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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