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Processing of respiratory tract specimen



Respiratory tract specimens can be categorized into upper respiratory and lower respiratory tract specimen


  1. Upper Respiratory Tract specimens:

 a. Throat Swab


Throat swab is collected to diagnose mainly pharyngitis caused by beta hemolytic Streptococci; however diphtheria, Vincent’s angina, thrush and gonococcal pharyngeal infection could also be diagnosed on special request.


Specimen collection procedure:


  1. Turn the patient’s face against the light and ask the patient to open the mouth wide and say ah, gently depress the tongue with the tongue depressor so that throat is well exposed.
  2. Guide a swab over the tongue into the posterior pharynx.
  3. Rub the swab firmly over the back of the throat, both tonsils and any area of inflammation, exudation or ulceration.

Care should be taken to avoid touching the tongue, cheeks or lips with the swab.

  1. Place the swab in the transport medium and send to the laboratory.



Cooling box is not necessary if the specimen reach the lab within one day.


Culture Inoculation:


  1. Media /Reagents


  • Media:

Sheep blood agar (SBA) or Blood agar with supplemented colistin and nalidixic acid (BCNA)

  • Reagents:

3% Hydrogen peroxide for catalase test

Lancefield grouping (Patho DX kit).

  1. Procedure


  • Firmly roll swab over agar surface, and streak in four quadrants for isolation to minimize overgrowth by other microorganisms.


  • Incubate it in CO2 incubator for overnight (18-24Hrs).


  • After overnight incubation next day observe for beta hemolytic Streptococcal colonies, if there is no such type of colonies then reincubate for further 24 hours in CO2


  • If there are beta hemolytic colonies (beta-hemolytic streptococci colonies are small, transparent, or translucent and dome shaped, has entire edge, and is surrounded by a relatively wide zone of complete hemolysis) then pick one or two colonies and perform catalase test. If negative then proceed with confirmatory identification.


  • In final reporting at 48 hours


  1. If there is no growth on plates report “no growth”.
  2. If there is growth on 1st quadrant report “few”
  3. If there is growth on 1st & 2nd quadrant report “moderate”
  4. If colonies present in the primary and secondary areas of inoculation report “heavy”.




Infections of the lower respiratory tract are the leading cause of cause of mortality world wide. Streptococcus pneumoniae is the leading bacterial agent of community acquired pneumonia along with Hemophilus influenzae and Moraxella catarrhalis

Specimen collection and transportation

Possible specimens from lower respiratory tract are; Expectorated Sputum, Tracheal Aspirates, Endotracheal aspirate, Bronchoalveolar Lavage (BAL), Bronchial Wash, Bronchoscopy specimens, Bronchial brush.



  • BCNA ( with 5%sheep Blood Agar) or SBA
  • MacConkey agar
  • Chocolate agar



  • Gram stain reagents
  • 3%hydrogen peroxide
  • Oxidase reagents
  • Kovac’s reagent


Equipments & Supplies:

  • 5% CO2 incubator of 37oC
  • Atmospheric incubator of 37oC
  • Frosted end slides
  • Inoculating loop and Needle
  • Conventional Microscope with oil immersion lens
  • Immersion Oil


Microscopic Examination


Prepare a gram stain smear for all lower respiratory tract specimens to determine the presence of oropharyngeal contamination (indicated by squamous epithelial cells) and lower respiratory tract secretions (indicated by WBCs) as well as to identity the most likely pathogens (Indicated by the predominant organisms associated with WBCs).



Reporting of Gram Stain Results:


Squamous Epithelial Cells

  1. If no squamous epithelial cell are found, report “ No epithelial cells seen”
  2. If only a few epithelial cells are found report “ Few epithelial cells seen”
  3. If abundant epithelial cells seen, indicating oropharyngeal contamination, such specimens are graded as unsatisfactory sample.



  1. If no WBC are found report “No WBCs seen”
  2. If WBCs are present in any amount state as few, moderate or numerous WBCs seen.



Report the bacteria seen few/moderate/numerous.




None        Few         Moderate             Numerous


Squamous epithelial cells/ LPF*          0             1-9           10-24                      >25

Neutrophils/LPF*                             0             1-9           10-24                      >25




Type / Number of organisms / HPF**

Gram-positive cocci

Gram-negative cocci

Gram-negative rods

Gram-positive rods


LPF*: (low power field) x 10 (examine 10-20 fields)

HPF**: (high power field) oil immersion




  1. Sputum Expectorated:

Instruction for procedure:

  • Specimen collection:
  1. The patient should be standing, If possible or sitting upright in bed.
  2. He or she should take deep breath to full the lungs, and empty then in one breath, coughing as hard and as deeply as possible.
  3. Sputum brought up should be spit into screw capped container.
  4. Visually inspect the specimen.
  5. Tighten the cap of the container and send immediately to lab.


  • Transportation:

Send immediately or if delay more then one hour is suspected then place in cooling box.


Procedure for sputum washing:

  1. Pour the specimen in sterile dry empty Petri dish.
  • Note the appearance of the specimen as purulent, mucoid, watery bloody or brownish.
  1. Fill three Petri dishes with sterile saline.
  2. Wash carefully in the first dish purulent of the specimen with a sterile loop.
  3. Transfer this respective part of washed specimen to the second dish and wash carefully.
  • Repeat step#5 in third dish with the same loop without heating and transporting the specimen in the lid.
  • Make a smear on a glass slide with a part of this washed sample; evaluate the washing with x10 objective.
  1. If pus cell are present and epithelial cell absent inoculate the media. Make gram stain on the slide.
  2. If pus cell are present with moderate to numerous epithelial cells, repeat washing with better specimen.
  3. If no pus cells are present or after repeated washing the epithelial cells can not be removed ask for another sample.


Culture processing:

  • Streak the specimen on chocolate agar, MacConkey agar, BCNA or SBA
  • Incubate chocolate and BCNA or SBA in CO2 incubator at 37o with 5-7% CO2 and MacConkey incubator in atmospheric incubator at 37o for 24hrs.
  • Exam all plates after 24hours of incubation.
    1. If there is no growth on plates report “no growth” and re-incubate for further 24hours.
    2. If there is growth on 1st quadrant report “few”
    3. If there is growth on 1st & 2nd quadrant report “moderate”
    4. If colonies present in the primary and secondary areas of inoculation report “heavy”.


Note: If numerous epithelial cells on gram stain and growth of predominant organisms is not observed on culture plates no further evaluation is required.

*Don’t process sputum specimens with numerous epithelial cells.



  1. Tracheal aspirate:

Aspirate the specimen into a sterile sputum trap or leak proof cup. These could be processed either quantitatively (See below) or semi quantitatively (Processing and reporting is same as that of sputum)

  1. Bronchoscopy specimens:  collected by a pulmonologist or other trained physician.

Bronchoscopy specimens include bronchoalveolar lavage (BAL), bronchial washings, protected specimen brushings (PSB), & transbronchial biopsy specimens. Quantitative cultures are performed on BAL samples (See below).


Transport the specimens as early as possible to the lab, if delay is anticipated then store at 2 to 8oC.


«Note: A delay in processing of more then 1 to 2 hours may result in loss of recovery of fastidious pathogens, such as of S. pneumoniae, and over growth of oropharyngeal flora.




Process specimens in biological safety cabinet, as aerosol can result in laboratory-squired respiratory infections.

Process all specimens as rapidly as possible, especially specimen from emergency department, and inpatients. Select the most purulent or most blood-tinged portion of the specimen. Significant growth above the cutoff should be reported; however if more than one pathogen is isolated than it is suggestive of oropharyngeal contamination and clinical correlation should be done before reporting the samples.


Tracheal aspirate (TA):

  1. Add an equal volume of Sputasol (Dithiothreiotol) to the specimen and digest the sputum by mixing on vortex mixer. This should normally take about 20-30 seconds. Incubate at 37Co for 15 minutes.
  2. When digestion is complete dilute 100µl (0.1ml) of the digested sputum into 9.9ml of ¼ strength Ringers Solution (sterile). Mix properly.
  3. Form this diluted well mixed sputum, transfer 10µl in each plate of Chocolate agar, SBA, & MacConkey agar (MAC). Each colony on the plate is equal to 10,000 cfu/mL.
  4. Incubate CHOC & SBA in CO2 incubator and MAC in atmospheric incubator at 37oC.



Any count of ≥ 105 is considered as significant. Therefore growth of 5 or more than 5 colonies is identified and reported.


Broncho-Alveolar Lavage:

  1. Vertex 30seconds
  2. Prepare smear on clean gress free glass slide for gram stain.
  3. Using 0.001 loops inoculate specimen on SBA, CHOC, & MAC labeled x1000. Each colony on this plate = 1000cfu/ml
  4. Streak the inoculum in four quadrants.
  5. Incubate SBA & CHOC in CO2 incubator & MAC in atmospheric incubator at 37oC.


Any count of ≥ 104 is considered as significant. Therefore growth of 10 or more than 10 colonies is identified and reported.

Culture examination

  1. observe plates at 24 h.
  2. Incubate plates for an additional 24 to 48 h, which is useful to detect molds and slow-growing, fastidious gram-negative rods, such as Bordetella spp.
  • Even if there was growth at 24 h, examine plates again at 48 h for morphologies not seen at 24 h.
  1. Use the Gram stain result as a guide to interpreting the culture.
  2. Use the presence of inflammatory cells and bacteria in deciding and the extent of processing the culture.
  3. If the culture dose not match the smear results, review the smear a second time
  4. Follow table #1 for processing and reporting significant microbioata.



* Culture for fungi of for legionella should be made before adding of sputasol (digestion)

* If AFB investigation is required, separate a portion for mycobacterial culture of AFB Smear prior to processing

* Smears are prepared before adding Sputasol.




Action Organisms

Examine for and always report.











Always report, but do not make an effort to find low numbers, unless they are seen in the smear.



Report if present in significant amounts, even if not predominant.








Report if present in significant amount and if it is the predominant organism in the culture, particularly if suggests infection with morphology consistent with isolate.






Report as “Enteric gram-negative rods.”




Report as “Non-glucose fermenting, gram-negative rods.”

1.   Streptococcus pyogenes

2.   Group B streptococci in pediatric population

3.   Francisella tularensis

4.   Bordetella spp., especially Bordetella bronchiseptica

5.   Yersinia pestis

6.   Nocardia spp.

7.   Bacillus anthracis

8.   Cryptococcus neoformans

9.   Molds, not considered saprophytic contaminants

10.              Neisseria gonorrhoeae



1.   Streptococcus pneumoniae

2.   Hemophilus influenzae report beta-lactamase




1.   Moraxella catarrhalis

2.   Neisseria meningitides

Report the following for nosocomial infections:

3.  Pseudomonas aeruginosa

4.  Stenotrophomonas maltophilia

5.  Acinotobacter spp.

6.  Burkholderia spp.


1.   Staphylococcus aureus

2.   Beta-hemolytic streptococcus B (adults), C, or G

3.   Single morphotype of gram-negative rod (especially Klebsiella pneumoniae)

4.   Fastidious gram-negative rods; usually report beta-lactamase

5.   Corynebacterium spp. if urea positive or from ICU

6.   Rhodococcus equi in immunocompromised patients



More than one morphology of gram-negative rods that grow on MAC and are oxidase negative and are either indole positive, lactose positive, or spreading.


More than one morphology of gram-negative rods that either are oxidase positive and grow on MAC or are nonreactive on KIA or TSI.


1– Henry D Isenberg. Clinical Microbiology Procedure Handbook, 2nd ed. Vol. 1 ASM press.