Bacteriology Specimen Collection

ทนพญ. สุวฒันา กาญจนหฤทัย

งานจุลชีววิทยา

กลุมงานเทคนิคการแพทย

โรงพยาบาลราชวิถี

งานจุลชีววิทยา กลุมงานเทคนิคการแพทย

โรงพยาบาลราชวิถี

ตึกสิรินธร ช้ัน12 โทร.3142-3

ผูรวมงาน

นักเทคนิคการแพทย 8

เจาพนักงานวทิยาศาสตรการแพทย 4

พนักงานหองทดลอง 6

ผูชวยเหลือผูปวย 2

เวลาทํางาน

เวลาราชการ 8.30 - 16.00 น.

วันหยุดราชการ 8.30 - 16.00 น. (เปดทุกวัน)

Microscopic Examination: Gram stain

Acid-fast stain / Modified acid-fast stain

India ink preparation

KOH preparation

Culture : Aerobic(Blood, Urine, Sputum, CSF, Stool,Genital, Pus,Throat)

Helicobacter pylori

Mycobacteria

Anaerobic

Fungus

Antimicrobial Susceptibility Test: Disk diffusion method, MIC (E-Test)

Other: Clostridium difficile Toxin B gene

Microbiology Laboratory Test Services

Gram stain Semiquantitative Designation for Bacteria and Cells

Numerous = > 10 per oil immersion field

Moderate = 5 - 10 per oil immersion field

Few = < 5 per oil immersion field

Acid fast stain (AFS)

•Direct smear

•Concentrate smear

Sputum + NALC-NaOH

Centrifuge at 3,500Xg,20 min

Suspend sediment in 1-2 ml sterile saline

Inoculate in LJ Smear and fix it in hot air oven 65-750C, 2 h

Modified acid - fast stain • Negative :- -No partially acid - fast organism found.

• Positive :- -Partially acid - fast branching filaments,

suggestive Nocardia spp.

-Positive for Cryptosporidium spp.

Cryptosporidium spp. Nocardia spp.

India ink preparation

CSF

• Report : Encapsulate budding yeast,

suspected Cryptococcus spp.

Specimen Workup

Bacterial Colonies on Culture Media

Grading : Numerous = > 200 colonies

Moderate = 50-200 colonies

Few = < 50 colonies

Bacterial Identification

MALDI-T0F Mass Spectrometry

Bacterial Identification

Biochemical Test

C.difficile Toxin B Gene DNA Detection BD MAXTM Automated System

(Real-Time PCR)

Antimicrobial Susceptibility Test (Clinical and

Laboratory Standards Institute ;CLSI)

Disk diffusion method MIC (E-Test)

Report

Collection of Blood culture • Disinfect culture bottle with 70% alc. to rubber

stopper and wait 1 min.

• Palpate for the vein first.

• Clean site with 70% alcohol, then swab concentrically, starting at the center, with 2% tincture iodine or chlorhexidine and allow the iodine to dry.

• Collect blood (adults 10-20 ml/set, infant 1-10 ml/set).

• Inject blood into culture bottle, mix well.

• After venipuncture, remove iodine with alc.

• Transport ≤ 2 h, RT. Store ≤ 24 h, RT.

Acute sepsis : 2 sets from separate site , all ≤ 10 min.

Endocarditis : Acute : 3 sets from 3 separate sites ≤ 1-2 h. Subacute : 3 sets from 3 separate sites, > 1 h apart

. If negative at 24 h, obtain 3 more sets.

Fever of unknown origin : 2-4 sets from separate sites >1 h apart.

If negative at 24-48 h, obtain 2 or 3 more sets.

NOTE :

• Should be collected before antimicrobial agents are administered.

• Should be collected during fever spike.

Collection of blood cultures

Blood (Haemoculture) The successful recovery of microorganism

1. Types of bacteremia: Transient, continuous, or intermittent

2. Specimen collection method

3. Blood volume: Higher volume most productive

• Septic adults have fewer than 30 CFU/ ml of blood.

• Septic children have more than 10 CFU/ml of blood.

4. Number of blood culture ( at least 2 blood cultures )

• Endocarditis: A single blood culture will be positive in 90-95%.

• Bacteremia: The first two culture will be positive in 90-99%.

5. Timing of collection

• Not important as other factors.

Culture Techniques (BACTEC System: fluorescent technique)

Hemoculture bottle

1. PLUS+Aerobic/F

2. PEDS PLUS/F

3. MYCO/F Lytic

• The anticoagulant used in all blood culture

system is sodium polyanethol sulfonate

(SPS).

• SPS is known to be inhibitory to

N.meningitidis, N.gonorrhoeae,

Peptostreptococcus anaerobius,

Streptobacillus moniliformis and

Gardnerella vaginalis.

Intravascular Catheter Tip

• To determine the source of bacteremia.

• The most common technique is the semi- quantitative

method.

• The 5-cm of the catheter is rolled across a sheep blood

agar(SBA) plate 4 times.

• Growth > 15 colonies is considered significant.

• Collect catheter in sterile screw cap container.

• Transport ≤ 15 min, RT., store ≤ 2 h, 4 °C

Collection of Urine Culture • Midstream Urine

• A first voided morning urine is optimal.

• Cleanse the urethral area with soap and water.

• Rinse with gauze pads.

• While holding the labia apart, begin voiding.

• After several ml (20ml) has passed, collect a midstream portion

without stopping the flow of urine.

• Collect urine in sterile, wide mouth container.

• Straight Catheter

• Cleanse the urethral area.

• Aseptically insert a catheter into bladder.

• After allowing 15 ml to pass, collect urine in sterile container.

Indwelling Catheter

Disinfect the catheter with 70% alc.

Use needle and syringe to collect 5-10 ml of urine.

Transfer in sterile leak-proof, container.

Foley catheter: Do not culture since growth represents

distal urethral flora

Volume: > 1 ml

Transport ≤ 2 h, RT. Store ≤ 24 h, 4 °C Once/Day

Collection of Urine Culture

Collection of Sputum • Collect before eating, drinking or smoking.

• For TB culture or AFS,collect early morning sputum on three

consecutive days.

• Rinse/gargle with water to remove flora.

• Cough deeply, collect in sterile wide-mouth container.

• For pediatrics, physician should collect via suction.

• For detection of Mycoplasma pneumoniae, Legionella spp.,and

M. tuberculosis, all specimens are considered acceptable.

The best specimen < 10 squamous cell/LPF

Transport ≤ 2 h, RT, store ≤ 24 hr. 4 °C. Only once/ Days

Lower Respiratory Tract Infection Specimen Collection and Processing

• Bronchoalveolar lavage,Bronchial washing

– Place the specimen into sterile container.

– Colony count is performed on CA and SBA and the specimen is also inoculated on MAC.

– The recovery of organism

>105 bacteria/ml potential pathogen

104-105 bacteria/ml gray zone

< 104 bacteria/ml contamination

– A smear is prepared by centrifugation for gram stain. Transport ≤ 2 h, RT,store ≤ 24 h, 4 °C

Other Body Fluids • Pleural, peritoneal, peritoneal dialysis and synovial fluid

Collection of specimen

• Disinfect skin with 2% tincture iodine.

• Collect specimen by needle aspiration or surgery.

• Inoculate directly into blood culture bottle or sterile container or anaerobic bottle.

• Submit as much fluid as possible (1-5 ml).

• Transport ≤15 min, RT. Store ≤ 24 h, RT. Specimen processing

• Concentrate by centrifugation (1,500xg,15 min).

• Sediment is inoculated to CA, BA, MAC and Thioglycolate broth.

• Gram stained smear should be performed.

Oral Remove oral secretions, and sample the lesion. Tissue and

aspirates are better.

Nasal Insert a swab 2 cm into the nares, rotate against mucosa.

Anterior nare S.aureus carriers Nasopharynx insert a swab into posterior nasopharynx, rotate for

absorption. Direct media inoculation or swab transport.

Detect Bordetella spp. and N. meningitidis carriers.

Throat Sample posterior pharynx, tonsils ,and inflamed areas

For N. gonorrhoeae, should be plated ≤12 h.

Transport ≤ 2 h, RT. Store ≤ 24 h, RT. Only once/day.

Upper Respiratory Tract Collection of specimen

CSF CULTURE

Specimen collection and transport • Disinfect with 2% tincture iodine.

• Insert a needle with stylet at L3-L4, L4-L5, L5-S1 interspace.

• Collect 1-2 ml into leak-proof , sterile screw-cap tube.

• Transport ≤ 15 min, RT. DO NOT refrigerate.

• Store ≤ 2 h, RT.

• Minimum amount - Bact. 1 ml, AFB 5 ml, Fungus ≥2 ml.

1. Stuart transport medium MAC: Salmonella, Shigella,

VIbrio, Aeromonas, Plesiomonas

SS: Salmonella, Shigella

2. GN broth Modified Semi-solid Rappaport-Vassiliadis

(MSRV):Salmonella (except S.typhi and S.paratyphi A)

3. Alkaline peptone water TCBS: Vibrio

Stool culture or Rectal swab

Helicobacter pylori Specimen: 2 pieces of gastric biopsies (antrum and corpus)

Media: 5% HBA( vancomycin , polymyxin B , trimethoprim,and amphotericin B

Culture and Isolation procedure

• Mince and homogenize biopsy in a tissue grinder in 0.5ml saline.

• Inoculate the homogenate on 2 plate of media.

• Incubate under microaerophilic condition at 35-37 °C .

• Colonies appear as small, pinpoint 1-2 mm, translucent.

Identification

• Gram negative curved, straight rod, rare spiral in culture.

• Biochemical test: catalase+ ve, oxidase + ve, and rapid urease+ve.

Transport media : Stuart transport media, Sterile saline

Transport ≤ 2 h, 4 °C and store ≤ 24 h, 4 °C

Lesion Cleanse the lesion with sterile saline, and remove surface

with blade.

Sample exudate from the base of the lesion with a sterile

swab.

Transport ≤ 2 h, RT.

Store ≤ 24 h, RT. Only once / day.

Genital tract (Male or Female)

Collection of specimen

Collection of specimen Prostate Cleanse with soap and water. Massage prostate through rectum.

Collect fluid on a sterile swab / sterile tube.

More relevant results collected urine before and after massage, and

ejaculate may also be culture.

Urethra Insert urethrogenital swab 2-4 cm into urethra, rotate for absorption.

Transport ≤ 2 h, RT. Store ≤ 24 h,RT

Genital tract-Male

Collection of specimen Cervix

Remove mucus / secretions. Then swab endocervical canal.

Urethra

Collect 1 h after urination. Remove exudate.

Collect discharge on a swab by massaging the urethra

against pubic symphysis through vagina.

If no discharge, wash the external urethra with Betadine

soap , rinse water.

Then insert urethrogenital swab 2-4 cm into urethra and

rotate the swab for 2 sec.

Genital tract-Female

Vagina Wipe away old discharge.

Collect discharge from mucosal membrane with a sterile swab.

If a smear is also requested used a 2 nd swab.

For IUD , place entire device into a sterile container.

Transport ≤ 2 h,RT. Store ≤ 24 h,RT. Only Once / day.

Collection of specimen

Ear Collection of specimen Inner • For intact eardrum, clean ear canal (with soap solution), collect fluid via

syringe aspiration and transfer to sterile container or anaerobic bottle.

• For ruptured eardrum, collect fluid on swab.

Outer • Use swab to remove any debris from the ear canal.

• Obtain a sample by rotating the swab in the outer canal.

• Transport ≤2 h, RT and store ≤ 24 h, RT.

Specimen processing

• Gram stained smear of middle ear fluid is recommended.

• Specimen is inoculated to CA, BA and MAC .

• All isolates and their antimicrobial testing should be report.

• External ear specimen may be contaminated with normal flora (CoNS, diptheriods, and Viridans group streptococci)

Eye Types of specimens

• Conjunctival scraping obtained with a swab or sterile spatula.

• Corneal scraping collected with a sterile spatula.

• Vitreous fluid collected with aspiration.

Culture

• Direct inoculation of agar culture plates and preparation of

smears in the clinic is recommended.

• Multiple inoculations with the spatula or fluid are made to BA,

CA, Thioglycolate broth and Sabouraud Dextrose agar.

• Rapid transport the plates and smear to Lab.

• Remove surface exudate with sterile saline / 70% alcohol

• Tissue / fluid is better than swab

• For swab, collect two,one for stain &one for culture

• Collect once / day / source

• Transport ≤ 2 h, RT. Store ≤ 24 h, RT. • OPEN abscess

- aspirate or pass a swab deeply at the base / edge / wall

• CLOSED abscess

- aspirate abscess with needle and syringe.

- transfer into anaerobic transport medium.

Media; CA,BA, MAC,and Thyoglycolate broth.

Abscess and Wound