2. INTRODUCTION
Clinical microbiology is a science of interpretive judgment that is becoming
more complex, not less.
Even with the advent of laboratory automation and the integration of
genomics and proteomics in microbiology, interpretation of results still depends
on the quality of the specimens received for analysis.
Physicians and other advanced practice providers need confidence that the
results provided by the microbiology laboratory are accurate, significant, and
clinically relevant.
3. IMPACT OF SPECIMEN MANAGEMENT
It is the key to accurate laboratory diagnosis and confirmation, it directly
affects patient care and patient outcomes.
It influences
therapeutic decisions,
it impacts hospital infection control
patient length of hospital stay and laboratory costs
it influences antibiotic stewardship, and
it drives laboratory efficiency.
4. TENETS OF SPECIMEN MANAGEMENT
Specimens of poor quality must be rejected.
Microbiologists should act correctly and responsibly when they call
physicians to clarify and resolve problems with specimen
submissions.
Physicians should not demand that the laboratory report
“everything that grows.”
Background noise of commensal microbiota must be avoided
where possible.
5. The laboratory requires a specimen, not a swab of a specimen.
Tissue, aspirates, and fluids are always specimens of choice.
The laboratory must follow its procedure manual. The procedures
in the manuals should be supported by evidence based literature.
A specimen should be collected prior to administration of
antibiotics.
6. Susceptibility testing should be done only on clinically significant
isolates.
Microbiology laboratory results that are reported should be
accurate, significant, and clinically relevant.
The laboratory should set technical policy.Good communication
and mutual respect will lead to collaborative policies.
Specimens must be labeled accurately and completely so that
interpretation of results will be reliable.
7. APPROPRIATE COLLECTION TECHNIQUES
Specimens should be collected during the acute phase of an illness
(or within 2 to 3 days for viral infections).
Swabs generally are poor specimens if tissue or needle aspirates
can be obtained.
Provide clinicians with a collection manual or instruction cards
listing optimal specimen collection techniques and transport
information.
8. Information for the nursing staff and clinicians should include:
● Safety considerations
● Selection of appropriate anatomic site
● Collection instructions including type of swab or transport medium
● Transportation instructions including time and temperature
● Labeling instructions including minimum patient demographic
information
● Special instructions such as patient preparation
9. SPECIMEN TRANSPORT
Ideally, specimens should be transported to the lab within 2 hours
of collection.
All specimen containers should be leak-proof
The specimens should be transported within sealable, leak-proof,
plastic bags with a separate section for paperwork.
10. Many microorganisms are susceptible to environmental conditions
such as the presence of oxygen (anaerobic bacteria), changes in
temperature and pH .
Use of special preservatives or holding media for transportation of
specimens delayed for more than 2 hours is important to ensure
organism viability.
11. SPECIMEN PRESERVATION
Preservatives, such as boric acid for urine or polyvinyl alcohol (PVA)
and buffered formalin for stool.
Stuart’s medium and Amie’s medium are two common holding
media.
Anticoagulants are used to prevent clotting of specimens such as
blood, bone marrow, and synovial fluid because microorganisms will
otherwise be bound up in the clot.
12. SPECIMEN STORAGE
Several storage methods are used (i.e., refrigerator temperature
[4°C], ambient [room] temperature [22°C], body temperature [37°C],
and freezer temperature [either 20° or –70°C]), depending on the
type of transport media (if applicable) and the etiologic (infectious)
agents sought.
Specimens suspected of containing anaerobic bacteria, for
example, should never be stored in the refrigerator, while cerebral
spinal fluid (CSF) should always be kept at 37°C.
13. Urine, stool, viral specimens, sputa, swabs, and foreign devices
such as catheters should be stored at 4°C.
Serum for serologic studies may be frozen for up to 1 week at –
20°C, further storage and tissues or specimens for long-term storage
should be frozen at –70°C.
14. SPECIMEN LABELING
Specimens should be labeled at the very least with the:
Patient’s name,age and sex
Patient’s identification number
Source of specimen
Date/hour collected
Enough information must be provided on the specimen label so
that the specimen can be matched up with the requisition when it is
received in the laboratory
15. SPECIMEN REQUISITION
The specimen (or test) requisition is an order form that is sent to
the laboratory along with a specimen.
The requisition should contain as much information as possible
regarding the patient history, diagnosis, and immunization record.
Helps the microbiologist to work up the specimen and determine
which organisms are significant in the culture.
16. A complete requisition should include the following:
The patient’s name
Hospital number
Age or date of birth
Sex
Collection date and time
18. REJECTION CRITERIA
The information on the label does not match the information on the
requisition (patient name or source of specimen is different).
The specimen has been transported at the improper temperature.
The specimen has not been transported in the proper medium (e.g., specimens
for anaerobic bacteria submitted in aerobic transports).
The quantity of specimen is insufficient for testing.
The specimen container is leaking.
19. The specimen transport time exceeds 2 hours postcollection and the specimen
is not preserved.
The specimen was received in a fixative (formalin) which, in essence, kills any
microorganism present.
The specimen has been received for anaerobic culture from a site known to
have anaerobes as part of the normal flora (vagina, mouth).
The specimen is dried up.
Salivary specimen send for sputum culture.
20. CONTAINERS FOR THE COLLECTION OF
SPECIMEN
Re-usable glass containers are economical, but disposable glass or
plastic containers are generally used.
These are usually modifications of the old screw-capped, cylindrical
container with a flat base and a wide mouth (8cm high × 3 cm
diameter).
The screw-cap seal must be strong and entirely secure so that the
contents cannot leak or become contaminated.
21. 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 cap fitted) is recommended for
the collection of fluids that may coagulate on standing.
22.
23. SWABS
Suitable for taking specimens of exudate from the throat, nostril,
ear, skin,vagina,cervix, 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 nature & preparation of the swab can markedly influence the
viability of pathogens sampled on it, as inhibitory substances are
known to occur in swabs of cotton-wool or synthetic fibre.
24. Cotton swabs may contain residual fatty acids, and calcium alginate
may emit toxic products that may inhibit certain fastidious bacteria.
Swabs tipped with Dacron are better choices.
Steps to exclude or inactivate these inhibitors include boiling the
swab in phosphate buffer, or coating the swab with
serum/albumin/charcoal.
Sterilization of swabs in hot air oven is not recommended as
charring gives rise to substances that are inhibitory or toxic.
25.
26. SWABS FOR SPECIAL PURPOSES
Baby swab – very fine swab mounted on fine wire.
Pernasal swab – a small swab & a flexible swab-wire, used for the diagnosis of
whooping cough. The swab is passed along the floor of the nasal cavity to reach
& sample the secretions in the nasopharynx.
Post-nasal swab – to sample nasopharyngeal secretions for the diagnosis of
meningococcal carriage. The terminal 20mm of a long stiff metal swab-wire is
bent through an angle of 45˚, so that, when introduced through the mouth, it
carries the swab up behind the soft palate into the nasopharynx.
27. Laryngeal swab – to obtain a sample of bronchial secretion for the
diagnosis of tuberculosis in patients who cannot expectorate
sputum.
Resembles that of post-nasal swab, but the bent, swab bearing end
should be longer, about 50 mm, and be more sharply bent through
an angle of 60˚.
A very wide stoppered tube is required to contain it. The swab is
moistened with sterile water just before use, passed over the
dorsum of the tongue and introduced into the larynx, where it
stimulates coughing and collects the expelled secretion.
28. High vaginal & cervical swabs – for the diagnosis of gonorrhoea &
puerperal fever. Swab should be taken from the uterine cervix & its
lumen, rather than from the general area of the upper vagina.
A swab on a specially long, rigid swab-stick, preferably about 22
cm long, is required.
29. BLOOD
Blood culture is requested in:
Presence of fever or other signs and symptoms occuring in a case
with known/suspected local infection indicating bacteremia.
As part of investigation in PUO/FUO
30. PREPARATION OF THE SITE
Select the site of venipuncture. If the patient is unusually dirty,
wash the intended site with soap and water prior to venipuncture.
Apply a tourniquet, 3-4 inches above the intended site of
venipuncture, can be done after cleaning.
Put on examination gloves.
Vigorously cleanse with 70% isopropyl or ethyl alcohol to remove
surface dirt and oils.
31. Scrub the venipuncture site
gently but firmly with the cotton
beginning in the center and
continuing in an outward
direction circularly for an area of
4 to 5 inches in diameter.
Allow to dry.
32. Swab or wipe concentric circles of povidone/tincture of iodine, in a
similar manner as given earlier- beginning in the center and
continuing in an outward direction circularly for an area of 4 to 5
inches in diameter.
Allow the povidone iodine to dry (2 minutes).
Do NOT touch the site after cleaning.
Instruct patient to clench and unclench the fist.
33.
34. Inoculate blood into blood culture bottle containing media at
bedside itself.
Blood collected from indwelling iv or intraarterial catheter are not
ideal,increase risk of recovering skin commensals.
If an iv catheter sample is used a second sample should be drawn
by venipuncture for comparison.
35. BLOOD CULTURE BOTTLE
100ml broth with metal cap with a hole of
5mm diameter sealed with an inner tube
seal.
How to inoculate blood into media:
Ideally change needle before inoculating
blood into bottle to decrease contamination
but it increases the risk of needle stick injury.
36. SPECIMEN VOLUME
Sufficient sample volume is critical for isolation of organisms from
blood.
10-20ml of blood is collected as 2 sets is recommended for adults.
1-3ml blood is drawn in infants and small children.
37.
38. NUMBER OF BLOOD CULTURES
More than 1 blood culture is required because
Periodicity of microorganisms in bloodstream varies with different
diseases.
More chance of isolation of organism.
To differentiate true pathogens from contaminants.
If the volume is adequate usually 2-3 blood cultures taken at half
an hour intervals is sufficient.
39. IN INFECTIVE ENDOCARDITIS
Patients who have not received prior antibiotics :
A single blood culture is positive in 90-95% cases and 2nd blood
culture establishes the diagnosis in 98% cases.
If prior antibiotics were taken ,3 separate blood cultures of 10-20ml
each and an additional blood culture or 2 taken on the 2nd day will
be necessary for isolation.
40. TIMING OF BLOOD CULTURE
Blood for culture should be drawn before the use of antibiotics if
possible.
Blood collected at the time of fever spikes have increased chance
of isolation as presence of fever indicates the entry of bacteria into
blood stream.
41. DILUTION OF SAMPLES
1:10 ratio of blood to medium.
To reduce the concentration of natural antimicrobial constituents
of blood.
Para aminobenzoic acid added to blood culture media to neutralize
sulphonamides.
Broad spectrum β lactamase may be added if patient is receiving
penicillin or cephalosporins.
42. ANTICOAGULATION
Blood drawn for blood culture should not be allowed to clot as
bacteria may get entrapped within the clot and their presence may
go undetected.
Therefore blood is inoculated directly into blood culture bottle
containing anticoagulant and transported to lab for subsequent
inoculation.
Sodium polyanethol sulfonate 0.025%-0.03% is most suited for
blood cultures.
43. SPS has anticomplementary, antiphagocytic,antilyposomal activities
and interferes with antimicrobial agent activity especially
aminoglycosides.
But SPS can inhibit growth of Neisseria sp,Gardenella
vaginalis,Streptobacillus moniliformis,peptostreptococcus.
44. TRANSPORT
In case of delay keep in incubator or at room temperature.
Blood for serological investigation- plastic tubes with screw caps
without anticoagulation.
53. MIDSTREAM URINE SPECIMEN
COLLECTION:
Urine collection from women by this
technique requires special care.
Periurethral and perineum are first
cleansed with 2 or 3 gauze pads
saturated with soap water using forward
to backward motion.
Followed by a rinse with sterile saline
or water.
54. Labia should be held apart during voiding and the first few
milliliters of urine passed should not be taken for culture as it
contain the normal flora from urethra.
The midstream portion of urine is then collected in a sterile wide
mouthed container with a tight fitting lid.
55. CATHETER URINE SPECIMENS
Straight catheterized urine specimen
more invasive, requires health personal.
catheterization for the purpose of obtaining urine specimen for
culture should be avoided because of risk of introducing infection
(Iatrogenic UTI).
It should be limited to patients who are unable to produce an
adequate midstream urine specimen.
56. Indwelling catheter
Urine should be collected from the rubber tubing and not from the
collecting bag .Express out the urine collected in the tube.
Clamp the catheter tubing and wait 2 hours to allow collection of
freshly voided urine.
Clean the site with 70% ethanol and aspirate (5-10ml) urine from
the catheter tube using a needle and syringe under aseptic
precautions.
57.
58. Urine specimen from the collection bag is inappropriate because
the organism can multiply in urine collected.
Catheter tips are also inappropriate specimen as they are
contaminated with urethral flora or colonizing organisms.
59. SUPRAPUBIC ASPIRATION
Primarily in neonates and small children.
The procedure is done when the bladder is full.
The skin above pubic symphysis is disinfected using 70% isopropyl
alcohol.
An 18G spinal needle is inserted into the bladder and appoximately
10ml of urine is aspirated using syringe.
60.
61.
62. IN CHILDREN
Collection of urine in a bag held over the genitalia using
an adhesive tape-yields contaminated specimen.
But a negative culture in such cases can rule out UTI.
Stimulating urine flow by tapping first above the pubic
symphysis using 2 fingers,1 hr after a feed.1 tap per
second for 1 min.
An interval of 1 min is allowed.Then continue tapping.
This will stimulate urine flow in neonates and young
children.
63. SUSPECTED TUBERCULOSIS INFECTION
Complete early morning urine sample for 3 consecutive days are
collected. Centrifuged and the deposit is taken for culture.
This is to increase the chance of isolation of Mycobacterium
tuberculosis.
500 ml sterile wide mouthed container is used.
64. SUSPECTED URETHRITIS AND PROSTATIS
Instead of midstream urine sample the initial part of urine is
collected for culture.
Terminal urine is collected in case of Schistosoma haematobium
65. TRANSPORT
Urine acts as a good culture medium. Therefore bacterial overgrowth occurs
contaminating the specimen.
Freshly voided urine sample should be transported to the lab as soon as
possible or atleast within 2 hrs of collection.
If delay is unavoidable:
Refrigerate the specimen(at 4 deg celsius)-upto 24hrs and transport to lab in
ice packs.
Collect urine in containers with preservatives like boric
acid(1.8%),glycerol,sodium formate.
66. These preservatives act as as bacteriostatic agents and prevents
overgrowth and can be used for storage of urine upto 24hrs.
Disadvantage:
Boric acid may inhibit growth of certain pathogenic organisms.
67. DIPSLIDE METHOD
Dipslide is a small plastic tray carrying
a layer of agar on either side.
It is attached to the cap of a screw
capped container.
Urine is passed into a sterile container
and the dipslide is dipped into it and
then drain off excess urine and replace
the dipslide in the container and
transport to the lab.
68. CSF
Collection of CSF is done by doing lumbar puncture.
Other methods-shunt,ventricular aspirate.
The patient lies on his side with kness flexed.
Skin over the lumbar spine is disinfected and a spinal needle(23G)
is inserted between the L2 and L3 vertebrae into the spinal canal.
3-4ml of CSF is collected into a sterile container with tight fitting
lid.
69.
70. Physicians should be instructed to sequentially collect 2.0 ml of CSF
into three sterile calibrated tubes if only routine chemistry (total
protein and glucose), bacteriology (culture & susceptibility), and
hematology (cell count) are required.
CSF must be send to the lab as quickly as possible.
Any delay may result in the death of delicate pathogens like
meningococci.
Do not refrigerate the specimen as it will kill organisms like
H.influenzae.
71. VENTRICULAR SHUNT FLUID
Clean the reservoir site with antiseptic solution and alcohol prior to
removal of fluid to prevent introduction of infection.
Remove fluid by aspiration of CSF from the Ommaya reservoir or by
collection from the ventricular drain or shunt.
An initial CSF sample should be collected prior to antimicrobial
therapy for highest diagnostic sensitivity.
Subsequent CSF samples are then collected every 2 to 3 days once
antimicrobial therapy is started to monitor for resolution of the
infection
72. SPECIMENS FROM GIT
Gastric aspirate:
For AFB culture in children
Collected in early morning before patient
eats or gets out of bed.
Collected in a sterile,screw capped
container and transported to the lab within
15 mins.
73. GASTRIC BIOPSY
Gastric biopsy:
For detection of H.pylori infection.
Gastric biopsy specimen is collected in a sterile screw capped
container with sterile normal saline.
Should be transported within 1hr at room temperature.
Can be stored at 4 degree celsius for upto 24 hrs.
74. DUODENAL ASPIRATES
For detecting giardiasis,strongyloidiasis.
Collected using a naso-jejunal tube or by using string test
(enterotest).
It is a weighted gelatin capsule containing tightly wound string, one
end of which is taped to the patients' cheek and the capsule
swallowed.
After 30-60mins capsule reaches duodenum,string is removed and
mucous adhering to the string is taken for microscopic examination.
75. STOOL
Stool should be collected in a clean wide
mouthed screw capped container with a
spoon projecting from the underside of the
cap.
Collect 1-2ml of stool in the spoon and
insert it into the container and close the lid.
Stool should not be mixed with urine or
disinfectants.
Transport within 1 hour if unpreserved.
76. In case of delay collect stool in container holding about 6ml of
buffered glycerol saline or other transport media like Cary-Blair
media(upto 24hrs)
For detection of ova and parasites, a small portion of stool sample
must be placed in preservatives like polyvinyl alcohol,10%
formalin(to maintain the integrity of trophozoites and cysts).
Stool for viral culture can be refrigerated if not inoculated within 2
hrs.
77. RECTAL SWAB
In newborn, debilitated adults.
Insert swab approx 2.5cm past anal sphincter
Specimen should be collected from the faecal material held in the
rectum.
Swabs must be placed in transport media to avoid drying.
78. EXUDATE
Most often submitted specimen in case of wounds, abscess or
cellulitis is a swab taken from the site.
Swabs are inefficient sampling devices because:
Tend to dessicate the specimen
Inadequate quantity of specimen
Surface wound are most often colonised with environmental
bacteria and swabs taken without proper cleaning may yield
colonisers instead of true pathogen.
79. If swabs are used, certain precautions must be taken.
Cotton swabs may contain residual fatty acids and calcium alginate
may emit toxic products that may inhibit fastidious bacteria.
Swabs tipped with Dacron are better choices.
Swabs should be placed in a moist container or a transport medium
to prevent drying and death of bacteria.
80. Ideal specimen in case of abscess,wounds etc are biopsy of the
infected tissue, curettage of a draining wound or aspiration of
loculated fluid/pus from the depths of abscess.
The site should first be cleansed with 70% isopropyl alcohol to
disinfect the skin.
Fluid or pus must be aspirated from the depths of the abscess.
Tissue must be excised from the depth of wound.
81. The specimen should be transported in a sterile container with tight fitting lid.
The aspirating syringe itself is often used as a transport container.
This procedure is discouraged now due to risk of transmission of blood borne
diseases by needle prick injury.
But if only a small amount of fluid is obtained and the entire specimen is within
the needle the needle should be recapped carefully and whole syringe and
needle is placed in a puncture proof container and send to lab.
And needle is rinsed with broth to obtain material for culture
82. ANAEROBIC CULTURE METHODS
Collect the aspirate in a sterile container with airtight lid.If possible
fill the container upto the brim.
If the aspirate is send in the syringe itself instead of a needle
cap,use a rubber seal or cork.
Tissue specimen are better specimen for isolation of
anaerobes.They are collected in sterile container with airtight lid.
Ideally the specimen should be inoculated into anaerobic media
like RCM at the site of collection itself and only then transported to
lab.
83. IN CASE OF ABSCESS/CLOSED WOUNDS
Disinfect the skin using 2% chlorhexidine with alcohol
3-5ml of fluid is aspirated using a syringe and needle and placed in
a sterile container with tight fitting lid.
84. IN CASE OF OPEN WOUNDS
Remove overlying debris. Clean the superficial area thoroughly
with sterile saline.
Collect tissue sample from the base or advancing margin of the
lesion.
Collect a portion of the specimen in anaerobic media like RCM for
anaerobic culture.
Tissue/biopsy specimen should be sent in a sterile container with
saline.
85. BODY FLUIDS FROM STERILE SITES
Collected by percutaneous aspiration
under strict aseptic
conditions.Eg:Pleural,pericardial,peritoneal,
synovial fluid.
Synovial fluid and peritoneal fluid may be
immediately inoculated into BCB,retaining
about 0.5ml in syringe for gram staining.
Other fluids are collected in a sterile
container or blood collection tube without
preservatives.
87. THROAT SWAB
Patient is asked to tilt his head back,open mouth and breathe
deeply.
Tongue is depressed using a tongue depressor.
Mucosa of the tonsillar pillars,behind the uvula and posterior
pharynx is swabbed in a gentle sweeping motion.
Do not touch other areas of mouth to decrease contamination with
normal flora.
88.
89. Swab should be transported immediately to the laboratory to
prevent drying.
Transport media like Amies and Pikes media can be used.
90. NASOPHARYNGEAL SWAB
For detection of Bordetella pertussis, RSV, influenza virus,
meningococcal carrier.
Specimen from nasopharynx is collected using a thin flexible
nasopharyngeal wire swab passed along the floor of nasal cavity.
A long swab with terminal 20mm bent at an angle 45 degree
introduced through mouth up behind soft palate into nasopharynx.
Keep it in place for 5 seconds.
91. SPUTUM
Simplest and least expensive.
More often contaminated with saliva.
Brush teeth and gargle with water before
sputum collection.
Early morning sputum sample is ideal because
the pooled overnight secretions in which
pathogenic bacteria are more likely to be
concentrated is coughed out in the morning.
Collected in a sterile wide mouthed container of
50-100 ml volume, with a screw capped lid.
92. 24 hour sputum sample is not ideal because:
Increased chance of overnight growth of normal flora.
Bacterial pathogen in 1 sample may become diluted with addition
of subsequent more watery specimen.
93. TRANSBRONCHIAL BRUSHING AND
BIOPSY
Collected during bronchoscopy
A telescoping double catheter plugged with polyethylene glycol at
the distal end to protect a small bronchial brush is used for collecting
bronchial brushings.
94. BRONCHOALVEOLAR LAVAGE
Inject 30-50ml of sterile normal
saline through the bronchoscope
into the bronchioles and then re-
aspirate the fluid.
Ideal for diagnosing pneumonia
in intubated patients.
95. In patients with difficulty in sputum production sputum can be
induced by:
Inhalation of nebulized saline
Chest physiotherapy and postural drainage
In children who tend to swallow sputum early morning gastric
aspirate sample can be used.
Transport within 2 hrs.
96. ENDOTRACHEAL ASPIRATE
Collected by aspirating
tracheal secretions through
endotracheaal tube or
tracheostomy tube.
More representative of lower
respiratory tract specimen.But
oral secretions can dribble
down the ET tube and
contaminate the specimen.
97. OCULAR SPECIMEN
Premoistened swabs from conjunctiva and lid
margins(blepharitis).
Culture specimens if needed should be
obtained from both eyes separately.
Conjunctival/corneal scrapings in case of
bacterial keratitis taken using sterile scalpel.
Aqueous/vitreous humor.(Endophthalmitis)
Specimens collected by ophthalmologists and
immediately inoculated into culture media
98. TRANSPORT MEDIA
TRANSPORT OR HOLDING MEDIA is a non-nutrient media, that
maintain the viability of microorganisms present in the specimen
without supporting the growth of any of the organisms.
This maintains the organisms in a state of suspended animation so
that no organism overgrows another or dies out.
99. These media are essentially solutions of buffers, with
carbohydrates, peptones and other nutrients & growth factors
excluded, designed to preserve the viability of bacteria during
transport without allowing their multiplication.
SODIUM THIOGLYCOLLATE may be added as a reducing agent to
improve recovery of anaerobic bacteria.
SUCROSE-PHOSPHATE-GLUCONATE is a good transport buffer
medium for recovery of certain viruses, such as Herpes virus.
100. For convenience, this method requires the use of swabs on
wooden sticks that can be broken easily.
If thin wire handles are used, they may be cut off or pressed down
into the container.
Transport media are available with different ingredients and in
different containers for special purposes, such as deep thioglycollate
medium for anaerobes.
Sterile disposable swab kits incorporating a transport medium are
supplied commercially.
101. The transport medium which is made semi-solid with agar, may be
used in a screw-capped universal container or smaller bottle, or a
hermetically stoppered test tube.
Immediately the specimen has been taken, the swab is plunged
into the depths of the transport medium and the cap or stopper is
firmly applied. The container is then sent to the laboratory.
Even the delicate pathogens may remain alive for a day or two at
ambient temperature.
102. STUART’S TRANSPORT MEDIA
This soft agar medium is used to maintain the viability of gonococci
on swabs during their transmission to a laboratory.
Na thioglycollate 1gm
Na glycerophosphate 10 gm
CaCl2 0.1gm
Agar 6gm
Methylene blue 1% 4ml
Distilled water 1 litre
103. Dissolve all the solids in the distilled water at 100˚C.
Adjust the pH to 7.3-7.4
Swabs: boil in PO4 buffer,1% charcoal,sterilize
104. AMIE’S TRANSPORT MEDIUM
Charcoal is incorporated into the media to absorb fatty acids present in
specimen that could kill fastidious organisms such as N. gonorrhoea & B.
pertussis.
106. Dissolve the chemical salts & the agar in the distilled water at
100˚C. Add the charcoal. Adjust the pH to 7.2
Dispense, with regular stirring to keep the charcoal in suspension,
into bijou bottles filled nearly full.
Autoclave at 121˚C for 15 min and cool.
Frequent inversion of the bottles during cooling is needed to
distribute the charcoal.
107. PIKE’S MEDIUM
To preserve Streptococcus pyogenes, Pneumococci and
Hemophilus influenzae in nose & throat swabs.
It is blood agar containing crystal violet 1-in-10,00,000 and sodium
azide 1-in-16,000 distributed as for stab cultures in tubes or bottles.
108. GLYCEROL SALINE TRANSPORT MEDIUM
If there is likely to be a delay of several hours before
specimens of faeces for culture to reach the
laboratory, this transport medium prevents other
intestinal microorganisms from overgrowing the
typhoid bacilli.
Glycerol 300ml
Sodium chloride 4.2g
Disodium hydrogen phosphate(anhydrous) 10g
Phenol red, 0.02%(aqueous) 15ml
Water 700ml
109. Dissolve the sodium chloride in water and add the glycerol.
Add the phosphate and steam to dissolve it.
Then add enough phenol red to give a purple-pink color.
Distribute in 6 ml amounts in universal containers and autoclave at
115˚C for 20 min.
The fluid should not be used if it becomes acidic, indicated by a
change in color to yellow.
110. CARY BLAIR MEDIUM
Salmonella and shigella may survive upto
48hrs
Campylobacter for about 6 hrs.
Insert a faecal swab into this semisolid
transport medium,break off the swab stick
projecting out of the bottle and replace the
cap tightly.
112. COLLECTION AND TRANSPORT OF
VIROLOGICAL SPECIMENS
Specimen selection depends on the specific disease syndrome, viral
etiologies suspected, and time of year.
Specimen selection based on virus suspected is complicated by the
fact that similar clinical syndromes can be caused by many different
viruses.
The laboratory should always be notified if rare agents
representing a danger to laboratory workers, such as SARS
coronavirus, H5N1 avian influenza virus, hemorrhagic fever viruses,
and the like, are suspected.
113. Serum for serologic testing may be necessary, and some viral
disease need only to be considered during certain months because
appearance is seasonal.
Specimen should be collected as early as possible following the
onset of symptomatic disease. Virus may no longer be present as
early as 2 days after the appearance of symptom
114.
115.
116.
117. TRANSPORT AND STORAGE OF
VIROLOGICAL SPECIMENS
Ideally, all specimens collected for detection of virus should be processed by
the laboratory immediately.
Specimens should be placed in ice and transported to the laboratory at once.
If a delay is unavoidable, the specimen should be refrigerated, not frozen, until
processing occurs.
For storage up to 5 days, hold specimen at 4°C.
Storage for 6 or more days should be at –20° or preferably at –70° C.
Specimens for freezing should first be diluted or emulsified in viral transport
medium.
118. If a commercial kit is being used, specimens for molecular testing should be
transported and stored according to the manufacturer’s instructions.
Blood for viral culture, transported in a sterile tube containing anticoagulant,
must be kept at refrigeration temperature (4° C) until processing.
Blood for viral serology testing should be transported to the laboratory in the
sterile tube in which it was collected. Serum should be separated from the clot
as soon as possible.
Serum can be stored for hours or days at 4° C or for weeks or months at –20° C
or below before testing
119. VIRAL TRANSPORT MEDIUM
Viral transport medium (VTM) prevents specimen drying, maintains
viral viability and retards the growth of microbial contaminants.
VTM contains gelatin and antimicrobial agents in a buffered salt
solution.
Tubes containing 2-3 mL VTM are used for swab specimens, while
those with 5-7 mL VTM are suitable for tissue samples.
120. VIRAL TRANSPORT MEDIA
In general it contains:
Saline(adequate ion concentration)
Protein(albumin or gelatin)
Buffer(adequate pH)
Antibiotics and fungicides
121. MODIFIED HANK’S BALANCED SALT
SOLUTION
Bovine serum albumin
Sucrose
Glutamic acid
Gelatin
Phenol red
pH 7.3
Amphotericin
Colistin
Vancomycin
122. MYCOLOGY SPECIMEN COLLECTION
The principal goals of a sound clinical mycology laboratory are to isolate
efficiently and to identify accurately the suspected etiological agents of fungal
infection.
The following points need to be emphasized: -
Appropriate sample/specimen collection
Prompt transportation
Correct processing of the specimen
Inoculation of specimens onto appropriate culture media and incubation at
suitable temperature
123. Specimens should be collected aseptically, placed in sterile containers,
delivered to the laboratory within 2 hours, processed, and then inoculated to
primary isolation media within a few hours of collection.
Viability may decrease with prolonged specimen storage.
Swabs are not encouraged; however specimens from certain body sites such as
the ear canal, nasopharynx, throat, vagina and cervix are not readily collected by
other means.
Swabs for collection of material from open wounds or draining lesions are
frequently contaminated with environmental microorganisms.
124. TRANSPORT OF MYCOLOGY SPECIMENS
Specimens should be transported in sterile, humidified, leak-proof
container.
Dermatological specimens should be transported in a dry container.
Transport medium should not be used unless the specimen can be
easily and completely retrieved from the medium.
Specimens should be processed and inoculated to primary isolation
media as soon as possible after collection, ideally within few hours.
125. If processing is to be delayed for more than several hours, it is
recommended that specimens be stored under refrigeration at 4°C
with the following exceptions:
Blood and cerebrospinal fluid are stored at 30-37°C
Dermatological specimens are stored at 15-30°C
Editor's Notes
Must be neutralised with sodium bicarbonate within 1 hr of collection.
Organisms that are present in concentration 10^4ml CFU/ml and specimen that demonstrate intracellular bacteria in >25% of inflammatory cells are indicator of pneumonia
By collecting only specimens needed to detect a specific virus, other important etiologies may be missed.
HiViralTM Transport medium is a specially formulated
medium for collection, transport and long term freeze
storage of viruses. It is designed to maintain the optimum
viability and virulence of the viral sample.
HiViralTM Tra
