A. Blood:
(1). Collection and transport Purpose:
To reduce blood culture contamination rate, collection may be improved by taking the following precautions.
Note: This is an emergency procedure.
The sample has to be processed and reported immediately. The results of the smear should be informed to the concerned clinician and documented in the critical alert register.
a. Prepare the site:
Select the site of venipuncture.
If the patient is unusually dirty, wash the intended site with soap and water prior to venipuncture.
Apply a tourniquet, 3-4 inches above the intended site of venipuncture. Alternatively this can be done after cleaning.
Put on examination gloves. Vigorously cleanse with 70% isopropyl or ethyl alcohol to remove surface dirt and oils.
Scrub the venipuncture site gently but firmly with the cotton beginning in the center and continuing in an outward direction circularly for an area of 4 to 5 inches in diameter.

Allow to dry. Swab or wipe concentric circles of povidone/tincture of iodine, in a similar manner as given earlier- beginning in the center and continuing in an outward direction circularly for an area of 4 to 5 inches in diameter.

Allow the povidone iodine to dry (2 minutes). Do NOT touch the site after cleaning. Instruct patient to clench and unclench the fist.

Perform phlebotomy using the needle and syringe. Release the tourniquet and withdraw the needle. Apply pressure to the site of venipuncture and place a bandage over the puncture site.

Povidone I2 Chlorhexidine Gluconate (2%)/ tincture I2 (10%)
Drying period 2 minutes ~ 30 seconds

• Skin preparation with either alcohol, alcoholic Chlorhexidine (2%), or tincture of iodine (10%) leads to lower blood culture contamination rates than does the use of povidone-iodine
 For pediatric patients
 < 2months
 Omit the iodine step
 Clean two additional times with separate preparation pads saturated with 70% isopropyl alcohol or ethyl alcohol
 2 months
 Chlorhexidine Gluconate as a skin antiseptic is approved for use in pediatric patients two months of age and older
b. Prepare the bottle:
 Prepare the septum of the blood culture bottle and the rubber stoppers on bottles or tubes.
 Label the bottles with the patient’s name and the date and time of draw. Site of draw may be listed.
• In particular, please mention whether blood is collected from a central line or from peripheral venipuncture.
• Collection through an intravenous line: It is not necessary to discard the initial volume of blood or flush the line with saline to eliminate residual heparin or other anticoagulants.
• Vigorously wipe septa with 70% alcohol and allow drying completely, for 30 to 60 seconds.
• Pediatric bottles are not to be used for adult patients except for those elderly patients in whom it’s difficult to obtain larger amounts of blood.
c. Recommended total volume and numbers of blood cultures:
Adult patient (50 kg):
 10-20 ml, divided between two blood cultures from separate, peripheral venipuncture sites.
Pediatric patient:
 6-10 ml, divided between two blood cultures.
 Usually, a properly collected paired sample of blood culture need not be repeated up to 5 days. Initially obtain three blood culture sets within a 30 minute period before administration of empiric antimicrobial agents from patients presenting with possible infective endocarditis.
 If those sets are negative at 24 hours, obtain two more sets of cultures, for a total of five sets overall.
d. Timing of blood cultures :
Although drawing blood cultures before or during the fever spike is optimal for recovery, volume is more important than timing in the detection of agents of septicemia. Thoroughly mix bottles to avoid clotting.
Don’t forget: After phlebotomy, remove residual tincture of iodine from the patient’s skin by cleansing with alcohol to avoid skin irritation.
Manual blood culture inoculation:
 For conventional blood culture method, blood culture for bacterial infections to be carried out in two bottles containing 50 ml each of tryptone soya broth and bile broth (Hi-Media Labs, India).
 After removing the Kraft paper, inoculate the blood culture bottles. Incubate at 37oC and examine daily for 7 days for evidence of growth, indicated by turbidity, hemolysis, gas production, discrete colonies, or a combination of these.
e. Transport of blood culture bottles: In case of delay between collection and processing, never refrigerate the bottle. Preferably keep the bottle in a 35ºC incubator, if available. Otherwise, leave the bottle at room temperature.
(2). Processing of blood cultures:
a. Safety:
(i) Keep the culture bottles within a bio-safety cabinet or behind a shield, or wear a face mask.
(ii) Always wear gloves, because blood cultures contain material from patients who may harbor blood-borne pathogens.
(iii) Use needleless transfer devices or safety needles, and never recap them. iv. Dispose off needles and syringes in puncture-proof container.
b. Incubate blood cultures for the predetermined period at 35°C (usually 5 days, unless quality monitors indicate less time).
c. Maintain incubation conditions to allow recovery of microorganisms (follow manufacturer’s instructions) and maintain rotation or agitation of the media if possible.
d. Examine the cultures at least daily, whether detection of positives is by visual inspection or by an automated system. For visual inspection, observe for hemolysis, turbidity, gas production, pellicle formation, “puffballs,” and clotting, which are indicative of microbial growth.
Visible signs of growth caused by organisms commonly encountered in blood cultures
Microscopic observation Associated microorganisms
Hemolysis Streptococci, staphylococci, Listeria spp., Clostridium spp., Bacillus spp
Turbidity Aerobic gram-negative bacilli, staphylococci, Bacteroides spp
Gas formation Aerobic gram-negative bacilli, anaerobes
Pellicle formation Pseudomonas spp., Bacillus spp., yeast cells
Clotting Staphylococcus aureus
Visible colonies, “puffballs” Staphylococci, streptococci
e. For manual broth systems, perform at least one blind subculture to solid agar from visually negative bottles. Perform blind subculture after overnight incubation, and on 5th (final) day post-incubation on sheep blood agar and McConkey agar (MAC). In between, examine the bottles daily and subculture on solid media whenever there is a visible sign of growth in the bottle.
NOTE: Subculture of automated systems has little clinical utility.
f. In special circumstances when cultures appear to be negative, perform a Gram stain, wet mount, or acridine orange stain from the culture or its sediment to determine the presence of organisms.
g. Discard positive and negative bottles safely after autoclaving at 121ºC for 30 minutes. After autoclaving, open the automated system bottles with an opener and discard the inoculated medium in a designated shank for biomedical waste. For manual systems, open the screw-capped bottles, and discard the cultured media.

(3). Further processing of positive blood cultures:
a. Gram-stain a thin smear from the broth or agar immediately when suggestive of growth, for optimal patient care.
b. Subculture to agar media and put up biochemical tests based on the Gram stain results.
c. Follow-up workup of positive blood culture isolates.
(4). Reporting results:
a. For “No growth” cultures, indicate the length of incubation: “No growth after x days of incubation” for both preliminary and, final reports (automated systems – 5 days, manual systems – 5-7 days).
b. Positive cultures:
(i). Immediately report Gram stain results of all positive cultures to the physician incharge, with as much interpretive information as possible.
(ii). Follow immediately with a written or computer-generated final report including the following:
 AST result.
 Date and time of collection and receipt.
 Date and time positive result is reported and whether it was from a catheter draw or a peripheral draw.
NOTE: Such information is useful in the diagnosis of catheter-related bloodstream infections (CRBSI).
(5). Interpretation:
a. The report of a positive culture generally means that the patient is bacteremic. However, skin microbiota may contaminate the culture, causing a false-positive result, or pseudo-bacteremia; the latter has many other causes too.
b. Mixed cultures can be present and account for a small but significant number of bacteremias. Be aware of this when examining smears and plates.
c. For diagnosing CRBSI, differential time to positivity (DTP) is noted that includes growth of microbes from a blood sample drawn from a catheter hub at least 2 hours before microbial growth is detected in a blood sample obtained from a peripheral vein, which best defines CRBSI.
(6). Limitations:
a. Low levels of organisms may not be detected in the incubation interval of the culture.
b. The media used may not support the growth of all organisms. Use of multiple formulations increases the yield.
c. Sodium polyanethol sulphonate (SPS) may inhibit the growth and viability of some organisms.
d. Other diseases can present similarly to bacteremia, since there are many causes of fever of unknown origin.
e. Bacterial metabolism may not produce sufficient CO2 for detection in automated systems.
f. There are a number of fastidious microorganisms that infect the blood but cannot be grown in routine blood culture.
(i). Collection and transport Purpose: To identify the organisms causing pyrogenic meningitis.
Note: This is an emergency procedure. The sample has to be processed and reported immediately. The results of the smear should be informed to the concerned clinician and documented in the critical alert register.
a. Specimen collection:
Lumbar puncture Cap, face mask, gown and gloves for physician drawing CSF are useful adjuncts to infection prevention.

Disinfect the puncture site with antiseptic solution and alcohol in a manner identical to phlebotomy skin preparation for blood culture to prevent specimen contamination and introduction of infection.

Insert a needle with style t at the L3-L4, L4-L5, or L5-S1 interspace. When the subarachnoid space is reached, remove the style t; spinal fluid will appear in the needle hub.

Measure the hydrostatic pressure with a manometer. Collect the CSF into five calibrated sterile labeled tubes.

Physicians should be instructed to sequentially collect 2.0 ml of CSF into three sterile calibrated tubes if only routine chemistry (total protein and glucose), bacteriology (culture & susceptibility), and hematology (cell count) are required.
Ventricular shunt fluid
 Clean the reservoir site with antiseptic solution and alcohol prior to removal of fluid to prevent introduction of infection.
 Remove fluid by aspiration of CSF from the Ommaya reservoir or by collection from the ventricular drain or shunt.
 Collect CSF into a minimum of three sterile calibrated tubes if only routine chemistry (total protein and glucose:
Tube no. (1), bacteriology [culture & susceptibility]; tube no. (2), and hematology [cell count]; tube no.(3) are required. An initial CSF sample should be collected prior to antimicrobial therapy for highest diagnostic sensitivity. Subsequent CSF samples are then collected every 2 to 3 days once antimicrobial therapy is started to monitor for resolution of the infection.
b. Specimen transport Submit to laboratory as soon as possible and alert laboratory that specimen is in transit. Do not refrigerate. Each sterile calibrated tube containing CSF must be properly labeled with the patient’s name, unique identification number, and the date and time of collection. Requisition must be complete with demographic and specimen collection information. Record the patient diagnosis for proper processing of specimen.
c. Rejection criteria Call physician to prioritize requests if there is insufficient volume. Specimens in leaky containers must be processed, but alert the physician of the possibility of contamination.
(ii). CSF Processing:
Performance specifications:
The procedure described below is suited to identify common aerobic bacteria/fungi listed below:
Neonates E.coli, Group B streptococci
< 2 Months Group B streptococci and Haemophilus influenzae
< 10 Years H. influenzae, Streptococcus pneumoniae
Adult S. pneumoniae,Neisseria meningitidis
Immunocompromised Cryptococcus neoformans
CNS shunt infection Coagulase negative staphylococci (CoNS)

Day 1
a. CSF: gross appearance; Clear; slightly turbid; cloudy; purulent; Contains blood; Contains clots Xanthochromic
b. Centrifuge: CSF If 1 ml volume, centrifuge at 3000 g for 20 minutes /cytocentrifuge (1000 rpm for 10 minutes)
c. Wet mount: Look for pus cells, RBCs, bacteria, yeast cells.
d. India ink preparation: Look for capsulated organisms like Cryptococcus neoformans, S. pneumoniae.
e. Gram’s smear:
 Place 5-6 drops of sample or if the specimen is cloudy prepare the smear by placing 1-2 drops of CSF. Allow the drop(s) to form one large drop. Do not spread the fluid. Air-dry the slide.
 Fix smear with methanol or heat. Perform Gram’s stain. Interpret CSF microscopy immediately. Any bacteria seen are considered significant.
Report: Number of WBCs Bacteria – describe the morphology
f. Culture Chocolate agar (CHOC), blood agar, McConkey agar and RCM broth:
 Incubate at 37ºC for 48 hours; examine daily. If no growth in plates but RCM is turbid, subculture from RCM on blood agar, McConkey agar and chocolate agar.
Day 2

Culture examination Examine all plates and broth media for macroscopic evidence of growth at 24 hours. If no visible growth is observed on the culture media, reincubate.
 Cultures with growth: Notify physician of positive culture findings. Identify all organisms, using the rapid tests. Perform rapid bile solubility spot test on all alpha hemolytic streptococci to identify S. pneumoniae. If positive, report it as S. pneumoniae.
 Perform catalase and Gram stain of organisms growing on BAP and/or CHOC. Triple antigen test for S. pneumoniae, N. meningitidis, H. influenzae is also performed directly on CSF, on request of clinician.


C. Body fluids from sterile sites:
1. Collection and transport
Specimen collection:
 Body fluids from sterile sites are collected by percutaneous aspiration for pleural, pericardial, peritoneal, amniotic, and synovial fluids.
 Use care to avoid contamination with commensal microbiota.
 Clean the needle puncture site with alcohol, and disinfect it with an iodine solution [1-2% tincture of iodine or a 10% solution of povidone iodine (1% free iodine)] to prevent specimen contamination or infection of patient (if tincture of iodine is used, remove with 70% ethanol after the procedure to avoid burn).
 Aseptically perform percutaneous aspiration with syringe and needle to obtain pleural, pericardial, peritoneal, or synovial fluid. Use safety devices to protect from needle exposure.
 Immediately place a portion of the joint fluid or peritoneal fluid collected from patients with CAPD or SBP into aerobic and anaerobic blood culture bottles, retaining some (0.5 ml) in syringe for Gram stain and direct plating.
 Use the minimum and maximum volumes recommended by the bottle manufacturer (generally up to 10 ml is the maximum for each bottle).
 Alternatively, inoculate the blood culture bottles after receipt in the laboratory.
 Submit other fluids and the remainder of specimens after inoculation of blood culture bottles in one of the following:
• A sterile, gassed-out tube or a sterile blood collection tube without preservative; however, fluids in such tubes may clot during transport.
• Anaerobic transport vial.
• Specimens received by the laboratory in a syringe with the needle still attached should be rejected because of the risk of a needless sharp exposure by laboratory staff. The physician should be immediately contacted to recollect the sample and send it in proper container.
Note: Establish a policy for the proper collection and transport of clinical specimens not collected on swabs. Educate the physicians that needles must be removed from the syringe and the syringe cap secured prior to transport to avoid leakage.
• Syringes that have been capped with a Luer-Lock (with needle removed) prior to transport may be accepted for culture provided the specimen has not clotted inside the syringe and there is no leakage during transport which could result in contamination of the culture. The laboratory may reject specimens that have clotted in a capped syringe because they cannot be processed for culture without inadvertently contaminating the specimen.
Specimen transport:
 Submit to laboratory as soon as possible and, if from a normally sterile site, alert laboratory that specimen has been submitted.
 Do not refrigerate.
 Label specimens with patient demographics and date, time, and site of collection,e.g. left knee joint fluid.
 Record the patient diagnosis for improved processing of specimen.
Rejection criteria:
 If specimens inoculated into blood culture bottles are received, Gram stain cannot be performed.
 Collect prior to antimicrobial therapy for greatest diagnostic sensitivity.
 Do not submit specimens from drains after they have been infused with antimicrobial agents.
 Call physician when fluid specimens are received on a swab.
 Contact physician if specimen is insufficient for the number of tests requested.
NOTE: Swabs constitute the least desirable sample for culture of body fluids and should be discouraged, since the quantity of sample may not be sufficient to ensure recovery of a small number of organisms.
 Routine bacterial culture is sufficient for culture for Candida species, if blood culture bottles are used or specimen is centrifuged.
2. Processing Day:
a. Gross appearance: Clear; slightly turbid; cloudy; purulent; Contains blood; Contains clots
b. Gram smear: Place 5 to 6 drops of sample, or if the specimen is cloudy; prepare the smear by placing 1 or 2 drops of fluid. Allow the drop(s) to form one large drop. Do not spread the fluid. Air-dry the slide. Fix smear with methanol. Perform Gram’s stain and interpret immediately. Report: Number of WBCs Bacteria- describes the morphology
c. Culture Inoculate blood agar, MAC and RCM: Incubate at 37ºC overnight. Body fluid samples should also be incubated anaerobically, examined after 48 hours. Body fluids can also be inoculated in BACTEC bottles like CSF.
d. Culture examination: Examine all plates and broth media for macroscopic evidence of growth after 24 hours. If no visible growth is observed on the culture media, re-incubate.
Cultures with growth: Notify physician of positive culture findings. Correlate culture results with those of the direct Gram stain microscopy. Identify all organisms, using the rapid tests. Do not perform complete identification if the physician indicates that the organism is a probable contaminant or that the isolate is one or two colonies of a Coagulase negative staphylococcus on one medium with no growth in the broth.
NOTE: Empiric antimicrobial therapies are selected for the treatment of gastrointestinal tract microbiota, including anaerobes, enteric gram negative bacilli, and Enterococcus. To attempt to isolate and report each of these agents is labor-intensive and does not add to the requirement to treat the patient with agents that are effective against all the usual microbiota. Hold positive culture plates or tubes for at least 7 days or, preferably, freeze isolates for long-term retrieval.
D. Ocular Specimens:
1. Specimen collection and transport
NOTE: Most eye specimens are collected by an ophthalmologist. These specimens are inoculated onto culture media at the bedside, in the clinic or the physician’s office. A variety of techniques are used to collect material from different parts of the eye. The conjunctiva is constantly contaminated by various bacteria from the environment and ocular adnexa. Therefore, specimens from the conjunctiva serve as a control when compared with specimens collected by more aggressive or invasive techniques.
Specimen collection
 Provide fresh media to the clinical areas routinely collecting ocular cultures, and instruct physicians to immediately transport inoculated media and slides to the laboratory.
 Obtain viral and chlamydial samples before topical anesthetics are instilled.
 Obtain samples for chlamydial cultures with calcium alginate swabs.
 For viral cultures, use Dacron or cotton swabs with non-wood shafts.
 Collection by anatomic site Conjunctiva (bacterial conjunctivitis) and lid margin (blepharoconjunctivitis suspected) Obtain the specimen with a sterile, pre-moistened cotton or calcium alginate swab.
 Roll the calcium alginate or cotton swab over the conjunctiva before topical medications are applied. Culture both eyes with separate swabs.
 Immediately inoculate the material at the bedside onto BAP and CHOC. Inoculate the swab from the right conjunctiva in horizontal streaks, and inoculate the swab from the left conjunctiva in vertical streaks, each on one half of the same agar plate.
 Inoculate specimens from the right and left lid margins, if collected, by making an R and an L to represent the respective sites on another agar plate.
 Obtain conjunctival scrapings for a smear preparation as follows:
• Instill 1 or 2 drops of proparacaine hydrochloride. Using a Kimura spatula, gently scrape across the lower right tarsal conjunctiva.
• Smear the material in a circular area 1 cm in diameter on a clean glass slide. Prepare at least two slides. Immerse the slides in 95% methyl alcohol or 100% methanol for 5 minutes. Repeat steps for the left conjunctiva.
Cornea (bacterial keratitis):
• Instill 1 or 2 drops of proparacaine hydrochloride. Obtain conjunctival samples as described above, and then obtain corneal scrapings from the advancing edge of the ulcer by scraping multiple areas of ulceration and suppuration with a sterile Kimura spatula, using short, firm strokes in one direction (keep the eyelid open, and be careful not to touch the eyelashes).
• Obtain approximately three to five scrapings per cornea. Inoculate each set of scrapings onto BAP and CHOC, using a ‘C’ formation for each scraping.
• Prepare smears by applying the scrapings in a gentle circular motion over a clean glass slide or by compressing material between two clean glass slides and pulling the slides apart.
Bacterial endophthalmitis:
• Collect an aspirate of the vitreous fluid or perform a paracentesis of the anterior chamber using a needle aspiration technique to collect intraocular fluid.
• Collect specimens for conjunctival cultures along with the fluid to determine the significance of indigenous microbiota.
• If a small volume of fluid is collected, inoculate cultures at the bedside by inoculating 1 or 2 drops of fluid onto culture media.
• Specimen processing:
Direct smears Gram stain Prepare two or three smears from the clinical material if glass slides do not accompany the specimen. If cheesy tissue bits are present, crush them onto a slide for Gram stain.
• Place slides in 100% methanol for 5 – 10 minutes to fix material. Perform a Gram stain. Examine the stained smear for the presence of somatic cells and extra- and intracellular organisms. The presence of PMNs suggests a bacterial infection. The presence of mononuclear cells may indicate viral conjunctivitis.
NOTE: Pigment granules that resemble gram-positive cocci may be present on the Gram-stained smear. They can be differentiated from cocci because they are large, oval, and brown.
Culture inoculation, examination, and interpretation: Inoculate culture media – blood agar and McConkey agar
If a scanty specimen of intraocular fluid is submitted in a syringe, wash out the syringe by drawing up a small amount of broth.
Use the broth to inoculate plate media with 2 drops per plate. Place the remainder of the broth and specimen in broth culture tube.
Avoid creating an aerosol. Incubate cultures at 35ºC in 5-7% CO2 for 24 hours. Examine daily for the presence of microorganisms.
Estimate and report the number of each organism on each plate. The presence of moderate numbers of colonies or many colonies on one or more culture plates should indicate the bacterial etiology of the infection.
Quantitation of C streaks:
(i): Less than half of the C streaks are positive per plate
(ii): More than half of the streaks, but not all, are positive
(iii): All streaks are positive for bacteria.
3. Post-analytical considerations:
Reporting Results:
Convey positive reports from invasively collected specimens to the physician as soon as possible.
Report the relative number and morphology of all microorganisms seen, the presence and numbers of somatic cells (especially PMNs), and whether the organisms were observed intracellular as well as extracellular.
Report the quantity and organism identity for each morphological type observed on culture media. Interpretation All organisms grown in any amount from critical eye specimens (i.e., aqueous and vitreous fluid) should be identified and AST results reported.
All organisms present in the direct smear that grow on primary culture plates are considered clinically significant and should be worked up.
The following criteria may assist the laboratory to determine the clinical significance of bacterial isolates from critical eye specimens that may otherwise be considered indigenous microbiota.
Coagulase-negative staphylococci, diphtheroids, P. acnes, or viridians group streptococci growing on more than one medium are generally considered significant. Indigenous commensal isolates may also be clinically significant when growth occurs only on one medium or in broth. In such cases a comment may be added to the final report indicating that the clinical significance of organisms that are part of the commensal microbiota must be determined by clinical correlation.
E.Respiratory specimens:
Purpose: To isolate and identify the potentially pathogenic organisms from upper and lower respiratory tracts (URT and LRT) aiding in the diagnosis of infections.
Sputum cultures are done primarily to identify the pathogens that cause pneumonia or bronchopneumonia: community-acquired or hospital-acquired.
This procedure is for the isolation and identification of the common respiratory pathogens such as S. pneumoniae, H. influenzae, S. aureus, Gram-negative bacilli, including P. aeruginosa and other non-fermenters, Moraxella catarrhalis, Streptococcus pyogenes, etc.
Appropriate specimens for diagnosis of bacterial infection in upper and lower respiratory diseases.
Clinical condition Primary pathogen(s) Specimen(s)
Chronic bronchitis S. pneumoniae. Lower respiratory
S. aureus: including MRSA; H. influenzae; Gram-negative bacilli.
Pneumonia Community-acquired pneumonia Lower respiratory
S. pneumoniae
S. aureus, including MRSA H. influenzae K.pneumoniae
Anaerobes (if aspiration)
Agents of bioterrorism, including Bacillus anthracis, Brucella spp., Francisella tularensis, Yersinia pestis, and Burkholderia pseudomallei
Hospital-acquired pneumonia, including ventilator associated pneumonia (VAP)
Gram-negative bacilli, including P. aeruginosa and other non-fermenters
Anaerobes (if aspiration)
S. aureus
Lung abscess S. aureus Lung aspirate or biopsy sample
S. pyogenes
K. pneumoniae
P. aeruginosa
Sinusitis Acute Sinus aspirate
S. aureus, S. pneumoniae, H. influenzae. catarrhalis
Agents of acute sinusitis
Gram-negative bacilli, including P. aeruginosa
Staphylococcal carriage S. aureus, including MRSA Nasal swab
Cystic fibrosis Pseudomonas aeruginosa Deep throat (young children) Lower respiratory
S. aureus
Burkholderia cepacia complex
Aspergillus spp.

Specimen collection and transport
Primary specimen Sputum Spontaneous:
Early morning specimen generated after a bout of cough.
Having the patient brush his or her teeth and gargle with water immediately before obtaining the sputum specimen reduces the number of contaminating oropharyngeal bacteria.
Collect specimen resulting from deep cough in a sterile screw-cap cup or other suitable sterile collection assembly of about 100 ml capacity.
To prevent contamination of the outside of the container, the patient should be instructed to press the rim of the container under the lower lip to catch the entire expectorated cough sample.
Tightly screw on the cap of the container.
Wipe off any spilled material on its outside with a tissue moistened with disinfectant, but take care not to let any disinfectant enter the container.
Such communication with patients can be rewarding. In addition, patients should remove dentures during the specimen collection.
 Early-morning sputum samples should be obtained because they contain pooled overnight secretions in which pathogenic bacteria are more likely to be concentrated. Twenty four hour collections should be discouraged. Deliver the specimen to the laboratory as quickly as possible, preferably within 2 hours, for delicate bacterial, viral and mycoplasma pathogens may die out during longer delay.
Sinus aspirate:
Collection of specimens from patients with sinusitis is performed by otolaryngologists who perform nasal endoscopy or sinus puncture and aspiration.
Endotracheal aspirate (ETA): Endotracheal aspiration is done with a sterile technique using a 22 inch, 12F suction catheter. The catheter is introduced through the endotracheal tube for at least 30 cm. Gentle aspiration is then performed without instilling saline solution. The first aspirate is discarded. The second aspirate is collected after tracheal instillation of 5 ml saline in a mucus collection tube. [If very little secretion is produced by the patient, chest vibration or percussion for 10 minutes is used to increase the retrieved volume (> 1 ml)]. The specimens are sent to the laboratory and cultured within 1 hour of collection.
Bronchoalveolar lavage (BAL) collection: In this procedure 100-300 ml of saline is infused into a lung segment through the bronchoscope to obtain cells and protein of the pulmonary interstitium and alveolar spaces. A portion of it is sent to the laboratory.
Type of Container:
Collect in a sterile leak proof screw-cap container.
Rejection Criteria:
For sputum and endotracheal aspirate specimens:
• Reject duplicate specimens received on the same day unless the initial sample was inappropriate for culture according to microscopic evaluation.
• Do not accept repeat cultures at intervals of less than every 48 hours. Reject the following specimens for diagnosis of lower respiratory tract disease:
• 24 hours sputum collection Contaminated sputum and endotracheal specimens as per Gram stain.
• Specimens that are visually saliva only.
• Specimens that are visibly contaminated with toothpaste or other substances.
• Nasal washes or swabs of nares to diagnose sinusitis.
• Sputum samples are highly contaminated with normal anaerobic flora of the upper respiratory tract. Therefore, anaerobic culture should not be done.
For BAL specimens, lung aspirates and open-lung biopsy (OLB) specimens: BAL specimens, lung aspirates, and OLB specimens should never be rejected by the laboratory, since the patient has undergone an invasive procedure for their collection. For specimens delayed in transit more than 2 hours without refrigeration, indicate on the report that the delay in transit may compromise the culture results.
 Anaerobic culture should be performed on lung aspirates, pleural fluid, and OLB specimens by request or when the original specimen Gram stain demonstrates morphotypes suggestive of anaerobic infection.
Media and Reagents
• Blood agar and
• MAC (optional); add when indicated by the consultant.
• Gram’s stain
Sputum and ETA, note the following:
 Features of the specimen: purulent, mucopurulent, mucoid, blood tinged saliva.
Microscopic examination:
Gram staining (G/S): Examine 20 to 40 fields from sputum smears under low power and endotracheal smears under both low power and oil immersion. Average the number of cells in representative fields that contain cells. Reject the following for culture, as poorly collected or not consistent with a bacterial infectious process.
Sputum: >10 SECs/LPF.
NOTE: If the number of WBCs is 10 times the number of squamous epithelial cells (SECs) and there is 3+ to 4+of a single morphotype of bacteria, accept the specimen for culture.
ETA from adults: If >10 SECs/LPF or no organisms seen; from pediatric patients: if no organisms seen.
BAL: Microscopic examination of cells from lavage has also been used to diagnose pneumonia. When more than 5% of cells from BAL contain bacteria, pneumonia has been diagnosed with sensitivities of up to 90% and specificities of 89% to 100% (Principles and Practice of Infectious Diseases, 2005).
Limitations in studying the sputum specimens by Gram staining include:
 Not all patients can provide an adequate sample. This may be either due to an inability to produce a sample, or because the sample is of poor quality.
 Interpretation is observer dependent. Atypical pathogens, common either singly or as co-infecting agents, cannot be seen.
 The definition of “positive” varies from study to study, and a positive result for pneumococcus is poorly predictive of the ability to recover that organism from a sputum or blood culture (Guidelines, ATS, 2001).
 CA, BA with Staphylococcus streaking,
 MAC Incubate in candle jar at 37ºC aerobically.
 Suction tips (ST)
 These are received in sterile test tubes. Wash the tips (including the bore) using approximately 0.5 ml sterile normal saline. This fluid is used to inoculate BA & MAC with a 4 mm diameter nichrome wire loop. Subsequent procedure is same as for sputum.
 Endotracheal aspirate (ETA), Bronchoalveolar lavage (BAL) and lung aspirates inoculation ETA and BAL received are processed for a semi-quantitative estimation; a standardized wire loop is therefore a must for inoculation. A wire loop of 1.2 mm diameter (1µl volume) is used for the purpose. The inoculation is done on the blood agar and MAC. The plates are incubated aerobically overnight at 37°C

Day 2

Examination and reporting:


Report the growth of:

Streptococcus pneumoniae. 



Gram-negative bacilli, including aeruginosa and other non-fermenters.

Moraxella catarrhalis.

Streptococcus pyogenes.


A CFU count of 105/ml (e. 100 colonies) or 104/ml (i.e. 10 colonies) respectively, is considered significant.

Antibiotic sensitivity testing: As per the procedure described for the clinically significant isolates.


This procedure is not suitable for the isolation of all respiratory pathogens g. M. tuberculosis, Legionella, chlamydiae, Mycoplasma, which can also cause pneumonia.

Assay time 1 hour

Turnaround time (TAT) 48 hours

Safety Precautions: Specimen should be processed in the bio-safety cabinet IIA.


Reference Link:        

  1. http://www.icmr.nic.in/guidelines/Standard_Operating_Procedures.pdf.
  2. https://www.pharmaguideline.com/2011/02/sop-for-operation-of-bacteriological.html