Various methods, including fluorescent antibody stain reagents, enzyme immunoassays, and nucleic acid

amplification methods are also commercially available to detect numerous viral agents.

 Culture of Streptococcus pyogenes (Beta-Hemolytic Group A Streptococci) are usually beta-hemolytic, with

less than 1% being nonhemolytic. Three variables must be taken into consideration regarding successful culture

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of group A streptococci from pharyngeal specimens: medium, atmosphere, and duration of incubation. Kellogg

recommended four combinations of media and atmosphere of incubation for throat specimens.

 Regardless of the medium and atmosphere of incubation employed, culture plates should be incubated for at

least 48 hours before reporting as negative for group A streptococci. In addition, the incubation of sheep blood

agar in 5% to 10% CO2 was strongly discouraged.

Drawbacks to culture include an extended incubation time of 24 to 48 hours for visible colony formation with

additional manipulations of the beta-hemolytic organisms for definitive identification. If sufficient numbers of

pure colonies are not available for identification, a subculture requiring additional incubation is necessary. By

placing a 0.04-unit differential bacitracin filter paper disk, available commercially directly

on the area of initial inoculation, presumptive identification of S. pyogenes can be made after overnight

incubation (all of group A and a very small percentage of group B streptococci are susceptible). However, use

of the bacitracin disk in the primary area of inoculation reduces the sensitivity and specificity of culture and

identification of S. pyogenes.

 Sometimes growth of too few beta-hemolytic colonies or overgrowth of other organisms makes interpretation

difficult. Therefore, using the bacitracin disk as the only method of identification of S. pyogenes is not

recommended.

 New selective agars, such as streptococcal selective agar, have been developed that suppress the growth of

almost all normal flora and beta-hemolytic streptococci except for groups A and B and Arcanobacterium

haemolyticum. Direct antigen or nucleic detection tests or the PYR test can also be carried out on isolated betahemolytic colonies.

Corynebacterium diphtheriae

If diphtheria is suspected, the physician must communicate this information to the clinical laboratory. Because

streptococcal pharyngitis is included in the differential diagnosis of diphtheria and because dual infections do

occur, cultures for Corynebacterium diphtheriae should be plated onto sheep blood agar or streptococcal

selective agar, as well as onto special media for recovery of this agent. These special media include a Loeffler’s

agar slant and a cystine-tellurite agar plate.

Recovery of this organism is improved when culturing specimens from the throat and nasopharynx of

potentially infected patients. In addition to culture, rapid toxigenicity assays, including immunoassays and

polymerase chain reaction, may be used to assist in the diagnosis. Caution should be used when interpreting

molecular assays, because positive results have been associated with related species of Corynebacteria.

Bordetella pertussis

Freshly prepared Bordet-Gengou agar was the first medium developed for isolation of Bordetella pertussis.

Today, Regan-Lowe or charcoal horse blood agar is recommended for use in diagnostic laboratories.

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Neisseria

Specimens received in the laboratory for isolation of Neisseria meningitidis (for detection of carriers) or N.

gonorrhoeae should be plated to a selective medium, either modified Thayer-Martin or Martin-Lewis agar.

After 24 to 48 hours of incubation in 5% to 10% carbon dioxide.

Epiglottitis:

Clinical specimens from cases of epiglottitis (swab sobtained by a physician) should be plated to sheep blood

agar, chocolate agar (for recovery of Haemophilus spp.), and a streptococcal selective medium. Staphylococcus

aureus, Streptococcus pneumoniae, and beta-hemolytic streptococci are all potential etiologic agents of this

disease.

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Lecture 17

Eye Diagnostic Microbiology

When we reject the specimens

1. Inappropriate specimen transport device

2. mislabeled specimen

3. Unlabeled specimen

4. specimen received after prolonged delay (usually more than two hour)

5. specimen received in expired transport media

Eye pathogens and commensals

Common pathogens commensals

Streptococcus pyogenes Staphylococcus epidermidis

Pseudomonas aeruginosa Lactobacillus spp

Chlamydia trachomatis Propionibacterium spp

Streptococcus pneumoniae Staphylococcus aureus

Haemophilus influenzae Various Enterobacteriaceae

Haemophilus aegyptius Various streptococcus spp

Specimen collection

1. Pull down the lower eyelid so that the lower conjunctival fornix is exposed.

2. Swab the fornix without touching the rim of the eyelid with the sterile cotton swab.

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3. Place the swab immediately in a bacterial transport medium or, if the specimen is brought to the laboratory immediately, in a

sterile test tube with 0.5 mL of buffered saline (pH 7)

Time relapse before processing the sample

Eye specimen should be processed immediately because tears contains lysosomes which may kill the organism

Media used in eye swab culture

A. Blood Agar plate

B. Chocolate Agar plate

C. MacConkey’s Agar plate

Result reporting

1) Report Gram stain finding as an initial report.

2) Report the isolated pathogen and its sensitivity pattern as a final report.

Turnaround time

1) Gram stain results should be available 1 hour after specimen receipt.

2) Isolation of a possible pathogen can be expected after 2-3 days.

3) Negative culture will be reported out 1-2 days after the receipt of the specimen.

Notes

a. All bacteria isolated in fair amounts and not resembling contaminants will be identified and tested for antibiotic

susceptibility, including susceptibility to chloramphenicol.

b. If trachoma is suspected, conjunctival scraping should be smeared onto a microscopic slide, air-dried and fixed in absolute

methanol. Chlamydia antigen detection systems are available for this purpose

c. Chlamydia trachomatis, an exclusively human pathogen, is a non-motile, Gram negative intracellular bacterium having a

unique developmental cycle in the infected cell. Metabolically inactive elementary body (EB) which is infectious enters the host

cell (columnar epithelial cell) and develops into a reticulate body (RB) which divides rapidly by binary fission leading to a

release of infectious

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Figure shows immunoflorescence staining showing the reticulate bodies of Chlamydia trachomatis grown on McCoy cell line

culture

Ear Diagnostic Microbiology

Etiological diagnosis of external or media otitis by aerobic and anaerobic culture with identification and susceptibility test of the

isolated organism(s).

Types of specimen

Pus from the external or middle ear.

Criteria of specimen rejection

1. Inappropriate specimen transport device

2. Mislabeled specimen

3. Unlabeled specimen

4. Specimen received after prolonged delay (usually more than two hour)

5. Specimen received in expired transport media

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Common pathogens Commensal flora at external canal

Staphylococcus aureus Staphylococcus epidermidis

Streptococcus pyogenes Lactobacillus spp.

Pseudomonas aeruginosa Propionibacterium spp.

Other Gram negative bacilli Staphylococcus aureus

Streptococcus pneumoniae Various Enterobacteriaceae

Haemophilus influenzae Various Streptococcus spp

Anaerobic bacteria Candida spp. other than albicans

Proteus spp. Occasion Pseudomonas aeruginosa

Specimen collection

1. Collect a specimen of the discharge on a thin, sterile cotton wool or Dacron swab.

2. Place the swab in a container with the transport medium, breaking off the swab stick to allow the stopper to be replaced tightly.

3. Label the specimen and send it to the laboratory.

Time relapse before processing the sample

Not more than 2 hours

Media used in eye swab culture

1) Blood Agar plate

2) Chocolate Agar plate

3) MacConkey Agar plate

Interfering factors

Patient on antibiotic therapy.

Improper sample collection.

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Result reporting

Report Gram stain finding as an initial report.

Report the isolated pathogen and its sensitivity pattern as a final report.

For external ear infections only Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, and Aspergillus will

be looked for and reported.

For middle ear infections only Pneumococcus, Streptococcus pyogenes, Haemophilus influenzae and Staphylococcus aureus will

be reported with a susceptibility test.

For the chronic discharging ear, Bacteroides species and fungi will also be reported in addition to the organisms reported for

middle ear infections.

Para nasal Sinus Diagnostic Microbiology

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Pathogens infect the sinuses

Viruses

Rhinovirus

Adenovirus

Influenzae virus

Parainfluenzae virus

Bacteria

Streptococcus pneumoniae

Haemophilus influenzae

Moraxella catarrhalis

Staphylococcus aureus

Streptococcus pyogenes

Most sinus infections are viral, and only a small proportion develops a secondary bacterial infection. Rhinoviruses, influenza

viruses, and Parainfluenzae viruses are the most common causes of sinusitis.

The most common bacteria isolated from pediatric and adult patients with community-acquired acute purulent sinusitis are

Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pyogenes. Staphylococcus aureus

and anaerobic bacteria (Prevotella and Porphyromonas, Fusobacterium and Peptostreptococcus spp.) are the main isolates in

chronic sinusitis.

Pseudomonas aeruginosa and other aerobic and facultative gram-negative rods are commonly isolated from patients with

nosocomial sinusitis, the immunocompromised host, those with HIV infection, and in cystic fibrosis.

Fungi and Pseudomonas aeruginosa are the most common isolates in neutropenic patients. The microbiology of sinusitis is

influenced by the previous antimicrobial therapy, vaccinations, and the presence of normal flora capable of interfering with the

growth of pathogens.

The nasal cavity must disinfect with Povidone solution / chlorhexidine solution, swabs were taken from the infected sinuses. Two

swabs were taken, one for aerobic and fungus, and another for anaerobic microorganisms.

Swabs were inoculated in MacConkey agar and Chocolate agar. The specimens were incubated at 35° C in a 5% carbon dioxide

environment. The plates were evaluated daily for at least two days for any microbial growth. For anaerobic culture, swabs were

transported via thioglycolate broth and later inoculated in blood agar plates. These were incubated anaerobically at 35° C, and

evaluated for any microbial growth daily for at least five days. Fungal analysis was done by KOH mount and culture on

Sabouraud Chloroamphenicol agar plate.

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Skin (wound, abscess, burns) Diagnostic Microbiology

Types of specimen

Swabs from the infected area or aspiration from deep wounds

Swabs in anaerobic transport media for the isolation of anaerobes

Criteria of specimen rejection

1) Inappropriate specimen transport device

2) Mislabeled specimen

3) Unlabelled specimen dried samples and specimen received after prolonged delay

(Usually more than 72 hours)

4) specimen received in expired transport media

Pathogenic bacteria Commensals bacteria

Pseudomonas aeruginosa Alpha haemolytic streptococci

Proteus spp Corynebacterium spp.

E. coli Coagulase negative Staph.

Klebsiella spp Propionibacterium spp.

Morganella Bacillus spp.

Providencia

Streptococcus pyogenes

Staphylococcus aureus

Enterococcus spp.

Clostridium perfringens

Fusobactrium spp

Peptostreptococcus spp

Mycobacterium tuberculosis

Nocardia spp.

Actinomyces israelii

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Specimen collection

Pus from abscess is best to collected at the time; the abscess is incised and drained. Using sterile technique, aspirate or collect

from drainage tube up to 5ml of pus, transfer to sterile container. If pus is not being discharged use sterile cotton wool swab to

sample from the infected site, extend the swab deeply into the depth of the lesion. Immerse the swab in container of transport

medium, label it and send to the laboratory as soon as possible.

Time relapse before processing the sample

30 minutes

Storage

Maintain specimen swab at room temperature. Do not refrigerate

Media used in culture

1. Blood Agar,

2. Chocolate Agar,

3. MacConkey Agar

4. Thioglycollate broth

Culturing procedure

Streak one blood agar plates, one chocolate, MacConkey and inoculate thioglycollate broth tube. Gram stain to check the

presence or absence and if present the type or types and the predominant organisms.

Interfering factors

 Patient on antibiotic therapy

 Improper sample collection

Result reporting

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Report Gram stain finding as an initial report

Report the isolated pathogen/s and its sensitivity pattern as a final report

Turnaround time

Gram stain results should be available 1 hour after specimen receipt. Isolation of a possible pathogen can be expected after 2-3

days. Negative culture will be reported out 1-2 days after the receipt of the specimen.

Note below

Contamination of the specimen with normal flora is one of the major obstacles in obtaining good results. Care should be taken to

avoid contaminating the specimen with normal flora. This could e accomplished by swabbing superficial infected wounds with

70% alcohol before we take our swab.

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