Chapter One

Chapter One
Specimens Collection and Processing
Diagnostic Microbiology Dr. Walid Abu Rayyan 1

Medical Microbiology: revision

chapter # 1: INTRODUCTION
 Basic Principles of Specimen Collection :
 The laboratory can make accurate and useful determinations only if a

specimen has been collected properly.
 The specimens to be analyzed are likely to contain living organisms; the goal
of the specimen collector must be to maintain the viability of these organisms
with minimal contamination

1- If possible, collect the specimen in the acute phase of the infection and before
antibiotics are administered.
2- Select the correct anatomic site for collection of the specimen.
3- Collect the specimen using the proper technique and supplies with minimal
contamination from normal biota (normal flora).
4- Collect the appropriate quantity of specimen.

5- Package the specimen in a container designed to maintain the viability of the

organisms and avoid hazards that result from leakage.
6- Label the specimen accurately with the specific anatomic site and the patient
information's.
7- Transport the specimen to the laboratory promptly or make provisions to store

the specimen in an environment that will not degrade the suspected organism(s).

 Collection Procedures:
- Specimens for microbiology cultures should be collected in sterile containers,

Except for stool specimens, which can be collected in clean, leak proof
containers.
- In general, swabs are not recommended for collection because they do not
provide sufficient quantity , easily contaminated, and can become dried out,
leading to a loss of organisms.

- Swabs are appropriate for specimens from the upper respiratory tract,
external ear, eye, and genital tract.
- The tips of swabs may contain cotton, Dacron, or calcium alginate.
- Cotton-tipped swabs tend to have excessive fatty acids, which may be toxic
to certain bacteria.
- Dacron or polyester swabs have a wide range of uses.

- Swab collection systems are available that provide transport media and
protect the specimen from drying.
- The term wound is not an appropriate specimen label, and the exact anatomic
site must be provided.
- The specimen is collected from the advancing margin of the lesion and
should be collected by needle aspiration rather than by swab.

- Before the specimen is collected, the area should be cleansed to eliminate as

much of the commensal flora as possible.
- Aspirated material should be placed into a sterile tube or transport vial and
not “squirted” onto a swab.

- For COVID 19, Zymo media was used as a transport

media

 Patient-Collected Specimens:
- Certain specimens are collected by the patients themselves. Ideally, this
should be done with assistance from medical personnel following thorough
instructions.
- It should never be assumed that the patient knows how to collect a particular
type of specimen.
-
- Attaching printed instructions in multiple languages to a collection device
does not ensure that patients will read them or understand them.
- The most effective method is to provide verbal and written instructions.

 Specimen Collection Guidelines:
• Blood culture
Disinfect skin with alcohol and iodine, Blood

culture media set (aerobic and anaerobic,
bottles) or Vacutainer tube with SPS (sodium
polyanethol sulfonate) /adults, 20 mL per set:
children 5 to 10 mL per set

• Body fluids :(abdominal, amniotic,

ascites, bile, joint, pericardial, pleural)
Disinfect skin before needle aspiration Sterile,

screw-cap tube ≥1 mL
Catheter tips, IV:

Disinfect skin before removal Sterile, screw-cap
container

• Cerebrospinal fluid
Disinfect skin before aspiration
Sterile, screw-cap tube/bacteria ≥1 mL, fungi, ≥2 mL, AFB ≥2 mL, virus ≥1 mL
•Ear
1. Inner: Clean ear canal with mild soap, Aspirate fluid, with needle, if
eardrum intact; Use swab, if eardrum ruptured, Sterile, screw-cap tube or
anaerobic transport system
2. Outer: Remove debris or crust from ear canal with saline moistened swab;
rotate swab in outer canal, Swab Transport system
•Eye:
1. Conjunctiva Sample both eyes; use separate swabs moistened with sterile
saline.
2. Corneal scrapings Instill local anesthetic, scrape with sterile spatula, and
inoculate directly to agar, Agar available at bedside

• Feces
Collect directly into container, avoid contamination with urine,

Clean, leakproof container or enteric transport system.
A rectal swab can be submitted for bacterial culture, but it must show feces. A
single specimen is not usually sufficient to exclude bacteria or parasites.
If a bacterial infection is suspected, three specimens should be collected, one a

day for 3 days.
• Feces
If parasites are suspected, three specimens collected within 10 days should be

sufficient for microscopic detection of ova and parasites.
The newer methods detect parasite antigens, and one sample is usually
sufficient.
Commercial systems are available with preservatives for bacteria and parasites.
The appropriate ratio of stool to preservative is 1:3
• Fungal scrapings
Wipe nails or skin with alcohol

Clean, screw-cap container
a) Hair: 10-12 hairs with shaft intact

b) Nails: Clip affected area
c) Skin: Scrape skin at outer edge of lesion

• Genitalia
A. Cervix/vagina: Remove mucus before collection; do not use lubricant on

speculum; swab endocervical canal or vaginal mucosa, Swab transport system
or JEMBEC transport system
B. Urethra: Flexible swab inserted 2-4 cm into urethra for 2-3 sec or collect
discharge, Swab transport system or JEMBEC transport system

• Lesion/wound/abscess
 Wipe area with sterile saline or alcohol

 Superficial Swab along outer edge,
 Swab transport system, Deep Aspirate with needle and syringe, use
Anaerobic transport system

• Respiratory tract: LRT
Bronchial specimens
 - Sputum, Rinse mouth or gargle with water, instruct to cough deeply into
container Sterile, screw-cap container.
 Or the patient should rinse the mouth with water and expectorate with the
aid of a deep cough directly into a sterile container (expectorated sputum).

Bronchial specimens
 Patients with dentures should remove the dentures first.
 A single specimen should be adequate for detection of bacterial lower
respiratory tract infection.
 If fungal or mycobacterial infections are possible, three separate early
morning specimens (collected on successive days) are appropriate.

Bronchial specimens
 Specimens may be collected through aerosol-induction in which the

patient breathes aerosolized droplets of a solution that stimulates cough
reflex (induced sputum).

• Respiratory tract: URT
1. Nasal: Insert premoistened swab with sterile saline 1 inch into nares, Swab
transport system
2. Nasopharynx: Insert flexible swab through nose into posterior nasopharynx,

rotate for 5 sec, Swab transport system or direct inoculation to media
3. Throat Swab, posterior pharynx, tonsils, and inflamed areas, use swab
transport system
• Tissue
• Disinfect skin; do not allow tissue to dry out; if necessary, moisten with
sterile saline, Anaerobic transport system or sterile screw-cap container

• Urine
1. Clean-catch midstream:
Clean external genitalia; begin voiding and after several mL have passed;
collect midstream without stopping flow of urine, Sterile, screw-cap container,
2-3 mL.
The first portion of the urine flow washes contaminants from the urethra, and
the midstream portion is more representative of that in the bladder.
• Urine
2. Catheter: Clean urethral area, insert catheter, and allow first 15 mL to pass;
then collect remainder ,Sterile, use screw-cap container or urine transport kit
3. Indwelling catheter: disinfect catheter collection port, aspirate 5-10 mL with

needle and syringe Sterile, use screw-cap container or urine transport kit
4. Suprapubic aspirate: disinfect skin, aspirate with needle and syringe through
abdominal wall into full bladder, use sterile, screw-cap container or anaerobic
transport system Diagnostic Microbiology Dr. Walid Abu Rayyan 28
• Urine

 Shipping Infectious Substances:
- The shipment of clinical specimens and cultures of microorganisms is

governed by a complex set of national and international guidelines
- The regulations specify the way potentially infectious substances must be

packaged to prevent leaks or spills and how packages must be labeled to
caution handlers and other parties about their hazardous content.
- An infectious substance is assigned to a risk group with a number from 1

(low risk) to 4 (high risk).

- Patient specimens or culture isolates must be triple packaged before being

shipped. The material is placed into a primary receptacle that must be
watertight.
- Absorbent material is placed around the primary receptacle, and it is then
placed into a secondary container that is also watertight.
- The secondary package is sealed and placed into an outer container
constructed of fiberboard.
- Specific instructions must be followed for labeling the container as
“Hazardous Material.”


 Anticoagulants:
- Anticoagulants are used to prevent clotting of specimens, including blood,
bone marrow, and synovial fluid. The type of anticoagulant used, and the
concentration are important because some anticoagulants have antimicrobial
properties.

 Anticoagulants:
1- Sodium Polyanethol Sulfonate (SPS) is the most common anticoagulant used

for microbiology specimens. The concentration of SPS must not exceed
0.025% (wt/vol) because some Neisseria spp. and certain anaerobes are
inhibited by higher concentrations
2- Heparin is often used for viral cultures and for isolation of

Mycobacterium spp. from blood.
3- Citrate and ethylenediamine tetracacetic acid (EDTA) should not be used

for microbiology specimens.
 Holding or Transport Media:
- These usually contain substances that do not promote multiplication of

microorganisms but ensure their preservation and are available in swab
collection systems.
- Stuart’s or Amie’s transport medium are commonly used.

 Holding or Transport Media:
- Some transport systems contain charcoal to absorb fatty acids given off by
the swab that can be detrimental to the survival of Neisseria gonorrhoeae and
Bordetella pertussis.
- In certain situations, direct inoculation to culture media at the time of

specimen collection (bedside inoculation) is optimal for isolation of the
pathogen.

 SPECIMEN RECEIPT AND PROCESSING:

Specimen Priority
- lists clinical samples and the ways each can be prioritized in a four-level
system.
- Level 1: Critical/invasive (Amniotic fluid, Blood, Brain Cerebrospinal fluid,
Heart valves, Pericardial fluid
- Level 2: Unpreserved (Body fluids, Bone, Drainage from wounds, Feces,
Sputum, Tissue)
- Level 3: Quantitation required (Catheter tip, Urine, Tissue for quantitation)
- Level 4: Preserved (Feces in preservative, Urine in preservative, Swabs in
holding medium, (aerobic and anaerobic)

- Specimens should be transported to the laboratory ideally within 30

minutes of collection, preferably within 2 hours.
- Adverse environmental changes in oxygen, pH, and temperature can prevent

the recovery of certain microorganisms and allow overgrowth of others.


Specimen Storage
- Some specimens, such as urine, stool, sputum, swabs (not for anaerobes),
foreign devices such as catheters, and viral specimens can be maintained at
refrigerator temperature (4° C) for 24 hours.
- Pathogens that are cold sensitive may be found in other specimens, and those
specimens should be kept at room temperature if culture is to be performed.
This includes samples that might contain anaerobic bacteria as well as most
other sterile body fluids, genital specimens, and ear and eye swabs.
- If cerebrospinal fluid is not processed immediately, it can be stored in a 35° C

incubator for 6 hours. Diagnostic Microbiology Dr. Walid Abu Rayyan 39

Preservatives
Two specimen types in which preservatives can be used are urine and stool.
Boric acid is used in commercial products to maintain accurate urine colony

counts.
The systems are designed to maintain the bacterial population in the urine at
room temperature for 24 hours and thus are useful for collection of urine
specimens at distant locations.


Preservatives
Stool specimens for bacterial culture that are not transported immediately to the
laboratory can be refrigerated, but if the delay is longer than 2 hours, the
specimen can be added to Cary-Blair transport media.
Stools for Clostridium difficile toxin assay should be collected without a

preservative and can be refrigerated; if the delay is longer than 48 h, the
specimen should be frozen at -70° C


Preservatives
• Formalin is the preservatives for ova and parasite (O & P) examinations that
maintain the morphology of trophozoites and cysts.
• Specimens for acid-fast bacillus (AFB) that need to be digested and
decontaminated can be refrigerated and processed once per day.
• Stool specimens for O & P that are in preservative also can be processed in
batch format.
• Specimens for viral culture that are collected in viral transport media also
can be batched

Unacceptable Specimens and Specimen Rejection
1. The information on the requisition does not match the information on the
specimen label. If the patient's name or source does not match, the specimen
should be collected again.
2. The specimen is not submitted in the appropriate transport container, or the

container is leaking.
3. The quantity of the specimen is not adequate to perform all tests requested.


4. The specimen transport time is more than 2 hours, and the specimen has not
been preserved.
5. The specimen is received in a fixative such as formalin (Stools for ova and
parasites are exceptions).
6. Specimen is dried up.


7. More than one specimen from the same source was submitted from the same
patient on the same day, except for blood cultures.
8. One swab was submitted with multiple requests for various organisms.


9. An anaerobic culture is requested on a specimen in which anaerobes are

indigenous.
10. Expectorated sputum in which the Gram stain reveals fewer than 25 white
blood cells (WBCs) and more than 10 epithelial cells per low-power field (LPF)
and mixed bacterial flora.


In certain situations, it may be necessary to process a suboptimal specimen.

Specimens that are impossible to recollect or that would require the patient to
undergo another invasive procedure (bone marrow, spinal fluid, or surgery) may
need to be processed regardless of the situation.


Macroscopic Observation
Notations from the macroscopic observation should include the following:
• Stool consistency (formed or liquid)

• Blood or mucus present
• Volume of specimen
• Fluid: clear or cloudy


Macroscopic Observation
• Areas of blood and mucus are selected for culture and direct microscopic
examination.
• Anaerobic cultures may be indicated if gas, foul smell, or sulfur granules are
present.
• The diagnosis is evident if adult helminths or tapeworm proglottids are

present in the specimen.


Microscopic Observation
A direct microscopic examination is a useful tool that provides rapid
information.
(1) It can be used to determine the quality of the specimen. Sputum specimens
that represent saliva rather than lower respiratory secretions can be determined
by the quantitation of WBCs or epithelial cells.
(2) It can give the indication of the infectious process involved. Gram stain of a
sputum specimen revealing WBCs and gram-positive diplococci is indicative of
Streptococcus pneumoniae.

(3) The routine culture workup can be Guided by the results of the smear.
(4) It can dictate the need for nonroutine or additional testing. The presence of
fungal elements in a specimen for bacterial culture will alert the technologist to
notify the physician to request a fungus culture


In certain specimen types, the direct microscopic examination does not provide
useful information and therefore is not appropriate. This includes throat and
nasopharyngeal specimens.
Gram stains for N. gonorrhoeae on specimens from the vagina, cervix, and anal
crypts are not recommended because these sites contain other bacteria that can
have the same morphology, although Gram stain direct smears are
recommended to diagnose bacterial vaginosis.


Gram stains on stool specimens are not usually routine, although they can be
useful to determine whether the patient has an inflammatory diarrhea based on
the presence of white blood cells.
Although a direct Gram stain on a urine specimen provides useful information,

many laboratories do not perform them routinely because they are time-
consuming and preliminary culture results are available within 24 hours.


Culture Media Selection
1. The selection of media to inoculate is based on the type of specimen

submitted for culture and the organisms likely to be involved in the
infectious process.
2. Specimens in which fastidious pathogens are more likely involved require

media with appropriate nutrients to aid in their recovery.


Culture Media Selection
3. Specimens that are collected from a site containing normal biota will require
types of media to diminish the normal biota while allowing the pathogens to be
detected
4. Some specimens require a single plate, whereas others will require a battery
of several plates.


Specimen Preparation
Most specimens arrive in the laboratory in one of three forms:
swab, tissue, or fluid.
Specimens such as sterile body fluids, pus, and urine, are inoculated directly
onto selected media.


1- Large volumes of sterile body fluids (peritoneal, pleural, continuous

ambulatory peritoneal dialysis [CAPD] are concentrated to increase the
recovery of bacteria.
Centrifugation or filtration are methods for concentration.
If the volume of fluid is greater than 1 mL, the specimen can be centrifuged for
20 minutes at 3000 g.
The sediment is then used to inoculate media and to prepare smears.


2- If the specimen consistency is thin enough to avoid filter clogging, filtration

with a Nalgene filter unit can be performed.
3- Some laboratories place the swab into 0.5 to 1.0 mL of broth or saline and
then vortex the specimen to loosen material from the swab and produce an even
suspension of organisms. A sterile pipette is then used to dispense the inoculum
onto plates and broth.


4- Tissues can be prepared for culture by homogenization, in which the tissue is

ground in a tissue grinder.
Because homogenization can destroy certain organisms, in some situations the
tissue is minced with sterile scissors and forceps into small pieces suitable for
culture.

Thanks for your attention!
Diagnostic Microbiology 61 Dr. Walid Abu Rayyan

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Chapter One

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Chapter One

Specimens Collection and Processing

Diagnostic Microbiology Dr. Walid Abu Rayyan 1

Diagnostic Microbiology Dr. Walid Abu Rayyan 2

 Basic Principles of Specimen Collection :

 The laboratory can make accurate and useful determinations only if a

Diagnostic Microbiology Dr. Walid Abu Rayyan 3

 Basic Principles of Specimen Collection :

2- Select the correct anatomic site for collection of the specimen.

4- Collect the appropriate quantity of specimen.

 Basic Principles of Specimen Collection :

5- Package the specimen in a container designed to maintain the viability of the

7- Transport the specimen to the laboratory promptly or make provisions to store

Diagnostic Microbiology Dr. Walid Abu Rayyan 5

- Specimens for microbiology cultures should be collected in sterile containers,

Diagnostic Microbiology Dr. Walid Abu Rayyan 6

- The tips of swabs may contain cotton, Dacron, or calcium alginate.

- Dacron or polyester swabs have a wide range of uses.

Diagnostic Microbiology Dr. Walid Abu Rayyan 7

Diagnostic Microbiology Dr. Walid Abu Rayyan 8

- Before the specimen is collected, the area should be cleansed to eliminate as

Diagnostic Microbiology Dr. Walid Abu Rayyan 9

- For COVID 19, Zymo media was used as a transport

Diagnostic Microbiology Dr. Walid Abu Rayyan 10

- The most effective method is to provide verbal and written instructions.

 Specimen Collection Guidelines:

Disinfect skin with alcohol and iodine, Blood

Diagnostic Microbiology Dr. Walid Abu Rayyan 12

 Specimen Collection Guidelines:

• Body fluids :(abdominal, amniotic,

Disinfect skin before needle aspiration Sterile,

Catheter tips, IV:

Diagnostic Microbiology Dr. Walid Abu Rayyan 13

 Specimen Collection Guidelines:

 Specimen Collection Guidelines:

Diagnostic Microbiology Dr. Walid Abu Rayyan 15

 Specimen Collection Guidelines:

Collect directly into container, avoid contamination with urine,

If a bacterial infection is suspected, three specimens should be collected, one a

 Specimen Collection Guidelines:

If parasites are suspected, three specimens collected within 10 days should be

 Specimen Collection Guidelines:

Wipe nails or skin with alcohol

a) Hair: 10-12 hairs with shaft intact

Diagnostic Microbiology Dr. Walid Abu Rayyan 18

 Specimen Collection Guidelines:

A. Cervix/vagina: Remove mucus before collection; do not use lubricant on

Diagnostic Microbiology Dr. Walid Abu Rayyan 19

 Specimen Collection Guidelines:

 Wipe area with sterile saline or alcohol

Diagnostic Microbiology Dr. Walid Abu Rayyan 20

 Specimen Collection Guidelines:

• Respiratory tract: LRT

Diagnostic Microbiology Dr. Walid Abu Rayyan 21

 Specimen Collection Guidelines:

• Respiratory tract: LRT

Diagnostic Microbiology Dr. Walid Abu Rayyan 22

 Specimen Collection Guidelines:

• Respiratory tract: LRT

 Specimens may be collected through aerosol-induction in which the

Diagnostic Microbiology Dr. Walid Abu Rayyan 23

 Specimen Collection Guidelines:

• Respiratory tract: URT