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close this bookBasic Laboratory Procedures in Clinical Bacteriology (WHO; 1991; 128 pages)
View the documentPreface
View the documentIntroduction
open this folder and view contentsQuality assurance in microbiology
close this folderPart I. Bacteriological investigations
open this folder and view contentsBlood
open this folder and view contentsCerebrospinal fluid
open this folder and view contentsUrine
open this folder and view contentsStool
close this folderLower respiratory tract infections
View the documentIntroduction
View the documentThe most common infections
View the documentCollection of sputum specimens
View the documentProcessing of sputum in the laboratory (for non-tuberculous infections)
View the documentCulture for Mycobacterium tuberculosis
View the documentGeneral note on safety
open this folder and view contentsUpper respiratory tract infections
open this folder and view contentsSexually transmitted diseases
open this folder and view contentsPurulent exudates, wounds, and abscesses
open this folder and view contentsAnaerobic bacteriology
open this folder and view contentsAntimicrobial susceptibility testing
open this folder and view contentsPart II. Essential media and reagents for isolation and identification of clinical pathogens
View the documentSelected further reading
View the documentSelected WHO publications of related interest
View the documentBack Cover

Processing of sputum in the laboratory (for non-tuberculous infections)

Sputum should not be allowed to remain longer than approximately I hour at room temperature before being processed.

Macroscopic evaluation

The macroscopic appearance of the sputum should be recorded. Possible descriptions include:


purulent, green
purulent, yellow
mucopurulent (i.e., partially mucoid and partially purulent)
blood-stained, with green floccules
*grey, mucoid
*grey, frothy
*white, mucoid
*white, frothy
*white, mucoid, with some food particles
*watery (i.e., only saliva present)
*watery, with some food particles

Sputum specimens marked with an asterisk should not normally be examined for non-tuberculous infections.

Microscopic examination

A portion of the purulent or mucopurulent sputum should be used for the preparation of a Gram-stained smear.

If no floccules of pus can be seen (e.g., in a grey mucoid sputum sample), the Gram-stained smear may show only the presence of large, rather square, squamous epithelial cells, frequently covered with masses of adherent bacteria. This is an indication that the specimen consists mainly of mouth or throat secretions, and culture should not be carried out as it is not relevant, and usually highly misleading. An accepted guideline is to reject, for culture, any specimen that contains fewer than 10 polymorphonuclear neutrophils per epithelial cell.1


1 HEINEMAN, H. S. & RADANO, R. R. Acceptability and cost savings of selective sputum microbiology in a community teaching hospital. Journal of clinical microbiology, 10: 567 - 573 (1979).

In many patients with acute respiratory infections (e.g., pneumonia) and purulent sputum the emergency examination of a Gram-stained smear may provide guidance to the clinician in the choice of antimicrobial chemotherapy. Possible results include:


• Gram-positive diplococci surrounded by an empty space from the unstained capsules (suggestive of S. pneumoniae);

• small Gram-negative coccobacilli (probably H. influenzae);

• Gram-negative diplococci, intracellular and extracellular (suggestive of B. catarrhalis);

• Gram-positive cocci in grape-like clusters (suggestive of S. aureus);

• Gram-negative rods (suggestive of the presence of Enterobacteriaceae or Pseudomonas spp);

• large Gram-positive yeast-like cells, often with mycelia (suggestive of the presence of Candida spp).

Cultural procedures and interpretation

Select a floccule of purulent material (or of the most nearly purulent material available) and inoculate on to the various culture plates (see below).

A suggested routine set of culture media is as follows:


• blood agar, with an optochin disc placed in the middle of the secondary streaking;
• chocolate agar (or a selective blood agar medium for H. influenzae1);
• MacConkey agar.

1 The medium is prepared as follows:


Columbia agar base: 43 g
distilled water: 1000 ml

The medium should be dispersed in 100-ml quantities which can be autoclaved and stored in screw-capped bottles (120 °C, 15 minutes). Excessive autoclaving should be avoided. For use, 100ml of base medium are heated at 100 °C to melt the agar, and then cooled to 56 °C. The following are then added (per 100 ml of base):

defibrinated (horse) blood: 8 ml
bacitracin solution (200 units/ml): 2.5 ml
growth supplement: 1 ml

The growth supplement may be Supplement VX (Difco 3354, Difco Laboratories, Detroit, Ml, USA), or VITOX (Oxoid SR 90 Oxoid Ltd., Basingstoke, England) or IsoVitaleX (BBL Microbiology Systems Division, Becton & Dickinson & Co., Cockeysville, MD, USA) or PolyViteX (Biomérieux, Marcy l’Étoile, Charbonnières-les-Bains, France). After thorough mixing, the medium is poured into plates (15 ml per 9 cm Petri dish). Most strains of upper respiratory commensal bacteria are inhibited on this medium, but Haemophilus spp grow well; Gram-negative enteric rods and yeasts are not inhibited.

The blood agar and chocolate agar plates are incubated at 35-36 °C in an atmosphere containing extra carbon dioxide (e.g., in a candle jar) and the MacConkey plate is incubated in air.

If grape-like clusters of Gram-positive cocci were present in the stained smear, an extra mannitol salt agar (MSA) plate is suggested. The presence of Gram-positive, yeast-like structures in the stained smear may be an indication for the inoculation of a tube of Sabouraud dextrose agar (which needs to be incubated for at least 3 days at 35-37 °C). MSA and Sabouraud cultures do not need to be done routinely for all sputum specimens.

Cultures should be inspected after incubation overnight (18 hours) but reincubation for an extra 24 hours may be indicated when growth is less than expected from the microscopic findings, or when only tiny colonies are present.

Typical findings include the following:


• Flat, clear colonies with concave centres and zones of green (alpha) haemolysis, as well as a zone of inhibition of growth around the optochin disc, may be S. pneumoniae. If the reading of the optochin test result on the primary plate is inconclusive, the test should be repeated on a subculture. It should not be forgotten that other α-haemolytic colonies (the so-called viridans streptococci) are normally present in the flora of the mouth and throat.

• Tiny, water-drop colonies growing as non-haemolytic satellite colonies on the blood agar plate, but much larger clear colonies on the chocolate agar or enriched blood agar plates, suggest the presence of H. influenzae. These colonies are usually present in large numbers, generally more than 20 per plate. Some laboratories choose to confirm this by X and V factor dependence tests, but these have to be very carefully controlled and are not strictly necessary. Serological typing of respiratory strains is usually not helpful, as most of them are “rough” and untypable.

• Brittle, dry, grey-white colonies on both blood agar and chocolate agar plates may indicate B. catarrhalis. If desired, a set of sugar degradation tests may be set up (all test results negative), but most laboratories do not do this. Branhamella organisms are strongly oxidase-positive, and the colonial appearance is highly characteristic.

• Medium-sized, golden-buff colonies are formed by S. aureus. The coagulase and the mannitol fermentation tests are positive, although the slide coagulase test (“bound” coagulase test) is occasionally negative. If there is a contradiction between the appearance of the colonies and the slide test, then a tube coagulase (“free” coagulase) test should be performed.

• Colonies on MacConkey agar suggest that Enterobacteriaceae or Pseudomonas spp or Acinetobacter spp are present. These organisms are usually not clinically relevant.

• Whitish, round, matt colonies on the blood agar and chocolate agar plates may be Candida albicans, which will also grow within 2 - 3 days on a Sabouraud dextrose agar culture tube.

It should be stressed that rare colonies of any of the above organisms either stem from the normal commensal flora of the respiratory tract, or are a result of colonization (e.g., coliforms, yeasts). As they may not be relevant to the management of the patient, they should not be reported, or should be reported as colonizing flora.

Susceptibility testing

Susceptibility tests should be performed only when the amount of growth is considered significant, and not on every bacterial species present in small numbers in the culture. Interpretations of some possible results are presented in Table 8.

For Enterobacteriaceae and staphylococci the standardized disc-diffusion method (Kirby-Bauer) should be used. Strains of S. pneumoniae should be tested on Mueller-Hinton agar, supplemented with 5% sheep blood, for susceptibility to tetracycline, chloramphenicol, erythromycin, and penicillin. Conventional blood agar can also be used. For penicillin, instead of a disc containing penicillin itself, a disc containing 1 μg of oxacillin is preferred, as results with this agent agree better with the MIC value for penicillin; it is also more stable. Penicillin discs may deteriorate rapidly in hot climates and thus produce unreliable results.

H. influenzae strains should be tested (on chocolate agar) for susceptibility to tetracycline and chloramphenicol, and for β-lactamase production, using for example the Nitrocefin test. Rare strains of H. influenzae may be ampicillin-resistant without producing β-lactamase.

Table 8. Interpretation of susceptibility test results of fastidious organismsa


Total zone diameter (mm)





S. pneumoniae (blood agar medium)

tetracycline (30 μg)

≤ 14


≥ 19

erythromycin (15 μg)

≤ 13


≥ 23

oxacillin (1 μg)

≤ 19b


≥ 20

chloramphenicol (30 μg)

≤ 12


≥ 18

H. influenzae (chocolate agar medium)

tetracycline (30 μg)

≤ 19


≥ 23

chloramphenicol (30 μg)

≤ 25


≥ 29

B. catarrhalis (blood agar medium)

tetracycline (30 μg)

≤ 14


≥ 19

erythromycin (15 μg)

≤ 13


≥ 23


a National Committee for Clinical Laboratory Standards (NCCLS). Voluntary consensus standards for clinical laboratory testing. Villanova, PA, NCCLS, 1990.

b Resistant or intermediate.

B. catarrhalis isolates should be tested for β-lactamase production. Testing against tetracycline and erythromycin is optional.

Candida albicans cultures need not be tested against any antimicrobial agents.

Most laboratories give a semi-quantitative assessment of the bacteria cultured on solid media, which might be presented as follows:


(+) = few colonies
+ = light growth
+ + = moderately heavy growth
+ + + = heavy growth.
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