LECTURE NOTES

ON

SPECIMEN COLLECTION, HANDLING AND

PROCESSING

IN

MEDICAL MICROBIOLOGY AND PARASITOLOGY

LANGLEY .A ORUTUGU
INTRODUCTION
 It is critical that the laboratory provide complete guidelines for the
proper collection and transport of specimens to ensure quality patient
care.
All diagnostic information from the microbiology laboratory is
contingent on the quality of specimen received.
 Consequences of a poorly collected and/or poorly transported
specimen include;
I. failure to isolate the causative microorganism and recovery of
contaminants or normal microbiota, which can lead to improper
treatment of the patient.

 This Lecture therefore is aimed at providing guidelines on

collecting, handling and transporting quality patient specimens
WHY DO WE NEED PROPER COLLECTION OF SPECIMEN?

 The proper collection, handling and processing of patient

specimens in the medical microbiology and parasitology laboratory is
one most important step in the recovery of pathogenic organisms
responsible for infectious disease.

 A poorly collected specimen may lead to failure to isolate the

causative organism(s) and/or result in recovery and subsequent
treatment of contaminating organisms.

 The quality of a result is directly related to the quality of the

specimen being cultured. The best results are obtained when the
following guidelines are maintained.
WHO DOES THE COLLECTION OF SPECIMEN?

1. Clinicians
2. Nurses
3. Medical laboratory Scientist
4. Patients (Under Instruction)
5. Epidemiologist
6. Phlebotomist
7. Others
WHY DO WE NEED SPECIMEN?

1. To provide information relating to clinical diagnosis

2. To investigate the aetiology of a clinical condition

3. To study complications of a disease or its treatment

4. To provide prognostic information

5. To monitor natural history or response to treatment for a condition

6. To detect subclinical disease (screening)

7. For research purposes

KEY FACTORS TO BE CONSIDERED WHEN COLLECTING
SPECIMEN

1. Patient Identification (Mandatory)

2. Specimen Type
3. Availability Of Materials
4. Time of collection
5. Technical Know-how
Steps required for successful collection procedure
1. Identify the patient
2. Assess the patients physical disposition (i.e. Diet, exercise,
stress, basal state).
3. Check the requisition form for required test, patients information,
and any special requirement.
5. For body fluid collection, provide the patient with the suitable
container and collection instruction
6. FOR BLOOD
i. Select a suitable site for Venipuncture
ii. Prepare the equipment , the patient and the puncture site.
Steps required for successful collection procedure continued……

iii. Perform the Venipuncture

iv. Collect the sample in the appropriate container
v. Recognize complications associated with the phlebotomy
procedure
7. Assess the need for sample recollection and / or rejection
8. Label the collection tubes at the bedside or drawing area
9. Promptly send the specimen with the requisition to the laboratory
2. Patients preparation for specimen collection

A. Inform patient on
1. Specimen to be collected
2. The time of collection
3. Mode or method of collection
4. Conditions required for collection
 Diet
 Drugs / medications
Patients preparation factors
1. Therapeutic drug monitoring

2. Stress / anxiety: this may cause transient elevation in WBC,

adrenal hormone (cortisol, catecholamine) values. Anxiety that
results in hyperventilation may cause acid- base imbalance, and
increased lactate.

3. Effect of exercise (mostly chemistry parameters)

4. Postural : change in posture from supine to erect or sitting

causes a shift in fluid from intravascular to the interstitial space of
about 12%
i. From upright to supine can have a dilutional effect owing to an
increase in plasma volume. These effects are accentuated in
patients with oedema and low plasma protein values
Patients preparation factors continued…

5. Diurnal variations :
i. Serum cortisol levels are highest in early morning but are
decreased in the afternoon

ii. Serum sex hormones need special timing as regard cycle

iii. Other factors:

 Age
 Gender
 Pregnancy

Normal reference ranges are noted according to age and sex

3. Requisition form and Labeling

A. Each sample must have a label firmly attached to the specimen

container bearing the following information accompanied with a
requisition form
i. Date & Time of Collection
ii. Patient name and DOB
iii. Hospital number
iv. Culture site
v. Initials of Collector
 DEPARTMENT OF MEDICAL MICROBIOLOGY AND PARASITOLOGY
FACULTY OF BASIC MEDICAL SCIENCES
COLLEGE OF HEALTH SCIENCES
NIGER DELTA UNIVERSITY, AMASSOMA
WILBERFORCE ISLAND

LABORATORY REQUEST FORM

Name of Patient...........................................................Age............. Sex: Male/ Female. Date of

Request.................... Occupation...................................
Marital Status (Single), (Married), (Divorced), (Separated)
Hospital Number................................ Nature of Specimen........................... Time of
Collection.................................... Investigation Required ...........................................

Physician’s Name / SIGN......................

Requisition form and Labeling continued…

The requisition form must be reviewed to understand

i. What test are requested?
ii. What containers are required to collect the specimens?
iii. When the specimens are to be collected?
iv. What is the amount of specimen necessary for the testing?
v. What is the collection order of tubes?
vi. Source of specimen

Labelling rules
This should be done always immediately before leaving patient
NEVER;

1. NEVER Pre- label blood specimen tubes before drawing blood

2. NEVER Give specimen to another to label
3. Verify again that the information on the labelled specimen
matches that on the request form
Unlabeled specimen (s) or incorrectly Labeled specimen (s)

1. Repeat procedure (all blood specimen)

2. Discard (if sent to the laboratory)
3. If specimen is irreplaceable, the ordering physician must attest to
the correct patients identity and must authorize (on the specimen
labeling form) affixation of the correct label by an attendance
nurse.

Examples of irreplaceable specimen

a) Specimens from internal body sites or obtained as invasive
procedures (bone marrow, CSF, Bronchoscopy)
b) Critically timed specimen (prior to antibiotics or dosing schedules)
c) Neonatal specimens e.g. cord blood
4. Equipments For Collection
 These are dependent on specimen type and procedure, they
include but not limited to
a. Syringes and needles
b. Blades
c. Universal bottles
d. Anticoagulant bottles
e. Swab sticks
f. Media
g. Spatula
h. Applicator sticks
Collection tube additives

i. EDTA
ii. Heparin
iii. Citrate
iv. Serum separator
v. etc
Some common examples of specimen collected in the medical
microbiology and parasitology laboratory

I. Urine
II. Stool
III. Sputum
IV. Swab (Eye, Nasal, Throat, Ear, Wound, Vaginal, Urethral,
Cervical, etc).
V. Body fluids ( Blood, Peritoneal, Synovial,etc).
I. URINE SPECIMEN COLLECTION
GENERAL CONSIDERATIONS IN URINE SPECIMEN
COLLECTION:
 Never collect urine from a bedpan or urinal when culture is
required.
 Thoroughly clean the urethral opening (and vaginal vestibule in
females) prior to collection.
 Use sterile tubes or cups to collect and transport the urine.
 Use soap rather than disinfectants for cleaning the urethral area.
Urine Specimen Collection process
A sterile pre-labelled urine collection cup is given to the subject and the
following explanations are given before sample collection;
I. First ,wash hands with soap and water
II. Collection cup should not be opened until just before urinating
III. The cap should be turned up when urinating, and then immediately
recap the filled container tightly
IV. It is most important that the inside of the container and the cap not be
touched and come into contact with any body part, clothing or external
surface
V. Exposure to air should be minimized
Urine Specimen Collection continued …….

VI. Collect Urine with a wide mouthed

container from patients.

VII. A mid stream specimen is most ideal for

Processing

VIII. Female patients passes urine with the labia

separated and mid stream urine is collected
Types of urine specimens
I. Random Urine specimen
II. First morning (microscopic examination, HCG)
III. Clean catch specimen (MSU) for culture
IV. Supra-pubic specimen (especially for infants)
V. Catheter specimen
VI. Urine from children collected in bags with hypoallergenic skin
adhesive
VII. 24 hour urine for quantitative chemical determination in urine
Indwelling catheter urine collection:
I. Clean the catheter collection port with a 70% alcohol swab.
II. Using sterile technique, puncture the collection port with a needle
attached to a syringe.
III. Aspirate the urine and place it into a sterile container.
Straight catheter urine collection:
IV. Clean the patient's urethral opening (and in females the vaginal
vestibule) with soap, and carefully rinse the area with water.
V. Using sterile technique, pass a catheter into the bladder.
VI. Collect the initial 15 to 30 ml of urine and discard it.
VII. Collect a sample from the mid- or later flow of urine in a sterile
Clean catch specimen collection (male):
I. The person obtaining the specimen should wash their hands with
soap and water, rinse and dry.
II. If the patient is collecting the specimen, he should be given
detailed instructions.
III. Cleanse the penis, retract the foreskin (if not circumcised), and
wash with soapy water.
IV. Rinse the area well with water.
V. Keeping foreskin retracted; allow a few milliliters of urine to pass.
VI. Collect the midstream portion of urine in a sterile container.
Clean catch specimen (MSU) for male
Clean catch urine specimen collection (female):
i. The person obtaining the urine specimen should wash hands with soap and
water, rinse and dry.
ii. If the patient is collecting the specimen, she should be given detailed
instructions.
iii. Cleanse the urethral opening and vaginal vestibule area with soapy water
or clean gauze pads soaked with liquid soap.
iv. Rinse the area well with water or wet gauze pads.
v. Hold labia apart during voiding.
vi. Allow a few milliliters to pass.
vii. Collect the midstream portion of urine in a sterile container.
viii. Refrigerate if prolonged transport time
N/B. If disinfectants are introduced into the urine during collection
 They can inhibit the growth of microorganisms.
 Transport the specimen to the laboratory such that it will be plated
within two (2) hours of collection.
 Urines from clinics outside the main hospital campus should be
placed in tubes with preservative. These specimens can be held for
eight (8) hours.
Alternatively, urines can be refrigerated for 24 hours before plating.
 N CO LL E C T I O N
PE CI M E
2. BLOOD S
Indications for blood collection:

1. Culture.
Type of blood culture
 Routine blood culture
 Fungus blood culture
 Acid fast blood culture –

2. Microscopy
 Wet preparation
 Staining

Volume: to be determined by the investigation required

Blood collection methods:
i. Evacuated Tube Method,
ii. Syringe Method Venipuncture Locate a suitable vein before
cleansing the skin.
iii. Microtainer Collection Method (skin puncture heel or finger prick
3. Faecal specimens
Indications for test

Culture

Microscopy

Others
1. Collect specimen in a clean bedpan, commode specimen system,
or use plastic wrap placed between the toilet seat and the bowl.
Do not submit feces contaminated with urine or toilet water.
2. Transfer specimen into a clean, dry container.
3. Transport stool in Cary Blair media at ambient temperature within
1 hour of collection,
4. Diarrhea that develops after 3 days hospitalization is likely due to
Clostridium difficile toxin. Routine testing and OVA & Parasite
exams should not be performed on these patients.
5. Recommend that no more than 2 bacteriology specimens ( be
processed per patient without consultation. (2 separate bowel
movements)
6. Ova & Parasite – 3 specimens collected over a 10 day period is
optimum.
Notes:
• Only loose or diarrheal stools are recommended for routine stool
testing.

• Place the specimen in an appropriate preservative or transport

media, immediately after collection.

For ova and parasite, use 10% formalin and modified PVA;
for stool Biofire, use Cary-Blair transport media.
:
 4. RECTAL SWABS:
Collection Method
 Pass the tip of a sterile swab approximately 1 inch beyond the
anal sphincter.

 Carefully rotate the swab to sample the anal crypts and withdraw
the swab.

 Place the swab in transport medium.

If Neisseria gonorrhoeae is suspected,

inoculate a Thayer Martin plate at the bedside
and transport to the Laboratory immediately.
5. SPUTUM SPECIMEN
SPECIMEN COLLECTION
(ACCEPTANCE AND REJECTION CRITERIA)

I. All clinical specimens must be collected in clean sterile

containers, which must be properly sealed.
II. The outer portion of the container must not be Contaminate.
III. Optimal specimens are aseptically obtained
IV. Direct aspiration into a syringe is recommended for fresh pus,
fluid, or tissue that is rapidly and safely transported to the
laboratory.
REASONS FOR A REJECTED SPECIMEN
i. No test Requisition Form
ii. Test not written on (TRF)
iii. Submitted wrong specimen for test requested
iv. Quantity Not Sufficient (QNS)
v. Missing or inadequate identification
vi. Clotted Specimen
Common Avoidable Errors In Specimen Collection

i. Insufficient quantity
ii. Haemolysis
iii. Using incorrect specimen container for collection
iv. Inaccurate and incomplete patient guidance
v. Wrong labeling
vi. Failure to tighten specimen container lids, resulting in leakage
and / or contamination of specimen
SPECIMEN SAFETY CONSIDERATIONS
I. Follow universal precaution guidelines.
II. Treat all specimens as potentially biohazardous.
III. Laboratory workers should use appropriate barrier protection
when collecting or handling specimens.
IV. If splashing may occur, protective eye wear, face masks, and
aprons may be necessary.
V. Don't contaminate external surface of collection container and/or
its accompanying paperwork.
SPECIMEN SAFETY CONSIDERATIONS continued….
vi. Minimize direct handling of specimens in transit.
V. Use plastic sealable bags with a separate pouch for the
laboratory requisition orders or transport carriers (for example,
small buckets with rigid handles).
VI. Swabs should not be used if fluid can be obtained.
VII. Swabs without transport medium are not satisfactory since they
allow drying of the specimen and loss of viability.
VIII. All specimens should be transported to the laboratory in a sealed
zip lock bag.