_____________________________________________________________________________________

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 alpha-hemolytic
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.
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.

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 Result: any zone of inhibition--- Strep. pyogenes

F. Bile-esculin hyfrolysis 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 Staphylococcus  SIM- stab ¾
(CNS); (to identify Staph. epidermidis  LIM- stab halfway
in blood and CSF) 3. Aeromonas
 Urease- streak  Mannitol
 Mannitol (-)  Salicin
 Maltose  Arginine
 Sucrose  Lysine
 Trehalose (-)  Ornithine
 Ornithine  Arabinose
2. Enterococcus
 Arginine 4. OX: Taxo-N
 Arabinose [Efa (-), Efm (+)] **get colony from TSI or Mac, touch on oxidase
 Sorbitol strip or Taxo N disc. Observe change in color.
 Mannitol Negative- nochange
Positive= purple

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

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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); positive—yellow

6. Amino acid: emulsify organism to* and incubate.

*example—arginine, ornithine, lysine, salicin
Result: positive---( no change; purple)
Negative---(yellow)

7. BEA slant: streak; incubate.

Negative: no change ( khaki color)
Positive: blackening Differentiate entero from non-entero

8. 6.5 % NaCl tube broth: translucent colonies, mucoid

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

GUIDE IN IDENTIFICATION OF ORGANISMS

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. (-)

3
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 (+) decarb (-)
Organism: K. pneumoniae K. rhinoscleromatis
K. oxytoca
K. ozanae
Test Indole: K. oxytoca (+) Test urease: K. pneumo (+)
K. pneumoniae (-) K. ozanae (-)
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.

4
 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
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 Susceptibility plates Purity plates Incubation

Enterobacteriacae MH plain Mac 37 C
NFO MH plain Mac 37 C
Salmonella MH plain Mac 37 C
Vibrio MH plain TCBS 37 C

Susceptibility testing
Direct Method--for fastidious organismsStaph, Strep pneumo, Enterococcus, other Strep, Hemophilus
Procedure:
1. Growth from primary plate—inoculate itno NSS.
2. Compare turbidity with 0.5% Mac Farland standard
3. Streak to desired susceptibility plates

5
 4. Place desired sensitivity disks (refer to antibiotic chart)
5. Incubate in an inverted position (18-24 hrs)
6. Measure zone oh inhibition—18, IR

Organism Susceptibility plates Purity plate Incubation

Staph MH (2% NaCl) BAP 37 C
Strep pneumo
Other Strep BAP or MHA 5% blood BAP CO2
Moraxella
Enterococcus MH plain BAP 37 C
Hemophilus HTM or CA CA CO2

Enterococcus Non-enterococcus Aerococcus

Bile esculin + + v
6.5% NaCl G(+) cp,cch - G+ strongly tetrads
Bacitracin disk test s s r/s (v)
Arginine + + -
10C or 45C + + -
Hippurate HDH v v +

Enterococcus
Strep. avium Strep. durans E. faecalis E. gallinarum E .faecium
10C or 45C - +
Pyruvate + -
Arginine - + + + +
Arabinose + - - + +
Sorbitol + - + -/+ -/+
Hippurate - V
Aminoglycoside S S
s
Mannitol - -
BE + + +
6.5% NaCl + + +
Motility - + -

Non-enterococcus
Lactose Hemolysis
Strep equinis - Alpha, Gamma
Strep bovis + Alpha

X.V.

V. 1.5 X.
/
5
/
XV

6
 Positive: fine colonies
V.---Haemophilus parainfluenzae ( mousy, chlorox odor)
x.---Haemophilus aphrophilus
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 sulfide—blackening; 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: KOVAC’s 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

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 Catalase (+)
Coagulase (-)

Staphylococcus

Urine CSF, Blood

S. saprophyticus S. epidermidis

NOVOBIOCIN CNS SUGAR

**thiomartin

**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

8
 Non-lactose fermenting organism
w/ or w/o Hydrogen sulfide

Proteus species
Salmonella sp
Arizona/Citrobacter
E. tarda/E. coli

Urease+ L-deaminase
+ -

Proteus sp. Salmonella

Arizona
Indole Citrobacter
+ - E. tarda
P. vulgaris P. mirabilis E. coli
Salmonella typing
- +
Arizona Salmonella
Citrobacter
E. tarda
E. coli

Citrate
+ -
Arizona E. tarda
Citrobacter E. coli

L-decarboxylase ONPG & Mannitol

+ - + -
Arizona Citrobacter freundii E. coli E. tarda

9
 Schematic for Lactose fermenters (pink color)

Hydrogen sulfide
+ -
Arizona E. coli
Citobacter Klebsiella
Enterobacter
L-decarboxylase
Citrate
+ - + -
Arizona Citrobacter Klebsiella E. coli
Enterobacter, Hafnia

Motility
+ -
Enterobacter sp. Klebsiella sp.
Hafnia alvei (related germs)
Decarboxylation
Indole + -
+ - Kleb pneumo K. rhinoscleromatis
L-decarboxylase (-) E. aerogenes K. oxytoca
Pantoea agglomerans E. cloacae K. ozanae
E. sakazakii Hafnia alvei Vogues Proskauer
- +
Arginine L- decarboxylase K. ozanae K. pneumoniae
Ornithine + - K. oxytoca
+ - E. aerogenes E. cloacae
E. sakazakii P. agglomerans E. gergoviae Indole
Hafnia alvae + -
K. oxytoca K. pneumoniae
Sorbitol
+ -
E. aerogenes E. gergoviae
Hafnia alvae

Urease
Ornithine
+ -
E. gergoviae Hafnia alvae

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________________________________________________________
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

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 8. Chloramphenicol*
9. Refampicin
*not included in urine

STY Strep. pneumo culture in BAPcandle 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 important—enterobacter

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/Eye discharge--- ED Ear)
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
 Endocervical discharge (CVD)
 Urethral discharge (UD)  Folley catheter (FC)

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Specimen in order: Processing
1. Urine Specimen Media/broth Requirements
2. Body fluids Urine Mac, BAP 37 C
3. Exudates Exudates Mac, BAP, Thiomartin
4. Respiratory Blood Bactec vial, subculture: Mac, BAP, Bacte 9050, 37 C,
5. Blood CAP Candle jar
6. CVD BF Mac, TSB, BAP, CAP 37 C, candle jar
7. Stool Respiratory Mac, Thio (ETA colony), BAP, CAP
Stool Mac, SSA, TCBS, Alk. Peptone 37 C
water, Selenite F
CVD/UD CAP, Modified Thio-Martin Candle jar

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 violet1 min
2. Wash off with running water
3. Flood of Grams iodine1 min
4. Wash with running water
5. Decolorize--- acetone alcohol
6. Wash with running water
7. Counterstain---Safranin10-20 secs.
8. Wash with running water
9. Air dry

AFB
1. Flood with Carbol fuchsin
2. Heat till steam. Do not boil/dry5mins
3. Wash with running water
4. Acid alcohol
5. Wash with running water
6. Flood with methylene blue or malachite green10 sec
7. Wash with running water
8. Air dry

13
Smearing:
1. Don’t 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. Washing—gentle stream of running water
2. Tilt slide after washing to drain excess water
3. Decolorize completely before counterstain

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