procedures in Family Practice

Office Bacteriology in Children Louis W. Menachof, MD Santa Rosa, C a lifo rn ia

Office bacteriological procedures can be a valuable part of a physi­ cian’s diagnostic armamentarium when utilized within the framework of sound clinical judgment. This is particularly true in children, where infectious diseases represent the major portion of illnesses. The tech­ niques are simple and inexpensive. The expertise necessary is rapidly and easily acquired. The results can be gratifying and serve as a stimu­ lant for greater involvement in this technique.

Acute infectious diseases represent logic diagnosis can be a more intelli­ ria and gonorrhea are not clinical con­ the major share of childhood illnesses. gent approach to therapy. siderations, a throat culture is taken Although the diagnosis of most infec­ The most common diseases for for only one purpose, to determine the tious diseases is made on clinical which culture specimens are obtained presence of significant numbers of grounds only, a more specific etiologic include tonsillopharyngitis, urinary group A beta hemolytic streptococci diagnosis based on culture results can tract infection, and skin and soft tissue for which antibiotics are indicated. often be made by the judicious use of infections. This paper will discuss tech­ The absence of significant numbers of office culture techniques. While there niques and interpretations of cultures, this organism indicates a probable viral are pitfalls in the techniques and inter­ together with pitfalls which may be etiology without indication for antibi­ pretations of culture data, these can be encountered. Brief clinical comments otic therapy. kept to a minimum. Only a reasonable will be included where relevant to the The patient with streptococcal ton­ effort directed at gaining some exper­ discussion. sillopharyngitis often presents with an tise in this area is required. The end illness characterized by sudden onset, result of being closer to a specific etio- high fever, toxicity, and sore throat, Throat Culture often accompanied by headache and Tonsillopharyngitis, ie, infection in­ abdominal discomfort. Significant rhi- volving tonsils and/or pharynx, can be norrhea, cough, or respiratory symp­ caused by either viral or bacterial toms are so unusual in streptococcal Fi-om the Community Hospital, Santa Rosa, cahfom'a. Dr. Menachof is Assistant Cl ini- agents. Other than Corynebacterium tonsillopharyngitis that their presence » L • SSOr' Ambulatory and Community diphtheriae and Neisseria gonorrhoeae, makes this diagnosis unlikely. If signif­ Medicine and Pediatrics, University of Cali- .T ni.a: ?an Francisc°- Requests for reprints the only bacterium of importance in icant numbers of strep are grown from ° ldJ>e addressed to Dr. Louis W. Mena- this disease is group A beta hemolytic a patient with significant rhinorrhea, R °f; Community Hospital, 3325 Chanate noad, Santa Rosa, Calif 95402. streptococcus. Assuming that diphthe­ cough or respiratory symptoms, con-

TRE JOURNAL OF FAMILY PRACTICE, VOL. 2, NO. 2, 1975 135 sideration should be given to the prob­ (5) Place the “A” disc (containing Technique ability that the strep represents a carri­ bacitracin) in the streaked area adja­ er state, while the illness is actually (1) Swab the tonsillopharyngeal cent to the swabbed area where beta caused by a viral agent. An occasional area directly with a disposable sterile hemolysis is expected to be sufficient exception may be found in cases cotton applicator and inoculate heavi­ to detect the area of inhibition char­ where a streptococcal infection is su­ ly over approximately 20 percent of a acteristic of group A. perimposed on a preceding runny nose five percent sheep’s blood agar plate. (6) Following identification (label or cough unrelated to the streptococ­ (See Figure 1) the plate and not the lid) the plates are cal infection. (2) Heat the streaking loop until then placed (lid down) in the incuba­ Examination of the throat of a pa­ the wire is red, then cool. (It will cool tor at 35 to 37 degrees until the end of tient with streptococcal tonsillophar- quickly if the loop is jabbed into the the working day when they may be yngitis may or may not show exuda­ agar.) transferred to CC>2 for optimum tion but characteristically shows a cell­ (3) Streak the surface of the agar growth. ulitis of the tonsillar and adjacent with the loop in the direction designed structures, particularly the soft palate to progressively thin the inoculum. and uvula. A characteristic petechial or Since there are usually additional or­ Interpretation and Discussion ganisms grown from the throat beside hemorrhagic rash of the soft palate Accurate identification of group A beta hemolytic streptococci, beta he­ and uvula is often seen. The presence beta hemolytic streptococci can be molysis may be suppressed in the com­ of exudation on the tonsils, in the ab­ easily achieved by any physician. A sence of cellulitis of the tonsils and ad­ petition for nutrient in the initial, heavily swabbed area but will show good method of gaining expertise is to jacent structures, is almost always viral make quick rounds in the bacteriology clearly in the thinned area. in origin. Infectious mononucleosis is a laboratory of a hospital each morning (4) Cut the agar at an angle with good example of this. However, even and to look at various cultures on ap­ the loop in the heavily swabbed area. after 15 years of experience, this au­ propriate media. Laboratory personnel Beta hemolysis may show well sur­ thor’s accuracy with clinical diagnosis are anxious to help, and, in a short rounding this cut since streptolysin ac­ remains approximately 75 percent, so period of time, rapid and accurate tivity is greater at the depths of the culture, therefore, remains the best identification by gross appearance can agar plate. guide to diagnosis. be mastered. Beta hemolytic strepto­ cocci are identified by a small-sized colony with distinct, clear hemolysis on sheep’s blood. Beta hemolytic or­ ganisms are identified as belonging to group A by the zone of inhibition to beta hemolysis which the bacitracin- containing A discs impart. Since non­ group A beta hemolytic streptococci are only rarely associated with clinical tonsillopharyngitis, isolation of this or­ ganism is not an indication for antibi­ otic therapy. Beta hemolysis may often be ob­ scured by other organisms in areas of heavy growth and, for this reason, the heavy inoculum must be thinned out with the loop so that beta hemolysis can be recognized. Since many normal throat cultures will demonstrate small numbers of beta streptococci, it is im­ portant to estimate roughly the per­ centage of beta streptococci in com­ parison to the overall number of or­ ganisms grown. In most cases of signif­ icant acute streptococcal illness requir­ ing treatment and of the heavy carrier who may be at risk to others, throat cultures demonstrate large numbers of streptococci.1 In general, al least 25 percent of colonies isolated in the thinned-out areas are beta hemolytic streptococci in streptococcal pharyngi­ tis. An acute febrile illness is unlikely to be caused by the streptococcus if

136 THE JOURNAL OF FAMILY PRACTICE, VOL. 2 , NO. 2, 197S the culture does not contain at least sources4 and is beyond the scope of (5) This calibrated loop should be this number of streptococci. Studies this paper. If the physician believes used only to deliver the desired vol­ using similar criteria for diagnosis and that pyuria is an inaccurate indication ume of urine. treatment demonstrated that only one of infection and that gram stain and (6) A streaking loop (same as used of 53 ill children who had negative methylene blue stain of the urinary for throat cultures) is then used to throat cultures and who, therefore, sediment have intrinsic errors, he then thin the inoculum in order that the in­ were not treated, developed an in­ needs some method of quantifying dividual colonies can be counted. This crease in ASO titer.2 Although these bacterial growth to indicate the pres­ is done by streaking perpendicular to studies have not been performed using ence of urinary tract infection. Al­ the linear line of urine deposited by the streptozyme test, some prelimi­ though there are limitations to the uti­ the calibrated loop. This procedure is nary experiments indicate that the re­ lization of statistical chance as the cri­ repeated on the opposite side. sults will be similar. Selective blood terion for diagnosis, it has been estab­ (7) Biplates can be made inexpen­ agar media, designed to encourage lished5 that less than 10,000 colonies sively by any hospital or commercial identification of beta hemolytic strep per ml represents absence of infection microbiology laboratory and have the by inhibiting growth of other organ­ and greater than 100,000 colonies per advantage that they conserve incuba­ isms, have been recommended but ml represents infection. The intermedi­ tor space. However, individual agar have the disadvantage of failing to ate numbers are subject to individual plates containing these media can be quantify relative numbers of organ­ clinical interpretation, with perhaps an used if biplates are not available. isms. indication for repeat cultures using While I would not withhold antibi­ clean voided, catheterized or supra­ Interpretation and Discussion otic therapy from a seriously ill child pubic urine specimens. If the inoculum of urine is 0.001 with suspected streptococcal tonsillo- V arious methods of quantifying ml, the number of colonies counted on pharyngitis, it has been my experience bacterial growth in urine have been either side multiplied by 1,000 equals that most children with this illness do utilized including pour plates, minia­ the number of bacteria per ml of not require immediate therapy and are ture agar plates (testuria), dip inocu­ urine. These figures are based on the able to wait comfortably overnight for lum methods, and chemical indices theory that each bacterium develops culture results before the initiation of demonstrating color changes. This au­ into one colony. While this may not be therapy. There are several advantages thor has found that direct culture of completely accurate, since some of the to this approach. First, there is evi­ urine onto an agar biplate containing bacteria close together may develop in­ dence that starting antibiotic therapy five percent sheep’s blood and eosin- to a single colony, it is reliable enough early in the course of this disease may methylene blue (EMB) agars, utilizing when considered in combination with suppress natural antibodies. This may a calibrated loop, has several advan­ the clinical picture. Since it is unusual mean that following therapy the child tages. for children without chronic urinary remains susceptible to recurrence of tract disease to have mixed infections, infection with the same type of strep­ Technique the presence of more than one organ­ tococcus.3 Secondly, unnecessary ex­ (1) Voided urine specimens may be ism should alert the physician to the posure to antibiotics can be avoided obtained in any clean container. In probability of contamination. All of since the greater share of tonsillophar- older children, a midstream specimen the usual gram negative organisms will yngitis is caused by viral agents. Third­ may be obtained. Acceptability of grow well on EMB agar and can be ly, young parents, for the most part, voided urines in clean containers re­ readily identified grossly. Enterococ­ no longer expect or demand immedi­ quires that the urine be cultured with­ cus, a relatively common urinary tract ate antibiotic therapy. A few words of in ten minutes of voiding. This is not pathogen, is difficult to identify on explanation as to the advantages of difficult when office personnel are EMB agar because the colony is small awaiting a culture result are more of­ convinced of the necessity of this re­ and frail, but it will grow well and can ten interpreted by parents as being quirement. Urine samples from infants be easily identified on blood agar. In representative of thoughtful and ex­ may be obtained with a collection bag, most instances, interpretation of cul­ pert care. In addition, most physicians as long as the infant is observed closely tures can be done after overnight incu­ feel better about the quality of their enough to detect urination within sev­ bation, particularly when grown in a work when a selective approach to an­ eral minutes. C02 environment. tibiotic therapy is used. (2) A loop calibrated to deliver Bacteriological means of identifica­ 0.001 ml is placed into the urine speci­ tion of urinary tract infection are Urine Culture men and removed. Examine the loop ideally suited to the pediatric age The diagnosis of urinary tract infec­ to be certain that it contains the urine. group. The possibility of urinary tract tion, by definition, refers to the pres­ All loops should be heated and cooled infection is considered most often in ence of significant bacteriuria. The prior to use. children presenting with unexplained problems involved in determining sig­ (3) Immediately deposit the con­ fever or in asymptomatic children with nificant bacteriuria are many, includ­ tents of the loop with a single linear equivocal or abnormal urinary sedi­ ing method of collection, time interval motion on the surface of the agar on ment. In my experience, urine cultures between collection and examination, one side of the biplate. (See Figure 2) done for these purposes lead to a nega­ and numbers of organisms or colonies (4) Reinsert the loop in the urine tive result in over 90 percent. There­ on culture. A complete discussion of and repeat the procedure on the oppo­ fore, the physician need only be able these factors can be gained from other site side of the biplate. to recognize absence of colonies in or-

THE JOURNAL OF FAM ILY PRACTICE, VOL. 2, NO. 2, 1975 137 expensive penicillinase-resistant antibi otic is being considered. Staphylococcal disease in newborns has increased since routine use of hex achlorophene washes in the nursery has been discontinued. Isolation of staphylococci from newborn skin fc. sions has diagnostic and therapeutic value to the physician. In addition, it is important in determining the source of the organism, since antibiotic sensi­ tivity patterns of such staphylococci can often suggest a direct relationship between hospital-acquired staphylo­ cocci and clinical disease. The culture technique is identical with that used for throat culture. From both personal studies and recently published work.6 one can safely assume that an impetigo lesion yielding only staphylococci in pure culture will be resistant in vitro to penicillin in greater than 85 percent of instances, whether it be a communi­ ty or hospital-acquired organism. Iden­ tification of staphylococci grossly on five percent sheep’s blood agar is not difficult. Staph colonies are at least 20 times larger than the tiny strep colo­ nies. Staph colonies are granular in Figure 2. Technique for urine culture. Biplate containing five percent sheep's blood consistency and are easily picked up agar and EMB agar when a loop is streaked across the agai surface, in contrast to the slippery strep colonies which are pushed ahead der to be at least 90 percent correct in in the long-term follow-up of child­ of the loop without being picked up. interpretation. Of the less than ten hood urinary tract infection. It is my When picked up on the loop in this percent positive cultures in children, practice to follow an acute urinary manner, the yellow-orange color of approximately 90 percent will be tract infection in a child for one year most staph colonies will be apparent Escherichia coli. This allows the same with urine cultures done one week fol­ even though they may look white on physician to be nearly 99 percent ac­ lowing diagnosis while on therapy; one the blood agar plate. Staph colonies curate if he can identify E. coli. With week following discontinuation of bubble when hydrogen peroxide is only the minimal effort involved in vis­ therapy; three, monthly cultures; and dropped on them while strep colonies iting the microbiology laboratory, any follow-up cultures every three months do not. When these criteria are used, physician can become quickly profi­ up to one year. A total of eight cul­ confident identification will come cient in recognizing the common path­ tures during one year is equivalent in quickly. ogens causing urinary tract infection in cost to approximately two office vis­ Instant availability of culture me­ children. If any question exists as to its. dia, including EMB agar for identifica­ the identification of an organism or if tion of gram negative bacteria, can en­ sensitivity studies are desirable (very Miscellaneous Cultures courage most physicians to obtain rarely in children), the culture plate is Although the greatest number of more specimens for culture, thus add­ available for instant consultation with office cultures in children will involve ing a new dimension to their diagnos­ laboratory personnel. throat or urine, the physician has avail­ tic armamentarium. Other diseases in Once the diagnosis of urinary tract able to him the expertise and equip­ which culturing has been shown to be infection is made in a child, the choice ment to expand culture techniques to of practical value in selected cases in­ and duration of antimicrobial therapy many other areas, depending on the clude purulent conjunctivitis, otitis is probably not as important as the fol­ clinical situation and information to media following myringotomy and low-up to determine which children re­ be gained. At times, identification of chronic sinusitis. While some of these quire further evaluation because of the organism causing impetigo or ab­ require special media or techniques, continuing or recurrent infection. Of­ scess may be indicated, particularly the expertise is easily obtained by fice culture techniques allow for regu­ when it has failed to respond to initial someone who has the desire to expand lar culture follow-up at minimal cost antibiotic therapy, or when the origi­ his knowledge of office bacteriology. to the patient, since repeated physi­ nal lesion is thought to be staphylo­ An important word of caution is in­ cian examinations are rarely indicated coccal and the initial use of a more quired. Any laboratory test, including

138 THE JOURNAL OF FAM ILY PRACTICE, VOL. 2, NO. 2, 19JS culture results, should never be inter­ fice incubator. All cultures taken are r e d without consideration of the Table 1. Equipment for Office Bacte­ riology Laboratory placed in the office incubator until the clinical situation and must only be in­ end of the working day, when they are terpreted within this limitation. For placed in 0O2 for overnight growth. instance, the growth of a pure culture 1. Incubator $95.00 2. Loop for streaking 3.00 Although enhancement of growth by of staphylococcus from the throat of a 3. Loop, calibrated to deliver C02 allows more reliable interpreta­ child with an acute pharyngitis is al­ 0.001 ml 19.00 4. F ive percent sheep's tion when cultures are read in the blood agar plates most never significant and the patient @ .30 morning after 16 to 18 hours incuba­ should not receive antibiotic therapy 5. Eosin-methylene blue agar plates @ .30 tion, it is not a requirement. If a cul­ on that basis alone. The presence of 6. Alcohol lamp 1.50 7. " A " discs with dispenser ture is too young for adequate inter­ staphylococci along with streptococci (50) 1.75 pretation, leaving it to incubate longer 8. C 0 2 container (optional) 2.08 in an impetigo lesion in rarely signifi­ will allow for further growth and easi­ cant and will usually clear when only er interpretation. the streptococci are treated with peni­ cillin. There are numerous examples Since most bacteria with which we References where adherence only to the culture 1. Catanzaro FJ, et al: The role of the are concerned are aerobic or faculta­ streptococcus in the pathogenesis of rheu­ result will cause erroneous assump­ tively anaerobic, growth is enhanced matic fever. AM J Med 17:749-756, 1964 2. Moffet H L, et al: Group A strepto­ tions and improper treatment. by C 02 content of four to eight per­ coccal infections in a children's home. I. cent. In addition, beta hemolysis is al­ Evaluation of practical bacteriologic meth ods. Pediatrics 33:5-10, 1964 so enhanced at this C02 content be­ 3. Brock L L , et al: Studies on the pre cause streptolysin S is more active in a vention of rheumatic fever: The effect of Incubators the time of initiation of treatment of strep lowered oxygen environment pro­ tococcal infections on the immune response Any incubator is acceptable which of the host. J Clin Invest 32:630-632, 1953 duced by the increased CC>2 content. 4. Stamey TA : The diagnosis of bacte will reliably maintain the temperature While various means of producing this riuria. In Urinary Infections. Baltimore, Wil liams & Wilkins, 1972, ch 1, pp 1-30 of 35 to 37 degrees in which common C 02 content can be utilized, we have 5. Kass EH : Asymptomatic infections of bacteria grow best. These are readily the urinary tract. Trans Assoc Am Physi­ demonstrated that Alka-Seltzer and cians 69:56-64, 1956 available, occupy little space and are water in a plastic container* will pro­ 6. Ross S, et al: Staphylococcal suscep­ tibility to penicillin G. JA M A 229:1075 inexpensive. Table 1 lists the equip­ duce a C02 content of this magnitude 1077, 1974 ment and cost involved in carrying out simply, reliably, and inexpensively. the office bacteriology procedures dis­ This C02 container, holding ten agar *One half foil-wrapped Alka-Seltzer tablet added to ten cc water in a Tupperware con cussed in this paper. plates, comfortably fits into a small of­ tainer (Catalog No. 140)

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THE JOURNAL OF FAM ILY PRACTICE, VOL. 2, NO. 2, 1975 139