1. INNOCENT AFEKE SCH. OF ALLIED HEALTH SCIENCES UHAS
Collection and Transport of Specimens for Microbiological Examinations (Physician Assistants , Nurses & Public Health Students)
INNOCENT AFEKE
SCH. OF ALLIED HEALTH SCIENCES
UHAS

2. Philosophy:
The result of any laboratory examination is only as good as the sample received in the laboratory
Think about this

3. Upon completion of this topic, the student should be able to:
Emphasize that obtaining sensitive and specific microbiology results begins with the patient and not at the door of the microbiology laboratory
Highlight the importance of proper collection and transport of specimens in both local and referral environments
Stress the importance of timely communication between the Microbiology laboratory and those collecting specimens
Describe common pitfalls in specimen collection and transport
Discuss the 7 rules or principles one must be followed in order to collect microbiology specimens which will accurately reflect the pathogenesis of the microbiological agent

4. Introduction
Specimens submitted for Microbi testing require proper handling from the time of collection through all stages of transport, storage and processing
Nurses and PAs are most often delegated to carry out these processes and hence needed to understand the requirements and consequences of such procedures
There may be prior preparation of patient before collection of specimen
Once the specimen is collected, it should be IMMEDIATELY dispatched to the Lab
-delay in sending the specimen may cause delicate microbes to die from;
Lack of nutrition, lowered or raised temperature, action of enzymes etc.

5. Influences of Sample Management
Good sample management
Influences the
accuracy of laboratory diagnosis
Influences therapeutic decisions
affects patient care and outcome
Influences laboratory efficiency

6. Know the anatomical sites samples are collected for Microbiological investigations

7. Samples that are Collected for Microbiological Testing
BLOOD for:
Culture
-preparation of site: critical
-Multiple specimens: different sites
-Bottle kept at RT or 35-37°C (Do not refrigerate)
Serology
-Serum/ Plasma: choice of bottle
- Store frozen at -70°C

8. Field Work: Blood Collection & transport
Collection of blood from large communities for research
-Dried Blood Spot for serology
Blood film on slide for parasitology
Unless necessary, take blood into sample tubes

9. CEREBROSPINAL FLUID (CSF) for:
Cytology
-first tapped specimen
Biochemistry
-Glucose & Protein
-2nd tapped specimen
Microbiology; culture
-3rd tapped specimen
-about 3ml
NB CSF is emergency specimen and must be sent to lab immediately (No refrigeration except for viral cultures)
Specimen collection is done by specialist

10. URINE for:
Routine examination (RE) for microorganisms e.g.- Trichomonas vaginalis, Schistosoma ova, Candida etc.
-Collected into clean & dry universal bottle
Culture and Sensitivity Testing (CS)
-Patient preparation very critical
-Clean mid-stream catch urine
-Sterile universal bottle 2/3 full
-Immediately refrigerate the specimen and submit to the lab within 24 hours of collection (maintain at 2-8 ºC when transporting)
Immunology(ELISA) & Molecular testing (PCR)
-First early morning voided specimen
SPECIAL CASES:
Paediatric urine collection kits are also available
Suprapubic Aspiration Specimen: needle aspiration through the abdominal wall into the bladder
Catheter Collection Specimen can be used specially when patient cannot produce urine by him/her self

11. Field Work : Urine sample
For immunology test e.g. ELISA and for Molecular test e.g. PCR ; for organisms like
N. gonorrhoea, Chlamydia and viruses that are shared in urine
-Take sample into conning tube and store on ice whilst on the field
-Transport to the lab and store at -20oC till ready to work on them
Urine samples for parasitology e.g. Schistosoma ova
- Preserve with 10% formalin and store at RT
Conning Tubes

12. RE for parasites e.g. amoeba, Tape worm, Hookworm etc.
STOOL for:
RE for parasites e.g. amoeba, Tape worm, Hookworm etc.
-clean wide-mouth crew-capped container
-work on immediately or RT for months in fixative(buffered formalin)
C&S
-Freshly passed stool samples
avoid specimens from a bed pan unless sterile one
-Use sterile or clean container
do not clean with disinfectant
-Cary-Blair medium (transport medium)
-Storage &Transport: refrigerate at 4oC if testing within 48 hours
FIELD WORK
Immunology & PCR
-For Ag detection/PCR – no transport medium
-Transport on dry ice for Ag, PCR detection
Store at -15oC for Ag detection and PCR; , -70oC if longer
Rectal swabs
Advantage: convenient, adapted to small children, debilitated patients and other situations where voided stool sample not feasible
Drawbacks: no macroscopic assessment possible & less material available

13. GENITOURINARY TRACT SPECIMENS
Organism/Syndrome Investigated
Source of Specimen
Collection
Neisseria gonorrhoeae
Endocervical-women
Urethral-men
Direct culture
Amies Transport Swab
Yeast, Bacterial Vaginosis, T. vaginalis
Post Vaginal Vault
Direct examination
Syphilis/Treponema pallidum
Aspirates/Scrapings from ulcerative lesions
Transport immediately for dark-field microscopy
Prenatal Screening for Group B Streptococcus at weeks gestation
Combined Vaginal/Rectal

14. Sampling-FEMALE
Sampling-MALE

15. Lower Respiratory: Sputum for TB Culture
RESPIRATORY SPECIMENS
Lower Respiratory: Sputum for TB Culture
Do not rinse mouth with water as saprophytic mycobacteria in tap water may
produce false positive results
Collect an early morning specimen from a deep, productive cough on three
consecutive days into the empty 90 mL sterile or clean container (?Ghana)
Carefully and tightly replace the cap
Wash your hands after collecting the specimen
Do not pool specimens
Store refrigerated at 2-8°C
Submit to the PHL laboratory within 24 hours
of collection with a completed PHL requisition

16. FIELD WORK: TB Measurement of reaction spot
Use Mantoux test for screening

17. Throat swab (posterior pharyngeal swab)
b) Upper Respiratory:
Throat swab (posterior pharyngeal swab)
-Hold tongue away with tongue depressor
-Locate areas of inflammation and exudate in posterior pharynx, tonsillar region of throat behind uvula
-Avoid swabbing soft palate; do not touch tongue
-Rub area back and forth with cotton or Dacron swab
WHO/CDS/EPR/ARO/2006.1

18. Nasopharyngeal swab -Tilt head backwards c) Upper Respiratory:
-Insert flexible fine-shafted polyester swab
into nostril and back to nasopharynx
-Leave in place a few seconds
-Withdraw slowly; rotating motion
-This procedure is done by a trained physician

19. Naso-pharyngeal aspirate (Lavage)
d) Upper Respiratory:
Naso-pharyngeal aspirate (Lavage)
-Tilt head slightly backward
-Instill ml of sterile normal saline into one nostril
-Use aspiration trap
-Insert silicon catheter in nostril and aspirate
the secretion gently by suction in each nostril
-Procedure normal done by a trained Physician

20. Collection of Wound Swabs
Gentle cleansing of a skin wound prior to sample collection is recommended to reduce commensal flora contamination
Purulent exudates must be expressed onto swabs
Place the swab into the transport media
Label swab with patient’s full name, source of specimen and date and time of collection.

21. Maintain swabs at room temperature and submit to the lab within 24 hours of collection
Deep wound specimens (aspirates) are optimal as the predictive value of
superficial swabs is low
Investigation of deep wounds for anaerobes requires a special anaerobic collection kit
Anaerobic GasPak System
Anaerobic bags/ containers

22. Tissue for Culture
Tissue for routine C&S should be collected in a sterile container
For small samples add several drops of sterile saline to the container to maintain moisture
Maintain at room temperature and submit to the laboratory within 24 hours of collection

23. Surgical Specimens (Other Shared Specimens)
TISSUE
FLUID
Specimen size of pea or larger
Divide
Anaerobic transport tube
Hold
upright,
uncap,
insert specimen and
recap
Anaerobic
Culture
Keep moist by
adding 1 -
2 mL
sterile
saline
Aerobic culture
and Gram stain
Fungus culture
and stain
AFB Culture
and stain
Virus Culture
Specimen smaller than a pea
(i.e. Needle biopsy)
Put entire specimen into
On piece of
telfa
backed gauze
And stain
Viral Culture
Pleural Peritoneal
Abscess CSF, etc.
4 drops –
Transport
Tube 1
20 mL
Sterile Specimen tube
or cup Container
In Viral Transport
Medium
Indicate source
On requisition (Cath.,
cysto, bladder,
R ureter, L. ureter)
Maximum
2 hr transport
to lab
URINE
COLLECTION AND HANDLING OF OPERATING ROOM SPECIMENS FOR MICROBIO
LOGY
Mike/Mary

24. Know the 7 rules or principles must be followed in order to collect microbiology specimens which will accurately reflect the pathogenesis of the microbiological agent

25. Principle #1: The specimen must be collected with a minimum of contamination as close to site of infection as possible
BLOOD CULTURES:
-Improper cleaning of skin or catheter prior to drawing blood
-Acceptable blood culture contamination <3%
URINE CULTURES:
-Males have a lower contamination rate than females (11.2% Vs 22.8%)???
NB. patient preparation/education very important
Sputum for AFBs
-Sputum not saliva

26. Principle #2: A specimen must be collected at the optimal time(s) in order to recover the pathogen(s) of interest
Urine Bact. Culture: 1st morning Mid-Stream Clean Catch
Urine Gono/chlymedia PCR/RE: 1st voided , first stream urine of day
Blood Bact. Culture:
- Collect prior to admin. of antibiotics
- During Chills and To spikes
- Collect 2-3 bottles from different sites (times)??
Transient bacteraemia endocarditis/ pathogenic role of saprophytes e.g. S. epidermidis

27. Blood Parasites: Collect during febrile episode or every 6hrs for 24hrs
Sputum for AFBs Microscopy & Culture:
-3 consecutive specimen collected 8-24hrs apart
-At least one must be early morning specimen
-In Ghana, one early morning + One spot specimen at clinic/Lab.
Viral cultures: Collect as soon after onset of symptoms as possible
Stool specimen
For parasites ;vegetative form e.g. Entamoeba histolytica: freshly stool specimen
For ova, cysts and Oocyst: preserve stool within an hour of collection

28. Principle #3: A sufficient quantity of the specimen must be obtained to perform the requested tests
BLOOD
-Adult : 10ml for aerobic, 10ml for anaerobic bottle
-Child: 2-5ml
-Infant: ml why??
CSF
-At least 0.5ml in 1st bottle, 2ml each into bottles 2 &3
URINE
- At least 12ml (2/3 of the container, do not fill it)

29. Principle #4: Appropriate collection devices and specimen containers must be used to ensure recovery of specific organisms
Anaerobic Culture
-Best collected with metal; needle aspiration or with a scalpel
Viral culture sample must not be transported in bacterial transport media
Skin Parasites: place skin scraping in a clean dry container, cap tightly and transport to lab within 24hrs at RT
Blood culture from Heparin or EDTA tube
-Heparin is toxic to many organisms
-Transferring from these tubes increases risk of contamination

30. Principle #5: Collect all microbiology test samples prior to the institution of antimicrobial agents
Hair, Skin and Nails for fungal culture:
-Collect sample before antifungal therapy
-if already started, discontinue at least 5 days before taken sample
Urine Culture:
-Antibiotics may cause a transient decrease in bacterial concentration resulting in a false negative report
Blood Culture:
Take blood sample before antibiotic therapy
State on the lab form if you have started therapy before blood taken

31. Principle #6: The specimen container must be properly labeled and sealed prior to transport

32. Principle #7: Minimize transport time or maximize transport media
Principle #7: Minimize transport time or maximize transport media. There is always some loss of viability during transport
Minimize transport time and maximize use of transport media as
much as possible

34. Criteria For Rejection of Microbiological Specimens
Unlabeled or improperly labeled specimen
Prolonged storage or transport
Improper or damaged container
Specimen received in fixative
Oropharyngeal contaminated sputum
Duplicate specimens stools, sputum) within a 24 hour period. Exceptions cleared by the laboratory
Specimens unsuitable for culture request (anaerobic culture from not acceptable source, urine from Foley catheter)
Dry Swab
24-hr collection of urine or sputum for AFB or fungal culture