Although the casual organisms may be recovered from the bloodstream throughout the illness, they are most commonly found during the first 7-10 days of the clinical illness and during relapses; 5-10 ml of blood obtained by venepucture should be added to a blood culture bottle containing an appropriate broth.
Alternatively, a clot culture may be performed; here 5-10ml of venous blood is placed in a sterile universal container and the blood allowed clotting. The serum is then removed to perform a Widal test, the clotted blood digested with streptokinase or minced up with scissors and added to a bottle of bile-salt broth which is incubated and examined as in the blood culture techniques; the advantages of clot-culture over blood culture are that it may yield a higher percentage of positive result, it does not require a stock of special blood culture bottles to be maintained or to be taken to the patient and it allows a base-line titer of antibodies to be determined against which the results of further Widal tests can be judged.
Specimens of faeces and urine should be submitted for examination in an Endeavour to isolate the casual organism but it must be remembered that the isolation of a salmonella strain from either of these specimens does not necessarily mean that the patient is suffering from a clinical infection – he may be a carrier.
In the clinical case of a typhoid fever, stool cultures are usually positive from the 2nd week and urine cultures from the 3rd of infection. In paratyphoid-B infections the clinical course in much shorter (7-10 days) than in typhoid; diarrhea is usually early and stool cultures are often positive in the first week of illness.
After centrifugation, faeces or Urine are plated on DCA medium and are:-
Also inoculated into fluid enrichment media like tetrathionate or selenite broth; both culture is subcultured to a fresh plate of DCA medium after incubation at 370C for 18-24 hours.
Pale, non-lactose-fermenting colonies on the original DCA plate or on that inoculated from the enrichment broth are then tested for:-
(a) urease production which is negative for salmonellae.
(b) Motility which is positive.
(c) For ability to utilize certain sugar substrate with gas production. Except salmonella typhi, all genus produce gas.
Colonies that gives characteristic reactions are then identified by determining serologically their group (O) and type (H) antigens. Salmonella typhi and Salmonella paratyphi C possess an additional surface antigen, the Vi antigen, which may obscure agglutination with somatic antisera; identification of such organisms can be made with Vi antiserum or by removing the Vi antigen by boiling a suspension of the organisms before testing with O antisera.
Similarly, when attempting to determine the serotype within the O group to which the isolate belongs, the bacteriologist may be frustrated if the flagellar antigens are in a non-specific phase when they will react with several H antisera; on such occasions he should attempt to harvest the minority population which have type-specific flagellar antigens by the technique known as the Craigie tube method. This entails growing the organisms in a medium containing non-specific flagellar antiserum which will immobilize organisms in the non-specific phase and only those which possess specific flagelar antigens will be free to move away from their non-specific partners and be collected for subcultivation and re-testing against the relevant type specific antisera.
Tests for specific antibodies (O and H) in the patient’s serum can be performed but the interpretations of the results of such Widal tests are valid only if note is taken of certain findings which may otherwise cause false positive results. The level of enteric antibodies in the healthy population must be known and may be variable; again, previous inoculation with TAB vaccine can give relatively high titres of specific antibodies as can previous clinical or latent infection, although only the H antibodies tend to persists at detectable levels. Not frequently false-positive results in the Widal tests stem from the presence of non-specific antibodies such as fimbrial antibody. The usefulness of the Widal test is greatest when in testing a second specimen of the patient’s serum 4-7 days after a first a four-fold or greater rise in O and H antibody titres is detected.
In the search of typhoid carriers, either as part of the routine examination of food handlers or water –works employees or after an outbreak of typhoid fever, a useful screening test is to examine blood-serum specimens for specific antibodies; in particular, if the Vi antibody is present in a titre of 10 or higher, the individual may be a carrier and should have several stool examinations plus bile examination by duodenal aspiration, preferably in hospital.