Biology Specialist

Mudhafar Qader Saber

Bacteriology Division Official
MOH / CPHL
The laboratory diagnosis of an infectious disease begins with the collection of
a clinical specimen for examination or processing in the laboratory (the right
one, collected at the right time, transported in the right way to the right
laboratory). Proper collection of an appropriate clinical specimen is the first
step in obtaining an accurate laboratory diagnosis of an infectious disease.
Guidelines for the collection and transportation of specimens should be made
available to clinicians in a lucidly written format. The guidelines must
emphasize two important aspects:
➢ Collection of the specimen before the administration of antimicrobial
agents.
➢ Prevention of contamination of the specimen with externally present
organisms or normal flora of the body.
General rules for collection and transportation of specimens are
summarized in Table 1.
Apply strict aseptic techniques throughout the procedure.
• Wash hands before and after the collection.
• Collect the specimen at the appropriate phase of disease.
• Make certain that the specimen is representative of the infectious
process (e.g. sputum is the specimen for pneumonia and not saliva) and
is adequate in quantity for the desired tests to be performed.
• Collect or place the specimen aseptically in a sterile and/or appropriate
container.
• Ensure that the outside of the specimen container is clean and
uncontaminated.
• Close the container tightly so that its contents do not leak during
transportation.
• Label and date the container appropriately and complete the requisition
form.
• Arrange for immediate transportation of the specimen to the
laboratory.
Criteria should be developed by a laboratory on the 
basis of which the
processing of a specimen may not be done by the 
laboratory. The following
are some examples: 
➢ Missing or inadequate identification. 
➢ Insufficient quantity. 
➢ Specimen collected in an inappropriate container. 

➢ Contamination suspected. 
➢ Inappropriate transport or storage. 
➢ Unknown time delay. 
➢ Haemolysed blood sample 
Blood
Whole blood is required for bacteriological examination. Serum separated
from blood is used for serological techniques. Skin antisepsis is extremely
important at the time of collection of the sample. Tincture of iodine (1-2%),
povidone iodine (10%) and chlorhexidine (0.5% in 70% alcohol) are ideal
agents. However, some individuals may be hypersensitive to iodine present in
some of these. While collecting blood for culture,the following points must be
remembered:
➢ Collect blood during the early stages of disease since the number of
bacteria in blood is higher in the acute and early stages of disease.
➢ Collect blood during paroxysm of fever since the number of bacteria
is higher at high temperatures in patients with fever.
➢ In the absence of antibiotic administration, 99% culture positivity can
be seen with three blood cultures.
➢ Small children usually have higher number of bacteria in their blood
as compared to adults and hence less quantity of blood needs to be
collected from them (Table 2).
 Age Volume in 2 bottles

< 2 years 2 ml
2-5 years 8 ml
6-10 years l 12 ml
>10 years 20 ml
Examination of CSF is an essential step in the diagnosis of any patient with
evidence of meningeal irritation or affected cerebrum. Almost 3-10 ml of CSF
is collected and part of it is used for biochemical, immunological and
microscopic examination and remaining for bacteriological or fungal
examination. The following important precautions need to be taken for CSF
collection and transportation:
➢ Collect CSF before antimicrobial therapy is started.
➢ Collect CSF in a screw – capped sterile container and not in an
injection vial with cotton plug.
➢ Do not delay transport and laboratory investigations.
➢ Transport in a transport medium if delay in processing is
unavoidable.
➢ CSF is a precious specimen, handle it carefully and economically. It
may not be possible to get a repeat specimen.
➢ Perform physical inspection immediately after collection and indicate
findings on laboratory requisition form.
➢ Store at 37oC, if delay in processing is inevitable.
The characteristics of the appearance of CSF are outlined in Table 3
Clear and colourless Normal
Clear with Tyndall effect
(sparkling appearance against incident
light)
High protein content
Clear yellowish Old haemolysis
Clear red Fresh haemolysis
Turbid blood-stained Haemorrhage
Turbid white High cell or protein
content
Turbid clot (after overnight storage) Fibrin clots
aetiological investigation of Sputum is processed in the laboratory for
bacterial and fungal infections of the lower respiratory tract. It is of utmost
importance in the diagnosis of pulmonary tuberculosis.
➢ Select a good wide-mouthed sputum container, which is preferably
disposable, made of clear thin plastic, unbreakable and leak proof
material.
➢ Give the patient a sputum container with the laboratory serial
number written on it. Show the patient how to open and close the
container and explain the importance of not rubbing off the number
written on the side of the container.
➢ Instruct the patient to inhale deeply 2-3 times, cough up deeply
from the chest and spit in the sputum container by bringing it closer
to the mouth.
➢ Make sure the sputum sample is of good quality. A good sputum
sample is thick, purulent and sufficient in amount (2-3 ml).
Give the patient an additional container with laboratory serial number
written on it for an early morning specimen. Explain to the patient to rinse
his/her mouth with plain water before bringing up the sputum.
Under normal circumstances urine is sterile. The lower part of the urethra
and
the genitalia are normally colonised by bacteria, many of which may also
cause urinary tract infection. Since urine is a good growth medium for all
sorts of bacteria, proper and aseptic collection assumes greater importance
for this specimen.
For microbiological examination urine must be collected as a “clean
catch-mid-stream” specimen.
Urine specimens should be transported to the laboratory within one hour
for bacteriological examination, because of the continuous growth of bacteria
in vitro thus altering the actual concentration of organisms.
Faecal specimens for the aetiological diagnosis of acute infectious
diarrhoeas
should be collected in the early stage of illness and prior to treatment
with
antimicrobials. A stool specimen rather than a rectal swab is
preferred.
➢ The faeces specimen should not be contaminated with urine.
➢ Do not collect the specimen from bed pan.
➢ Collect the specimen during the early phase of the disease and as
far
as possible before the administration of antimicrobial agents.
➢ 1 to 2 gm quantity is sufficient.
➢ If possible, submit more than one specimen on different days.
➢ The fresh stool specimen must be received within 1-2 hours of
passage.
➢ Store at 2-8oC.
➢ Modified Cary and Blair medium (see chapter 5) is recommended as a
good transport medium. It is a very stable medium and can be stored
for use in screw – capped containers. It is a semi-solid transport
medium. At least two swabs should be inoculated. Most pathogens
will survive for up to 48 hours at room temperature. Specimens are
unacceptable if the medium is held for more than one week or if
there is detectable drying of the specimen.
Alternative transport media are Venkataraman-Ramakrishnan
medium (V-R fluid) or alkaline peptone water. VR fluid should be
prepared in 30 ml (1 oz) screw capped bottles (MacCartney bottles).
It preserves vibrios for more than six weeks and has also proved to
be a very convenient medium for transportation as it can be kept at
room temperature after collection of the specimen
➢ Depress the tongue with a tongue blade.
➢ Swab the inflammed area of the throat,
pharynx or tonsils with a
sterile swab taking care to collect the pus or piece
of membrane.
➢ Transport in sterile transport tube.
➢ Insert swab at least 2.5 cm beyond the anal 
sphincter so that it
enters the rectum. 
➢ Rotate it once before withdrawing. 
➢ Transport in Cary and Blair or other 
transport medium.
Preparation
Chlorinated water - add sodium thiosulphate (0.5ml of 10%
solution or a small crystal)
Tap/ pump
• remove attachments
• wipe, clean and flame outlet
• allow to flow (at least one minute)
Water course or reservoir - collect from a depth of at least
20 cm
Dug well - do not allow the bottle to touch the sides of the
well
At least 200 ml of water sample from the source

In sterile glass bottles OR autoclavable plastic

bottles
• tight screw capped lid

• securely fitting stoppers/caps

At least 200 ml of water sample from the source
In sterile glass bottles OR autoclavable
bottles plastic
Test the water sample within 3 hours of collection
• keep at ambient temperature

If delayed:
• pack sample on ice
• test refrigerated sample within 24 hours
Collect suspect food earliest
Collect aseptically - sterile tools, containers.

Solid Food
• cut 100-200 grams from centre with sterile knife
• raw meat or poultry - refrigerate in a sterile plastic jar
Liquids
• shake to mix, use sterile tube
As fast as possible
Keep perishable food at 2-8oC
Cool hot food rapidly - put containers under
cold running water
Pack samples to prevent spillage
Contact the laboratory regarding method of
transport and anticipated time of receipt
Allows organisms (pathogens and contaminants)
to survive
Non-nutritive - does not allow organisms to
proliferate
For bacteria – i.e., Cary& Blair
For viruses - virus transport media (VTM)
Epidemiologist sends:
Patient information
• age (or date of birth), sex, complete address
Clinical information
• date of onset of symptoms, clinical and immunization history, risk factors or
contact history where relevant, anti-microbial drugs taken prior to
specimen collection
Laboratory information
• acute or convalescent specimen
• other specimens from the same patient
Line listing – if large number of patients

Receiving laboratory records:

Date and time when specimen was received

Name and initials of the person receiving specimen

Record of specimen quality

Thank you