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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
open this folder and view contentsLower respiratory tract infections
close this folderUpper respiratory tract infections
View the documentIntroduction
View the documentThe normal flora of the pharynx
View the documentBacterial agents of pharyngitis
View the documentCollection and dispatch of specimens
View the documentDirect microscopy
View the documentCulture and identification
View the documentSusceptibility testing
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
 

Culture and identification

Culture for Streptococcus pyogenes

Immediately upon receipt in the laboratory, the swab should be rubbed over one-quarter of a blood agar plate, and the rest of the plate streaked with a sterile wire loop. The blood agar should be prepared from a basal agar medium without glucose (or with a low glucose content), e.g., tryptic soy agar (TSA). Acidification of glucose by S. pyogenes inhibits the production of haemolysin. Blood from any species, even human blood (fresh donor blood), can be used at a concentration of 5%. The plates should be filled to a depth of 4-5 mm. Sheep’s blood is preferred because it does not permit growth of haemolytic Haemophilus spp and it gives no haemolysis with the zymogenes variant of E. faecalis.

The recognition of (β-haemolytic colonies can be improved, and their presumptive identification hastened, by placing a co-trimoxazole disc (as used for the susceptibility test) and a special low-concentration bacitracin disc over the initial streaked area. Because S. pyogenes is resistant and many other bacteria are susceptible to cotrimoxazole, this disc improves the visibility of β-haemolysis. Incubation in a candle-jar will detect most β-haemolytic streptococci. A simple way to increase haemolysis is to stab the agar surface perpendicularly by inserting the loop deep into the medium to encourage growth of subsurface colonies. After 18 hours and again after 48 hours of incubation at 35 - 37 °C, the blood plates should be examined for the presence of small (0.5 - 1 mm) colonies surrounded by a relatively wide zone of clear haemolysis. After Gram-staining to verify that they are Gram-positive cocci, the colonies should be submitted to specific identification tests for S. pyogenes. For clinical purposes, presumptive identification of S. pyogenes is based on its susceptibility to a low concentration of bacitracin. For this purpose, a special differential disc is used containing 0.02-0.05 IU of bacitracin. The ordinary discs used in the susceptibility test, with a content of 10 units, are not suitable for identification. A β-haemolytic streptococcus showing any zone of inhibition around the disc should be reported as S. pyogenes. If the haemolytic colonies are sufficiently numerous, the presence or absence of an inhibition zone may be read directly from the primary blood agar plate. If the colonies are less numerous, one or two should be picked from the primary plate, streaked on another plate, and each inoculated area covered with a bacitracin disc. After overnight incubation, the subcultures should be read for inhibition zones.

In some laboratories this presumptive identification is confirmed by serological demonstration of the specific cell wall polysaccharide. This can be done either by the classical precipitin method, or more rapidly by using a commercial kit for the rapid slide coagglutination or latex agglutination tests. If desirable, bacitracin-resistant (β-haemolytic streptococci can be further identified using some simple physiological tests (see Table 9).

In reporting the presence of S. pyogenes in a throat culture, a semiquantitative answer should be given (rare, +, ++, or +++). Patients with streptococcal pharyngitis generally show massive growth of S. pyogenes, with colonies over the entire surface of the plate. Plates of carriers generally show fewer than 20 colonies per plate. Even rare colonies of β-haemolytic streptococci should be confirmed and reported.

Table 9. Differentiation of p-haemolytic streptococci

Species

S. pyogenes

S. agalactiae

E. faecalis var. zymogenesa

Others

Lancefield group

A

B

D

C, G, F

haemolysis

β

βb

β

β

zone around the differential bacitracin disc

+

0c

0c

0d

bile-aesculin agar (growth and blackening)

0

0

+

0

CAMP test

0 (+)

+

0

0

co-trimoxazolee susceptibility

0

0

0

+

 

aE. faecalis var. zymogenes produces β-haemolysis only on horse blood agar.
b 5% of B streptococci are non-haemolytic.
c 5% are positive.
d 10% are positive
e Same disc as in the Kirby-Bauer method.

Culture for Corynebacterium diphtheriae

Although the diphtheria bacillus grows well on ordinary blood agar, growth is improved by inoculating one or two special media:

 

Löffler coagulated serum or Dorset egg medium. Although not selective, both of these media give abundant growth of the diphtheria bacillus after overnight incubation. Moreover, the cellular morphology of the bacilli is more “typical”: irregularly stained, short to long, slightly curved rods, showing metachromatic granules, and arranged in a V form or in parallel palisades. Metachromatic granules are more apparent after staining with methylene blue or Albert’s stain than with the Gram stain.

A selective tellurite blood agar. This medium facilitates isolation when the bacilli are few in number, as is the case for healthy carriers. On this medium, colonies of the diphtheria bacillus are greyish to black and may be fully developed only after 48 hours. Suspicious colonies, consisting of bacilli with a coryneform morphology on the Gram-stained smear, should be subcultured to a blood agar plate to check for purity and for “typical” morphology. It should also be remembered that colonies of the mitis biotype of C. diphtheriae, which is the most prevalent, show a marked zone of β-haemolysis on blood agar.

A presumptive report on the presence of C. diphtheriae can often be given at this stage. However, this should be confirmed or ruled out by some simple biochemical tests and by demonstration of the toxigenicity. As the latter requires inoculation of guinea pigs and has to be performed in a central laboratory, only rapid biochemical identification will be covered here. C. diphtheriae is catalase- and nitrate-positive. Urea is not hydrolysed. Acid without gas is produced from glucose and maltose, generally not from saccharose. The fermentation of glucose can be tested on Kligler medium. Urease activity can be demonstrated on MIU and nitrate reduction in nitrate broth in the same way as for Enterobacteriaceae. For the fermentation of maltose and saccharose, Andrade’s peptone water can be used as a base with a 1% final concentration of each carbohydrate. Results can usually be read after 24 hours, although it may be necessary to reincubate for one night. It must be emphasized that the microbiology laboratory’s role is to confirm the clinical diagnosis of diphtheria. Therapy should not be withheld pending receipt of laboratory reports. More detailed information on the isolation and identification of C. diphtheriae is found in Guidelines for the laboratory diagnosis of diphtheria.1

 

1 Unpublished WHO document LAB/81.7; available on request from Health Laboratory Technology and Blood Safety, World Health Organization, 1211 Geneva 27, Switzerland.
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