Obtaining Specimens for Microbiological Evaluation

Obtaining Specimens for Microbiological Evaluation

Bacteremia I
* Most bacteremias are intermittent
* One blood culture is rarely sufficient
o Staphylococcus epidermidis
+ Frequent contaminant
+ Commonest cause of PVE
* Two blood cultures usually sufficient
o Three or four if suspect likely contaminant
o Antibiotic therapy

Blood Cultures - Volume
The magnitude of bacteremia may be low (<1cfu/ml)

Higher volumes have higher yield
Blood Cultures - Lab Aspects
* Additives (SPS, resins) increase yield
* Aerobic and anaerobic bottle = one blood culture
* Five days incubation sufficient
o Exception: Brucella, Histoplasma, Mycobacterium, Bartonella, Legionella
* Automated Systems detect CO2
o Subculture detected bottles

Aerobic/Anaerobic Blood Culture Bottles
AFB Blood Culture Bottle
Obtaining Blood Culture
* Locate the vein
* Prep kit
o Alcohol 5 sec. Dry 30-60 sec
o Tincture of Iodine-center to periphery. Dry 45-60 sec
* Remove caps, clean with alcohol
* Put on gloves
* Without palpating, draw 20 ml and put 10 in anaerobic and 10 in aerobic bottle
* Dispose of syringe in sharps container
* Label bottles and send to lab

Blood Culture Prep Kit
Sputum Culture Reliability
* Expectorated unreliable because of contamination
o Reliability  if physician observes
* Laboratory reliability screen
o > 25 PMN’s, < 10 oral squamous cells per hpf

Sputum Container
Sputum
* Gram stain
o Useful for immediate therapy
o May be more reliable than culture
+ Many PMN’s with single bacterial morphology
* AFB - first morning specimen
* Pneumocystis carinii - induced specimen

Nasal Cultures
* Virus
o Use wire swab
o Place in nose 1-3 cm, rotate, 10-15 sec
o Obtain viral transport medium from lab
* Bacterial
o Culturette with rigid or wire swab
o Suspect pertussis - special media

Wire Swab
Throat Cultures
* For Group A strept, diphtheria, gonorrhea
* Tongue blade - visualize pharynx and tonsils
* Rub swab over tonsils and pharynx
o INCLUDE ANY EXUDATE
* Insert into holder, crush vial

Swabs for Bacterial (red) and Viral (green) Cultures

Cerebrospinal Fluid
* Use sterile technique
* First or second tube to Microbiology
* Studies
o Gram stain - one drop cloudy fluid or sediment
o Aerobic culture - 1.0 ml
o Viral culture - 1.0 ml
o AFB or fungal culture - up to 10 ml

Wounds: General Principles
* Closed space infections provide reliable specimens
* Open wounds heavily contaminated
o May quantitate
* May obtain culture by aspirating advancing border
* Culture skin, soft tissue or wound abscesses for anaerobic and aerobic organisms
o Transport in capped syringe or special tube

Wound Culture
* Closed space abscesses
o Decontaminate skin
o Insert needle and aspirate or aspirate pus after incision
* Open wound
o Remove superficial exudate
o Aspirate through margin or swab (least reliable)
* Transport
o Capped syringe or anaerobic transport tube
o Rapidly to lab

Urine - General
* Collection method must avoid contamination
o Clean catch, midstream voided
o Catheterized urine
o Suprapubic aspiration
* Cultures performed quantitatively
o Less than 10,000 per ml suggest contamination

Clean Catch, Midstream Urine
* Cleanse periurethral area with soap and water
* Pass initial urine into toilet, then collect specimen in cup
* Instructions to patient are critical

Instructions for Patient
* Remove underpants completely so they will not get soiled.
* Sit comfortably on the seat, but do not leave your knees in front of you. Instead swing one knee to the side as far as you can.
* Spread yourself with one hand, and continue to hold yourself spread while you clean and collect the specimen.
* Wash—Be sure you wash well and rinse well before you collect your urine sample. Wash only the area from which you pass urine. You do not have to wash hard, but wash slowly. Be sure to wipe from the front of your body towards the back. Wash between the folds of skin as carefully as you can.
* Do not put sponges in the toilet. Put them back in the plate.
* Rinse—After you have washed with each soap pad, rinse with each moistened pad with the same front to back motion. Do not use any pad more than once.
* Hold cup by the outside and pass your urine into the cup. If you touch the inside of the cup or drop it on the floor, ask the nurse to give you a new one.

Catheterized Urine
* Cleanse periurethral area with soap and water
* DO NOT RECONTAMINATE
* Insert catheter into bladder
o Discard initial urine
o Collect specimen in sterile cup
* Chronic indwelling Foley catheter
o Clamp tubing below junction (or port)
o Disinfect with alcohol
o Insert needle (on syringe) through port or catheter wall and aspirate

Suprapubic Aspiration
* BE CERTAIN BLADDER IS FULL - PALPATE OR PERCUSS
* Prep skin with alcohol or iodine
* Anesthetize with lidocaine
* Introduce needle 2.0 cm above symphysis
* Aspirate 20 ml for culture

Suprapubic Aspiration
Wire Swab

Obtaining Specimens for Microbiological Evaluation.ppt

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Specimen collection Role of the Nurse

Specimen collection Role of the Nurse

Nurses often assume the responsibility of specimen collection

* Specimens consist
o Urine
o Stool
o Sputum
o Wound drainage
o Blood

What about the client?

* Comfort
* Privacy
* Questions
* Clear, concise directions
o NPO

The Nurse

* Check physician orders
* Keep it Simple directions to client
* Standard precautions
* Label specimen
* Timely
* C&S to lab ASAP or refrigerated
* Documentation

Urine Specimen

* Random
* Clean
* Female ? Menses (make note)
* Tested for:
+ Specific gravity
+ pH
+ Albumin
+ Glucose
+ Microscopic exam

Urine for C&S

* Culture = ? Bacteria growing
* Sensitivity = which antibiotics are effective
* Readings after 24; 48; 72 hrs.

Midstream Urine

Sterile Catheter Specimen

(never from bag)

Why a urine specimen for C&S

* ? Urinary Tract Infection (UTI)
o Frequency
o Urgency
o Dysuria
o Hematuria
o Flank pain
o Fever
o Cloudy, malodorous urine

Obtaining specimen

* Wash hands
* Clean meatus, female front to back
* Start stream, then stop, collect specimen
* Aseptic technique
* Bedpan/mexican hat
* To lab 15-20min post collection

Children

* Pediatric bags ( u Bag)
* Never squeeze diaper

Characteristics of Urine

* Color
* Clarity
* Odor

Specimen Collection

* Random Specimens
o Clean-not sterile
o Ordered for
+ Urinalysis testing
+ Measurement of specific gravity
+ pH
+ Glucose levels

Urine specimen collection

* Midstream Specimen
o Clean voided
o C&S
o 30-60 mls urine
* Sterile Specimen
o Indwelling catheter
o Drainage bag

Urine collection

* Timed urine specimens
o 2-72 hr intervals (24hr most common)
o Begin after urinating
o Note start time on container & requisition
o Collect all urine in timed period

Post Reminder Signs

Indwelling Catheter

* Strict aseptic technique
* Only from Bag if Brand new
* Sampling Port?
* Clamp 30 min. prior
* Wash hands – Glove
* Cleanse port with alcohol swab
* Sterile needle
* To lab 30 min (may refridge 2hrs)

Common Urine Lab Tests

* Routine Urinalysis
o Examine within 2hrs
o 1st voided specimen in AM
o Reagent strip
* Specific Gravity
o Concentration
o 1.010-1.025
* Urine glucose
o Diabetics
o Reagent strips
o Double void

Measuring chemical properties of urine=Urinalysis

* Glucose
* Ketones
* Protein
* Blood- hematuria
* pH
* Specific gravity
* Microscopic examination

Stool Specimen

Analysis of fecal material can detect pathological conditions ie: tumors, hemorrhage, infection

* Tests
o OB
o Pus
o Ova & Parasites

Fecal specimens

* ? Chemical preservatives
* Medical aseptic technique
* To lab on time
* Labelling
* Documentation

Guaiac Test

Colorectal cancer screening test

FOBT

Hemoccult slide test

Fecal Characteristics

* Color
o melena
* Odor
* Consistency
* Frequency
* Amount
* Shape
* Constituents

Guaiac Test

* Single positive test result does not confirm bleeding or colorectal cancer.
* Repeat test 3X
* Meat free, high residue diet

Vaginal or Urethral Discharge Specimens

* Normally thin, nonpurulent, whitish or clear, small in amount
* S&S STD’s, UTI
* Not Delegated
* Assess external genitalia
* If STD record sexual history
* Physician’s order- vaginal/urethral

Blood Specimens

* Lab techs
* ABG’s
* Blood Glucose

Respiratory Tract

* Tests to determine abnormal cells or infection
o Throat cultures
o Sputum specimens
o Skin testing
o Thoracentesis

Nose, Throat Specimens

* Upper respiratory/ throat infections
* Should Not be delegated
* Throat swabs
o ac meal or 1 hr pc meal
o Wash hands, glove
o Tilt head backward
o “ah” ( if pharynx not visualized, tongue depressor, anterior 1/3 of tongue)
o Don’t contaminate

Throat cultures

* Oropharynx & tonsillar
* Sterile swab
* Culture determines pathogenic microorganisms
* Sensitivity determines the antibiotics to which the microorganisms are sensitive or resistant

Method for throat culture

* Insert swab into pharyngeal region
* Reddened areas/ exudate
* Gag reflex if client sitting and leaning forward slightly
* Inform client re procedure

Nose culture

* Blow nose, check nostril patency
* Rotate Swab inflamed mucosa or exudate
* Swab must advance into nasopharynx to ensure culture properly obtained

Sputum specimens (3 major types)

Ordered to identify organisms growing in sputum

* C&S
* AFB
o 3 consecutive, early am
* Cytology
o Abnormal lung cancer by cell type
o 3 early am

Sputum collection

* May be delegated
* Cough effectively
* Mucus from bronchus
* Not Saliva
* Record
o Color
o Consistency
o Amount
o Odor
o Document date & time sent to lab.

Sputum collection

* No mouthwash/toothpaste-

viability of microorganisms and alter culture results

Skin testing

* Determines pulmonary diseases
o Bacterial
o Fungal
o Viral

Antigen injected intradermally

Injection site circled

Instructions not to wash site

Reading skin test

* Induration – palpable, elevated, hardened area around site. Edema and inflammation from antigen –antibiotic reaction. Measured in millimeters
* Reddened flat areas are neg.

The elderly freq. display false neg. or false positive TB skin test

If positive TB test

* Complete history risk factors
* Symptoms
o Weight loss
o Night sweats
o Hemoptysis
o Fatigue

Early am sputum for AFB

Chest xray

Thoracentesis

Insert needle through chest wall into pleural space

Aspirate fluid

* Diagnostic
* Therapeutic
* Biopsy

Gastric Secretions

* NG tube

Cultures

* Culturette/swab
* Wet/dry method
* Nose, throat, wound



Review procedure manual & fill in requisitions.

Nursing Functions for Specimen Collection

* Explain procedure, gain client’s participation
* Collect right amt. of specimen at the right time
* Place specimen in correct container
* Label container accurately

(addressograph), plastic bag

Nursing Functions for specimen collection

* Complete lab. Req.
* Place the specimen in the appropriate place for pick up.
* Document/record specimen sent and anything unusual about the appearance of specimen

Blood glucose levels

* Capillary Puncture
* Reduces Venipunctures
* Clients can perform
* Glucometers
* Chemical reagent strip
* Delegated to those instructed in skill if client’s condition stable

Glucose monitoring

* Ordered ac, pc, hs, fasting, before insulin (sliding scale)
* ? Risks for skin puncture
* Assess area of skin
o Sides of fingers, toes, heels
* Client’s ability
* Normal fasting Bld. Sugar

70-120 mg/100ml

Glucose Monitoring

* Wash hands, glove
* Client wash hands, warm water
* Follow instructions on meter
* Massage /milk finger or puncture site
* Antiseptic swab ( allow to dry completely)
* Wipe away first droplet of blood with tissue/cotton ball

Glucose Monitoring

* Dispose of lancet in sharps container
* Wash hands
* Check puncture site
o Can share reading with client
* Record results
* Proceed as indicated by results

The Value of Measurement

3 benefits to measuring progress and results

* Shows where we are now
* Tells if we are heading toward our goal
* Allows us to make improvements along the way

What we measure gets improved. Peter F. Drucker

* Heightens our awareness
* Helps us focus on what we value and where we are going
* Keeps us on track
* Gives info what is happening along the way and enables us to continue or change depending on desired results


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Laboratory specimen: collection, safe transport and biosafety

Successful laboratory investigations

* advance planning
* collection of appropriate and adequate specimens
* labeling and documentation of laboratory specimen
* storage, packaging and transport to appropriate laboratory
* the ability of the laboratory to accurately perform the diagnostic tests
* biosafety and decontamination procedures to reduce the risk of further spread of the disease
* timely communication of results

Specimen collection:
key issues

* Consider differential diagnoses
* Decide on test(s) to be conducted
* Decide on clinical samples to be

collected to conduct these tests
o consultation between microbiologists, clinicians and epidemiologists

Transport medium

* Allows organisms (pathogens and contaminants) to survive
* Non-nutritive - does not allow organisms to proliferate
* For bacteria – i.e., Cary Blair
* For viruses - virus transport media (VTM)

Some tips

* Laboratory investigation should start as early as possible
* Specimens obtained early, preferably prior to antimicrobial treatment likely to yield the infective pathogen
* Before doing anything, explain the procedure to patient and relatives
* When collecting the specimen, avoid contamination
* Take a sufficient quantity of material
* Follow the appropriate precautions for safety

Blood for smears Collection

Capillary blood from finger prick
+ make smear
+ fix with methanol or other fixative

Handling and transport
Transport slides within 24 hours
Do not refrigerate (can alter cell morphology)

Blood for cultures
Collection

Venous blood
+ infants: 0.5 – 2 ml
+ children: 2 – 5 ml
+ adults: 5 – 10 ml

Requires aseptic technique
Collect within 10 minutes of fever
+ if suspect bacterial endocarditis: 3 sets of blood culture

Blood for cultures
Handling and Transport

Collect into bottles with infusion broth
+ change needle to inoculate the broth
Transport upright with cushion
+ prevents hemolysis
Wrap tubes with absorbent cotton
Travel at ambient temperature
Store at 4oC if can’t reach laboratory in 24 hours

Serum Collection
Venous blood in sterile tube
+ let clot for 30 minutes at ambient temperature
+ glass better than plastic
Handling
Place at 4-8°C for clot retraction for at least 1-2 hours
Centrifuge at 1 500 RPM for 5-10 min
+ separates serum from the clot
Transport
4-8oC if transport lasts less than 10 days
Freeze at -20°C if storage for weeks or months before processing and shipment to reference laboratory
Avoid repeated freeze-thaw cycles
+ destroys IgM
To avoid hemolysis: do not freeze unseparated blood

Collection
o Lumbar puncture
o Sterile tubes
o Aseptic conditions
o Trained person

Cerebrospinal fluid (CSF)

CSF
Handling and transportation
Bacteria
+ preferably in trans-isolate medium, pre-warmed to 25-37°C before inoculation
OR
+ transport at ambient temperature (relevant pathogens do not survive at low temperatures)

Viruses
+ transport at 4-8°C (if up to 48hrs or -70°C for longer duration)

Rectal swabs
Advantage
o convenient
o adapted to small children, debilitated patients and other situations where voided stool sample not feasible

Drawbacks
o no macroscopic assessment possible
o less material available
o not recommended for viruses

Stool samples Collection:
Freshly passed stool samples
+ avoid specimens from a bed pan
Use sterile or clean container
+ do not clean with disinfectant
During an outbreak - collect from 10-20 patients

Stool samples for viruses
Timing
o within 48 hours of onset
Sample amount
o 5-10 ml fresh stool from patients (and controls)

Methods
o fresh stool unmixed with urine in clean, dry and sterile container

Storage
o refrigerate at 4°C; do not freeze
o store at -15°C - for Ag detection, polymerase chain reaction (PCR)

Transport
o 4°C (do not freeze); dry ice for (Ag detection and PCR)

Stool samples for bacteria
Timing
o during active phase
Sample amount and size
o fresh sample and two swabs from patients, controls and carriers (if indicated)

Method
o Cary-Blair medium
o For Ag detection/PCR – no transport medium

Storage
o refrigerate at 4°C if testing within 48 hours, -70°C if longer; store at -15°C for Ag detection and PCR

Transport
o 4°C (do not freeze); dry ice for Ag, PCR detection

Stool samples for parasites
Timing
o as soon as possible after onset

Sample amount and size
o at least 3 x 5-10 ml fresh stool from patients and controls

Method
o mix with 10% formalin or polyvinyl chloride, 3 parts stool to 1 part preservative
o unpreserved samples for Ag detection and PCR

Storage
o refrigerate at 4°C; store at -15°C for Ag detection and PCR

Transport
o 4°C (do not freeze); dry ice for antigen detection and PCR

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

Nasopharyngeal swab
Tilt head backwards
Insert flexible fine-shafted polyester swab into nostril and back to nasopharynx
Leave in place a few seconds
Withdraw slowly; rotating motion
WHO/CDS/EPR/ARO/2006.1

Nasopharyngeal aspirate
Tilt head slightly backward
Instill 1-1.5 ml of VTM /sterile normal saline into one nostril
Use aspiration mucus trap
Insert silicon catheter in nostril and aspirate the secretion gently by suction in each nostril
WHO/CDS/EPR/ARO/2006.1

Sputum
Collection
Instruct patient to take a deep breath and cough up sputum directly into a wide-mouth sterile container

o avoid saliva or postnasal discharge
o 1 ml minimum volume

Respiratory samples
Handling and Transport
All respiratory specimens except sputum are transported in appropriate media

o bacteria: Amie’s or Stuart’s transport medium
o viruses: viral transport medium (VTM)
Transport as quickly as possible to the laboratory to reduce overgrowth by oral flora

For transit periods up to 24 hours
o ambient temperature for bacteria
o 4-8°C for viruses

Collection
Biopsy relevant tissues
+ place in formalin for histopathology
+ place in transport medium for microbiological testing
+ place in sterile saline for isolation of viral pathogens

Post-mortem samples
Post-mortem samples
Handling and transportation
Fixed specimens can be transported at ambient temperatures

+ transport specimens in transport media within 24h at ambient temperature
+ transport specimens in sterile saline at 4-8oC within 48h

Specimen Transport
media
Storage condition
Purpose/ Lab investigation
Transport
Pending test
Throat swab
VTM
Isolation
Isolation, serology
Stool
Isolation
Urine
Clotted blood
Whole blood
Virologic Investigations
Specimen
Transport
media
Storage condition
Purpose/ Lab investigation
Transport
Pending test
Throat swab
Amie’s or Stuart’s TM
Isolation Visualization
Bacteriologic Investigations
Water for bacteriology
Preparation
Chlorinated water - add sodium thiosulphate (0.5ml of 10% solution or a small crystal)
Tap/ pump
+ remove attachments
+ wipe, clean and flame outlet
+ allow to flow (at least one minute)

Water course or reservoir - collect from a depth of at least 20 cm
Dug well - do not allow the bottle to touch the sides of the well

Water for bacteriology Collection
At least 200 ml of water sample from the source
In sterile glass bottles OR autoclavable plastic bottles
+ tight screw capped lid
+ securely fitting stoppers/caps
+ an overhanging rim

Handling and transportation
Test the water sample within 3 hours of collection
+ keep at ambient temperature

If delayed:
+ pack sample on ice
+ test refrigerated sample within 24 hours

Food samples
Collect suspect food earliest
Collect aseptically - sterile tools, containers

Solid Food
o cut 100-200 grams from centre with sterile knife
o raw meat or poultry - refrigerate in a sterile plastic jar

Liquids
o shake to mix, use sterile tube
o water used for cooking -- 1-5 liters

Contact surfaces (utensils and/or equipment) for food processing
o moisten swab with sterile 0.1% peptone water or buffered distilled water; put the swab in an enrichment broth

Food samples
* Handling and transportation
o As fast as possible
o Keep perishable food at 2-8 °C
o Cool hot food rapidly - put containers under cold running water
o Pack samples to prevent spillage
o Contact the laboratory regarding method of transport and anticipated time of receipt
o Seek help from environmental/veterinary microbiologist

Labeling specimens
* Patient’s name (or Patient Identifier)
* Unique ID number (Research/Outbreak)
* Specimen type
* Date, time and place of collection
* Name/ initials of collector

Patient’s Name/Identifier Unique ID Number
RRR-0023 001712643003
Date, Time, Place of Collection
Specimen Type
Serum
Collected by:
MDR

Glass slides for microscopy
Label slides individually
o use glass marking pencil
o ensure markings don’t interfere with staining process

Each slide should bear:
o patient name
o unique identification number
o date of collection

Some Tips
* Pre-print labels
* Permanently affix label to the specimen container.
* Glass slides for microscopy labeled individually
* One specimen – one lab request
* Each slide should bear the patient’s name, unique identifier, and date of collection
* Use line listing for multiple patients
* Original documents kept by investigation team


Case investigation form
Epidemiologist sends:
Patient information
o age (or date of birth), sex, complete address

Clinical information
o date of onset of symptoms, clinical and immunization history, risk factors or contact history where relevant, anti-microbial drugs taken prior to specimen collection

Laboratory information
o acute or convalescent specimen
o other specimens from the same patient

Line listing – if large number of patients

Case investigation form
Receiving laboratory records:
Date and time when specimen was received
Name and initials of the person receiving specimen
Record of specimen quality

Criteria for rejecting samples
Mismatch of information on the label and the request
Inappropriate transport temperature
Excessive delay in transportation
Inappropriate transport medium
o specimen received in a fixative
o dry specimen
o sample with questionable relevance

Insufficient quantity
Leakage
Reference

Most of the slides used in this presentation are developed by the Department of Epidemic and Pandemic Alert and Response of the World Health Organization with assistance from:

European Program for Intervention Epidemiology Training
Canadian Field Epidemiology Program
Thailand Ministry of Health
Institut Pasteur

References

* Communicable Disease Toolkit: Iraq Crisis. Guidelines for the collection of specimens for laboratory testing. WHO, 2003
* Guidelines for the collection of clinical specimens during field investigation of outbreaks, WHO, 2000
* The role of laboratories and blood banks in disaster situations, WHO publication, 2001
* Sampling during avian influenza investigations, 2006
* IDSR guidelines for specimen collection, 2003
* Laboratory Needs for Emergency Situations, 2003
* Overview of Laboratory Structure and Operational Needs for the Iraqi Crisis, 2003
* Costing for sampling materials and diagnostic reagents for the Iraq crisis, 2003

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