an Chapter 17 aeons fount pecinene MICROBIOLOGIC EXAMINATION a error i A iy BACTERIOLOGIC SPECIMENS | BG a] | (alle ta Principles of Collection and Handling a sea laa le A idol cotamisaing yoursel, Teteriale, and surrounding meus flail alalaislSle Sele reas. Treat sil specimens ax potential sources of i3- Regen [el a]2|slsislals e132 la fection, and collet ‘hem im closed serie container. 7 SEE CEE CETTE , Cottect specimens irom areas where suapected organ Re hoor ae ean {ome are most key to be found (e.g. spreading edge of : Teslon see hr’ py 278) and at fre when ther grow hae eet e EEE Ets Is'protable (ergs Blood culture when fever is rliog oF — - ot peak. Erels . Coltet scanty material on two stele cotton exabs. Uae faaner Php ll e Sie for allure, ‘From the other prepare enears By 20" {Ring owab lightly over ie. sae BG Pelle . Coilet apecrons prior 1 uae of antixeptics or antbi= oe BETTE F Giles when povsibie. Sulfonamides administered aystom= {Sally cam be antagonfaed in the specimen by using cul ease afolalel: fobslelatat Inedis containing & mgs para-sminobensoe ald per 100 7a : eaig IRI. The action of peril ie blocked Inthe specimen TT cutare media contin penicinae,Thlogiyealiste alshelobelelale ‘nediutn neutralizes antibioties to some extent, Ser Carbazide antagonizex steeptomycin (but not dinyaro~ Streptoasyein) BEL heel 2, Lal all specimens clearly and completely = F. Send to laboratory promptly after collection ual Routine Procedure When No Specific Technic {a Indicate: zt “A. Routine Smear: Prepare ameara and stain by Grams z eee EEL EPELE ‘ethod. Smear pus like Blood fim. Centrifuge Muids Sarasa ASseptically and amear and culture sediment, Grind Gotlds and tissues ia mortar aeeptically with sterile sand, +I PELL Serpend ground material in broth . Routine Culture: Culture all spocime In thioglycollate medium, Techie” Plate one loop of rE Bp Doth, of touch ewab to one comer of plate and streak from that comer with sterile wire loop. Place one drop214 Bacteriologic Specimens Special Technics May Be Indicated as Follows: SEs anging drop of liquid cuturse oF suspensions of acts for motiity B. Darkfield Microscopy: Objects in focus appear brightly ‘lluminated on dark background, This ia tie best method for spirochetes and some fungi. Special equipment Deeded is a darkfleld condenser (paraboloid or cardioid) {instead of an ordinary condenser. “Tunnel stop" in the oll immersion objectives, and a strong, concentrated source of parallel light. Always examine fresh, un Stained materiel, ¢.g., expreaged serum. Place d-op on ‘lean glass slide, press on cover slip. Place immersion ‘oil between condenser and slide and between cover slp and objective, nacrobic Cuitsres: Colturing bacteria tn the abeence af ‘nygen ts schieved by (1) displacing ai= with nitrogen oF Inyarogen, (2) absorbing oxygen with pyrogalie acid, oF () reducing substances (e.g., thiogycollate medium), Some organiome (¢.g., neisceriae, brucellae) prefer fan atmosphere of 10% CO, in ain. ‘This in easily ob= tsined by burning a candle sm cloned jar. The candie {fove ost when CO, concentration reackew 10%. Blood Agar Media for Hemolysis: ‘Bela hemolysis ‘means complete disealution of red blood cells; it resulta {in clensing of previously turbid blood agar plate oF blood broth. “Alpha” hemolysis means partial elterstion of pigment of red blood cells, resulting in a green zone round colonies. To establish type of hemolysis ‘tis Sten necessary to male blood agar pour plates in addi to sinface euture, Add blood and culture euspension to melted ager cooled to 45°C, (113°P,) and pour into Pete (Onidane Test for Neloseriae; Oxidase, an enzyme pro- duced by nelaseriae (and some other bacteria), canbe ‘ected by dyes. Drop 1% aqueous dimetnyl-paray diamine hydrochloride on suspected colonies on choco fo blood agar. Neisseria colonies will turn pink, max oon, and filly Black, ThEr test {9 uaed rowtlnely to detect colonies of gonacore. Coagulase Test for Pathogenic Staphylococcl: Production of coagulate by staphylococe! indicates pathogenicity; ‘44d Loopful of suspected colony trom blood plato t0 0.8 bil, of cltrated rabbit plasma diluted 1:5 with phystologl= ‘al saline, Place tbe in water bath st 31°C, (08.8°F) land examine st 30-minate intervals for signe of clotting, If's clot forms within two hours, the ongeniam is cong ‘lage positive, "Most such pathogente staphylococet are. eo hemolytic and reduce tellurite (Jet black colon fn tellurite agar). Fibrinolysin Test for B-Hemolytic Streptococci: The fibrindiscolving power of certain B-hemolytie acteriologle Specimens 275 ococe! is guide to their pathogenicity. Dilute 0.2 citrated human plasma (from sndividdala who have ino streptococcal infections recenty) with 0.8 mi ‘Add 0.5 mi. of actively growing broth culture of tobe tented, mix the two immediately, and Mix ‘mwater bath at 30. (98.8°F.). A solid should form in ten minutes. Observe frequently culture technic ~ Use great care with sterile and materials. Apply couralquel. Prepare ‘area in antecubital forea with weal (2 10 99) finc= af iodine. Remove iouine with 10% alcohol.” With "15 ml. of blood. Add 5 ml. of blood to sterile citrate, mixing well. and add remainder of fd to flask containing 80 to 100 mi. infusion brots. citrated blood to laboratory. Inoculate | ml. into ogiycollate medium. Pour two infusion agar plates ‘blood in each, incubating one aerobically, er anaerobically. Keep ail cultures et 37°C “E.), and examine every other day for three ‘making stained ameara and subcultures, "blood culture methods for the following or gsn= Tmeubate 10 ml. pacient's biocd in 10 ml. 4% © sterile sodium cltrate for 20 days at 37°C __(BB.6°F.) in atmosphere of 10% CO, ae sec. (32. F.) in water bath, then inoculate liver ‘agar or tryplcare agar slants, eal Bear C. (08.6°F.), la tularensis ~ Inoculate blood onto cystine blood agar slants and incubate for ten days ‘f blood cultures and amears - Cultures for the proper diagnosis and treatment bacferemias and septicemias, Blood cultures eal be taken in all cares of fever of unkaown origin blood cultures are commonly found fm n= swith tho organiame noted in the following chiefly early in the a216 Bacteriologic Specimens Intective Organisms Which May Be Found in Blood ‘Bacillus anthracis Bacteroides group ‘Klebsiella pneumoniae Malleomyces mallet Hemoplius tnfluencae Inga gramenegtive tii Po. aeruginosa, ete) Netaseria meningitidis} Pastesrella. Diplococeus pneumoniae Sia gow Staphylococe! Streptococci, hemolytic Streptococss, non-hemolae Caltare media mun Disease “nihrax (aepticemia) Septicemtia from pelvie ‘or abdominal foc, Ling abscess (anaerobe) Undue fer Parsmonia, meningitis Glanders (septicemia) Meningitis, preumonia, ot Septicemia, particulariy with ‘inary tract infections, [Meningiis, bacteremia Plague, tularemia Poeumonia, endocarditis, ‘bacteremia, meningitie Bnterie fevers, sepals Bacteremia, sepsis, osteo- ryelitie, endocarditis Septicomia, endocarditis TSubscute bacterial endo= —— omtain subsiances to neutral Jrulfonamides (PABA) or antbioties (eg, pea ‘illinase) which the patient may be receiving for ‘therapy. Whenever possible, blood cultures should be oblained prior to incituting therapy (see above). Poaltive blood smexrs are found often in the follow ing diseases: Leptospirosis Well’s isease), re lapaing fever, filariasis, kala-sear, malaria, ‘Fichinosis, and trypanosomiasis Poritive results from inoculation of blood snto ex following diseases: Leptospirosis, rat-bite fever, relapsing fever, rickettsial diseases (sce p. 295), ‘some viral diseases (e.g., psittacosis; 820 p. 287), Quantitative blood culture ~ Estimation of namber of organiema present per ml. of blood is important when qualitative culture is positive. For technic sce p. 215, 1B Bye Cultures: Collect materia from conjunctiva with small cotton swab or curet, avoiding contact with margina Oflids. Corneal specimens must be obtained by euret ‘Take ewsb or specimen in broth jube to laboratory. Plate on blood agur aerobically and in 10% CO, (candle sar) Har and Mastoid Cultures: Collect material on cotton swab and remove solid material ‘rom external canal for Bacteriologte Specimens 277 ‘study. Smear and culture geal Cultures: (Avoid external nares, ‘nose’ ) Introduce sterile glass ‘ube into nostril a= ‘sis comfortably possible. Pass sterile cotton ‘oh wire through Lube. Rotate sn nasopharynx and fw" Place into broth, Plate on Blood agar and Pass two sterile coton swabs over each sres and over posterior pharyngeal walls Smears and culture on blood agar. Sain smear Btfer's alkaline methylene blue for tentative diag- esis of diphtheria. If suggestive diphtheria-like organ- ‘serum slants ood agar platen Stein another smear with ‘iolet (one minute) for Vincent's ongensems (pale tea snd dark, cigar- shaped fusiform bacilli). Cultures: See p. 254. Fluid: Always collect three specimens in sterile fand send to laboratory promptly (cell count, chem- erology, and bacteriology). If fluid i cloudy, direct sear and stain with Grar's stan. I uid lear, centrifuge ten minutes, smear sediment, fon blood ager. in thioglycollate medium, and colate agtr in 10% CO, (candle jar). For exam= Tor tubercle bacilli, See p, 280, "and Uleera: Collect material on cotton awa. Biopaiex: Divide in half. Place one piece in for pathological sections, the other half 2 aline and take to laboratory,” Grind up with sand in mortar, suspend in broth, and culture fand enaerobleally, always including thio- Inject part of suspension subcutaneously Into pigs (for tubercle bacils) Caltures: Obtain stool specimen from defecation, ‘oF anal evab. Suspend small amount in broth and 4 blood agar plate snd a differential medium for i fermenting bacteria (0.g., $5 egar, desoxy- TRgar, conin-methylene blue agar, oF bismath ages). If typhoid is suspected, first enrich for 18 “peclonite F-medium, then slresk, Colorless cal~ Aifterental media are nonlactone fetmentera, pal Cultures: Stomach washings for tubercle face p. 280), bile drainage, and surgical speci- /afe treated like stool specimens, [Cont'a. on p. 281.)