Section-E Group4 WrittenOutput v.2

Cebu Doctors’ University
College of Nursing
1 Dr. P. V. Larrazabal Jr. Avenue, North Reclamation, 6014 Mandaue City, Cebu, Philippines
Tel. No. +63 (32) 238-8746 Web: www.cebudoctorsuniversity.edu E-mail: cdu-cn@cebudoctorsuniversity.edu
Collection, Labelling, Handling and Care of Specimen – Part 2
In Partial Fulfillment of MC3: Microbiology and Parasitology Laboratory Course
Submitted by:
1. Rivera, Franchesca Claire – Group Leader

2. Ang, Jorgen
3. Betonio, Krizza Jill
4. Capacao, Jamaica Rose
5. Castillano, Kristin Mikaela
6. Comille, Reina
7. Escabas, Joachim
8. Galeon, Aubrey Justine
9. Madrazo, Eiffel Jane
10. Oporto, Kyle Chleo
11. Perez, Jun Carmel
12. So, Sej Andrei
April 20, 2021
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Course Learning Outcomes
Group 4: Collection, Labelling, Handling and Care of Specimen – Part 2
1. Comprehensively discuss how to collect, package, transport, store the following
laboratory specimen for the following examinations:
1.1. Urine – urinalysis and culture
1.2. Sputum – DSSM/ AFB and MTB GeneXpert
1.3 Blood – CBC and Blood culture
1.4. Vaginal fluid
1.5. Seminal fluid
1.6. Pericardial fluid
1.7. Peritoneal fluid
1.8. Synovial fluid
1.9. Cerebrospinal fluid
1.10. Gastric fluid
1.11 Pleural fluid
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1. Comprehensively discuss how to collect, package, transport, store the
following laboratory specimen for the following examinations:
1.1. Urine – urinalysis and culture
URINALYSIS
COLLECT:
1. Routine or random sample: The patient is given a non-sterile collection container
and instructed to collect a midstream specimen in the container. This type of specimen
is routinely used for urinalysis and may not be used for a culture and sensitivity.
2. First voided specimen: The patient is given a urine container to take home and
instructed to collect a sample of the urine the first time he or she urinates in the
morning. Urine is not stable, the specimen should be returned to the laboratory within
one (1) hour of collection. If that is not possible, the specimen should be refrigerated
until it can be tested.
3. Timed specimen: Timed specimens are usually a 24 hour urine collection.
https://nursekey.com/3-urinalysis/
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A. The patient is given a large container (approximately 1 gallon) that is labeled
with the patient’s name and date.
B. Before issuing the 24 hour urine container the type of testing ordered is
checked for preservative requirements.
C. The test usually begins in the morning. The patient is told to empty their
bladder and discard the urine in the toilet and record the time on the label of the
urine container.
D. The 24 hour urine specimen is brought to the laboratory as soon as possible
as the 24- hour period is over
4. Clean-catch mid-stream specimen: Patients with orders for a urine culture and
sensitivity are given the proper mid-stream urine collection kit and the appropriate
instruction sheet.
A. Give the patient a sterile urine collection kit..
B. Explain to the patient that an instruction sheet is included in the kit.
C. Male urine culture collection instructions:
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1. Wash hands thoroughly with soap and water, rinse and dry.
2. Open the collection package but DO NOT TOUCH INSIDE OF CUP OR
RIM. Open the package of 3 towelettes. Retract foreskin if present. With the first
towelette, cleanse the urinary opening of the penis starting at the center and
work outward. Repeat the cleansing in the same manner with the two
remaining towelettes.
3. Remove lid carefully from the collection container, DO NOT TOUCH the
inside of the container or rim. Gently grasp the container.
4. Begin to void urine, letting the first 20-25 ml pass into the toilet.
Position
the cup in the stream of urine until the container is about half to two-thirds full.
Finish voiding into the toilet.
5. After obtaining the urine specimen, screw the lid on tightly again being
careful to avoid touching inside the container or lid.
6. Bring the specimen to the lab within 1 hour or collection or store
refrigerated for up to 24 hours.
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D. Female urine culture collection instructions:
1. Wash hands thoroughly with soap and water, rinse and dry.
2. Open the collection package but DO NOT TOUCH INSIDE OF CUP OR RIM.
Open the package of 3 towelettes. While seated on the toilet spread labia major
(outer folds). With the first towelette, wipe one side of the labia minora (inner
fold)
using a single downward stroke. Discard towelette. With the second towelette
repeat the procedure on the opposite side using a single downward stroke.
Discard towelette. With the third towelette, cleanse meatus (center area) with a
single downward stroke. Discard towelette.
3. Remove lid carefully from the collection container, DO NOT TOUCH the inside
of the container or rim. Gently grasp the container.
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4. Begin to void urine, letting the first 20-25 ml pass into the toilet. Position the
cup in the stream of urine until the container is about one-half to two-thirds full.
Finish voiding into the toilet.
5. After obtaining the urine specimen, screw the lid on tightly again being careful
to avoid touching inside the container or lid.
6. Bring the specimen to the lab within 1 hour of collection or store refrigerated
for up to 24 hours.
6. Catheterized specimen: These specimens are collected by specially trained
personnel only.
https://www.nursingtimes.net/clinical-archive/assessment-skills/specimen-collection-2-
obtaining-a-catheter-specimen-of-urine-10-07-2017/
PACKAGE:
Preferred Specimen Container
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https://www.vet.cornell.edu/animal-health-diagnostic-center/testing/how-to-send-
specimens/urine-culture
Urine Culture Transport Tubes (UTT) (gray top or yellow top) with appropriate volume
(4 mL /cc) are the preferred specimen container for culture. Results include a
quantitative assessment.
● The UTTs contain boric acid preservative which prevents overgrowth and allows
for a more accurate quantitative assessment. Volumes below the minimum risk
the preservative ratio being toxic to the organisms.
● Specimen should arrive to the lab as soon as feasible (the preservative allows
transport within 48 hours). Urine culture transport tubes should not be used for
urinalysis or cytology.
● The preservative allows for room temperature transport or these vacutainer
tubes come with a sterile straw transfer device.
Alternative Specimen Containers
If you don’t have a culture transport tube or you don’t have the minimum volume,
sterile containers can be used (send to lab same day or overnight). Results include a
quantitative assessment.
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● If a urine culture transport tube or the minimum volume is not available, a plain
sterile container (i.e. red top tube, specimen cup) can be used. This is suitable
for specimens that can be delivered immediately or overnight.
● These unpreserved liquid specimens should be kept cool during transport.
Alternative Specimen Containers
Alternative Specimen Containers: If the volume is too small for a urine transport tube
and the sample cannot get to the lab within 24 hours, send both the liquid urine and a
transport swab of the urine. (Results include a semi-quantitative assessment)
● For small volume samples Sterile Container and/or Transport Swabthat can’t get
to the lab within 24 hours, a culture transport swab can also be taken of the
urine. Both the swab and liquid specimen can be sent together.
● These specimens should be kept cool during transport

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TRANSPORT:
In order to ensure proper stability of the specimen these guidelines should be followed:
❏ Ensure the transport container lid is secure and leak resistant
❏ Utilize urine containers that are made of break-resistant plastic instead of glass.
❏ Verify that the specimen has been properly labeled, using at least two forms of
positive patient identification (eg, full name and hospital or medical record
number or for outpatients, full name and date of birth).
❏ Verify that the time the specimen was collected is documented (urinalysis
specimens must be analyzed within 2 hours of collection)
Specimen Transfer to Vacuette Urine Culture Transport Tubes
4mL Urine from syringe can be transferred directly to the Vacuette urine culture
tube
https://www.vet.cornell.edu/animal-health-diagnostic-center/testing/how-to-
send-specimens/urine-culture
NOTE: if urine is being submitted for urinalysis/cytology it must be submitted separately
in a plain tube or specimen cup without additives.
The transfer straw can be used to draw
up urine from cup or tube.
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Transfer Straw for Urine Culture https://www.vet.cornell.edu/animal-
health-diagnostic-center/testing/how-to-
Immerse the straw into the urine (4mL
minimum required)
https://www.vet.cornell.edu/animal-
While the straw is immersed, insert the
vacuette into the straw device to draw up
the urine. The tubes are pre vacuumed to
draw up the appropriate amount.
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Mix the specimen to dissolve the
preservative.
CAUTION The straw device has a needle
and should be disposed of as a medical
sharp.
STORE:
Store your urine from the 24-hour time period in a cool environment. It can be kept in
the refrigerator or on ice in a cooler. Label the container with your name, date, and
time of collection. After 24 hours of urine collection, the samples must be taken to a lab
for analysis.
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Urine Culture
COLLECT:
A. Urine Specimen:
● Clean catch midstream urine specimen is the most common technique for
urine collection. Specimens are preferably collected early in the morning upon
waking up (First morning urine).
○ Collection procedure:
■ Cleanse periurethral area with soap, sterile water, and sterile gauze
in a front-to-back manner.
■ Discard the first few milliliters of the urine to eliminate bacteria
from the urethra.
■ Collect the midstream portion into the sterile urine container with a
wide mouth and cover with a tight fitting lid.
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● Randomly voided urine specimen is not acceptable because of the presence of
contaminating bacteria from the periurethral and vaginal areas.
● Other specimens include:
○ Straight catheterized specimens are recommended for patients who
are unable to void or for patients who have been inserted with a catheter
due to a medical condition. This urine specimen will not contain any
bacteria due to the bladder’s sterile environment.
■ Collection procedure:
● Cleanse the urethral opening or vaginal vestibule (for
females) with soap and water.
● Insert the catheter into the bladder using a sterile technique.
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● Discard first 15 mL of the urine and then collect the latter
flow of urine in a sterile container.
○ Suprapubic aspirations are specifically used for obtaining anaerobic
bacterial culture. This invasive procedure may be done among infants,
young children, patients who are unable to void urine, and patients who
are catheterized, when emergency situations arise.
● Ensure that the patient’s bladder is full prior to doing this
procedure.
● Disinfect the skin over the urinary bladder and inoculate
local anesthesia on the area.
● Make a small incision using a scalpel and insert the needle
percutaneously above the symphysis pubis into the bladder
through the abdominal wall.
● Withdraw the urine by using a needle and syringe.
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○ Indwelling catheterized specimens are used for patients in hospitals
and nursing homes. These types of urine samples should be performed
with a proper aseptic technique.
● Clamp the catheter tubing above the port to obtain freshly
voided urine.
● Disinfect catheter collection port or the tubig wall with 70%
ethanol.
● Aspirate 5-10 mL of urine using a needle and syringe.
● Maintain the integrity of the closed drainage system to avoid
bacterial organisms to contaminate the bladder.
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B. Inoculation and Incubation of Urine Cultures
● Mix the uncentrifuged urine specimen thoroughly by immersing a
disposable sterile 1 µL (0.001 mL) loop vertically to obtain the desired
volume of urine.
● Inoculate the loopful of urine by making a straight line down the center of
the blood agar plate, and using the same loop, streak the sample by
making a series of horizontal streaks at 90º through the inoculum.
● Obtain a loopful of a well mixed uncentrifuged urine using a sterile
inoculating loop and inoculate it onto the other plated media. Streak the
inoculum following the clock method.
● Incubate the inoculated culture media plates at 35 to 37ºC for 18 to 24
hours.
● For specimens inoculated at a later time and cannot be interpreted
accurately the following day, incubate again for 1 day or interpret the
plate after a complete 24 hour incubation.
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PACKAGE
A. While in transport, urine specimen should be collected in the following
container depending on the type of specimen:
● Clean catch midstream:
○ Sterile container with a wide mouth and a tight fitting lid
○ Containers with chemical urinalysis preservatives may be used also.
● Straight catheterized specimens
○ Sterile, screw-cap container
● Suprapubic aspirate
○ Sterile, screw-cap container or anaerobic transporter
● Indwelling catheter
○ Sterile, screw-cap container
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B. While processing: Culture media
Bailey and Scott, Sir Flores notes
● 5% sheep blood agar plate (BAP) – enriched with unheated blood;
differentiates organisms through hemolytic patterns
Uninoculated plate S. pneumoniae (alpha hemolytic
streptococci)
● MacConkey agar plate (Mac) – for isolation and differentiation of lactose
(LF) and non-lactose fermenting (NLF) Gram negative bacilli
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Uninoculated plate K. pneumoniae (LF with pink mucoid
colonies)
● Columbia colistin-nalidixic acid agar (CNA) – for the isolation of
Staphylococci and Streptococci from mixed specimens
Uninoculated plate S. agalactiae (beta hemolytic
streptococci)
● Phenyl-ethyl alcohol agar (PEA) – for isolation of obligate Gram positive &
Gram negative anaerobes. It inhibits growth of enteric bacilli and
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swarming of Proteus
Uninoculated plate Gram pos: S. aureus Gram neg: K.
pneumoniae
● Cystine-lactose electrolyte-deficient (CLED) agar – for the isolation and
enumeration of bacteria in urine
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Uninoculated plate E. coli on CLED agar
● BD CHROMagar (Becton Dickinson, Heidelberg, Germany) – selective and
differential media specific for Staphylococcus aureus
Methicillin-resistant Staphylococcus aureus (MRSA) on CHROMagar
TRANSPORT:
● Urine transport tubes composed of boric acid, glycerol, and sodium formate
enable bacteria preservation of urine specimens with greater than 105 CFU/mL in
the absence of refrigeration up to 24 hours.
○ Example: BD Urine Culture Kit (Becton Dickinson Vacutainer Kits,
Rutherford, New Jersey)
■ These transport tubes can contain a minimum of 3 mL of urine and
inhibit certain growth of bacteria.
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○ Example: Starplex Scientific, Inc., Etobicoke, Cleveland, TN
■ These containers facilitate preservation of bacterial viability in the
specimen for 24 hours sans antibiotics.
● Uncultured urine should be delivered to the microbiology laboratory within
24 hours to lessen chances of bacterial multiplication.
● Straight catheterized specimens are to be transported to the laboratory
within 2 hours at RT.
○ If any delays occur (<24 hours), use appropriate preservatives (i.e.,
Boric Acid) and keep the specimen at RT.
● Suprabic urine specimens are to be transported to the laboratory
immediately at RT.
● Indwelling catheterized specimens should be delivered within 2 hours at
4ºC.
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○ If any delays occur (<24 hours), use appropriate preservatives (i.e.,
Boric Acid) and keep the specimen at RT.
STORAGE
● Refrigeration at 4ºC keeps the bacterial growth at constant for 24 hours.
● Uncultured urine should be kept at refrigeration temperature of 4ºC for 24
hours
● Straight catheterized should have refrigeration at 4ºC for 24 hours.
● Suprabic urine specimens must be plated immediately upon receipt.
● Indwelling catheterized specimens should be stored at 4ºC for 24 hours.
1.2. Sputum – DSSM/ AFB and MTB GeneXpert
DSSM / AFB
COLLECT:
A. The number of sputum specimens to be submitted depends on the reason for
examination and the test requested.
1. Two specimens, submitted within three working days, are required for diagnosis by
DSSM, and baseline testing by TB Culture and DST. If only one specimen was submitted
for diagnosis by DSSM, and the result turned out “0” (zero), it will be reported as
“Incomplete”, and a new set of specimens will have to be submitted. The specimens
can be collected either by:
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● Spot-Early Morning Collection – the first specimen (spot) is collected when
the presumptive TB presents to the clinic. He/she is then given a sputum
container for the second specimen that should be collected early morning the
next day, and submitted to the clinic promptly.
● Spot-Spot (Frontloading) Collection – these specimens are collected within
the same day, at least one hour apart.
2. Only one specimen, preferably an early morning one, is needed for follow-up
examinations, Xpert MTB/RIF Assay, and LPA.
Note: For hospital inpatients, it is better to collect a sputum specimen each morning on
two consecutive days.
B. Sputum collection may pose a risk and should be performed with extra
precaution. Thus, collection should only be done in any of the following areas:
❏ Open space (e.g. outside the DOTS facility, away from people and traffic)
❏ Well-ventilated area with patient facing away from the wind.
❏ Sputum collection booth – a specialized cubicle equipped with negative pressure,
HEPA filters and UV light used to prevent the spread of TB during collection.
Note: Never collect sputum specimens inside the laboratory or in closed spaces like
toilet cubicles, waiting rooms, reception rooms, and any other poorly ventilated area.
No one should be standing in front of the patient during expectoration.
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C. At least one sputum specimen should be collected under the supervision of a
health worker to guarantee correct identity of specimen and to ensure that the
patient closely follows the proper collection procedure.
Figure 2. Ideal sputum container
http://www.ntp.doh.gov.ph/downloads/publications/guidelines/NTRL_MCSTSTT_2nd_Ed
ition.pdf
D. The ideal sputum container (Figure 2) to be used should possess the following
characteristics:
● Volume capacity of 50 mL
● Made of transparent or translucent material
● Wide-mouthed (at least 35mm in diameter)
● Screw-capped
● Unbreakable and leak-proof
● Clean and sterile (preferably)
● Single-use, combustible material
● With walls that can be easily labeled
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E. Labeling of sputum container should be done after collection. Labels should be
placed on the container’s body, not on the lid to avoid specimen mismatch.
F. An early morning specimen is an optimal specimen. This is usually collected at
home. The site staff should instruct the patient on proper collection procedure,
including correct labeling of sputum container. Sputum collected at home is
collected early in the morning so that the specimen could be delivered to the
laboratory within the same day of collection.
G. Sputum production may be induced with the use of a nebulizer containing saline
solution (5-10% sodium chloride in water) in cases when patient is having
difficulty in producing sputum.
Figure 3. Purulent sputum Figure 4. Mucoid sputum
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Figure 5. Blood-stained sputum Figure 6. Salivary
sputum
ition.pdf
H. Good quality sputum specimens are those that are purulent (Figure 3), mucoid
(Figure 4) and blood-stained (Figure 5). However, grossly bloody or pure blood
specimens should not be examined.
I. Poor quality sputum specimens are those that are thin, watery and composed
largely of bubbles (Figure 6). When possible, the patient should be encouraged
to try collecting again. If not, these types of specimen can still be processed,
provided that the poor quality of the samples is reported on the result forms.
J. Prior to initiating sputum collection, the following should be observed:
❏ Identify the patient.
❏ Check the laboratory request form. Fill in any missing details.
❏ Discuss the collection procedure as well as the reason for examination with the
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patient
Figure 7. Patient-nurse discussion
ition.pdf
❏ If dentures are present, advise patient to remove them and rinse mouth with
water.
❏ The desired specimen is produced by a deep cough, and is thick, mucoid, white-
yellow and sometimes blood-tinged. It comes from the lower airways of the
lungs.
❏ Saliva or nasal secretions are not sputum, and therefore unsuitable specimens.
❏ An adequate sputum specimen with optimal quality is important to ensure
accurate and reliable test results.
❏ Demonstrate how to properly open and securely close the specimen container to
avoid contamination. Instruct patient not to touch the inside of the container or
its lid.
K. During collection, the following should be observed:
❏ If able, instruct the patient to stand. Give the patient a glass of water to rinse
the mouth free of food particles. Instruct the patient to rinse twice (Figure 8).
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❏ Instruct the patient to produce sputum by three repeated deep inhalation and
exhalation followed by a forceful cough to produce 3-5mL of sputum for each
sample.
❏ If patient is unable to cough spontaneously, instruct the patient to take several
deep breaths and hold breath momentarily. Repeating this several times may
induce coughing.
❏ Place the open container close to the mouth to collect the sputum (Figure 9).
Figure 8. Rinsing the mouth Figure 9. Collecting sputum
L. After collection, close the container with the screw-on lid without touching the
inside of the lid. Avoid spills or soiling the outside of the container.
M. Check the quality and quantity of the sputum produced. When possible, repeat
the procedure until desired specimen consistency and volume are met.
N. Label the body of the sputum container with the patient’s name, date & time of
collection, and specimen number (Figure 10).
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Figure 10. Preparing the container label
ition.pdf
Note: If patient is unable to cough spontaneously, instruct the patient to take several
deep breaths and hold breath momentarily. Repeating this several times may induce
coughing.
PACKAGE:
● Plastic leak-proof packaging
container
https://spectrumhealth.testcatalog.org
/show/LAB8770-1
● Disposable (paper) towels
https://recyclecoach.com/residents/re
cyclepedia/paper-towels/
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● 0.5% Chlorine solution for
disinfecting plastic leak-proof
packaging container
http://www.bioanalytic.de/files/bioanal
ytic/productimages/002529-
1010%20Natrium-
Hypochlorit%200.5%25%20%280800
x0600x72.080%29.jpg
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2
● Cooler or cold box, if sample
requires refrigeration
https://colemanphilippines.com/shop/c
oolers/hard-coolers/personal-
coolers/coleman-5-quart-personal-
cooler/
For the shipment of samples to the National Reference Laboratory follow sample
shipment packaging requirements (Follow WHO documents about how to safely ship
human samples from patients suspected to be infected with highly infectious
pathogens)
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https://www.who.int/csr/disease/plague/collecting-sputum-samples.PDF
Note: A designated Assistant wearing gloves should be available to help you. This
person should stand outside the patient room. He/She will help you prepare the sample
for transport. He/She will assist you with putting on the personal protective equipment.
He/She will provide any additional equipment you may need. He/She will monitor you
while you remove the personal protective equipment.
TRANSPORT:
A. Ensure that a completely filled-out laboratory request form is
available and corresponds with each specimen.
B. Transport specimens according to the triple packaging system.
C. Sputum specimens collected in the morning should be delivered to
the laboratory the same day they were collected (within 2 hours) as
much as possible. If this is not possible (e.g. due to late afternoon
collection, or long distance between sending and receiving
facilities), transport the specimens in coolers with ice packs
sufficient to contain the temperature similar to refrigeration.
STORE:
A. Check the container if it is tightly capped and properly labeled. Make
sure the specimen identification matches with the laboratory request
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form.
B. Store in a cool (2-10°C), dry place away from sunlight until ready for
transport to the laboratory to avoid liquefaction. Refrigeration also
reduces the growth of contaminants in the specimen. If refrigeration is
not possible, sputum can be contained in coolers with ice packs. Do not
freeze.
C. Do not use any chemical preservative for storage.
MTB GeneXpert
- The Xpert MTB/RIF assay is a new test that is revolutionizing tuberculosis (TB)
control by contributing to the rapid diagnosis of TB disease and drug resistance.
The test simultaneously detects Mycobacterium tuberculosis complex (MTBC)
and resistance to rifampin (RIF) in less than 2 hours.
COLLECT & PACKAGE:
A. Sputum Obtained Spontaneously
Collect raw sputum or sputum sediment samples following your institution’s standard
procedures. Two specimens are to be collected. When possible, specimens should be
collected outside in the open air and far away from other people.
1. The first sample is collected on the spot, at the consultation, when the patient is
identified as suspected TB case. If the patient has recently eaten, ask him/her to
rinse his/her mouth with water in order to avoid the presence of food in the
sample.
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2. The second sample is collected the day after, in the early morning, right after the
patient wakes up and before eating. The second sample may be collected at
home then the patient brings it to the health facility.
Alternatively, two sputum specimens can be collected one hour apart (frontloaded
microscopy).
Required Specimen Volume
Specimen Type Minimum Volume for 2 Minimum Volume for Test
Test and Retest
Sputum Sediment 0.5 mL 1 mL
Raw Sputum 1.0 mL 2 mL
Collection technique:
– The patient must be given a labelled sputum container (or a Falcon® tube, if the
sample is to be shipped by air).
https://ecatalog.corning.com/life-sciences/b2c/UK/en/Liquid-Handling/Tubes,-Liquid-
Handling/Centrifuge-Tubes/Falcon%C2%AE-Conical-Centrifuge-
Tubes/p/falconConicalTubes
– Have the patient take a deep breath, hold for a few seconds, exhale, repeat two or
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three times, then cough: sputum is material brought up from the lungs after a
productive cough. One or two minutes of chest clapping are of benefit.
– Collect at least 3 ml and close the container hermetically.
The quality of the sample determines the reliability of the result. Always check that the
sample contains solid or purulent material and not only saliva. Take a new sample if
unsatisfactory.
If the sample is collected at home, make sure that the patient has understood the
technique, including closing the container hermetically after collecting the sputum.
B. Sputum Induction
Sputum induction is sometimes used in children when sputa cannot be spontaneously
collected, and only in order to perform cultures or Xpert MTB/RIF.
Sputum induction must be performed under close medical supervision. The child should
be observed for respiratory distress during, and for 15 minutes after, the procedure.
Bronchospasm may occur. Salbutamol spray and oxygen must be ready at hand.
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Figure 1
procedure-for-obtaining-a-sputum-specimen-11-09-2017/
Equipment:
– Gloves and respirator
– Suction catheter (6, 7, 8F)
– Sputum container
– 50 ml syringe, needle
– Mask and tubing for nebulizer
– Holding chamber with child’s mask (to be sterilized between each patient)
– Sterile hypertonic solution of 5% sodium chloride (to be kept refrigerated)
– Sterile solution of 0.9% sodium chloride (for the specimen)
– Salbutamol spray
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– Oxygen
Procedure
The child should fast for at least 2 hours before the procedure.
- Prior to nebulization:
● Explain the procedure to the child and/or the person accompanying
him/her (this person must wear a respirator).
● Place the child in a sitting position in the adult’s arms.
● Administer 2 puffs of salbutamol via a holding chamber, 10 minutes
before nebulization.
● Prepare a sputum container.
- Nebulization:
● Fill the nebulizer with 5 ml of 5% hypertonic saline solution (sputum
inducer).
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● Place the nebulizer mask over the child’s mouth.
● Leave the child to inhale until the reservoir is empty.
- Nasopharyngeal suction:
● Do 1 to 2 minutes of clapping.
● Clean out the nasal cavity.
● During suction, the child is laid on his /her side, back to the operator, who
is behind him/her.
● Fit a suction catheter to a 50 ml syringe. Lubricate the end of the
catheter.
● Measure the distance from the tip of the nose to the angle of the jaw.
Insert the suction catheter to that depth.
● When inserting and withdrawing the tube, pull on the plunger of the
syringe to create suction.
● Once the syringe is filled with air and mucus, disconnect it from the
suction catheter and purge the air (tip facing upward), so that only mucus
is left in the syringe.
● To collect the mucus: draw 2 ml of 0.9% sodium chloride into the syringe
to rinse, then empty contents into the sample container.
C. Gastric Aspiration
Gastric aspiration is sometimes used in children when sputa cannot be spontaneously
collected nor induced using hypertonic saline, and only in order to perform cultures or
Xpert MTB/RIF.
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https://sentinel-project.org/2014/03/14/webinar-recap-demonstration-of-
gastric-aspiration-technique-in-children/
Equipment:
– Gloves and respirator
– Suction catheter (6, 7, 8F)
– Sputum container
– 50 ml syringe
– Sterile water
Procedure:
- Prior to inserting the suction catheter:

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● Explain the procedure to the child and/or the person accompanying
him/her (this person must wear a respirator);
● Place the child in a half-sitting or sitting position in the adult’s arms.
- Insert a nasogastric tube and check that it is correctly placed.
- First suction to collect the gastric fluid and place it in the sputum container, then
rinse the stomach with 30 ml of sterile water and suction again. Add the
suctioned fluid to the first sample.
- Start culture within 4 hours of collecting the sample. If there will be more than
four hours’ delay, neutralize with 100 mg of sodium bicarbonate.
TRANSPORT:
To a local laboratory
- Without CPC (Cetylpyridinium Chloride) transport medium: between 2 and 8°C
and protected from light;
- With CPC transport medium: should not be refrigerated because at low
temperatures the CPC will crystallize and ruin the sample. Specimens should be
kept at room temperature, protected from heat and light.
By air to a reference laboratory for culture
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● Samples are collected and shipped in 50 ml Falcon® conical tubes (Figure 1)
with screw caps. The tubes are labelled UN 3373, corresponding to Category B
infectious substances. If transport times are less than 12 hours, even specimens
without CPC can be transported at room temperature.
● Samples are triple-packaged, in accordance with IATA packing instruction 650:
1. Primary container holding the sputum sample: tube tightly closed and placed into a
latex glove;
2. Secondary container intended to protect the primary container: leak-proof box with
enough absorbent material to absorb the entire sample, should the primary container
break;
3. Outer packaging intended to protect the secondary container, with UN 3373 labelling.
Information to be provided:
- Primary container: label with the patient’s name or identification number and the
sample collection date and location;
- Outer package: indicate the name of the receiving laboratory, the complete
address (name, street, postal code, locality, country), and telephone number.
All samples must be accompanied by the corresponding laboratory test request form
(including clinical information).
STORE:
When examinations are not performed on the site of collection:
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Specimen for smear microscopy
Smears should be performed within three-four days of collection and in the meanwhile
stored refrigerated (2 to 8°C) and protected from light.
Contamination does not affect microscopy but heat makes specimens liquefy, with
selection of mucopurulent part of the sample more difficult.
Specimen for culture in liquid medium
Keep the specimen refrigerated (2 to 8°C), protected from light. Do not use
cethylpyrodinium chloride (CPC) as it is not compatible with MGIT.
The specimen should be processed as soon as possible.
Specimen for culture on Lowenstein-Jensen medium (LJ)
– Specimens that can be cultured in less than 3 days after collection:
Keep refrigerated (2 to 8°C) and protected from light until transport OR immediately
transport to the laboratory for processing.
− Specimens that will be cultured more than 3 days after collection:
Use Falcon tubes and add 1% CPC to preserve the specimen for up to 2 weeks.
Specimens with CPC should not be refrigerated, as the CPC will crystallize and be
ineffective.
Samples with CPC can be inoculated on LJ. For inoculation on agar, they require prior
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neutralization by neutralizing buffer (Difco).
CPC can be used for specimens tested by Xpert MTB/RIF.
1.3. Blood - CBC and culture
CBC
COLLECT:
Steps: Adult
1. Cleansing of skin area with antiseptic wipe (typically inside of the elbow or back
of hand)
2. Placement of elastic band or a tourniquet, around upper arm to swell vein with
blood
3. Insertion of needle and collection of blood into one or more vials
4. Removal of elastic band or tourniquet
5. Bandage of insertion point to prevent bleeding
https://www.limamemorial.org/health-library/HIE%20Multimedia-
TextOnly/1/003462
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https://medlineplus.gov/ency/presentations/100152_2.htm
*NOTE: Collect a minimum of 12 mL of blood using three 4-mL or larger vacuum–fill
only (unopened), non-gel, purple-top (EDTA) tubes; use four tubes if using 3-mL tubes.
Steps: Infant
1. Sterilization of the heel of foot
2. Prick site with lancet
3. Squeeze heel to collect small blood sample
https://science.sciencemag.org/content/324/5924/166
PACKAGING OF CBC
Consist of:
1. primary receptacles (blood tubes)
2. secondary packaging (materials used to protect primary
receptacles)
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3. outer packaging (polystyrene foam-insulated, corrugated fiberboard
shipper)
Secondary Packaging:
● To facilitate processing, package all blood tubes from the same patient together.
● Place absorbent material between the blood tubes and the first layer of
secondary packaging. Use adequate absorbent material to absorb the entire
contents of the blood tubes.
● Separate each tube of blood collected from other tubes to prevent tube-to-tube
contact.
● Wrap and seal the first layer of secondary packaging with absorbent material
● Seal one wrapped gridded box or alternative container inside a clear, leak-proof
biohazard polybag equivalent to Saf-T-Pak product STP-701, STP-711 or STP-731
● Place this bag inside a white Tyvek® outer envelope (or equivalent) and seal the
opening with a continuous strip of evidence tape initialed half on the packaging
and half on the evidence tape by the individual making the seal
● According to 49 CFR 173.199(b), if specimens are to be transported by air, either
the primary receptacle or the secondary packaging used must be capable of
withstanding, without leaking, an internal pressure producing a pressure
differential of not less than 95 kPa (0.95 bar, 14 psi). Verify in advance that the
manufacturer of either the blood tube or secondary packaging used in your
facility is in compliance with the pressure differential requirement.
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Outer Packaging:
● Use polystyrene foam-insulated, corrugated fiberboard shipper
● For cushioning, place additional absorbent material in the bottom of the shipper
● Add a single layer of refrigerator packs on top of absorbent material
● Place the packaged specimens on top of the refrigerator packs
● Use additional cushioning material to minimize shifting while the shipper is in
transit
● Place additional refrigerator packs on top of the secondary packaging to maintain
a shipping temperature of 1 °C – 10 °C
● Place blood shipping manifest in a sealable plastic bag and put on top of packs
inside the shipper. Place lid on the shipper
● Keep chain-of-custody documents for your files
● Place your return address in the upper left-hand corner of the shipper top and
put CDC’s receiving address in center
● Affix labels and markings adjacent to the shipper’s/consignee’s address that
appears on the shipper
● Place the UN 3373 label and the words “Biological Substance, Category B”
adjacent to the label on the front of the shipper
TRANSPORTING OF CBC
Standard precaution must be used
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● All samples treated as if they are infectious with HIV or hepatitis and other
pathogens of the such
● Must be transported in a sealed biohazard bag
STORING OF CBC
● Should not be kept in room temperature for > 8 hours
● Ideally be stored at 4-8 °C for max 7 days
● If kept for extended periods, maintained at -20°C or lower
Blood Culture
COLLECT:
1. Types of Blood Cultures available
a. Routine blood culture
b. Fungus blood culture
c. Acid Fast blood culture
d. CMV blood culture
2. Number and Timing - Most cases of bacteremia are detected by using 2 sets of
blood cultures, collected separately. A single blood culture may miss intermittent
bacteremia and make it difficult to interpret findings. Blood cultures ordered when a
patient is "spiking a temperature" are considered STAT and the first culture must be
obtained within 10 minutes of the time ordered.
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3. Volume - The volume of blood is critical because the concentration of organisms in
most cases of bacteremia is low.
4. Site selection - If multiple cultures are ordered, select a different phlebotomy site
for each culture drawn. Avoid drawing blood through indwelling intravascular catheters
unless blood cannot be obtained by venipuncture or unless the diagnosis of catheter
sepsis is suspected.
Procedure
1. Wash or sanitize hands before and after removing gloves.
2. Assemble necessary equipment for phlebotomy before preparation of the
patient's skin.
3. Apply tourniquet and identify the phlebotomy site
4. Cleansing the phlebotomy site using Chloraprep® One-Step Frepp
a. apply solution to skin using a side to side motion (no concentric circles
is necessary) for 15 seconds.
b. Reverse the direction and clean up and down for 15 seconds.
c. Allow to air dry for 30 seconds.
d. Avoid touching the chosen phlebotomy site after cleansing.
5. Using a sterile syringe or butterfly, draw 20 ml of blood sample.
6. Place 10 ml of sample into the Anaerobic bottle first, then 10 ml into the
Aerobic bottle.
PACKAGE:
Types of blood culture bottles and blood volumes required:
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● Yellow top – paediatric aerobic (0.5–4 mL blood)
● Green top – adult aerobic (5–10 mL blood)
● Orange top – anaerobic (5–10 mL blood)
● Black top – mycobacteria (5–10 mL blood)
● Silver top – mycoplasma (3–5 mL blood)
https://www.omnia-health.com/product/blood-culture-bottle-media-culture
TRANSPORT:
Ideally, inoculated blood culture bottles should be transported to a microbiology
laboratory immediately for overnight incubation at 35-37°C with ~5% CO2 (or in a
candle-jar) and subsequent culture onto a BAP and CAP. All inoculated blood culture
media should be protected from temperature extremes (not less than 18°C or more
than 37°C) with a transport carrier and thermal insulator (such as extruded polystyrene
foam).
STORE:
Blood cultures can be held at room temperature for up to 12 hours after
collection before placing in the BacTAlert. After 12 hours they can no longer be tested
in the analyzer. Blood culture bottles must be stored in the dark and stored upright, in a
cool, dry place (2-25°C), out of direct light. If the sensor on the bottle turns yellow,
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discard the bottle.
1.4. Vaginal fluid
COLLECT:
Note : The subject must refrain from any kind of sexual activity, douching, and
inserting any intravaginal products for at least 48 hours prior to the collection of
vaginal/cervical specimens. The participant should undress from the waist down and lie
on her back on the exam table for collection of these 16-Female Genital Secretions
Collection Processing-LTC-SOP-16v2.0-2015-08-12 Page 6 of 23 Female Genital
Secretions Collection Processing samples in the clinic. For some protocols, vaginal
swabs may be collected at home; specific instructions will be provided in the protocol
Manual of Operations (MOP).
1. Endocervical canal fluid collected by Tear-Flo™ strip wicking.
- The purpose of this collection procedure is to obtain endocervical canal
fluid for viral RNA quantification.
- Two Tear-Flo™ strips will be used as wicks to collect primarily cell-free
virions from the endocervical fluid. If excess mucus or menses clot has
accumulated near the cervical os, a large cotton-tipped swab may be used
to gently remove this material before inserting the strips.
- Use forceps (ring or sponge forceps work well) to hold two Tear-Flo™
strips on the squared end and gently insert the two strips simultaneously
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into the vagina, placing through the cervical os into the distal endocervical
canal and hold in place to adsorb sample.
2.Vaginal aspirate of ectocervicovaginal fluid.
- The participant should undress from the waist down and lie on her back
on
the exam table for approximately 15 minutes prior to beginning collection
of this sample.
- Partially peel the aspirator envelope open, exposing the plunger.
- Put on clean gloves.
- Remove the aspirator from the package. Do not discard the package (the
aspirator may be returned to the package after sample collection). Pull the
plunger away from the tip of the aspirator and then push it back towards
the tip.
Do this a few times to loosen the plunger and prevent it from sticking.
When
done, push the plunger all the way down, towards the tip.
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- While separating the labia with one hand, use the other hand to hold the
plunger-end of the aspirator between the thumb and forefinger. Insert the
rounded end (tip) of the aspirator into the participant’s vagina until it
touches the back of the posterior fornix (imagine trying to reach the area
below the cervix). Pull the entire aspirator out ever so slightly to avoid
suction of tissue.
- Hold the aspirator in place with one hand, and using your other hand,
very slowly pull the plunger out of the aspirator (away from the tip). This
will cause vaginal fluid to be drawn into the aspirator. Continue to pull the
plunger out with one hand while removing the aspirator from the vagina
with the other hand.
3. Vaginal Swab
- Insert a Dacron swab gently and rotate 360 degrees in all four quadrants
of the vaginal vault.
4. Ectocervicovaginal lavage (CVL).
- The purpose of this collection procedure is to obtain a washing of virus
and cells from the ectocervix and fluid from the posterior vaginal fornix for
viral and immunologic studies.
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- Draw up 10mL of either nonbacteriostatic normal saline (saline for
irrigation) or 1X phosphate buffered saline in a 10mL syringe.
- Introduce the syringe through the speculum to the opening of the cervical
os, but do not insert into the os .
- Aim a continuous stream of saline directly at and into the os to bathe the
cervix and the ectocervix.
5. Endocervical Swab
- Gently insert a Dacron swab 1cm into the cervical os and rotate 360
Degrees.
6. Endocervical canal cytobrush.
- The purpose of this collection procedure is to obtain primarily cells for
viral DNA quantification. Gently insert a cytobrush with a plastic shaft
1cm into the cervical os and rotate exactly 360 degrees. Note: bleeding
usually occurs with the cytobrush
7.Papanicolaou test (Pap test)
- Follow local instructions or protocol-specific instructions (if applicable) for
collecting and processing Pap test specimens as part of an ACTG protocol.
8. Cervical or Vaginal Biopsy (or both)
- A cervical and/or vaginal biopsy will provide tissue for immunologic,
pharmacologic, or virologic assessment. Generally, no prior preparation,
such as fasting or sedation, is required for a simple cervical or vaginal
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biopsy. The participant can take a pain reliever 30 minutes before the
procedure.
- Put participant in lithotomy position and insert a speculum to visualize the
vagina and cervix.
- Spray the cervix and/or vaginal fornices with topical 20% benzocaine
spray.
- Anesthetize the area of biopsy using a small needle to inject 2% lidocaine
solution.
- Tenaculum forceps may be used to hold the cervix steady for the biopsy.
The participant may feel some cramping when the tenaculum forceps is
applied.
PACKAGE:
1. Endocervical canal fluid collected by Tear-Flo™ strip wicking
- Place the round end of the two strips over and slightly inside one labeled
2mL cryovial. Cut the strips at the “15” mark with clean scissors, allowing
the round end to fall into the cryovial. Securely cap the cryovial. , Discard
the square end after cutting.
2. Vaginal aspirate of ectocervicovaginal fluid.
- Using the graduation marks on the barrel of the aspirator, determine the
volume of fluid that was collected. Be sure that air bubbles have been
eliminated before reading the volume. A minimum of 200 µL of fluid
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should be collected. If the aspirator did not collect enough vaginal fluid
with the first collection attempt, the aspiration may be repeated.
3. Vaginal Swab.
- Place the swab into a sterile 2mL cryovial. Break or cut the shaft short
enough to fit in the cryovial and allow the cap to be tightly sealed.
4. Ectocervicovaginal lavage (CVL).
- Use clean scissors to cut a sterile plastic transfer pipette just below the
bulb. Discard the bulb and place the pipette tip on the syringe.
Alternatively, a 14-gauge angiocath can be inserted over the tip of a 10mL
syringe. It may be helpful to seal the junction with parafilm
- Aspirate and transfer the fluid to a sterile 15mL conical polypropylene
tube.
5. Endocervical Swab
- Place the swab into a sterile cryovial. Break or cut the shaft short enough
to fit inside the cryovial and allow the cap to be tightly sealed.
- Place the brush in the vial, being certain that the brush is immersed in the
Digene transport medium (if applicable), and so that the scored area is
approximately even with the lip of the vial. Hold the vial containing the
swab upright with one hand and bend the shaft with the other hand,
snapping off the top of the swab handle. Firmly tighten the lid of the
cryovial.
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TRANSPORT:
1. Vaginal aspirate of ectocervicovaginal fluid
- If the sample will be transported to the laboratory to be dispensed into a
cryovial, carefully slide the aspirator back into the envelope, tip first,
without touching the outside of the envelope. Be careful that the tip of the
aspirator does not touch anything as it is put back into the envelope. The
plunger should remain extended.
- The sample may also be dispensed into the cryovial immediately after
collection. To do so, dispense the fluid into a sterile, screw top 1.8-2.0mL
cryovial by slowly pushing the plunger all the way down toward the tip.
Take care that the tip of the aspirator does not touch anything other than
the inside of the sterile tube before the sample is dispensed.
2. Vaginal Swab and Endocervical Swab
- Specimens should be transported to the laboratory within one hour.
3. Endocervicovaginal lavage
- Transport to the laboratory within 1 hour of collection.
- Cytobrushes in dry cryovials should be transported to the processing lab
within 4 hours and frozen within 6 hours. Clinicians should send the
sealed vial to the local laboratory for processing.
STORE:
1. Vaginal aspirate of ectocervicovaginal fluid
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- Deliver the sample to the laboratory for storage at -70°C until shipment
(refer to the Laboratory Processing Chart [LPC]).
2. Vaginal Swab, Endocervicovaginal lavage, and Endocervical Swab
- Place the specimen on wet ice or refrigerate at 4°C until transport, up to 4
hours. If the vial is to be placed on wet ice, seal the cryovial in a plastic
baggie or equivalent to keep exterior of vial dry.
3. Endocervical canal cytobrush
- Cytobrushes in the Digene fluid can remain at 4 degree Celsius for up to
72 hours.
1.5. Seminal fluid
Collect: Refrain from any sexual activity (including masturbation) for at least 2 days
and no more than 10 days. Longer or shorter periods of abstinence may result in a
lower sperm count or decreased sperm motility. A private room is provided for semen
collection within close proximity. It should be obtained through masturbation.
PACKAGE:
The specimen should be collected in a container provided by the ART lab. Ensure that
hands and penis are cleaned prior to collection. Avoid touching the inside of the cup. If
any semen is spilled, do not attempt to transfer it to the cup. Inform the lab personnel
about the spill. If the specimen was obtained outside of the collection room, bring the
specimen to the laboratory within one hour after ejaculation. Do not expose the
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specimen to extremes of temperature. Place specimen, container upright in a plastic
bag, with the lid securely tightened, and keep specimen close to body temperature by
transporting close to the body. The specimen should not be placed in a purse, pocket,
or briefcase. Sperms do not have a long life outside of the body and at different
temperatures. Delays in delivering semen and exposure to various temperatures will
result in lower overall motile sperm count and poor semen cryopreservation.
TRANSPORT:
The complex process of sperm transport through the female reproductive tract begins
at the time of ejaculation. During coitus, 1.5- to 5.0-ml of semen containing between
200 and 500 million sperm is deposited at the posterior vaginal fornix, leaving the
external cervical os partially submerged in this pool of fluid.
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STORE:
Semen should be stored at a constant temperature of 17°C±2°C in order to maintain
semen viability and maximise shelf life. Semen is extremely temperature sensitive: shelf
life is shortened at temperatures above 20°C; while temperatures below 15°C are likely
to reduce sperm viability. Semen doses should always be treated carefully to prevent
damage from rough handling and protected from exposure to light.
1.6. Pericardial fluid
COLLECT:
- Some people may have a cardiac monitor placed before the test to check for
heart disturbances. Patches called electrodes will be placed on the chest, similar
to during an ECG. A chest x-ray or ultrasound may be done before the test. The
skin of the chest will be cleaned with antibacterial soap. A health care provider
inserts a small needle into the chest between the ribs into the thin sac that
surrounds the heart (the pericardium). A small amount of fluid is removed. You
may have an ECG and chest x-ray after the test. Sometimes the pericardial fluid
is taken during open heart surgery.
PACKAGE:
- Samples of the fluid are placed on dishes of growth media to see if bacteria
grow. It can take a few days to several weeks to get
the test results.
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TRANSPORT:
- Pericardial fluid coloring studies report that the fluid distribution inside the cavity
is heterogeneous. The largest amount is inside the atrioventricular and the
intraventricular sulcus, the superior and the transversal sinus, especially on the
supine position. Nevertheless, there are some pharmacokinetic studies that show
that the pericardial fluid is stirring up constantly and thus the supplement's
composition is the same regardless of the position.
STORE:
- Room temperature - 7 days
- Refrigerated - 7 days
- Frozen - 28 days
- Cells may degenerate during storage. Therefore, the pericardial fluid sample for
cytopathology study should be sent to the laboratory as soon as possible in a
fresh state or refrigerated at 2-8º C.
1.7. Peritoneal fluid
COLLECT:
PROCEDURE:
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1. Urinary bladder should be emptied.
Emptying the bladder first helps prevent possible injuries to the bladder during the
procedure.
http://www.alchemyinmotion.com.au/incomplete-bladder-emptying/
2. Let the patient lie in a supine or semi reclined position.
This position allows patients to remain in a natural alignment while the procedure is
ongoing.
https://brooksidepress.org/basic_patient_care/lessons/lesson-2-positioning-the-
patient/2-03-positioning-the-patient/
3. Large bore I.V. needle is introduced in the midway between symphysis
pubis and umbilicus.
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A large bore IV needle may be used in order to collect higher volume of fluid or to
simply maximize fluid administration speed.
https://en.wikipedia.org/wiki/Paracentesis
4. The needle is connected with a rubber tubing which drains the fluid
into a container.
Connecting the needle to a rubber tubing simply allows stabilization of tube
engagement when drawing the fluid from the patient.
https://www.renax.com.tw/en/2-2008-160405/product/Peritoneal-Dialysis-Supplies-
id635184.html
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5. 20-50 mL of the fluid should be enough for the diagnostic procedures.
Usually, a minimum of 50 mL is collected as the sensitivity of detecting malignant cells
decreases with low-volume specimens.
https://www.healthline.com/health/peritoneal-fluid-culture
GRAM STAINING PERITONEAL FLUID
Peritoneal fluid analysis may be ordered when suspecting a condition or disease that is
causing peritonitis or ascites. The following is the procedure for the gram stain:
1. Drop a small amount of peritoneal fluid into the microscope slide.
2. First, crystal violet, a primary stain, is applied, giving all of the cells a purple
color.
3. Next, Gram’s iodine, a mordant, is added.
4. Next, a decolorizing agent is added, usually ethanol or an acetone/ethanol
solution.
5. Finally, a secondary counterstain, usually safranin, is added.
The purple, crystal-violet stained cells are referred to as gram-positive cells, while the
red, safranin-dyed cells are gram-negative. Take note that gram stains are useless in
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ascites fluid. The concentration of organisms just won’t be high enough to see
something on gram stain.
PERITONEAL FLUID CULTURE
A peritoneal fluid culture is a test that is performed on a small sample of peritoneal
fluid. It may also be called an abdominal tap or paracentesis. The laboratory examines
the fluid for any bacteria or fungi that may be causing an infection. Here are the
following steps in collecting such:
1. Let the patient empty their bladder before the procedure.
2. Clean the spot on their lower abdomen with an antiseptic and let the patient
receive local anesthesia to numb the area.
3. Insert a needle with 1-2 inches in length to the abdominal cavity. A small incision
may be necessary if there is difficulty inserting the needle.
4. Fluid is then removed through a syringe.
Specimen Processing:
Media
● Blood Agar (2 plates)
● Chocolate Agar
● MacConkey Agar
● Thioglycollate broth
Common Pathogens of the Peritoneal fluid:
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PACKAGE:
The preferred container is a sterile specimen container and make sure to refrigerate
or keep on wet ice until it is transported to the laboratory.
1. Without anticoagulant
2. With anticoagulant by addition of 3.0 ml of 20% solution sodium citrate to the
container prior to autoclaving.
Labeling
● Patient’s name
● Specimen type
● Unique ID number
● Date, time, and place of collection
● Name/initials of collector
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http://sterilmedical.com/product/sterile-container/
TRANSPORT:
Take note that it is a must for the peritoneal fluid to be transported to the laboratory as
soon as possible. Guidelines are also issued by national authorities which should be
strictly followed:
● Ideally specimens should be transported to the laboratory as quickly as possible
and processed in the laboratory within 1-2 hours after collection.
● In case of delay of more than 2 hours the specimen should be transported in
transport media or refrigerated.
Method of Transportation:
● For hand carried transportation over a short distance, the specimen should be
placed upright in appropriate racks.
● For long distance transportation, packaging is done in three containers.
STORE:
Turn Around Time STAT : within 2 hours
(upon the receipt of the laboratory) Routine : within 4 hours
Specimen Stability and Storage 16 hours at room temperature
Availability Daily (24 hours)
Special Processing Centrifuge remaining fluid for culture and
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Gram stain.
1.8. Synovial fluid
COLLECT:
PROCEDURE:
1. Clean the skin
A health care provider will clean the skin on and around the affected joint. Disinfecting
the skin with alcohol prior to invasive procedures is a
must to prevent infections caused by bacteria on the
skin
https://www.123rf.com/photo_80204071_disinfecting-of-the-skin-before-an-
injection.html
2. Numb the area with Local Anesthesia
The provider will administer an anesthetic and/or apply numbing cream to the skin to
ensure that the client is pain- free during the operation.
https://www.pnpspecialists.com/interventional-pain-therapies/shoulderkneehip-joint-or-
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bursa-injections/
3. Extract fluid by injecting
A large needle syringe is usually used when extracting knee synovial fluid from a
patient. Syringes 5mL, 20mL, 30mL and 60mL and needle gauge 18 or 20, and 25 or 27
are often used. However, the needle gauge and syringe depends on the amount of
synovial fluid needed. For morbidly obesed patients, a 21-
gauge spinal needle is used for arthrocentesis.
https://www.tedpella.com/Embedding_html/Disposable_Syringes.htm
https://www.stepwards.com/?page_id=24781
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GRAM STAINING SYNOVIAL FLUID
● After collecting synovial fluid specimens via injection (at least 1mL), it is then
placed in a red top tube container. The fluid sample is sent to a lab for further
testing.
Gram staining procedure:
1. Drop a small amount of synovial fluid onto a microscope slide.
2. Apply crystal violet solution (purple dye) on the specimen.
3. Apply iodine (mordant) after applying violet solution.
4. Decolorize by applying ethanol.
5. Apply safranin as a counterstain
https://laboratoryinfo.com/gram-staining-principle-procedure-interpretation-and-
animation/
Note: Gram positive will yield a violet color, while Gram negative will yield a pink
color.
Gram staining serves as an extremely important “clue” when attempting to learn the
identity or the species of bacteria found in a specimen
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SYNOVIAL FLUID CULTURE
Culture of the synovial fluid is a definitive method of diagnosing septic arthritis.
Patient Preparing:
1. Swab skin over the site of puncture with 2% tincture of iodine in concentric
circles
2. Iodine should remain in contact with skin for at least 1 minute prior to puncture
to ensure complete antisepsis
Specimen Collection:
1. Needle is inserted to the joint. Fluid is withdrawn
2. Specimens should be processed immediately. Centrifuge clear specimen and
inoculate plates and do staining from sediments. Note that turbid specimens may
not be centrifuged.
Specimen Processing
Media is through
● Blood Agar (2 Plates)
● Chocolate Agar
● MacConkey Agar
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Common Pathogens for Bones and Joints
Synovial Fluid Analysis
PACKAGE:
Normal synovial fluid will not clot; however, fluid from a diseased joint may contain
fibrinogen and form a clot. Therefore, both anticoagulant and non-anticoagulant
specimens should be collected.
Each specimen must be accompanied by a completed and matching Synovial fluid
cytology request form. All specimen containers must be clearly labelled with
1. Patient’s full name
2. Date of Birth
3. NHS and/or Hospital number
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4. Aspiration site
Recommended specimen containers:
EDTA tube (lavender) - for hematology cell counts, differentials and viscosity.
Immediately after collection, an aliquot of synovial fluid may be added directly from the
aspirating syringe into the tube.
https://www.allstarmedsupply.com/collection-tubes-needles/63-vacutainer-lavender-
top-4ml-tubes.html
Heparinized (Green) - for chemistry and immunologic tests. Fluids should be added
to the tube immediately after collection to avoid clot formation. Gently invert 5-10 times
to ensure adequate mixing of fluid and the anticoagulant.
https://www.cpllabs.com/clinicians/specimen-collection/tube-types/
Plain Sterile Tube - container must be preservative free for microbiology testing and
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crystal examination
https://www.amazon.com/Simport-Micrewtube-T335-4S-Polypropylene-Self-
Standing/dp/B008S296DE
Capped Syringe - for synovial fluids, these are
acceptable. Needle must be removed before submitting
to lab
https://imiweb.com/product/tamper-evident-cap-for-iv-syringes-5/
TRANSPORT AND STORE:
Samples taken at central site must be sent with the porter and not via the
pneumatic tube
Synovial fluid samples requiring transport on the public road must be packaged and
transported in compliance with the “The Carriage of Dangerous Goods and Use of
Transportable Pressure Equipment Regulations (ADR Regulations) 2011”. Specimens
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must be packaged according to P650 instructions with a UN3373 diamond point label –
Biological
Substance, Category B.
Please note instructions P650 requires three layers of packaging:
● Primary container (e.g. universal tube, vial)
● Secondary container (e.g. specimen bag)
● Outer packaging (e.g. rigid transport box)
The primary sample must be individually bagged in a secondary bag and sealed. If the
sample is liquid, enough absorbent material must be added to the secondary bag to
absorb a potential spillage of the sample. The request form must be placed in the
specimen bag’s separate pouch.
Specimens must then be placed in a rigid box and closed. The box must comply with
Transport Regulations. The outside must be clearly labelled Biological Substance
Category B, with a UN3373 diamond label. The laboratory address should be clearly
written.
Specimens must be delivered to the laboratory within 24 hours. If there is
unavoidable delay in sending the specimen, please keep it refrigerated at
4°C.
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1.9. Cerebrospinal fluid
COLLECT:
The collection of CSF is an invasive procedure and should only be performed by
experienced personnel under aseptic conditions. If bacterial meningitis is suspected,
CSF is the best clinical specimen to use for isolation, identification, and characterization
of the etiological agents.
a. Preparing for lumbar punctureIf possible, three tubes (1 ml each) of CSF should
be collected for microbiology, chemistry, and cytology. If only one tube of CSF is
available, it should be given to the microbiology laboratory. Because the
presence of blood can affect cultures of CSF, if more than one tube of CSF is
collected from a patient, the first tube collected (which could contain
contaminating blood from the lumbar puncture) should not be the tube sent to
the microbiology laboratory.The kit for collection of CSF should contain (Figure
1):
1. Skin disinfectant: 70% alcohol swab and povidone-iodine
■ Alcohol with concentrations greater than 70% should not be used
because the increased concentrations result in decreased
bactericidal activity. Do not use alcohol with glycerol added to it.
2. Sterile gloves
■ Be sure to check the expiration date.
3. Sterile gauze
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4. Surgical mask
5. Adhesive bandage
6. Lumbar puncture needle
■ 22 gauge/89 mm for adults
■ 23 gauge/64 mm for children
7. Sterile screw-cap tubes
8. Syringe and needle
9. Transport container
10. T-I medium (if CSF cannot be analyzed in a microbiological laboratory
immediately)
■ T-I should be refrigerated at 4°C and added to the kit immediately
before use in the field.
11. Venting needle (only if T-I is being used)
12. Instructions for lumbar puncture and use of T-I medium
13.
Figure 1. Kit for collection of cerebrospinal fluid (CSF)
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B. Lumbar puncture procedure. Follow all appropriate biosafety precautions (see
Section I).
1. Gather all materials from the CSF collection kit and a puncture-resistant
autoclavable container for used needles.
2. Wear surgical masks and sterile latex or nitrile gloves that are
impermeable to liquids and change gloves between every patient.
3. Label the collection tubes with appropriate information: patient’s name,
date and time of specimen collection, and Unique Identification Number.
Be sure this number matches the number on both the request and report
forms.
4. Ensure that the patient is kept motionless during the lumbar puncture
procedure, either sitting up or lying on the side, with his or her back
arched forward so that the head almost touches the knees in order to
separate the lumbar vertebrae during the procedure (Figure 2).
5. Disinfect the skin along a line drawn between the crests of the two ilia
with 70% alcohol and povidone-iodine to clean the surface and remove
debris and oils. Allow to dry completely.
6. Position the spinal needle between the 2 vertebral spines at the L4-L5
level and introduce into the skin with the bevel of the needle facing up.
■ Accurate placement of the needle is rewarded by a flow of fluid,
which normally is clear and colorless.
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7. Remove CSF (1 ml minimum, 3-4 ml if possible) and collect into sterile
screw-cap tubes. If 3-4 ml CSF is available, use 3 separate tubes and
place approximately 1ml into each tube.
8. Withdraw the needle and cover the insertion site with an adhesive
bandage. Discard the needle in a puncture-resistant, autoclavable discard
container.
9. Remove mask and gloves and discard in an autoclavable container.
10. Wash hands with antibacterial soap and water immediately after removing
gloves.
Figure 2. Collection of cerebrospinal fluid (CSF) by lumbar puncture
PACKAGE:
Specimens are usually collected in four sterile
tubes
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https://www.amazon.com/Simport-Micrewtube-T335-4S-Polypropylene-Self-
Standing/dp/B008S296DE
The tubes are usually disbursed for analysis as follows:
● Tube #1: Chemistry and serology tests. This tube should never be used for
Microbiology since it is most likely to contain skin contaminants.
● Tube #2: Microbiology
● Tube #3: Cell counts
● Tube #4: Miscellaneous or referral test request.Specimens must be transported
to the laboratory within 1 hour of collection to prevent deterioration of cells and
glucose.
TRANSPORT:
CSF specimens should be transported to a microbiology laboratory as soon as possible.
Specimens for culture should not be refrigerated or exposed to extreme cold, excessive
heat, or sunlight. They should be transported at temperatures between 20°C and 35°C.
For proper culture results, CSF specimens must be plated within 1 hour (See figure
below)
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https://www.cdc.gov/meningitis/lab-manual/images/chapt5-figure04.gif
STORE:
● The remaining CSF should be kept in the collection tube. It should not be
refrigerated, but should be maintained at room temperature (20-25°C) before
Gram staining and other tests.
● CSF samples can be stored in the refrigerator (2 – 6.0° C) for up to one week.
● CSF samples can be stored frozen (≤ -60.0 ° C) indefinitely
1.10. Gastric fluid
Gastric aspiration is a diagnostic procedure used to diagnose cases of pulmonary
tuberculosis in children and adults. The procedure involves obtaining gastric juices from
the stomach and testing them for an active tuberculosis infection. Gastric aspiration is
mostly used in children under six years old who are unable to produce sputum.
However, it is also used in adults who have negative results for tuberculosis (TB) on
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sputum smear examinations.
The following equipment is needed:
–gloves
– nasogastric tube (usually 10 French or larger)
– syringe of capacity 5, 10, 20 or 30 ml, with appropriate connector for the
nasogastric tube
– litmus paper
– specimen container
– pen (to label specimens)
– laboratory requisition forms
– sterile water or normal saline (0.9% NaCl)
– sodium bicarbonate solution (8%)
– alcohol/chlorhexidine.
The Procedure:
1. Find an assistant to help.
2. Prepare all equipment before starting the procedure.
3. Position the child on his or her back or side. The assistant should help to hold the
child.
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4. Measure the distance between the nose and stomach, to estimate how far the
tube will need to be inserted to reach the stomach.
5. Attach a syringe to the nasogastric tube.
6. Gently insert the nasogastric tube through the nose and advance it into the
stomach.
7. Withdraw (aspirate) gastric contents (2–5 ml) using the syringe attached to the
nasogastric tube.
8. To check that the position of the tube is correct, test the gastric contents with
litmus paper: blue litmus turns red in response to the acidic stomach contents.
(This can also be checked by pushing some air (e.g. 3–5 ml) from the syringe
into the stomach and listening with a stethoscope over the stomach.)
9. If no fluid is aspirated, insert 5–10 ml sterile water or normal saline and attempt
to aspirate again. If still unsuccessful, repeat the procedure. (Even if the
nasogastric tube is in an incorrect position and water or normal saline is inserted
into the airways, the risk of adverse events is still very small). Do not repeat
more than three times.
10. Withdraw the gastric contents (ideally at least 5–10 ml).
11. Transfer gastric fluid from the syringe into a sterile container (sputum collection
cup).
12. Add an equal volume of sodium bicarbonate solution to the specimen (in order to
neutralize the acidic gastric contents and so prevent destruction of tubercle
bacilli).
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COLLECT:
The doctor will then insert a tube into the nostril and guide it down towards the
stomach. An empty syringe is attached to the other end of the tube to aspirate the
stomach contents. Aspiration is performed three times with the patient being placed in
different positions.
At least 5ml of aspirate stomach contents needs to be retrieved. If the amount is
insufficient, the doctor may need to perform a gastric lavage procedure. This involves
passing at least 10 ml of sterile water down the tube and leaving it inside the stomach
for around 3 minutes. The contents of the stomach will then be aspirated until sufficient
amount of gastric juices is obtained.
Figure 3.1 - Visualization of Gastric Aspiration
PACKAGE:
Usually contained in a sterile container (sputum collection cup).
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Figure 3.2 - Sputum collection cups
TRANSPORT:
Transport the specimen to the microbiology lab. If a special bicarbonate containing tube
or cup is not available, the lab must neutralize the stomach acid with bicarbonate within
1/2 hour.
STORAGE:
● Gastric juice collected for analyses should be stored on ice until the analyses can
be performed. If analysis is delayed for more than 4 h, the samples should be
stored in a refrigerator.
● Samples collected for IF and protein determination should be stored on ice until
they can be depepsinized and the assay performed or stored in a refrigerator.
The samples can be stored for 5 days with only 7% loss of IF activity.
● Samples for the estimation of pepsin should be stored in a refrigerator for not
more than 1 week to ensure retention of significant peptic activity.
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● For periods greater than 1 week, gastric juice mixed with glycerol can be stored
at −20°C for many months without loss of peptic activity.
1.11 Pleural Fluid
A pleural aspiration is a procedure where a small needle or tube is inserted into the
space between the lung and chest wall to remove fluid that has accumulated around
the lung. This space is called the pleural space.
Pleural aspiration is usually carried out to determine why there is fluid around the lung
(diagnostic procedure) or to improve symptoms (therapeutic procedure), as the fluid
around the lung may be causing symptoms such as cough, shortness of breath or chest
pain.
Equipment required for pleural aspiration
○ Ultrasound machine and an operator who is at least level one competent at
pleural ultrasound
○ Sterile ultrasound probe cover
○ Sterile gloves
○ Sterile field and dressing
○ Chlorhexidine cleaning solution
○ Lignocaine
○ Remember 3mg/kg is the maximum safe dose
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○ 5mls of 2% preparation contains 100mg lignocaine. The max
dose for a 70kg person is therefore approximately 10mls 2%
lignocaine.
○ 50ml syringe and green needle
COLLECT:
A small cut is made in the skin and a needle or thin plastic tube is inserted into the
space between your lung and chest wall to remove the fluid. The doctor may use an
ultrasound to see the inside of your chest on a screen, as this can help find where the
largest area of fluid is located.
Most frequently, this area is at the back of your chest or to the side. This is the reason
why the doctor stands behind you while doing the pleural aspiration. You may be asked
to hold your breath by the doctor who is carrying out the procedure.
GRAM STAINING OF PLEURAL FLUID
A sample of the fluid can be removed for testing. This process is called thoracentesis.
One test that can be done on the pleural fluid involves placing the fluid onto a
microscope slide and mixing it with a violet stain (called a Gram stain). A laboratory
specialist uses a microscope to look for bacteria on the slide.
If bacteria are present, the color, number, and structure of the cells are used to identify
the type of bacteria. This test will be done if there is concern that a person has an
infection involving the lung or the space outside the lung but inside the chest (pleural
space).
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https://nationalpost.com/other/pleural-fluid-gram-stain
CULTURE OF PLEURAL FLUID
A pleural fluid culture is a test used to see if this fluid contains any bacteria, viruses, or fungi.
Procedure:
 Once the sample is extracted, it is placed in a petri dish to observed if bacteria or
any other microorganisms grow which can take several days.
 Inoculate 2 to 5 mL pleural fluid into aerobic and anaerobic blood culture
bottles. This increases the sensitivity of bacterial cultures by 20%
 For pleural tuberculosis, the inoculation of 5 mL into liquid culture media is as
beneficial (50% positivity) as the use of larger volumes
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https://www.healthing.ca/other/pleural-fluid-culture
Media:
● Blood Agar (2 Plates)
● Chocolate Agar
● MacConkey Agar
Common Pathogens for Pleural Fluid
Package:
The fluid usually drains through a collection bottle or a sterile specimen container.
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Figure 3.2 - Sputum collection cups
TRANSPORT:
Ideally, fluid specimens should be sent immediately to the laboratory and processed
within 2 hours.
STORAGE:
If a delay is expected, the sample should be maintained at 4ºC until analysis, except for
microbiological cultures. A delayed analysis of more than 48 hours is unacceptable,
although the cytomorphological features of refrigerated samples are well preserved for
at least 72 hours
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Cebu Doctors’ University

Collection, Labelling, Handling and Care of Specimen – Part 2

In Partial Fulfillment of MC3: Microbiology and Parasitology Laboratory Course

1. Rivera, Franchesca Claire – Group Leader

April 20, 2021

Group 4: Collection, Labelling, Handling and Care of Specimen – Part 2

1. Comprehensively discuss how to collect, package, transport, store the following

laboratory specimen for the following examinations:

1.1. Urine – urinalysis and culture

1.2. Sputum – DSSM/ AFB and MTB GeneXpert

1.3 Blood – CBC and Blood culture

1.4. Vaginal fluid

1.5. Seminal fluid

1.6. Pericardial fluid

1.7. Peritoneal fluid

1.8. Synovial fluid

1.9. Cerebrospinal fluid

1.10. Gastric fluid

1.11 Pleural fluid

following laboratory specimen for the following examinations:

1.1. Urine – urinalysis and culture

1. Routine or random sample: The patient is given a non-sterile collection container

until it can be tested.

3. Timed specimen: Timed specimens are usually a 24 hour urine collection.

with the patient’s name and date.

checked for preservative requirements.

D. The 24 hour urine specimen is brought to the laboratory as soon as possible

as the 24- hour period is over

A. Give the patient a sterile urine collection kit..

B. Explain to the patient that an instruction sheet is included in the kit.

C. Male urine culture collection instructions:

2. Open the collection package but DO NOT TOUCH INSIDE OF CUP OR

inside of the container or rim. Gently grasp the container.

Finish voiding into the toilet.

careful to avoid touching inside the container or lid.

6. Bring the specimen to the lab within 1 hour or collection or store

refrigerated for up to 24 hours.

single downward stroke. Discard towelette.

of the container or rim. Gently grasp the container.

Finish voiding into the toilet.

to avoid touching inside the container or lid.

6. Catheterized specimen: These specimens are collected by specially trained

Preferred Specimen Container

the preservative ratio being toxic to the organisms.

● The preservative allows for room temperature transport or these vacutainer

tubes come with a sterile straw transfer device.

Alternative Specimen Containers

for specimens that can be delivered immediately or overnight.

● These unpreserved liquid specimens should be kept cool during transport.

Alternative Specimen Containers

transport swab of the urine. (Results include a semi-quantitative assessment)

● These specimens should be kept cool during transport

❏ Ensure the transport container lid is secure and leak resistant

number or for outpatients, full name and date of birth).

specimens must be analyzed within 2 hours of collection)

Specimen Transfer to Vacuette Urine Culture Transport Tubes

NOTE: if urine is being submitted for urinalysis/cytology it must be submitted separately

in a plain tube or specimen cup without additives.

The transfer straw can be used to draw

up urine from cup or tube.

Immerse the straw into the urine (4mL

While the straw is immersed, insert the

vacuette into the straw device to draw up