
 Elek test is an in vitro
immunoprecipitation
(immunodiffusion) test to
determine whether or not a strain
of Corynebacterium diphtheriae is
toxigenic. A test strip of filter
paper containing diphtheria
antitoxin is placed in the center of
the agar plate.
 Strains to be tested (patient’s isolate), known positive and negative
toxigenic strains are also streaked on the agar’s surface in a line
across the plate and at a right angle to the antitoxin paper strip.

16. 16
ELEK TEST PROCEDURE
1. Mix a tube of melted nutrient agar with 2 ml of sterile horse serum.
2. Rotate the tube to mix the serum and agar. Do not shake the tube.
3. Pour the mixture into a sterile Petri dish.
4. Using lightly flamed forceps, lay the strip of anti-toxin impregnated filter paper
across the centre of the petri dish allowing it to sink beneath the agar surface.
5. Allow the agar to set, then lift one corner of the lid and let the plate dry for 30-
45 min in the incubator.
6. When dry inoculate with a toxinogenic strain of C. diphtheriae by streaking a
single line of inoculum across the plate and paper strip at right angles to the
strip.
7. Repeat this about 1 inch away from the C. diphtheriae inoculum with a test
strain.
8. Incubate the plate for 24 hrs. and observe the results.

17. 17
DIFFERENTIATION CHART
DiphtheroidsC. jeikeiumC. diphtheriaeCharacter
+ve+ve+veCatalase
γ-hemolysisγ-hemolysis
Very small zone
of β-hemolysis
Hemolysis
variable– ve+ veNitrate
variable+ve+ veGlucose
variablevariable+ veMaltose
variable– ve– veSucrose
variable– ve– veUrease

18. 18
TAXONOMY
Scientific nameRank
• ListeriaceaeFamily
• ListeriaGenus
• L. monocytogenes
Species
(medically important
spp.)

19. 19
Listeria monocytogenes
 Samples: cerebrospinal fluid (CSF), blood
 Small Gram +ve rods (could be coccobacilli)
 BAP (grayish-white colonies with small zone of β-hemolysis)
 Catalase +ve, Oxidase (-ve)
 Esculin hydrolysis +ve
 Hippurate hydrolysis +ve
 CAMP test +ve
 TSI: A/A, H2S (-), gas (-)
 Motility:
 wet preparation: tumbling motility
 25 oC semisolid media: inverted umbrella
Gram stain 100X

20. 20
Listeriosis
 Uncommon disease, restricted to elderly, pregnant women,
immunocompromised
 Healthy children and adults: AS carriage
 Pregnant moms:
 AS carriage, septicemia, neonatal disease
 Pregnancy renders mom more susceptible (immune
suppressed)
 Effect on mom usually minimal, can be devastating for fetus
or newborn
 Immunocompromised:
 AS carriage, meningitis, septicemia, other infections

21. 21
L. monocytogenes
Purple (+ve) Colorless (-ve)
Hippurate hydrolysis
Umbrella motility
Semi-solid media