Sample

collection
Dr.Sujeet Kumar tiwari
PG resident 1st year
(microbiology)
SBHGMC Dhule
 Introduction
● The samples are collected and handled may directly affect the
outcome of microbiological analysis

● Specific guidelines on sample collection are necessary for optimal use

of microbiology services

● Sample collection requires withdrawing blood, cerebrospinal fluid,

collecting urine, sputum, pus or swabs from mucosal surfaces.

● Sample collection is performed using aseptic techniques to ensure

sterility of the sample and avoid contamination.
 Objectives for sample collection
● Accuracy -To ensure that analytical results obtained are
representative of actual analyte conc. in patient and, thus of
his/her physiological/pathological state.

● Safety

● Minimize patient discomfort and complications

● Avoid recollection
Factors affecting quality of lab results
Pre-analytical
● Appropriate samples
>Test method
> Clinical details
● Sample collection
>Labelling
>Transportation / storage
Analytical -
equipments, reagents, calibration,
specificity, precision, accuracy,
updated knowledge, technique,
Quality Control.
Post-analytical - transcription, communication
 Sample management
● Sample collection

● Sample transport

● Sample receipt

● Sample rejection

● Sample storage

● Sample disposal
 IMPACT OF SAMPLE MANAGEMENT ON PATIENT CARE

• Key to accurate laboratory diagnosis

• Directly affects patient care and patient outcome

• Influences therapeutic decisions

• Impacts hospital infection control

• Impacts patient length of stay, hospital costs, and laboratory costs

• Influences laboratory efficiency

Information for the nursing staff For sample
collection
• Safety considerations

• Selection of the appropriate

anatomic site and specimen

• Collection instructions, including the

type of swab or transport medium

• Transportation instructions, including

time and temperature constraints
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• Labeling instructions, including patient demographic information

(minimum of two patient identifiers)

• Special instructions, such as patient preparation

• Sterile versus nonsterile collection devices

• Minimal acceptable quality and recommended quantity

 Fundamentals for sample collection
1. The material must be from the actual site of infection,collected with a minimum of
contamination from the adjacent tissues,organs,or secretions.

Situations where an improperly collected specimen may produce misleading results:

a. Failure to culture the depths of a wound or draining sinus without touching the
adjacent skin

b. Inadequate cleansing of the periurethral tissue and perineum before collecting a

clean-catch urine sample from a woman

c. Contamination of an endometrial sample with vaginal secretions

d. Failure to reach deep abscesses with aspirating needles or cannulas

2. Optimal times for collection of specimens must be established to provide the
best chance of recovering causative microorganisms.
 3. A sufficient quantity of specimen must be obtained to perform the tests
requested:

● Frequently 0.5mL or less of material labels “sputum”or “bronchial

washings” is delivered to the laboratory with a request for routine, acid-fast
bacilli (AFB), and fungal cultures such specimens may not represent
pulmonary secretions from the site of infection, and the low volume may
be insufficient to enable performance of all the procedures requested.

● Tubes containing holding broth such as physiologic saline (non nutrients )

or phosphate yeast glucose (nutrient) can be provide.

● The report should indicate that the material submitted for examination was
scanty.
 4. Appropriate collection devices, specimen containers, swab and
culture media must be used to ensure optimal recovery of
microorganisms:

Containers

● Containers must be leak proof

(universal container)

● Unbreakable.

● Tightly Sealed.

● Wide-Mouthed such as for Sputum sample.

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● For cultures sterile containers is a Must.

● Microbiology specimens should never be sent in formalin .

● For Virology/serology blood should be collected in Red color

tubes.

● Screw-capped, cylindrical glass universal container with a flat base

and a wide mouth (c. 8 cm high x c. 3 cm in diameter).
 SPECIAL CONTAINERS INDICATIONS:

● For feces: the universal container with a small metal or wooden spoon or a
plastic spoon that is attached to the inside of the screw cap. The spoon is
placed in the clean container and sterilized with it. The user removes the
screw-cap and grasps it with his fingers. Patient picks up a single spoonful
of feces, drops it with the spoon into the container and replaces the cap
tightly. When there is likely to be a delay of several hours under warm
conditions then before laboratory examination neutral glycerol saline
should be added to the container.

● For Urine:- The diagnosis of UTI, the universal sterile container is used. For
larger quantities, e.g. complete early morning specimens for the diagnosis
of renal, tuberculosis, 20 oz (250 ml) wide-mouthed screw capped bottles
are convenient.
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● For Blood: for serological examination blood is submitted (without
anticoagulant) in special glass or plastic containers with screw caps. Flip-top
lids are to be avoided as these are insecure. They also produce aerosols on
opening and this may be dangerous.

● For blood culture bottles : This is unique because transport & 20 culture
media both are same, 50 ml of liquid culture medium. The metal cap had a
central hole, about 5 mm diameter, which was covered on the inside by an
intact rubber liner. The cap is covered with plastic to maintain sterility. For
automated like BACTEC blood culture bottles containing a range of special
media are available.
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• For the collection of serous fluids: e.g, pleural fluid, the universal
container is suitable. The addition of 0.3 ml of a 20% solution of sodium
citrate to the container prior to autoclaving (with the cap fitted) is
recommended for the collection of fluids that may coagulate on
standing.

• Sputum should not be collected in a universal container, as it is very

difficult for the patient to expectorate into the container without soiling
its outside. Squat, wide-mouthed disposable containers should be used.

• For parasitological examination blood in EDTA tubes is suitable.

 SWAB:

● The swab is suitable for the taking sample from the throat, nostril,
ear, skin, wounds and other accessible lesions consist of a sterile
pledget of absorbent material, usually cotton-wool or synthetic fibre,
mounted on a thin wire or stick.

● The swab is a convenient but relatively inefficient sampling device. It

influences the viability of pathogens sampled on it as inhibitory
substances are known to occur in swabs of cotton-wool or synthetic
fiber. Steps to inactivate these inhibitors include boiling the swab in
phosphate buffer or coating the swab with serum or albumin or
charcoal.
Special swab indications:

• Baby swabs: A very fine swab mounted on fine wire so that small orifices,
such as the aural meatus.

• Pernasal swabs: Used for the diagnosis of whooping cough. The swab is
passed along the floor of the nasal cavity to reach and sample the
secretions in the nasopharynx. A small swab and a flexible swab-wire are
required to minimize the risk of damage to the nasal tissues.

• Post-nasal swabs: Used to sample nasopharyngeal secretion for the

diagnosis of meningococcal carriage. The terminal 20 mm of a long stiff
metal swab-wire, bent through an angle of 45 degrees, when introduced
through the mouth, it carries the swab up behind the soft palate into the
nasopharynx.
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● Laryngeal swabs:
Used to obtain a sample of bronchial secretion for the diagnosis of tuberculosis in
patients who cannot expectorate sputum. It is bent, swab-bearing end should be
longer, about 50 mm & be more sharply bent through an angle of 60 degrees to its
original direction.

● Nasopharyngeal washings and throat washings:

It may yield much better specimens for virological and other microbiological
investigations of the upper respiratory tract, endotracheal and bronchial sampling
may be used for special investigations.

● High vaginal and cervical swabs:

For the diagnosis of gonorrhea and puerperal fever, a swab should be taken from
the uterine cervix and its lumen, rather than from the general area of the upper
vagina. A swab on a specially long, rigid swab-stick, preferably about 22cm long.
Comparison of aspirated pus and swab specimens:

To detect the etiologic agent of infection is much higher with an aspirate or

biopsy that the sample will yield meaningful results. In contrast, a swab may
have been used to sample either for an inflammatory process(e.g.,the tonsillar
exudate) or colonizing microbiota (e.g.,the side of the mouth).
Blood Collection For Culture:
• Venous Blood ( During High Peak Fever) :
Infant: 0.5-2 ml
Children: 2-5 ml
Adults :5-10ml
• The Goal in blood collection is avoiding contamination during
withdraw.
• Request Slip must contain relevant patient information.
• Send immediately to laboratory with request slip.
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• Take proper precaution (gloves)

• Avoid contamination

• Palpate vein

• Apply disinfectant

• Use sterile needle and syringe

 5. Whenever possible, obtain cultures before the administration of
antibiotics:

● Obtaining cultures before the use of antibiotics isparticularly

recommended for recoveryof organisms that are usually highly
susceptible to antibiotics such as β-hemolytic streptococci from
throat specimens, N.gonorrhoeae from genitourinary samples, or
H.influenzae and Neisseria meningitidis from cerebrospinal fluid.

● It is also particularly important that a full complement of blood

cultures are drawn on patient suspected of having endocarditis
prior to the administration of antibiotics.
6. Smears should be performed in addition to cultures in most
instances:

● There are occasions when the smear is arguably more useful than
the culture, as in examination of expectorated sputum.

● Smears allow assessment of the inflammatory nature of the

sample and provide an indication as to whether the results of
culture are meaningful clinically.

For example, sputum sample contaminated with oral microbiota, as

evidenced by excessive squamous epithelial cells,should not be
cultured.
 7. The sample container must be properly labeled:

Each sample container must have a legible label, with the following
minimum information:

● Patient name

● Patient identification number

● Source of specimen

● Clinician

● Date/hour collected
SAMPLE REQUISITION BY PHYSICIAN:

A complete requisition should include the following:

• The patient’s name with Father’s name

• Hospital identification number

• Age and date of birth

• Mobile No.

• Sex
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• Ordering physician

• Collection date and time

• Exact nature and source of the specimen

• Diagnosis (may be ICD-10-CM code)

• Current antimicrobial therapy

• Serological investigation is required, relevant details of previous

immunization.
 SAMPLE COLLECTION FOR FUNGAL DISEASES

● Container: Clean, screw-top tube

● Patient preparation: Nails or skin: wipe with 70% alcohol

● Collection:
Hair: Collect hair with intact shaft
Nails: Send clippings of affected a nail
Skin: Scrape skin at leading edge of lesion

● Transport: Within 24hrs

 SAMPLE COLLECTION FOR LEPROSY
● Generally Six samples taken– four skin (forehead, cheek, chin and
buttock), one from ear lobe and nasal mucosa by nasal blow/scraping

● Slit skin smear to collect skin and ear lobe specimens tissue pulp from
below the epidermis

● Nasal specimens: Nasal blow - early morning mucus material

● Nasal scraping - mucosal scraper to scrape the nasal septum

● Biopsy- thickened nerves and nodular lesions

 SAMPLE COLLECTION FOR PARASITOLOGICAL DISEASES
● FRESH STOOL SAMPLES> should be collected in a suitable, clean, wide
mouthed container like a plastic container with a light fitting lid, waxed
cardboard box, or match box.

● The sample should not be contaminated with water, urine, or

disinfectants. Liquid stools should be examined or preserved within 30
minutes of passage. Soft stools should be examined or pre served
within 1 hour of passage and formed stool should be examined or
preserved within 24 hours of passage.

● • NIH swab, cellophane swab, or scotch tape swab method is used to

collect the eggs from the perianal skin.