medical microbiology ii lecture 1

MEDICAL MICROBIOLOGY II

LECTURE 1

Collection and Examination of Specimen for Microbiological

Investigation

Importance of Proper Specimen Collection

Specimen collection is the backbone of the investigative process

Isolation and identification of the microorganism - first step in determining proper treatment

Microorganism needs to be grown, isolated and identified

Before all this can happen, a specimen must be COLLECTED


Diagnostic results depend on quality of specimens

Poorly collected / transported specimens will result in:

1. Failure to isolate causative microorganism

2. Recover contaminants / normal flora

3. Waste of time, resources and man-power

4. Prolongs accurate diagnosis


5. May lead to harmful therapy

6. Inconvenience or painful for patient to recollect another specimen

Specimen Collection

A very important step - result of a culture of bacteria is only as reliable as the method used to collect and transport the specimen

Knowledge of microorganism growth requirements are crucial

Specimen should be collected BEFORE starting on antibiotics

Important questions before collecting a specimen

Are you suspecting an infection?

If so, what is the nature of infection (e.g.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 do I need to send the specimen to the laboratory promptly?

If not, what should I do?

Factors Influencing Specimen Collection

Several factors must be considered:

1. The specimen must be from the actual site of infection

2. The specimen must be collected with minimal contamination from adjacent tissues, organs, or secretions

3. Sufficient quantity must be obtained to perform the tests requested

Factors Influencing Specimen Collection

4. Appropriate collection devices, specimen containers and culture media must be used to ensure optimal recovery of microorganism

Common Specimen Types

These are common specimen seen in microbiology laboratory:

1. Urine

2. Blood

3. Swab (wound, throat, vaginal, urethral, high vaginal swab, nasal swab)

4. Sputum

5. Faeces

Less Common Specimen Types

These specimen types are less common:

1. Cerebrospinal fluid

2. Tissue

3. Other body fluid (ascites fluid, synovial fluid, pleural fluid, peritoneal fluid)

4. Tips (Foleys tip)

Criteria of Containers

Leak-proof

Unbreakable

Sterile (MUST for culture)

High risk specimens should be labeled (e.g.sputum specimen suspected with tuberculosis, faecal specimen suspected cholera or typhoid)

Screw-cap or firmly stopped

Factors affecting quality of result

1. Pre-Analytical

Appropriate samples

Test method

Clinical details

Sample collection

Labeling (incorrect specimen identification)

Transportation / storage

Specim

en co

llection


2. Analytical

- Equipment - Precision

- Accuracy - Calibration

- Updated knowledge - Reagent

- Technique - SOPs

- Quality Control - Specificity


3. Post-Analytical

Transcription

Communication

Specimens from Various Body Sites Specimen for microbiology can be collected

from various body sites:1. Blood2. Sputum3. Throat4. Nasopharynx 5. Cerebrospinal fluid6. Body fluids (synovial, pericardial, peritoneal

etc.) 7. Urine8. Stool9. Abscess aspirates or exudates

Blood culture

One of the most important investigation

An appropriate procedure in collection, processing, identifying and timely reporting can be life saving

Blood collection has to be free of contamination

2 - 3 cultures should be collected at random times during 24hrs period

Blood culture

Palpate the skin for a good puncture site

Skin is disinfected with 70% alcohol, followed by iodine

The disinfectant is allowed to dry

The puncture site should not be palpated after disinfection

Ideally, 20 - 30mL of blood per culture is collected from an adult (1 - 5mL from infants and small children)

Blood culture Iodine should be cleaned

from the puncture site with alcohol following the venipuncture

If collecting a set of tubes and blood culture, the firstsample should be blood culture

Clean the top of blood culture bottle (with 70% alcohol)

Blood Culture

DO NOT:

Palpate the skin after disinfection

Use iodine to clean the top of bottle

DO:

Only palpate before disinfection

Only use alcohol / chlorhexidine

Respiratory Samples

Common respiratory infections are localised in oropharynx, nasopharynx, and nasal cavities

Causes: sore throat, nasal discharge, fever, sinusitis, whooping cough, influenza, measles, infectious mononucleosis

Most throat infections are due to virus, but some will have secondary infection by bacteria

Among the common respiratory samples are sputum, ETT aspirates, throat swab, and bronchial lavage

Sputum

Mixture of bronchial exudates and inflammatory exudates coughed up into the mouth and expectorated

Most common specimen sent for lower respiratory infection investigation

Difficulties in collecting suitable specimen - busy or uninstructed staff may collect saliva only

Sometimes, repeat specimens may be required to isolate the causative pathogen

Sputum

Patient instruction

Sputum (expectorated)

Rinse mouth and gargle with water before collection

Quality of sample

Good quality sample: good isolated bacterial growth

Unacceptable sample: salivary, clear fluid

Sputum

Collection should be made in a disposable, wide-mouthed screw capped plastic container of 50 - 100 mL capacity

Collect sputum before antibiotics are given

Ideal to collect specimen when patient wakes up, and with the first cough

Throat Swab

Sample collection

The tongue should be depressed and need to ensure to not touch cheek, tongue, and teeth

A plain cotton swab can be used to collect as much exudates as possible from tonsils, posterior pharyngeal walls and other areas that are inflamed

The swab has to be returned to its tube with care not to soil the rim

Throat Swab

If it cannot be transported immediately, it should be refrigerated (2 - 8 C)

One swab / investigation

Nose swab

Nasal swabs are taken to diagnose healthy carriers

e.g. for H1N1 screening

A deep nasal swab generally yields the same information as throat swab

CSF

Collected aseptically by a physician

Collect about 3 - 5mL in a labeled sterile screw cap container

Must be transported immediately to lab as it needs to be processed immediately

Delay may kill the pathogen or disintegrate leukocytes

Specimen cannot be exposed to heat or refrigeration

CSF

If delay is unavoidable, do not refrigerate (will kill H. influenza)

If delay is anticipated, keep at room temperature.

3 sets: for Microbiology, Biochemistry and Haematology

First sample (or most turbid) is for Microbiology

Greater volume increase the chance of organism recover

Body Fluids

Collected by a physician with a needle and syringe or aspirated into a sterile container

The use of swabs may inhibit growth of anaerobes or increase the likelihood of contamination with indigenous bacterial flora from adjacent tissues

Greater volumes increase the chances of organism recovery

Urine

Clean catch, mid-stream urine (MSU) -

most common collection method

Do not stop flow of urine

Proper cleansing of the urethral area is important, especially in women to ensure urine is not contaminated with commensal bacteria that colonize in the urethral orifice and perineum area

Urine

Collect specimen in a disposable wide-mouthed plastic container with screw cap

Transport immediately, otherwise refrigerate at 2 - 8 C

Stool

The specimen may be collected from faeces passed in a clean bed pan or other clean receptacle

NEVER collect stool specimen from the toilet

Ensure faeces not mixed with urine, disinfectant or heavily soiled toilet paper

Specimen should be collected into a disposable, sterile, wide-mouthed, screw cap plastic container

Stool

Transport immediately to lab

If the specimen cannot be plated within 1 hour of collection, it should be mixed with a transport medium (e.g. buffered glycerol saline, Cary-Blair transport medium)

The change in pH and temperature over time is detrimental to Shigella spp.

High Vaginal Swab / Vaginal Swab

Specimen commonly collected for diagnosis of vaginitis, vaginosis, or uterine sepsis

Also used for examination for gonorrhea

The swab is inserted into the cervix and rotated several times before withdrawing it

Transport immediately or use Amies transport medium

Never refrigerate

Swab from Wound, Skin and Deep Sepsis

Swab from wound, skin or deep sepsis are usually sent to the lab

Swabs are actually inefficient sampling device and tends to desiccate the specimen and traps the bacteria which are then not released onto the plate

The ideal sample is pus or exudates

Swabs should be collected in a small screw capped bottle, firmly stopped tube or syringe, or sealed capillary tube

Pus

Surgically obtained pus / fluid by a physician

Pus better than swab

Aspirates from closed abscesses (syringe aspirates better than swab)

Must be immediately transported to lab

Tissue

Surgically obtained

Tissue in sterile container with small amount of sterile saline or,

Tissue in sterile container without any additional fluid

Immediate transport to the lab

Anaerobic Culture

Acceptable specimens:

1. Pus from closed abscesses

2. Blood

3. CSF

4. Peritoneal fluid

Anaerobic Culture

Unacceptable specimen:

1. Gastrointestinal specimens

Faeces

Rectal swab

Gastric / small intestine contents

2. Urogenital specimens

Urine

Urethral swab

Vaginal swab

3. Respiratory specimens

Throat swab

Coughed sputum

Anaerobic Culture

Collection technique

Exclusion of air

Blood (anaerobic) culture before all other samples

Rapid transport to the laboratory

Specimen for Viral Studies

Specimen are best collected during the time of first presentation of symptoms

Specimen should be collected with a Dacronor rayon swab

Cotton swabs are toxic to viruses

Specimen must not dry out

Usually will be transported in a modified Stuart transport medium to the laboratory


Other specimens should be collected into viral transport media such as Hanks balanced salt solution, 0.2M sucrose-phosphate, or 2% fetal calf serum with Eagles minimum essential medium

Some viral transport medium contain:

Antibiotics: inhibit growth of normal bacterial or fungal flora

Gelatin media: stabilize protein


Urine and stool should be collected in clean containers with no preservatives

Blood should be collected into heparin or EDTA tubes

All specimen for viral studies/recovery should be transported at refrigerated temperatures (0 - 4 C), regardless of source

Labeling of Specimen Container

Label specimen container and form:

1. Patient name

2. Identification number

3. Medical record number

4. Source / site

5. Date / time of collection

6. Collector

Rejection Criteria

Leaking container

Unlabeled container, wrong labeled, mismatched samples

Specimen received in fixative

Specimen unsuitable for request

Specimen received in non-sterile or contaminated containers

Prolonged transport

Rejection Criteria

24 hrs urine or 24 hrs sputum collections

Saliva instead of sputum

Unrefrigerated urine specimens 2 hrs or more post-collection

Dried specimen

Anaerobic culturing of MSU, upper respiratory tract specimens, superficial skin or stool specimens

Transportation

All specimens must be transported to the lab promptly

Anaerobic specimens must be transported in an anaerobic transport system

If delay in transportation, most specimens can be kept at 2 - 8 C

Exception are specimens that likely contain temperature-sensitive organisms (e.g.Neisseria), blood culture bottles, and cerebrospinal fluid

Transportation

Generally, swabs are the least desirable collection and transport method

However, organisms can be successfully cultured if the swab is handled and transported properly (e.g. not allowed to dry out)

Swabs are inappropriate for culturing anaerobes

Transportation

If swabs are used, it is essential that an anaerobic culturette be used

Use of aerobic culturette for the culture of anaerobes is criteria for specimen rejection

Transport Medium

A transport medium should be:

non-nutritive

semi-solid

reductive

able to hamper self-destructive enzymatic reactions within the cells

must inhibit toxic oxidation reactions

Generally, 2 commonly used: Stuarts transport medium and Amies transport medium

Transport Medium

Transport Medium1. Stuart transport medium - semisolid medium,

used to transport and preserve specimens for the cultivation gonococci, streptococci, Enterobacteriaceae, etc.

2. Amies transport medium - modification of Stuart's transport medium by replacing glycerophosphate with an inorganic phosphate buffer and adding charcoal to the medium. This modified medium gave a higher percentage of positive results than Stuart transport medium

Transport Medium

For the collection of the specimens, use sterile cotton-tipped swabs or wooden sticks

Push the swab down one third of the medium depth

When the cap is screwed down, the swab is forced to the bottom of the medium

Transport Medium

The cap should be firmly screwed

The medium has to be kept cool during transportation but do not freeze

The cultures on transport swabs must not be kept at room temperature for more than 24 hours

For optimum results, the time lapse between sample collection and inoculation onto culture medium should be reduced to the minimum

THE END

medical microbiology ii lecture 1

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