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____________________________________________________________________________ Bench B Guide in plate reading -all plates are read after 24 hours incubation: (MAC, BAP, CA,

GBA, MTM, SSA, TCBS) -growth in MAC, SSA, TCBS: do Biochemical test -growth in BAP, CA, GBA (non-enterobacteriaceae): do GS BAP: white, yellow, alpha-hemolytic, beta-hemolytic colonies CAP: translucent colonies If no growth after 24 hours: 1. Urine specimen- re-incubate for another 24 hours. Total incubation- 48 hours. 2. Respiratory- re-incubate for another 24 hours. Total incubation 48 hours. Examine fluid thioglycollate, if cloudy subculture broth to new desired plates. 3. Exudates- re-incubate for another 24 hours. Total incubation 48 hours. Examine fluid thioglycollate broth, if cloudy sub culture broth to new desired plates. 4. Body fluids- re-incubate 24 hours. Total incubation 72 hours. Examine tryptic soy broth (TSB). If cloudy sub culture broth to new desired plates. 5. Blood: discard plates 6. Stool: Discard. Subculture broths to respectie selective medium. 7. CVD: chocolate agar plate: re-incubate 24 hours. T.i.= 48 hours MTM: re-incubate 24 hours. T.i.= 72 hours BENCH B WORK UP GS: place one drop distilled water on a slide. Get colony from primary plate (BAP or CA). smear. Air dry, heat fix then stain. Results: g(+) cocci= catalase test: (+)coagulase If coagulase (-) do taxo P, Taxo A, BEA, 6.5 % NaCl with proper cosideration to morphological and colonial chars. ---fungus: germ tube--- 10 drops serum & colony from priamary plate incubate 4 hours. Mount on a slide and screen for C. albicans or non-albicans. ---gram (-) rods: do X, V factor Procedure: A. Catalase: slide= 1 drop Hydrogen peroxide & touch suspected colony from primary plate---(+) bubbles. B. Coagulase: 10 drops plasma & colony from primary plate. Incubate 4 hours.---(-) no clot. (+) coagulxn contol: 10 drops plasma & colony from ATCC staph aureus stock. Incubate 24 hours. C. X. V. factor. Incubate 24 hours growth from primary plate (CA) to NSS, compare with 0.5 % Mac Farland standard. Streak on HTm or CA plate, put X, V, X+v factor disks. Incubate 18-24 hours + carbon dioxide. Observe growth. D. Optochin disc sensitivity test ( Taxo-P): differentiates Strep. Pneumoniae from alphahemolytic streptococci. Procedure: inoculate overlapping strokes one alpha hemolytic colony on BAP, place taxo P disc at the center of the inoculated portion. Incubate @ 37 C under carbon dioxide for 18-24 hours. Result: 14-16 mm zone of inhibition= Strep. Pneumoniae No zone of inhibition or <14 mm= Strep. Viridans E. Bacitracin disc--- sensitivity test (taxo A): differentiate B- hemolytic group A Strep. (Strep. pyogenes) from other b-hemolytic streptococci. 1

Procedure: inocukate overlapping strokes on B-hemolytic colony on BAP, place taxo A disc @ the center of the inoculated portion. Incubate at 37 C under carbon dioxide for 18-24 hours. Result: any zone of inhibition--- Strep. pyogenes F. Bile-esculin hydrolysis test (BEA): for Group D Strep. Procedure: streak on slant of BEA one suspected colony. Incubate @ 37 C. Result: Blackening of agar---positive No change (khakicolor)---negative G. 6.5 % NaCl (salt tolerance test) for group D Streptococci (enterococcus) Procedure: inoculate suspected colony to 6.5 % NaCl broth, incubate @ 37 C for 24 hours. Result: Growth or turbidity of medium--- positive No growth or clear--- negative H. CAMP ( Christie, Atkins, Munch, Peterson): for group B strep (Streptococcus agalactiae) Procedure: make an initial streaking of Staph aureus down the center of a BAP. Then steeak the organism perpindicular to the Staph aureus about 1 cm apart. Incubate 37 C for 24 hours. Result: positive---arrowhead pattern of hemolysis adjacent to the Staphylococcal streak. negative---no arrowhead I. DNAse test: confirmatory test for identification of staph aureus (If coagulation test is not available) and workup test for Moraxella. ***gram neg. diplococci Procedure: streak test organism and positive control (SAU) on a DNAse plate in a parallel position. Incubate room temperature for 18-24 hours. Cover surface of plate with 1N HCl. Observe cleaning arround colonies---positive: clear; ---negative: no clear

J. Germ tube MANNER OF STREAKING***for stool specimen*** TSI (red)= stab/streak; LIM (violet)= stab halfway ____________________________________________________________________________ BENCH C 1. Coagulase Negative SIM- stab Staphylococcus (CNS); (to identify LIM- stab halfway Staph. epidermidis in blood and 3. Aeromonas CSF) Mannitol Urease- streak Salicin Mannitol (-) Arginine Maltose Lysine Sucrose Ornithine Trehalose (-) Arabinose Ornithine 2. Enterococcus 4. OX: Taxo-N Arginine **get colony from TSI or Mac, touch on oxidase strip or Taxo N disc. Observe Arabinose [Efa (-), Efm (+)] change in color. Sorbitol Negative- nochange Mannitol Positive= purple 2

OF tube (green tube)---get colony from TSI or Mac; stab 3x on OF tube; incubate 24 hours. Observe. No change---negative; yellow---positive.---OX/OF= Flavobacterium, Acinetobacter, and Pseudomonas Novobiocin disk (to identify Staph. saprophyticus in urine). CVD if SAU, do STY Sputum TSI: +sheen in Muiller-Hinton agar positive for Pseudomonas if agar turns green LIA: +sheen OF (+)/OX (-)Acinetobacter baumanni *workups: STYPAE or NFO PAE (not capable of fermenting TSI, usually when sheen is seen Pseudo is reported); STYENTERO; DIRECT STY---for fastidious organisms like Moraxilla, Staphylococcus and Streptococcus. Flavobacterium do OX/OF to differentiate the two organisms Acinetobacter Pseudomonas---positive if LIA have sheen (TSI: K/K) but still do OX/OF for confirmation ***Follow-ups: STY/NFOPAE to MH agar---green is Pseudo because of pyoverdin 5. Sugars: stab once to bottom, incubate mannitol, maltose, sucrose, trehalose Result: negative--no change in color (red); positiveyellow 6. Amino acid: emulsify organism to* and incubate. *examplearginine, ornithine, lysine, salicin Result: positive---( no change; purple) Negative---(yellow) BEA slant: streak; incubate. Negative: no change ( khaki color) Positive: blackening


Differentiate entero from non-entero translucent colonies, mucoid

8. 6.5 % NaCl tube broth: Emulsify then incubate. Negative: clear Positive: turbid

9. OF tube (green tube)---get colony from TSI or Mac; stab 3x on OF tube; incubate 24 hours. Observe. No change---negative; yellow---positive.---OX/OF= Flavobacterium, Acinetobacter, and Pseudomonas. WORKUPS: ***Enterococcus sugars [do if colony is BEA(+), 6.5% NaCl cloudy (Enterococcus faecalis, E. faecium, E. gallinarium) ***Staphylococcus (CNS): all sugars are positive except trehalose and mannitol. ***faecium, galinarum motile ***faecalis Non-motile


A. 1. TSI--- A/A or K/A, gas +, sulfide neg Possible organisms: E. coli, Klebsiella sp., Enterobacter sp. 2. CITRATE--- E. coli (-)/ Enterobacter sp. (+), Klebsiella sp. (+) 3. MOTILITY--- Enterobacter sp. (+), Klebsiella sp. (-) ENTEROBACTER SP. 1. LIA--- if decar. (+) Possible organisms: E. coli Enterobacter spp. Klebsiella spp. 2. Citrate--- E. coli (-) Enterobacter (+) Klebsiella (+) 3. Motility--- Enterobacter spp. (motile) Klebsiella spp. (non-motile) Enterobacter spp LIA--- decarb (+) Possible organisms: Enterobacter aerogenes Enterobacter gergoviae Hafnia alvei Test-sorbitol: E. aerogenes (+) E. gergoviae (-) H. alvei (-) Test urease:/ornithine: E. gergoviae (+) H. alvei (-) LIA--- decarb (-), indole (-) Possible organism: Enterobacter cloacae LIA---decarb (-), indole (+) Possible organism: Pantoea agglomerans Enterobacter sakazakii Test: arginine/ornithine: E. sakazakii (+) Pantoea agglomerans (-) Klebsiella species LIA: if decarb (+) Organism: K. pneumoniae K. oxytoca K. ozanae Test Indole: K. oxytoca (+) K. pneumoniae (-) K. ozanae (-)

decarb (-) K. rhinoscleromatis Test urease: K. pneumo (+) K. ozanae (-)

B. TSI: A/A, K/A, K/K, gas (+), sulfide (+) LIA: deam (+) Organism: Proteus spp. Test indole: Proteus vulgaris (+) Proteus mirabilis (-) C. TSI: A/A, K/A, gas (+), sulfide (-) LIA: deam + Organism: Providencia spp. Morganella morganii Test urease: Prov. stuartii (+) penneri (+) rettgerii (+) alkalifaciens (-) M. morganii (+) Test Citrate: Prov. rettgerii (+): adonitol (+) Stuartii (+): adonitol (-) M. morganii (-): indole (+) P. penneri (-): indole (-) D. TSI: K/K, K/A, gas (+), sulfide (+) LIA: deam (-) Organism: Salmonella spp. Arizona/Citrobacter Edwardsiella tarda/E. coli Test: Salmonella typing: Salmonella spp. (+) polyvalent 5

Arizona/Citrobacter--- refer to chart E. tarda/E. coli--- chart

Kirby Bauer Susceptibility Testing Growth method: for Enterobacteriaciae, NFO, Sne. Vibrio cholerae Procedure: 1. Mac/TSI growth--- inoculate to TSB to exceed turbidity of 0.5% Mac Farland std. 2. Incubate 3-4 hours. 3. Adjust turbidity of inuculum by adding few drops of TSB to NSS until it matches with .5% Mac Farland. 4. Streak to desired susceptibility plates 5. Place STY disks 6. Incubate inverted 37C, 18-24 hours 7. Measure zone of inhibition S, I, R.

Organism Enterobacteriacae NFO Salmonella Vibrio

Susceptibility plates MH plain MH plain MH plain MH plain

Purity plates Mac Mac Mac TCBS

Incubation 37 C 37 C 37 C 37 C

Susceptibility testing Direct Method--for fastidious organismsStaph, Strep pneumo, Enterococcus, other Strep, Hemophilus Procedure: 1. Growth from primary plateinoculate itno NSS. 2. Compare turbidity with 0.5% Mac Farland standard 3. Streak to desired susceptibility plates 4. Place desired sensitivity disks (refer to antibiotic chart) 5. Incubate in an inverted position (18-24 hrs) 6. Measure zone oh inhibition18, IR 6

Organism Staph Strep pneumo Other Strep Moraxella Enterococcus Hemophilus Bile esculin 6.5% NaCl Bacitracin disk test Arginine 10C or 45C Hippurate HDH

Susceptibility plates MH (2% NaCl) BAP or MHA 5% blood MH plain HTM or CA Enterococcus + G(+) cp,cch s + + v Strep. avium + + + S -

Purity plate BAP BAP BAP CA Non-enterococcus + s + + v

Incubation 37 C CO2 37 C CO2 Aerococcus v G+ strongly tetrads r/s (v) + E .faecium

10C or 45C Pyruvate Arginine Arabinose Sorbitol Hippurate Aminoglycosides Mannitol BE 6.5% NaCl Motility

Enterococcus Strep. E. faecalis durans + + + + V S + + + + Non-enterococcus Lactose + X.V. V. 1.5 / 5 / XV X.

E. gallinarum

+ + -/+

+ + -/+

+ Hemolysis Alpha, Gamma Alpha

+ + -

Strep equinis Strep bovis

Positive: fine colonies V.---Haemophilus parainfluenzae ( mousy, chlorox odor) x.---Haemophilus aphrophilus 7

XV.--- Haemophilus influenzae *characterized by a small g(-) rods (coccobacilli) on MH or TSA= 18-24 hours CO2 (candle jar) A/A, indole+, gas+, CITRATE-**E. coli=> STY/ Entero K/K, Sulfide+, gas+, deam+, INDOLE-**Proteus mirabilis**INDOLE+P. vulgaris Commonly seen in the laboratory: G+ rods: Bacillus; Lactose fermenters: E. coli

BIOCHEMICAL REACTIONS TSI---K(red)/A(yellow); Hydrogen sulfideblackening; gas----red tube: stab/streak LIA--- deam (-), decarb (+) yellow----violet: stab/streak/stab SC--- prussian blue; green: streak Urease--- light pink (weak+); dark pink (+); light pink: streak SIM---Motility: turbidity; Sulfide: blackening; Indole: KOVACs or p-dab (+ pink ring); light yellow:stab 3/4 URINE--- g(-) bacilli- Mac and do Bio g(+) cocci- BAP Taxo P- optochin---alpha hemolytic: BAP Taxo A- bacitracin disks---beta hemolytic: BAP Blood, CSF: possible organism is Staphylococcus epidermidis Urine: possible organism is Staphylococcus saprophyticus Catalase (+) Coagulase (-) Staphylococcus Urine S. saprophyticus NOVOBIOCIN **thiomartin CSF, Blood S. epidermidis CNS SUGAR

**Colonies on CAP is gray. Possible organism: Entero **TCBS [Thiosulfate-Citrate-Bile-Sucrose Agar (For the selective isolation and cultivation of vibrios) V. cholerae]. **0.5% MacFarland standard---compare turbidity to a sample before streaking to MH agar **LIM: Lysine Indole Motility

Non-lactose fermenting organism w/ or w/o Hydrogen sulfide Proteus species Salmonella sp Arizona/Citrobacter E. tarda/E. coli Urease+ + Proteus sp. Indole + P. vulgaris P. mirabilis Salmonella Arizona Citrobacter E. tarda E. coli Salmonella typing Arizona Salmonella Citrobacter E. tarda E. coli Citrate (- ) Arizona Citrobacter L-decarboxylase + Arizona tarda Citrobacter freundii + E. tarda E. coli ONPG & Mannitol E. coli E. (+) + L-deaminase -

Schematic for Lactose fermenters (pink color) Hydrogen sulfide + Arizona Citobacter L-decarboxylase + Arizona Citrobacter Citrate + Klebsiella Enterobacter, Hafnia Motility + Enterobacter sp. Klebsiella sp. E. coli E. coli Klebsiella Enterobacter

Hafnia alvei (related germs) Decarboxylation Indole + rhinoscleromatis L-decarboxylase (-) Pantoea agglomerans E. sakazakii Arginine pneumoniae Ornithine + E. sakazakii Indole K. oxytoca pneumoniae Sorbitol + E. aerogenes E. gergoviae Hafnia alvae K. E. aerogenes E. cloacae Hafnia alvei L- decarboxylase + Kleb pneumo K.

K. oxytoca K. ozanae Vogues Proskauer K. ozanae

+ K.

+ E. aerogenes E. cloacae P. agglomerans E. gergoviae Hafnia alvae +

K. oxytoca

Urease Ornithine + E. gergoviae Hafnia alvae

________________________________________________________ ANTIBIOTICS Vibrio cholerae disks

1. Ampicillin 3. Chloramphenicol 2. Cotrimexazole 4. Tetracycline Salmonella/Shigella disks 1. Ampicillin* 5. Cipro/Levofloxacin* 2. Cefroperazone 6. Cotrimoxazole* 3. Cefotoxime 7. Chloramphenicol 4. Ceftriaxone 8. Nalidixic acid* *for fecal isolates only **for extra intestinal sites like blood, CSF Enterococcus disks 1. Ampicillin or penicillin 2. Vancomycin 3. Chloramphenicol 4. Erythromycin 5. Gentamicin High level 6. Rifampicin 7. Streptomycin High level ***Add urine---tetracycline +++Hydrogen sulfide (-), slunt/butt (+), gas (-) Vibrio cholerae ++if LIM (+), it is reported as Not Important Pathogen Isolated or NIPI Other Strep. Disks: Streptococcus viridans 1. Ampicillin or Penicillin* 2. Erythromycin 3. Chloramphenicol 4. Clindamycin** 5. Vancomycin 6. Ceftriaxone 7. Cefepime 8. Cefloxacin* *for beta-hemolytic Streptococci only Streptococcus pneumoniae disks 1. Cotrimexazole 2. Erythromycin* 3. Oxacilin 4. Clindamycin* 5. Ofluxacin/Levo 6. Tetracycline 7. Vancomycin 8. Chloramphenicol* 9. Refampicin *not included in urine STY Strep. pneumo culture in BAPcandle jar ****OF(+)acinetobacter sp OX(+)Flavobacterium **Serratia work-up--- TSI: K/A; Citrate+; Motile **Moraxella catarrhalis workup GS--- gram (-) cocci or diplococci

Oxidase (+) DNAse (+) *S. liquefaciens, S. marcescens, S. rubideae-- DNAse---all (+) Ornithine---Siq (+), Sma (+), S. rub (-) Arabinose---Siq (+), Sma (-), S. rub (-) Sputum important organism--- Klebsiella pneumoniae **not importantenterobacter CHECKPLATE: if NIPI; the same as STY; if Pseudo--- MH turns green; NFO--- yellow ______________________________________________________ SPECIMEN GROUPING A. RESPIRATORY Sputum--- S Throat swab--- TS Endo trachial aspirate (ETA) Nosopharyngeal aspirate---NA B. EXUDATE/TRANSUDATE Wound discharge--- WD Abscess--- A Dialysate--- D Scrapings (Endometrial, Skin, Ear) Ear/Eye discharge--- ED C. URINE D. OTHER BODY FLUIDS Pleural fluid (PF) Perricardial fluid Synovial fluid (SF) Vitreous aspirate Peritoneal fluid (PF) E. CVD/UD Vaginal discharge (VD) Cervico Vaginal discharge (CVD) Endocervical discharge Folley catheter (FC) Urethral discharge (UD)

Specimen in order: 1. Urine 2. Body fluids 3. Exudates 4. Respiratory 5. Blood 6. CVD

Specimen Urine Exudates Blood BF Respiratory Stool CVD/UD

Processing Media/broth Mac, BAP Mac, BAP, Thiomartin Bactec vial, subculture: Mac, BAP, CAP Mac, TSB, BAP, CAP Mac, Thio (ETA colony), BAP, CAP Mac, SSA, TCBS, Alk. Peptone water, Selenite F CAP, Modified Thio-Martin

Requirements 37 C Bacte 9050, 37 C, Candle jar 37 C, candle jar 37 C Candle jar

7. Stool

Streptococcus agalactiae resistance to bacitracin (Taxo A) resistance to SXT Strep. Groups A & B= Resistance to SXT Other than groups A&B= Sensitive to SXT Strep. Pyogenes Taxo A= sensitive SXT resistance= perform as Taxo A Non group A Taxo A= sensitive SXT= sensitive..perform as taxo A Other than beta-hemolytic organism: Taxo A= R SXT= S STAINING PROCEDURES GS 1. Flood with crystal violet1 min 2. Wash off with running water 3. Flood of Grams iodine1 min 4. Wash with running water 5. Decolorize--- acetone alcohol 6. Wash with running water 7. Counterstain---Safranin10-20 secs. 8. Wash with running water 9. Air dry AFB 1. Flood with Carbol fuchsin 2. Heat till steam. Do not boil/dry5mins 3. Wash with running water 4. Acid alcohol 5. Wash with running water 6. Flood with methylene blue or malachite green10 sec 7. Wash with running water 8. Air dry Smearing: 1. Dont mix spx 2. Pick solid particles (purulent, bloodstained, mucoid) 3. Spread selected portion evenly 4. 2x3 cm Drying/fixation: 1. Air dry completely 2. Fix by passing over flame 2-3 times 3. Never pass over flame when still wet

Staining: Decolorize completely before counterstaining **Notes: 1. Washinggentle stream of running water 2. Tilt slide after washing to drain excess water 3. Decolorize completely before counterstain