Specimen Collection In Microbiology

Dr.T.V.Rao MD Professor of Microbiology

Dr.T.V.Rao MD

NABH Standards for Hospital

The programme is made to fulfill major objectives of the AAC.7

Clause C
on Policies and procedures guide collection, identification, handling safe transportation, processing and disposal
Dr.T.V.Rao MD

MICROBIOLOGY
Information derived from the results has impact on :
Diagnosis of infectious diseases Antibiotic prescribing Formulation of local antibiotic policy Public health impact eg food handlers Infection Control measures eg MRSA,
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Specimen collection in Microbiology to isolate and identify the causative agents forms back bone of the investigative procedures. In developing world, lack of awareness and casual attitude among junior staff hampers the definitive diagnosis. Specific procedures in collecting specimens will certainly improve the quality of services of Microbiology Departments
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Why specimen collection is Important in Microbiology

Successful laboratory investigations

advance planning collection of appropriate and adequate specimens labeling and documentation of laboratory specimen storage, packaging and transport to appropriate laboratory biosafety and decontamination procedures to reduce the risk of further spread of the disease timely communication of results
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Collection & transposition of Specimen

Collection & transportation of Good quality specimen for microbiological examination is crucial
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Some tips
Laboratory investigation should start as early as possible Specimens obtained early, preferably prior to antimicrobial treatment likely to yield the infective pathogen Before doing anything, explain the procedure to patient and relatives When collecting the specimen, avoid contamination Take a sufficient quantity of material Follow the appropriate precautions for safety

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Specimen collection: key issues

Consider differential diagnoses Decide on test(s) to be conducted Decide on clinical samples to be collected to conduct these tests

consultation between microbiologists, clinicians and epidemiologists highly helpful

Dr.T.V.Rao MD

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Important questions before collecting a specimen

Are you suspecting an Infection ? If so what is the Nature of infection, eg Bacterial, Viral, Mycological or Parasitological Which tests are your priority ? When to collect the specimen ? How to collect the specimen ? Am I choosing the correct container ? Why to send the specimens promptly if not what I should do ?

Dr.T.V.Rao MD

Fishing for Diagnosis in Laboratories, Is it worth?

The physicians and Microbiologists should be aware of the clinical manifestations, before undertaking the test. Microbiological tests are expensive and technically demanding Causal testing of Microbiological tests are counterproductive.
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Policies on Specimen Collection.

Every laboratory should assist extra examinations, outwith the standard procedures may be required if specifically requested by the Physician or if the clinical information provided on the request form suggests that an unusual infection may be present.
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Why Proper written Request

Your request is a legal document. Identifies all the outcome of test. No interchange of results. Short forms are dangerous Signature of the Doctor / Nurse is essential in legible form, can help to contact in case of results which can save a patient.
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An Ideal Request form

Name xxxx Age Sex IP/ OP No xyz Time Date Ward xx123 Urgent / Routine Nature of specimen Investigation needed Doctor/Staff Contact No 1234567
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Tele contact is crucial in serious patients

When the patient is serious, write a

Tele contact number which

can help in prompt delivery of results
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When one Expects the Results

On sending the sample the Physician will be anticipating the early reports, the Microbiologists should promptly dispatch results in all life saving investigations. However the Doctors must be made aware limitation of the investigations and discuss the pros and cons of the

Laboratory reports
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When to Collect the Earliest Specimen

Start collection of specimens for all cultures before starting an Antibiotic.

The advice is ideal but may not be possible, as many prescribe Antibiotics before considers the Microbiological diagnostic options.
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When to Request Transport Medium

When facilities are not available to perform the desired tests at the place of collection or laboratory located far away, request the Diagnostic laboratories to advice on transportation of specimens, and consider how to preserve and transport in ideal medium before it is processed
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What containers to use

Containers must be leak proof,
Unbreakable
For cultures sterile containers a Must

Microbiology specimens should never be sent in formalin

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Label High risk Specimens

Sputum with suspected Tuberculosis Fecal samples suspected with Cholera, Typhoid, Anthrax ?
Serum when suspected with HIV/ HBV/HCV, infections
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Request for Gram Staining

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Cultures That Should Include a Gram Stain

CSF or sterile body fluid (cytospin) Eye Purulent discharge Sputum or trans tracheal aspirate All surgical specimens Tissue Urethral exudates (male only, intracellular gonococcus)) Vaginal specimens Wounds
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Blood for Culturing

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Blood for cultures Collection

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

Requires aseptic technique Collect within 10 minutes of fever

if suspect bacterial endocarditis: 3 sets of blood culture

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Blood Collection for Culturing

Most important investigation An appropriate procedures in collection and processing, identifying and timely reporting can be Life saving
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Collection of Blood
A scientific approaches and dedicated staff participating in blood collection will eliminate the basic failure as Contamination Improper handling of syringes increases chances of contamination Contamination hampers the ideal reporting, A valuable time is lost The goal in blood collection is avoiding the contamination

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Collecting the Blood for Culturing

Teach the staff how to collect the Blood. The nurse are advised on principles of aseptic precautions by self as washing hands and wearing gloves Proper areas of disinfection with good antiseptic solutions.
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Hygienic precautions will decrease contamination

The staff should be advised how to disinfect the skin over vein, to use a fresh sterile syringe for the venepuncture with fresh sterile needle before inoculating culture bottle The staff should disinfect their hands before doing the procedure.
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Proper handling of Syringe is

essential to obtain a blood specimen
The staff should hold the needle by its butt, not shaft. Either with sterile forceps or with fingers covered with a dry sterile rubber glove, and protect self with potentially infective pathogens
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Do not collect from existing or indwelling catheters

The staff are warned that contamination is very likely if the specimen is collected from an indwelling peripheral venous catheter instead of from a fresh venepuncture.
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Always collect the Blood specimens in Hygienic areas

All procedures in relation to processing of the samples should be done in a sterile environment, or bacteria free areas. Despite insistence on aseptic precautions, most laboratories report finding contamination in 1-5% of the blood cultures.
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Collection

Serum

Venous blood in sterile tube

let clot for 30 minutes at ambient temperature glass better than plastic

Handling
Place at 4-8C for clot retraction for at least 1-2 hours Centrifuge at 1 500 RPM for 5-10 min
separates serum from the clot

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Transport 4-8oC if transport lasts less than 10 days Freeze at -20C if storage for weeks or months before processing and shipment to reference laboratory Avoid repeated freeze-thaw cycles destroys IgM To avoid hemolysis: do not freeze unseparated blood
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Serum

Cerebrospinal fluid examination

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Specimen collection for CSF Examination

Lumbar puncture to collect the CSF for examination to be collected by Physician trained in procedure with aseptic precautions to prevent introduction of Infection.
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Procedure to collect CSF

The trained physician will collect only 3-5 ml into a labeled sterile container Removal of large volume of CSF lead to headache,
The fluid to be collected at the rate of 4-5 drops per second. If sudden removal of fluid is allowed may draw down cerebellum into the Foramen magnum and compress the Medulla of the Brain

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CSF needs a New and Sterile container

Fresh sterile screw capped container to be used. Reused containers, not to be used, contamination from the previous specimens misrepresent the present specimen.
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Lumbar puncture for CSF collection

The best site for puncture is inter space between 3 and 4 lumbar vertebrae
( Corresponds to highest point of iliac crest )

The Physician should wear sterile gloves and conduct the procedure with sterile precautions, The site of procedure should be disinfected and sterile occlusive dressing applied to the puncture site after the procedure.
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Transportation to Laboratory
The collected specimen of CSF to be dispatched promptly to Laboratory , delay may cause death of delicate pathogens, eg Meningococcal and disintegrate leukocytes
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Preservation of CSF
It is important when there is delay in transportation of specimens to Laboratory do not keep in Refrigerator, which tends to kill H. Influenza If delay is anticipated leave at Room Temperature.
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Upper Respiratory Infections

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What are Upper Respiratory Infections

The commonest respiratory infections are localised in Oropharynx, Nasopharynx, and nasal cavity, Causes Sore thraot,nasal discharge and often fever. Infect larynx, otitis media,sinusitis,conjunctivitis or keratitis. May present with serious diseases whooping cough, influenza , measles and infectious mononucleosis.
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Aetiological agents in Upper Respiratory Infections

In most cases the primary infections are caused by virus, difficult to isolate. But many infections are caused by concomitant carriage or secondary infection with one of the potential pathogens present in the Nasopharynx Pneumococcus .Haemophilus influenza, Staphylococcus aureus, and Streptococcus pyogenes. Drug resistant coli form bacilli or yeasts may dominate the throat flora in patients receiving antibiotics.
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Specimen collection in Throat Infections

A plain cotton wool swab should be used to collect as much exudates as possible from tonsils, posterior pharyngeal wall and other area that is inflamed or bears exudates
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Cooperation of the patient and ideal techniques contributes better results

If cooperated by patient, the swab should be rubbed with rotation over one tonsillar area of the soft palate and uvula, the other tonsillar area and finally the posterior pharynx
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Collecting the Swab

An adequate view of throat should be ensured by good lighting conditions and the use of a disposable wooden spatula or a tongue depressor to pull outwards and so depress the tongue.
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SPECIMEN COLLECTION IN INFLUENZA ( H1 N1 )

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Collection should be done caution

The following should be collected as soon as possible after illness onset: nasopharyngeal swab, nasal aspirate or a combined nasopharyngeal swab with oropharyngeal swab. If these specimens cannot be collected, a nasal swab or oropharyngeal swab is acceptable. For patients who are intubated, an endotracheal aspirate should also be collected. Bronchoalveloar lavage (BAL) and sputum specimens are also acceptable.
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Sent in Transport Medium

Specimens should be placed into sterile viral transport media (VTM) and immediately placed on ice or cold packs or at 4C (refrigerator) for transport to the laboratory. Recommended infection control guidance is available for persons collecting clinical specimens in clinics and other clinical settings and for laboratory personnel.
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Specimens Stored at ..
All respiratory specimens should be kept at 4C for no longer than 4 days.
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Transportation of Throat Swabs

The swab should be replaced in its tube with care not to soil the rim If it cannot be transported immediately to laboratory it should be placed in a refrigerator at 4c until delivery or preferably submitted in a tube of transport medium
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Shipping specimens.
Clinical specimens should be shipped on wet ice or cold packs in appropriate packaging. All specimens should be labeled clearly and include information requested by your state public health laboratory.
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Nasal specimens
A deep nasal swab

generally yields the same information as throat swab. Nasal swabs are taken to detect healthy carriers than diagnose deep infection Deep nasal are taken to diagnose S.pyogenes and Diphtheria bacillus.
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Nasopharyngeal swab
Tilt head backwards Insert flexible fineshafted polyester swab into nostril and back to nasopharynx Leave in place a few seconds Withdraw slowly; rotating motion
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WHO/CDS/EPR/ARO/2006.1
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Nasopharyngeal aspirate
Tilt head slightly backward Instill 1-1.5 ml of VTM /sterile normal saline into one nostril Use aspiration mucus trap Insert silicon catheter in nostril and aspirate the secretion gently by suction in each nostril
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WHO/CDS/EPR/ARO/2006.1

Specimens in sinusitis
Pus collected or aspirated from sinus, or a saline wash out should be examined in a Gram film and by culture on aerobic and anaerobic blood agar plates.

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Acute otitis Media as long as eardrum remains intact, none of the infected exudates can be collected on an ear swab , though culture of the throat swab may give a provisional indication of casual organism

Collection of Ear Swabs Acute otitis Media

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Chronic suppurative otitis media

Swabs of the discharge in the external meatus should be cultured to guide the choice of antibiotics for systemic and topical therapy.
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Otitis externa
A swab should be taken from the meatus and cultured aerobically on blood agar and MacConkey agar plates for the bacteria. All specimens should also cultured on Sabourauds agar plate with Nystatin 50 units for Candida and Aspergillus.
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Eye Swabs
Obtaining a adequate specimen is difficult. It is best to make smears and seed culture plates beside the patient immediately after collecting the material from the eye.
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Collection of Eye swabs

It is ideal to pick up the material with a loop or on the smoothly rounded tip of a thin glass rod or on the thin serum coated swab Clinical material from Conjunctiva, i.e. from everted eyelid, The margin of the eyelid should be avoided.
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Specimens for Lower Respiratory Infections.

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Sputum
Collection Instruct patient to take a deep breath and cough up sputum directly into a wide-mouth sterile container
avoid saliva or postnasal discharge 1 ml minimum volume
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Lower Respiratory Infections

Sputum is the material from the lower respiratory infections most commonly submitted for bacteriological examination. The sputum is a mixture of bronchial secretions and inflammatory exudates coughed up into the mouth and expectorated
There are several difficulties both in collecting a suitable sample and interpreting the results of the culture Busy and uninstructed staff may send collection of saliva to the laboratory. On several occasions repeat sample may be required to isolate the causative agent.
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Instruction for collecting sputum

Make the collection in a disposable and wide mouthed screw capped plastic container of 50 100 ml capacity. Collect sputum before antibiotics are given. Ideal to have when patient wakes up and with first cough.
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Precautions in handling the specimens

Avoid spilling the material over the rim.
Tightly screw on the cap of the container. Wipe off any spilled material on its outside with tissue paper Deliver the specimen quickly to laboratory
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Sputum Examination for Tuberculosis

Specimen should be collected with biosafety precautions. Several specimens should be collected before coming to negative conclusions.

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Specimens for Urinary Tract Infections

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Collecting Urine for examination

Collect the Mid stream specimens of Urine Do not collect spontaneously passed urine without instructions, which can lead to contamination with commensals bacteria Colonized on urethral orifice and perineum
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Specimen Collection
The urine collected in a wide mouthed container from patients A mid stream specimen is the most ideal for processing Female patients passes urine with a labia separated and mid stream sample is collected
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How Urine Specimens collected in young and infants

Non invasive methods are safe and ideal Follow the Broom hall method, By tapping just above the pubis with two fingers placed on supra pubic region after 1 hour of feed, tapping on at the rate of 1 tap/second for a period of 1 minute, if not successful tapping is repeated once again. The child spontaneously pass the Urine and to be collected in a sterile container
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Transport of Urine for Culturing

All collected specimens of urine to be transported to laboratory with out delay Delay of 1 2 hour deter the quality of diagnostic evaluations.
If the delay is anticipated the specimens are at preserved at 40c In field conditions Boric acid can be added at a concentration of 1.8 %

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Genital Tract Infections

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Genital Infections in women

Genital infections present with, urethritis, vaginitis, genital ulceration, cervicitis, uterine sepsis, salphingits, oophoritis, and pelvic inflammatory disease.

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Collection of specimens
The specimen commonly collected for the diagnosis of vaginitis's, vaginosis or uterine sepsis is high vaginal swab The swab is inserted into upper part of the vagina and rotated there before withdrawing it.
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Specimen collection in Gonorrhoea

An endocervical swab must be collected for examination for gonococci. A vaginal speculum must be
used to provide a clear sight of the cervix and swab is rubbed in and around the introitus of the cervix and withdrawn without contamination from vaginal wall.

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Specimens from other genital areas

Other swabs should be collected from any exudate discharged from the meatus of the urethra or a Bartholin's gland. Rectal or pharyngeal swabs should be considered depends on sexual habits of the patient
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Transportation of specimens
All the swabs to be promptly transported to laboratory, in cases of delay or in cases of delicate microbes to be transported in Amie's transport medium. If possible two swabs to be collected and submitted for each site.
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Specimen collection in Men

The infection in men are mostly caused by the same organism as in women.
Urethritis is commonest presentation may be caused by Gonococci or Non-gonococcal. May present with Genital Ulcers.
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Gonococcus infection in Men

The specimen is collected by milking the urethra and urethral discharge is smeared on slides and inoculated on warmed plates of heated blood agar or selective medium for isolation of Gonococci
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Other Genital infections

When prostatitis is suspected and there is no spontaneous discharge from urethra, massage of the prostate per rectum may express some exudate for examination, and culture.
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Collection of specimens in Chancres

The examination of chancre requires the careful collection of exudates and its preparation for dark ground microscopy. Many patients need clotted blood for specific serological investigation.

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Wound, Skin, and Deep Sepsis

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Collection of specimens
Pus or exudates is often
submitted on a swab for laboratory investigation. The swabs are inefficient sampling device and tends to desiccate the specimen and trap the bacteria which are then not released on to culture plate
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Ideal samples from wounds

The ideal sample is pus or exudates should be submitted in a small screwcapped bottle in firmly stoppered tube or syringe or a sealed capillary tube. Fragments of excised tissue removed at wound toilet or curettings from infected sinuses and other tissues should be sent in a sterile container.
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Gastrointestinal Infections
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Stool samples
Collection: Freshly passed stool samples
avoid specimens from a bed pan

Use sterile or clean container

do not clean with disinfectant

During an outbreak - collect from 10-20 patients

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Timing within 48 hours of onset Sample amount

5-10 ml fresh stool from patients (and controls)

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

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

4C (do not freeze); dry ice for (Ag detection and PCR)
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Collection of specimens
Whenever possible, a specimen of faeces should be collected. A rectal swab is unsatisfactory, unless it is heavily charged and visibly stained with faeces collected from rectum, not anus
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Collection of Faeces
The specimen may be collected from faeces passed into a clean bed pan, not mixed with urine, or disinfectant or from the surface of heavily soiled toilet paper. The specimen is collected into 25 ml screw capped wide mouthed disposable container.
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Stool samples for parasites

Timing as soon as possible after onset Sample amount and size

at least 3 x 5-10 ml fresh stool from patients and controls

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

Storage refrigerate at 4C; store at -15C for Ag detection and PCR Transport 4C (do not freeze); dry ice for antigen detection and PCR

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Transportation of specimen
Collect 1-2 ml of faeces, and apply the cap tightly. Take care not to soil the rim or outside of the bottle. Transmit the container quickly to laboratory. If delay is unavoidable and particularly when the weather is warm collect the specimens in a container holding 6 ml buffered glycerol saline transport medium
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Specimen collection for Virological diseases

Direct virus diagnosis depends on the detection of virus particles, viral antigen or viral nucleic acid in specimen taken from the site of infection. Specimens should be delivered promptly to the laboratory so that no deterioration in the quality of the cells occur. However the specimen collection techniques alter depending on the Aetiological agent and site of involvement.
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MICROBIOLOGY
Information derived from the results has impact on :
Diagnosis of infectious diseases Antibiotic prescribing Formulation of local antibiotic policy Public health impact eg food handlers Infection Control measures eg MRSA, PfA target for reduction of C difficile, MSSA and MRSA bacteraemia
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Lab reports: No senders address or not collected

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On many occasions less than ideal sample is received in laboratory. The rejection of clinical samples should be done with great care and wisdom of only senior staff who should take the responsibility. In the welfare of the patient samples can be repeatedly collected for better diagnosis, as we need repeated isolation to confirm uncommon pathogens.
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When to Repeat Diagnostic Tests

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Criteria for rejecting samples

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

Insufficient quantity Leakage

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Biosafety Precaution
All the Technical staff should follow the Universal and other Biosafety Precautions while handling and Disposing the Microbiology Specimens

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Programme created by Dr.T.V.Rao MD for resources for Laboratory Personal in the Developing World email
doctortvrao@gmail.com

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