Blood culture - GLOBE Network

Blood Culture 

Primary pathogens: Salmonella, Brucella, Other Gram – rods, Staphylococcus aureus, 

Streptococcus pneumoniae, Haemophilus influenzae 

NOT pathogens unless recovered from >1 separate blood culture: Bacillus sp., coagulase – staph, 

viridans streptococci, coryneform Gram + rods 

#2 

#3 

Subculture at 18 h 

Make Gram stain & 

subculture when 

bottle shows growth 

#1 

Collect 10 ml per bottle 

from 2 separate skin sites 

Incubate at 35°C in air up to 

5 days 

Check & invert bottles daily 

Gram + cocci → BAP 

+ Optochin disk 

Example: Pneumococci 

#6 

Identify & perform 

Susceptibilities on 

significant pathogens 

#5 

Incubate up 

to 72 hours 

in CO 2 

#4 

Call 

significant 

Gram stain 

results to 

Doctor 

ASAP 

Gram – rods → BAP + Mac 

Gram – diplococci or 

coccobacilli → Choc 

& BAP 

Example: Klebsiella 

pneumoniae 

© Ellen Jo Baron 2007; Use with proper attribution

# 1 

Doctor collects 

CSF via sterile 

skin puncture 

(lumbar 

puncture) into 

sterile tubes. 

Tube #2 or #3 is 

for Microbiology 

Cerebrospinal Fluid (CSF) Culture 

Primary pathogens: Grp. B Beta streptococcus (newborns), 

Haemophilus influenzae (2 months – 2 years old), 

Streptococcus pneumoniae, Neisseria meningitidis 

1 mL minimum; 

>5 mL best 

# 7 

Place round drop of 

sediment on slide. 

Do NOT spread out 

drop. Let dry & Gram 

stain. If many PMNs, 

infection more likely. 

# 2 

Centrifuge 15 

min at 1500 x g 

(if possible) 

# 3 

Remove supernatant 

with pipette down to 

1 cc CSF sediment 

# 6 

Incubate 

up to 72 

hours in 

CO 2 

# 5 

Place 1 drop sediment 

onto chocolate & 

blood agar plates 

© Ellen Jo Baron 2007; Use with proper attribution 

Note: Do NOT pour off 

# 4 

Pipette up & 

down 10 X to 

mix sediment

Stool (Feces) Culture 

Important pathogens: Salmonella, Shigella, Campylobacter, Vibrio cholera, Vibrio 

parahemolyticus, Toxigenic E. coli & Clostridium difficile (may not be detected in basic labs) 

#1 

Culture feces within 30 

minutes of collection or 

place into Cary-Blair 

transport medium 

#6 

#2 

Choose areas with 

blood or pus to plate 

Plate onto Campy agar – or use filter 

paper method. Incubate in reduced 

oxygen at 37-42° C up to 72 h 

#3 

Plate onto BAP, MacConkey, 

Hektoen or DCA, & TCBS if Vibrio. 

Incubate at 37° C in air up to 48 h 

#4 

Lactose neg GNRs on Mac, 

HEK, or DCA need workup. 

Campy agar 

Gram stain shows curved 

or spiral shaped rods 

Campylobacter sp. 

Oxidase +, moist, 

beige colonies 

#5 

Oxidase + GNRs on BAP need 

additional workup. 


Plate to TCBS & grow in broth 

with salt concentrations 0-6%.

Genital Culture 

Primary pathogens: Neisseria gonorrhoeae, Trichomonas, Yeast & Bacterial Vaginosis 

syndrome in females, Group B Beta streptococcal carriage in pregnant females 

A. Cervical, anal swab from female 

B. Urethral swab from male 

# 1 

BAP & Thayer-Martin 

# 1 Thayer-Martin & Gram stain 

Yeast on BAP 

C. Vaginal & anal swab from 

female 35-37 wks pregnant 

# 2 

Incubate up to 48 hours in CO 2 

GC on T-M 

Incubate up to 72 hours in CO 2 

Look for N. gonorrhoeae on T-M 

Look for yeast on BAP 

# 1 

# 2 

Incubate overnight in 

enrichment broth 

Subculture to BAP to look for 

group B strep 

D. Vaginal swab from female 

# 1 

BAP for yeast 

T-M for GC 

# 1 

Gram stain for BV 

NOT PATHOGENS 

Staphylococcus aureus 

Any Gram negative rods, 

including E. coli, Proteus 

Beta streptococcus group B (GBS) 

Report for pregnant women only ! 


# 2 

Swab in 0.5 ml saline to examine 

for Trichomonas within 30 min 

Roll swab on slide to 

make smear 1 cell 

layer thick; look for 

“clue cells”

Pus, Aspirate, or Tissue Specimen 

Primary pathogens: Staphylococcus aureus, Streptococcus pyogenes, other beta 

streptococci, Pseudomonas aeruginosa, other Gram negative rods 

NOT pathogens: Coagulase neg staphylococci, small numbers of coryneform Gram + rods 

#1 

•Cleanse skin around wound 

with alcohol 

•Aspirate or tissue is best 

•Swab worst 

#2 

•Inoculate BAP & Mac 

•If aspirate or tissue, make 

Gram stain & call Doctor 

with significant results 

#3 

Incubate up 

to 48 hours 

in CO 2 

#5 

When 3 or more pathogens, report as “mixed 

flora” and list Pseudo, S. aureus, Proteus, 

Enteric GNRs descriptively if present 

Klebsiella 

pneumoniae 

#4 

Identify & perform 

susceptibilities on 1 or 2 

significant pathogens 

Beta streptococci 

Staph aureus 

predominant 

Mixed enteric Gram neg rods - 

2 types: report both; full workup 

Pseudomonas 

aeruginosa 


Sputum Specimen 

Primary pathogens: Streptococcus pneumoniae, Klebsiella species, Haemophilus 

influenzae, Staphylococcus aureus, Pseudomonas aeruginosa 

NOT pathogens: Yeast, viridans streptococci, coagulase negative staphylococci 

#1 

Patient should rinse mouth 

with water, cough from 

deep in lung, do not spit 

Strep. pneumo 

#2 

• Make Gram stain to screen if 

acceptable for culture 

• Call significant Gram stain 

results to Doctor ASAP 

Reject: Many 

squamous 

epithelial cells 

Good: Rare squamous 

epithelial cells 

Optochin disk 

Staph aureus 

Optochin + 

Klebsiella pneumoniae 

Bile solubility + 

#5 

Identify & perform susceptibilities 

on significant pathogens 

#3 

Plate to Choc, 

BAP, Mac 

#4 

Incubate up 

to 48 hours 

in CO 2 

Haemophilus influenzae 


# 1 

# 9 

Throat Culture 

Primary pathogen: Grp. A Beta streptococcus 

Optional report: Arcanobacterium haemolyticum 

Swab # 2 

Swab pharynx and 

tonsils. Avoid tongue 

and cheeks. 

Rub swab on 1/5 area of 

blood agar plate (sheep 

or horse) 

# 8 

Report results: 

# 3 

Loop 

Streak in 3 or 4 

quadrants 

1+ to 4+ Group A Beta strep 

or 

1+ to 4+ Beta strep, not group A 

or 

No beta strep detected 

# 4 

Place Co-trimoxazole (SxT) 

disk next to Bacitracin disk in 

first quadrant 

# 5 

Bacitracin 

& SxT disks 

Incubate candle jar at 37° C 

overnight and examine 

plates; if negative, incubate 

another night. 

Total = 48 h incubation 

Bacitracin 

SxT 

DO NOT REPORT 

Non-pathogens in pharyngitis: 

• Staphylococcus aureus 

• Streptococcus pneumoniae 

• Haemophilus influenzae 

• Moraxella catarrhalis 

• Alpha hemolytic strep 

• ANY gram negative rod 

• ANY yeast 

# 7 

Confirm Group A 

Strep by positive PYR 

# 6 

Beta hemolytic colonies, > 0.5 

mm, resistant to SxT & sensitive 

to Bacitracin = Group A Strep 

(catalase negative) 


Urine Culture 

Primary pathogens: E. coli, Proteus, Klebsiella, other Gram negative rods, 

Enterococcus, Staphylococcus saprophyticus, Staph aureus (if pure) 

a 

b 

a 

b 

#2 Plate onto BAP and 

MacConkey 

0.001 ml loop 

#1 

Collect midstream urine 

#3 

Incubate up to 24 h 

in air incubator 

GPC 

#6 

Criteria for evaluating urine cultures 

Patient Specimen type Significant pathogen CFU/ml 

Asymptomatic Voided, clean-catch 100,000 

or indwelling cath 

Symptomatic 

ambulatory 

Voided, clean-catch 10,000 1-2 species of potential 

pathogen. If > 2 species, urine 

considered to be "contaminated" 

Symptomatic, 

sexually active 

female 

Note: •Gram negative rods 

grow well on both 

BAP & Mac 

•Gram + cocci grow 

well on BAP only 

Voided, clean-catch 

100 pure culture 

Enterobacteriaceae 

Males Voided, clean-catch 1,000 potential pathogen 

All 

Obtained by straight 

catheterization 

100 any number of species of 

potential pathogens 

All 

Surgery or bladder 

aspirate 

Any numbers (broth enrichment, 

anaerobes) 

All Controversial Any isolates of yeast 

#5 

GNR 

If ≤ 2 pathogens, 

perform identification & 

susceptibility on each 

If >2 pathogens, report 

“mixed flora” 

50,000 cfu/ml 

75,000 cfu/ml 

100,000 cfu/ml 

#4 

Count colonies 


Fungus Culture 

Important pathogens: Cryptococcous neoformans, Blastocystis hominis, Dermatophytes, and 

other fungi depending on source of specimen and status of patient 

#1 

Important fungi: Tissue 

biopsy, sputum, CSF, 

Blood cultures 

Dermatophytes: skin 

scraping, nail clipping, 

hair pulled from root 

Do not grind up tissue: 

use scalpels to mince it 

into small pieces 

#2 

Swabs OK ONLY for 

isolation of yeast – plate 

onto BAP and Sab Dex 

#3 

Routine: Plate onto Sabouraud 

Dextrose and Mycobiotic. Incubate 

at 30° C in air up to 4 weeks 

For dermatophytes: Plate onto 

Mycobiotic and DTM (derm test 

medium) if available. 

If tissue, skin scraping, or nail, push 

one small piece of intact sample into 

agar surface halfway. 

#4 

Tape plates with porous 

tape or tape on two sides 

#6 

Examine plates daily for 

7 days, then 2x /week 

Smooth colonies = yeast 

Fuzzy growth = mold 

#5 

Place material in drop of 

10% KOH, add coverslip, 

and examine for fungal 

elements.

Blood culture - GLOBE Network

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