Bull. Org. mond. Sante) 1959, 21, Bull. Wid Hlth Org. 247-277 Laboratory Diagnosis of Shigella and Salmonella Infections * E. HORMAECHE, M.D.' & C. A. PELUFFO, M.D.2 In recent years a great deal of work has been We are aware that experienced bacteriologists devoted to the study of enteric diseases and, in will prefer some of the methods they are using to consequence, many methods have been developed the ones we recommend. This study is not in- for the isolation of their most common agents, tended for them, however, but rather as a guide to Shigella, Salmonella and some pathogenic Esche- people who are just starting work on enteric diseases richia, while at the same time new tests have been diagnosis. For more detailed information, we devised to differentiate the " groups " of the Entero- recommend the books of Kauffmann (1954) and bacteriaceae family, so that the beginner, especially, Edwards & Ewing (1955), of which we have made will be faced with the difficulty of selecting from ample use. among them those to be used. Investigation of enteric pathogens will sometimes Each author has his preferences according to his be the ultimate purpose, as for the public health own experience, and it is regrettable that up to now officer, who is primarily interested in finding them no serious effort has been made to standardize for the potential danger they represent to the com- techniques, which would save a lot of time and dis- munity. In clinical medicine, however, the role couraging failures for the bacteriologist unex- played by the bacteriologist does not end there. He perienced in this field and also avoid contradictory will have to act as an adviser to the clinician, who results. It is not our intention to review the profuse needs more information in order to arrive at a correct literature on the subject, but rather to limit ourselves diagnosis, follow the course of the case and indicate to describing such methods as have been used by us treatment, and for this purpose different materials for many years in our laboratory in Montevideo, must be examined (blood, urine, pharyngeal exu- and which we can recommend as requiring a dates, middle-ear discharges, etc.); certain other minimum of equipment and as saving time and tests will also be needed, such as agglutination labour. In some cases, though, we have thought it reactions and antibiograms of the isolated cultures. convenient to offer alternative methods which are Phage typing of S. typhi and some other Salmonella known to be reliable and which are in use in many types has proved important from the epidemiological countries. Some tests are also described which are point of view to trace the source of infection in the not generally needed but are of importance in certain course of outbreaks. Such a subject, though, does particular cases. not properly fall within the scope of the present work. Isolation and complete identification of all enteric * This is one of a series of studies on the laboratory pathogens require a lot of manual work, culture diagnosis of various diseases which, it is hoped, will eventually be revised and published in monograph form. An effort is media and agglutinating sera, which make it difficult made to ensure that the diagnostic methods recommended for the average hospital laboratories to cope with in these studies are as internationally representative and the task. should at acceptable as possible by securing the co-operation of a They aim, however, allocating number of experts from different countries. A list of the to their proper sub-groups all Shigella cultures, and reviewers of the study presented here is given in the Annex on at identifying most of the locally isolated Salmonella page 276. To all of these, and to the two authors, the World Health Organization is greatly indebted.-ED. strains. Unidentifiable cultures should be sent to 1 Emeritus Professor of Bacteriology; Late Director, national centres, of which there should be at least Institute of Hygiene, School of Medicine, Montevideo, one in every country, and newly described types Uruguay should be forwarded to one of the three international ' Professor of Bacteriology; Head, Department of Bacteriology, Institute of Hygiene, School of Medicine, centres in Copenhagen, Chamblee, Ga., USA, or Montevideo, Uruguay London, for final verification. 820 247- 248 E. HORMAECHE & C. A. PELUFFO In our opinion all routine laboratories should laboratories and used as standards, but the bacterio- endeavour gradually to prepare their own culture logist nevertheless needs personal experience in media, reagents for biochemical tests and, especially, the preparation of all the tools in his hands, in agglutinating sera. When starting work, type order to obtain adequate knowledge of their proper cultures and sera should be obtained from specialized use. COLLECTION OF SPECIMENS Stools samples have to be mailed to some distant labora- Fresh, recently evacuated stools obtained as tory, was recommended by Lie Kian Joe et al. (1954) early as possible after the onset of symptoms are from Djakarta University (see Varela, 1955) and the best for examination. When mucus and pus are consists in drying specimens on filter paper. The present they should also be collected, as in acute swab used for collection is rubbed gently on a cases they often yield a pure culture of the causative small piece of filter paper, impregnating a surface agent. of about 2 cm x 2 cm; this is allowed to dry at room It is a common observation that antibiotic treat- temperature and then wrapped in sterile paper. For ment of intestinal infections may prevent the growth examination, the impregnated zone is cut, put into of the causative agent on culture media, and there- a tube with 3 ml of broth and macerated by means fore it is advisable to examine stools immediately of a glass rod, and the liquid is collected for examina- after admission of patients and before treatment is tion. (See A, p. 249). started. Unfortunately, such treatment is fre- Urine quently given at home and only when it fails are patients sent to hospitals. Catheterization is needed to obtain aseptic urine When dealing with communities where many from women, but with men, when examination is persons have to be examined, the rectal swab has to be carried out immediately, it is sufficient to reject proved to be especially useful in the search for the first portions of the urine specimen. Urine should carriers. Sterilized swabs are introduced into the be centrifuged at high speed and the sediment used rectum, gently rubbed on the surface of the mucosa, for examination. If the examination is not done at and used for the direct inoculation of media. As once, the alternative is either to keep the urine in the introduction of swabs is sometimes painful, it is the ice-box, or to add a preservative as when dealing often advisable to submerge them in broth or some with stools (particularly when the specimen is being similar sterilized liquid, before use. In the case of sent to another laboratory). infants, whose stools are sometimes not easy to Blood collect, either because they are liquid or for some Blood should be collected for culture as early as other reason, the use of rectal swabs is highly recom- possible after the onset of symptoms and 1 % sodium mended. It is also excellent in cases of ulcerative citrate or some other anticoagulant (e.g., heparin) chronic colitis, as generally the lesion on the added, as liquid blood is easier to handle than mucosa can be reached in the course of recto- coagulated samples. If absolutely necessary, how- sygmoidoscopy. ever, the clot can be used for culture. When examination has to be delayed for more than four hours, it is convenient to add a preservative Other materials to prevent the multiplication of normal intestinal Pharyngeal and vaginal exudates and middle-ear bacteria which has a destructive effect on the discharges are best collected with a swab; bile by pathogens, partly owing to bacterial competition and duodenal catheterization. partly owing to pH changes. The simplest reliable Any solid foods are cut into small pieces with preservative is Sachs' buffered glycerol solution sterile instruments and then ground with sand in a (Sachs, 1939). Bangxang & Eliot's formula (1940) mortar. A few millilitres of saline are added and is also recommended. Stools, in a proportion of mixed thoroughly with the pestle, and the liquid is 1/10 should be well emulsified in the solutions to collected with a pipette. ensure preservation. Swabs can also be submerged As a general rule any specimens not to be examined in the same preservative. An excellent method for at once should be kept in the ice-box, or, better still, preserving stools, which is especially useful when in a deep-freeze, where they can be preserved for years. LABORATORY DIAGNOSIS OF SHIGELLA AND SALMONELLA INFECTIONS 249 CULTURE MEDIA AND BIOCHEMICAL TESTS A. PRESERVATIVE SOLUTIONS To this basic, still-hot medium, add 0.2 ml of bril- liant-green solution (0.5 %), lactose 1.5 g (sterilized 1. Sachs' (1939) buffered solution: water solution) (see D, p. 251). Kauffmann (1954) Glycerol 30 ml has recently recommended the use of broth pre- Sodium chloride 0.42 g pared with human placenta instead of beef extract. Dipotassium phosphate (anhydrous) 0.31 g Monopotassium phosphate (anhydrous) 0.1 g Phenol-red solution: Distilled water 70 ml Phenol red 0.2 g NaOH (N/10) 8 ml Distilled water 92 ml Add phenol-red solution to give a pink colour. Sterilize at 120°C for 15 minutes; pH should be 8; 2. Leifson's (1935) desoxycholate-citrate agar ifthe colour changes the solution should be discarded.