MODULE TITLE : Collecting and Processing Medical Samples

Unit Two
Sample Collection and Processing and Transportation
2.1 Purpose of sample collection

There is a high risk of communicable disease outbreaks in emergency situations. Outbreaks must be
recognized and controlled rapidly in order to minimize their impact.
Sample collection is useful for:
• early detection and reporting of suspect cases
• Rapid epidemiological investigation
• Rapid laboratory confirmation of the diagnosis
• Implementation of effective control measures.
Rapid identification of the causative agent and the likely source or mode of transmission is essential.
The initial investigation involves two important processes; collection of information on suspect cases
and collection of clinical specimens for laboratory diagnosis.
Successful laboratory confirmation of a Disease depends on:
 Collection of appropriate and adequate specimens
 correct packaging and rapid transport to an appropriate laboratory
 the ability of the laboratory to carry out the diagnostic tests
 Proper biosafety and decontamination procedures to reduce the risk of further spread of the
disease.
Outcomes of Improper Collection

 delays in reporting test results

 unnecessary re-draws/re-tests

 decreased customer satisfaction

 increased costs

 incorrect diagnosis / treatment

 injury

 death

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2.2 Sample collection requirement

1. Specimen collection manual

 Provides written instruction for proper and adequate collection of specimens capable of giving
timely and medically important data.
 A complete and up-to-date collection procedure manual, covering every test the laboratory offers,
should be distributed to all health professional in charge of collecting the specimens.
The manual should contain the following information:
1. Test name: The name of each test with alternate names and commonly used abbreviation are listed in
alphabetical or some other logical order.
2. Patient preparation: how the patient should be prepared prior to collection of the sample should be
detailed here. For example, should the patient be fasting or two hours prandial? Should the patient be in
the sitting or recumbent?
3. Type of specimen: Could be blood, stool, Urine, Other body fluids, & Tissue samples and indicate
which specimen is preferable for which test …etc.
4. Special timing for collection: should the sample be a random one or timed to coincide with the
administration of a drug or with some other pathologic event. This is especially important when looking
for specific responses to treatment, such as glucose tolerance test.
5. Preservatives or anticoagulants: to maintain the specimen in an acceptable state, it may be
necessary to use an anticoagulant, preservative or even to store the specimen on melting ice or ice water.
6. Transportation requirement: special handling between the collection time and delivery to the
laboratory may be required. Chemicals can be altered, microorganisms may die and the specimen altered
if improperly handled during transportation. Transportation considerations include:
A. Time: the sample should be removed to the laboratory as short as possible. If it cannot be processed
within a reasonable time period (this vary from sample to sample), steps must be taken to prevent
undesirable changes.
B. Temperature: All specimens should be stored at optimum temperature depending on the type of the
specimen & the analyte being investigated.
Either chilling or heating may adversely affect the outcome of the analysis. Overheating of the samples
may destroy enzymes, may lyses cells, kill some microorganisms and accelerate the growth of others.
Other samples need to be placed in ice water (2-8) during transportation to the laboratory to maintain
their integrity. The tightly stoppered collection tube should be placed up right in the container and

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completely immersed in the crushed ice or ice water. Crushed ice or iced water is used to establish good
contact between the ice and the sample so that there is complete chilling.
C. Exposure to light: exposure to light will result in the degradation of certain plasma analytes,
especially bilirubin. Wrap samples in aluminum foil or provide light tight containers.
D. Excessive vibration & rough handling: vigorous handling of specimen may result in hemolysis of
red cells, spillage of the specimen, or the breakage of the collection tubes or specimen containers.
E. Labeling procedure: specific requirements for labeling the specimen should be clearly defined. A
minimum patient name, identification number, collector’s initials and the date and time of collection
should be labeled on the sample. Some specimens may require more information; microbiological
samples need to have the site of the sample collected.
7. Required requisition: specific requisition with test name and reference ranges.
8. Specimen rejection Criteria:
A specimen should be rejected for any of the following:
1. Improper transport temperature
2. Improper transport container or medium
3. Prolonged transport time
4. Unlabeled or mislabeled specimen
5. Broken or cracked container
6. Leaking specimen
7. Dried-out specimen
8. Inappropriate specimen for test requested
9. Inadequate volume
10. Specimen in fixative (for culture)
11. Duplicate sample in 24-hr period (for urine, sputum, feces culture)
9. Performing laboratory: Each test should be listed with the phone number and hours of operation of
the performing laboratory. If the physicians and nurses staff who will collect the samples know where
the samples should go and the hours of operation of the performing laboratory, then improperly timed
specimens may be avoided.

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2. Laboratory Requisitions Paper

The following items should be included on the lab requisition:

1. Full name: middle name should be included to avoid confusion in the event that there is
another patient with the same first and last name.
2. Location: inpatient, room, unit, outpatient, address.
3. Patient's identification number: this identification can be very useful for instance in the
blood bank.
4. Patient age and sex: in evaluating laboratory results, the reference values may differ for
age and sex; disease prevalence may be age- or sex-linked.
5. Name(s) of the physician(s): name all of the physicians on the case; "panic values"
should be called to the attention of the physician ordering the test; a physician may have
some specific test guidelines for his patients.
6. Name of the test and the source: reference values may be different for the different
biologic specimens (e.g., serum and CSF glucose); in microbiology, it is essential to
know the source of the swab.
7. Possible diagnosis: essential for evaluating laboratory results and selecting appropriate
methodology; (media selection in microbiology).
8. The date and time the test is to be done: some tests must be scheduled by the laboratory;
blood transfusions may require ample advance notice; patient preparation and diet
regulations need to be considered.

3. Sample Register or Log

A register should include:

 date and time of collection

 date and time of receipt
 sample type and tests to be performed
 patient name and demographics as required
 laboratory assigned identification

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2.3. Basic Concept in Specimen Collection

1. Site selection

Clinician: should locate right anatomic site & select appropriate tests & specimens based on:

• Physical examination (sign & symptoms)

• Radiological examination
Laboratory personnel: should collect specimens from actual infection site with little external
contamination by using aseptic technique

• To prevent contamination of specimen &

• To protect the patient from infection
General approach approaches to avoid contamination:

• Careful patient education

There are occasions when patients participate actively in specimen collection (e.g. sputum,
urine). Therefore, they must be given full instructions & cooperation by the care giver

• Educating the clinicians

How to collect & transport specimens through written document & make available at every
patient care unit

2. Volume of specimens

• Collecting & processing too little specimen lower sensitivity

Collecting adequate volume of sample enhances recovery of the pathogen and enables to perform
all procedures required or to permit complete examination

For example:

Sputum: 5 -10 ml for mycobacterium examination

Blood: Serology: minimum 2 - 3 ml

Culture: 10 – 20 ml (adult) & 1-5ml (infant)

3. Time of collection - Provide best chance of recovery of the causative agent

Sputum & urine - early in the morning soon after the patient awaken

Blood - when the patient’s temperature begins to rise

4. Collect specimens before the administration of antimicrobial

Because antimicrobials limits recovery of pathogens

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5. Age of specimens- age of the specimen directly influences the recovery of protozoan
organism

6. Stage of the disease at which the specimen is collected

Enteric pathogens are present in great numbers during the acute or diarrheal stage of intestinal
infection

7. Labeling

• Make sure that you are collecting/drawing the right person first.
Then label with:

• Patient name
• Unique identification number
• Patient demographic information
• Specimen collection date
• Specimen collection location
During Labeling:

• Make sure that container label & the requisition match

• Label should be on the container not on the lid, since the lid can be mistakenly placed on
a different container
• Ensure the labels on the containers are adherent under refrigerated conditions

2.4 . Type of specimen

 Stool
 Urine
 Blood
 Body fluid; eg. Sputum, CSF…
 Vaginal discharge and urethral discharge
 Tissue sample
 Swabs
 Semen

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2.4.1. Collection and processing of fecal specimen for parasitic examination

2.4.1.1 Stool sample collection
Note
 For clinical purposes, a fresh fecal specimen is required. It should be uncontaminated
with urine and collected into a suitable size, clean, dry, leak-proof container.
 The container do not need be sterile but must be free of all traces of antiseptics and
disinfectants (also a bedpan if this is first used to collect a specimen from an inpatient).
 A large teaspoon amount of faeces is adequate or about 10 ml of a fluid specimen.
Several specimens collected on alternate days may be required to detect parasites that are
excreted intermittently, e.g. Giardia.
 Specimens must be labelled correctly and accompanied by a correctly completed request
form
 Unsuitable containers: Avoid using containers made from leaves, paper, or cardboard
(including matchboxes) because these will not be leak-proof, may not be clean, and can
result in the faecal contamination of hands and surfaces. If no leak-proof container is
available, a non-leakproof container can be used providing the specimen is first sealed in
a plastic bag. This, however, is not recommended for the routine collection of faeces
because the plastic bag is difficult to label and can be a hazard to laboratory staff.
 Dysenteric and watery specimens must reach the laboratory as soon as possible after
being passed (within 15 minutes) otherwise motile parasites such as E. histolyticaand G.
lamblia trophozoites may not be detected. Other specimens should reach the laboratory
within 1 hour of being collected.
 Faecal specimens, like other specimens received in the laboratory, must be handled with
care to avoid acquiring infection from infectious parasites, bacteria, or viruses.
 Faeces may contain:
– infective forms of parasites such as S. stercoralis, E. vermicularis, T. solium, G. lamblia,

E.histolytica, or C. parvum.
– bacteria such as V. cholerae, Shigella or Salmonella species.
– viruses including hepatitis viruses, and rotaviruses.

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Required material for stool collection

- Suitable size, clean, dry, leak-proof container.
- Applicator stick
- Pasteur pipette
- Labeling material
- Slide and cover slide
- Disinfectant (eg. Alcohol, Hypochlorate)
Procedure for stool sample collection
1. Take match stick head size or 2 gm of formed stool or use pasture pipette for diarrheal
stool by including all portion.
2. Put in a leak-proof container.
3. Label with basic information’s including patient name, ID No, time of collection and
name of sample collector.
4. Record or report the appearance of stool
5. Prepare the stool for analysis. Eg. Prepare wet mount for direct microscopy
examination.
During collection of stool, the precautions include:

 NO contamination with H2O - could bring free-living protozoans

 NO contamination with urine - destroys trophozoites
 Collect before barium, Antidiarrheal substances &
Antacids like MTs - obscures organisms (7+ days)

 Collect before antibiotics - may decrease number of organisms

Normal constituents of stool include:

 Water (about 75%).

 Rest constitute:
 dead bacteria that helped us digest our food, living bacteria,
 undigested food residue (known as fiber),
 cellular linings, sloughed epithelial cells
 substances released from the intestines (such as mucus) and the liver.

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 fat, protein, dried constituents of digestive juices (e.g., bile pigments),

 inorganic matter (e.g., calcium, phosphates)
Abnormal constituents of stool include:

 Pus: bacterial infection

 Mucus: inflammatory condition
 Parasites
 Blood: gastrointestinal bleeding
 Large quantities of fat: malabsorption
 Foreign objects: accidental ingestion
Reporting the appearance of faecal specimens
Before processing the specimen of faeces, it should be visually examined. Its colour, consistency
and presence of blood, pus, mucus or parasites should be reported.

Color

Normally, stool is brown in colour and it may be:

 New born infants: - Black(meconium)

 Breast feed infants: - scrambled egg
 Infant feed on animal milk: - “curd like”
Variations from this color may occur under certain conditions.

 Reddish color may be due to bleeding from the lower gastro-intestinal (G.I.) tract.
Consumption of beet-root may also give a red colour to the stool.
 Black-tarry color may be due to bleeding from the upper gastro intestinal tract or due
to consumption of iron.
 Clay coloured stool is seen in obstructive jaundice or after barium sulphate meal.
 Green stool may result from the consumption of leafy vegetables such as spinach or
sometimes due to oral antibiotic therapy
Consistency

 A normal stool specimen is formed or semi formed in consistency.

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 Abnormally Hard (Dehydration or decreased intestinal motility resulting from lack of

fiber in diet), Diarrhic, Watery, loose mixed with mucus and blood.
 Loose or watery stool may be seen in diarrhea. Trophozoites of intestinal protozoa are
usually seen in loose or watery specimen while cysts are found in formed and semi
formed ones. Eggs of helminths may be found in any consistency. Large amounts of
mushy, foul smelling, frothy specimen are seen in giardiasis and other conditions
associated with malabsorption.
 1-Hard- resists puncture
 2-Formed- can be punctured
 3- Soft-can be cut with applicator
 4- Mushy- can be reshaped
 5- Loose- shaped in to container
 6- Diarrhea- can flows
 7-Watery- can pour
Blood

The presence of blood in or on the specimen must always be reported.

 Fresh, bright red blood is often from the lower gastro-intestinal tract whereas
 Dark red color may indicate bleeding from the upper gastro-intestinal tract. When very
small amounts of blood are being passed in feces, it may not be visible macroscopically.
Such specimens are said to contain occult (hidden) blood. Some of the causes for the
presence of occult blood include iron deficiency anemia, peptic ulcer or cancer of the
gastro-intestinal tract. In suspected cases, a special request is sent for occult blood test.
Pus

 Fairly large quantities of pus may be passed out in stools of patient with inflammation
of the G.I. tract as in bacillary dysentery or chronic ulcerative colitis.
Mucus

 A normal stool does not contain mucus. Mucus may be associated with blood and pus in
many inflammatory conditions of the G.I. tract, and other conditions such as neoplasm.

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Parasites

 Adult worms such as Enterobius vermicolarisor Ascaris lumbricoides, and tape worm
segments may be found on the surface or in the stool. They should be identified and
reported.
Preservatives and fixatives for faecal parasites
The preservation of parasites in stool specimens are required:
- For the control of microscopically parasitology tests and for training purposes.
- When specimens are sent to peripheral laboratories as part of an external parasitology
quality assessment program.
- When specimens need to be sent to a Reference Laboratory for parasite identification.
- Specimens needed for teaching purpose
- When specimens are collected in the field, for example during epidemiological surveys.
For teaching purposes and external QA programmes it is useful to be able to preserve parasites in
faecal specimens, particularly eggs and cysts. To preserve and identify E. histolytica
trophozoites, special fixatives and staining procedures are required.
Fixative for egg and cyst of parasite in faeces
1. Bayer’s solution is recommended because it preserves well the morphology of faecal
eggs and cysts. It is a modified formalin solution containing copper chloride, glacial
acetic acid, and formalin.
The stock solution is stable indefinitely at room temperature.
Bayer-preserved faeces can be examined unstained in direct preparations (sampling from the
sediment) or examined after concentration by the formol ether technique.
Bayer’s solution is used as follows:
1. Prepare a working solution by diluting Bayer’s stock solution 1 in 10, e.g. use 1 ml stock
solution and 9 ml of distilled or filtered water.
2. Emulsify about 1 g of faeces in 3-4 ml of Bayer’s working solution. Use a leak-proof screw
cap container.
2. 10%v/v formol saline: can be used, but this will not preserve faecal cysts and eggs as
well as Bayer’s solution nor for as long a period.

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3. Merthiolate – Iodine formaldehyde (MIF): Contains iodine and eosin as a stain.

4. Poly Vinyl alcohol (PVA): This fixative is toxic and recommended in trichrome
staining. (1 parts of feces are emulsified with 3 parts of PVA)
5. 1% domestic bleach: 1ml added to 10ml of urine can preserve eggs of S.haematobium.
Fixation of faeces for E. histolytica trophozoites
Routinely, E. histolytica trophozoites are usually identified in dysenteric specimens by their
motility and ingested red cells. Only occasionally is it necessary to fix and stain faeces for E.
histolytica. The choice of fixative depends on the staining method used. Some of them are;
1. SAF (sodium acetate, acetic acid, formaldehyde): is fixative, commonly used when an
iron haematoxylin staining method is used
2. PVA (polyvinyl alcohol) or absolute methanol when using a trichrome staining method.
Preservation of worms
When required specimens can be fixed and preserved as follows:
1. Wearing protective gloves and eyeshields; make up the fixative solution as follows:
95% v/v ethanol . . . . . . . . . . . . . . . . . . . . .50 ml
Distilled or boiled water . . . . . . . . . . . . . . .45 ml
Formaldehyde solution, concentrated . . .. .10 ml
Glacial acetic acid . . . . . . . . . . . . . . . . . . . . .5 ml
2. Transfer the fixative solution to a beaker and heat to 60–65 °C.
3. If the specimen is a fluke or tapeworm segment, flatten it between two slides held loosely
together with an elastic band.
4. Place the specimen in the fixative and stir gently for a few minutes. Leave the specimen in the
fixative overnight.
5. Transfer the specimen to a container of 5% v/v glycerine in 70% v/v ethanol. Transport or
store the specimen in this solution.
Caution: Formaldehyde and glacial acetic acid have an irritating vapour, therefore ensure the
laboratory is well ventilated. The 95% ethanol solution is flammable; therefore keep it well away
from any open flame.
2.4.1.2. Preparing/processing stool samples for testing
Reasons for examining faecal specimens for parasites
 In district laboratories the examination of faecal specimens for parasites is requested:

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To identify the parasitic causes of blood and mucus in faeces and differentiate
amoebic dysentery from bacterial dysentery.
To identify intestinal parasitic infections that requires treatment.
Direct examination of faeces for parasites
 Routinely faecal specimens are examined in district laboratories by direct technique. This
involves:
Reporting the appearance of the specimen and identifying any parasitic worms or
tapeworm segments.
Examining the specimen microscopically for:
 Motile parasites.
 Helminth eggs,
 Cysts and oocysts of intestinal protozoa.
Reporting the appearance of faecal specimens
Report the following:
●Color of the specimen.
● Consistency, i.e. whether formed, semi formed, unformed, watery.
● Presence of blood, mucus, or, pus. If blood is present note whether this is mixed in the
faeces. If only on the surface this indicates rectal or anal bleeding.
● Whether the specimen contains worms, e.g. A. lumbricoides (large roundworm), E.
vermiculari (threadworm) or tapeworm segments, e.g.T. solium, T. saginata.
Microscopically examination of faecal specimens
 Examine immediately those specimens containing blood and mucus and those that are
unformed because these may contain motile trophozoites of E. histolytica or G. lamblia.
1. Direct wet mount method
A. Examination of dysenteric and unformed specimens
1. Using a wire loop or piece of stick, place a small amount of specimen, to include blood
and mucus on one end of a slide. Without adding saline, cover with a cover glass and
using a tissue, press gently on the cover glass to make a thin preparation.
2. Place a drop of eosin reagent on the other end of the slide. Mix a small amount of the
specimen with the eosin and cover with a cover glass.

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Value of eosin: Eosin does not stain living trophozoites but provides a pink background which
can make them easier to see.
3. Examine immediately the preparations microscopically, first using the 10× objective
with the condenser iris closed sufficiently to give good contrast. Use the 40× objective to
identify motile trophozoites, e.g. E. histolytica amoebae.
Note: The eggs of Schistosoma species and T. trichiura, and the trophozoites of B. coli can also
result in specimens containing blood and mucus.
B. Examination of semi-formed and formed faeces
1. Place a drop of fresh physiological saline on one end of a slide and a drop of iodine on
the other end. To avoid contaminating the fingers and stage of the microscope, do not use
too large a drop of saline or iodine.
2. Using a wire loop or piece of stick, mix a small amount of specimen, about 2 mg,
(matchstick head amount) with the saline and a similar amount with the iodine. Make
smooth thin preparations. Cover each preparation with a cover glass.
Important: Sample from different areas in and on the specimen or preferably mix the faeces
before sampling to distribute evenly any parasites in the specimen. Do not use too much
specimen otherwise the preparations will be too thick, making it difficult to detect and identify
parasites.
3. Examine systematically the entire saline preparation for larvae, ciliates, helminth eggs,
cysts, and oocysts. Use the 10× objective with the condenser iris closed sufficiently to
give good contrast. Use the 40× objective to assist in the detection and identification of
eggs, cysts, and oocysts. Always examine several microscope fields with this objective
before reporting ‘No parasites found’.
4. Use the iodine preparation to assist in the identification of cysts.

2. Faecal concentration techniques

Need for faecal concentration techniques
 The direct examination of faeces is essential to detect motile parasites and is usually
adequate to detect significant helminth infections.
 Important exceptions are Schistosoma species because only a few eggs are usually
produced even in moderate and severe infections, therefore a concentration technique

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should be performed when intestinal schistosomiasis is suspected and no eggs are found
by direct examination.
Concentration techniques may also be required:
– To detect Strongyloides larvae, the eggs of Taenia, cysts of G. lamblia, and to make it easier to
detect small parasites, e.g. small fluke eggs, or the oocysts of intestinal coccidia prior to staining.
– To check whether treatment has been successful.
– To quantify intestinal parasites.
 The following techniques are commonly used to concentrate faecal parasites in district
laboratories:
1. Sedimentation techniques in which parasites are sedimented by gravity or centrifugal
force, e.g. formol ether concentration method which is the most frequently used
technique because it concentrates a wide range of parasites with minimum damage to
their morphology.
2. Floatation techniques in which parasites are concentrated by being floated in solutions of
high specific gravity, i.e. solutions that are denser than the parasites being concentrated.
Examples include the zinc sulphate method and saturated sodium chloride method.
Choice of concentration technique
The method to use will depend on:
 Why the technique is being performed, the species of parasite requiring concentration,
and how well its morphology is retained by a particular technique.
 The number of specimens to be examined and time available.
 The location, e.g. field or laboratory situation and equipment available.
 Experience of staff performing the technique.
 Health and safety considerations.
A. Formal ether concentration technique
 This is recommended for use in district laboratories because it is rapid and can be used to
concentrate a wide range of faecal parasites from fresh or preserved faeces.
 Eggs that do not concentrate well by this technique are those of Fasciola species and
Vampirolepis nana but concentration of these parasites is not usually required.
 Risk of laboratory acquired infection from faecal pathogens is minimized because
organisms are killed by the formalin solution.

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 The technique, however, requires the use of highly flammable ether or less flammable
ethyl acetate.
Principle
 In the Ridley modified method, faeces are emulsified in formol water, the suspension is
strained to remove large faecal particles, ether or ethyl acetate is added, and the mixed
suspension is centrifuged. Cysts, oocysts, eggs, and larvae are fixed and sedimented and
the faecal debris is separated in a layer between the ether and the formol water. Faecal
fat is dissolved in the ether.
Required
– Formal water, 10% v/v.
– Diethyl ether or ethyl acetate.
– Sieve (strainer) with small holes, preferably 400–450 µm in size.*
*The small inexpensive nylon tea or coffee strainer available in most countries is suitable (can be
used many times and does not corrode like metal sieves).
Method
1. Using a rod or stick, emulsify an estimated 1 g (pea-size) of faeces in about 4 ml of 10%
formol water contained in a screw-cap bottle or tube.
Note: Include in the sample, faeces from the surface and several places in the specimen.
2. Add a further 3–4 ml of 10% v/v formol water, cap the bottle, and mix well by shaking.
3. Sieve the emulsified faeces, collecting the sieved suspension in a beaker.
4. Transfer the suspension to a conical (centrifuge) tube made of strong glass, copolymer,
or polypropylene. Add 3–4 ml of diethyl ether or ethyl acetate.
Caution: Ether is highly flammable and ethyl acetate is flammable, therefore use well away from
an open flame. Ether vapour is anaesthetic, therefore make sure the laboratory is well-ventilated.
5. Stopper* the tube and mix for 1 minute. If using a Vortex mixer, leave the tube
unstoppered and mix for about 15 seconds (it is best to use a boiling tube).
* Do not use a rubber bung or a cap with a rubber liner because ether attacks rubber.
6. With a tissue or piece of cloth wrapped around the top of the tube, loosen the stopper
(considerable pressure will have built up inside the tube).
7. Centrifuge immediately at 750–1 000 g (approx. 3000 rpm) for 1 minute.

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After centrifuging, the parasites will have sedimented to the bottom of the tube and the faecal
debris will have collected in a layer between the ether and formol water as shown in Fig. 2.1.
8. Using a stick or the stem of a plastic bulb pipette, loosen the layer of faecal debris from
the side of the tube and invert the tube to discard the ether, faecal debris, and formol
water. The sediment will remain.
9. Return the tube to its upright position and allow the fluid from the side of the tube to
drain to the bottom. Tap the bottom of the tube to re suspend and mix the sediment.
Transfer the sediment to a slide, and cover with a cover glass.

Ether and
dissolved fat
Faecal debris

Formol water

Sediment containing parasites

Fig. 2.1: Formal ether sedimentation concentration technique, after centrifugation.

10. Examine the preparation microscopically using the 10× objective with the condenser iris
closed sufficiently to give good contrast. Use the 40×objective to examine small cysts and
eggs. To assist in the identification of cysts, run a small drop of iodine under the cover
glass.
Although the motility of Strongyloides larvae will not be seen, the non-motile larvae can be
easily recognized.

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11. If required, count the number of each species of egg in the entire preparation. This will
give the approximate number per gram of faeces.
B. Zinc sulphate floatation technique
 The zinc sulphate technique is recommended for concentrating the cysts of G. lamblia
and E. histolytica/E. dispar, and the eggs of T. trichiura and Opisthorchis species.
 Other nematode eggs are concentrated less well.
 The technique is not suitable for concentrating eggs or cysts in fatty faeces.
 Adequate safety precautions should be taken because faecal pathogens are not killed by
zinc sulphate.
Principle
 A zinc sulphate solution is used which has a specific gravity (relative density) of 1.180–
1.200. Faeces are emulsified in the solution and the suspension is left undisturbed for the
eggs and cysts to float to the surface. They are collected on a cover glass.
Required
– Zinc sulphate solution, 33% w/v, specific gravity, 1.180–1.200.
Adjust with distilled water or more chemical if required.
– Test tube (without a lip) of about 15 ml capacity which has a completely smooth rim.
– Strainer (nylon coffee or tea strainer is suitable).
Method
1. Fill the tube about one quarter full with the zinc sulphate solution. Add an estimated 1
gram of faeces (or 2 ml if a fluid specimen). Using a rod or stick, emulsify the specimen
in the solution.
2. Fill the tube with the zinc sulphate solution, and mix well. Strain the faecal suspension to
remove large faecal particles.
3. Return the suspension to the tube. Stand the tube in a completely vertical position in a
rack.
4. Using a plastic bulb pipette or Pasteur pipette, add further solution to ensure the tube is
filled to the brim.
5. Carefully place a completely clean (grease-free) cover glass on top of the tube. Avoid
trapping any air bubbles.
6. Leave undisturbed for 30–45 minutes to give time for the cysts and eggs to float.

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Note: Do not leave longer because the cysts can become distorted and the eggs will begin to sink.
7. Carefully lift the cover glass from the tube by a straight pull upwards. Place the cover
glass face downwards on a slide.
Caution: Avoid contaminating the fingers. Mature E. histolytica and G. lamblia cysts are
infective when passed in the faeces.

8. Examine microscopically the entire preparation using the 10× objective with the
condenser iris closed sufficiently to give good contrast. Use the 40× objective, and run a
drop of iodine under the cover glass, to identify the cysts.
9. Count the number of T. trichiura eggs to give the approximate number per gram of
faeces.
Note: Parasites can also be recovered from the surface of the floatation fluid after centrifuging. If
however, a centrifuge is available, the safer formol ether technique is recommended for
concentrating eggs and infective cysts from faecal specimens.
C. Saturated sodium chloride floatation technique
 The saturated sodium chloride technique is a useful and inexpensive method of
concentrating hookworm or Ascaris eggs, e.g. in field surveys.
Method
 The technique is the same as that described for the zinc sulphate floatation technique
except that a saturated solution of sodium chloride (specific gravity of 1.20) is used and a
flat bottomed vial (50 mm tall and 20 mm wide) is used instead of a tube. A glass slide is
used to recover the eggs instead of a cover glass. Eggs can be recovered after about 20
minutes.

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 MODULE TITLE : Collecting and Processing Medical Samples

2.4.2 Collection and Processing of Urine Specimen:

2.4.2.1 Collection of urine specimens
- In order to make Urinalysis reliable the urine must be properly collected. Improper
collection may invalidate the results of the laboratory procedures, no matter how
carefully and skillfully the tests are performed.
- There are many types of containers used for collecting urine. Before specimens are
collected, the containers must be cleaned and thoroughly dried.
- Special polyethylene bags are available for collection of urine from infants and children
who are not toilet trained.
- A freshly voided urine specimen is adequate for most urinalysis except the
microbiological culture.
- The patient should be instructed to void directly into a clean, dry container or a clean, dry
bedpan so that the specimen can be transferred to an appropriate container.
- If a urine specimen is likely to be contaminated with vaginal discharge or menstrual
blood, this period has to be avoided and the patient must be informed to bring a clean-
voided specimen.
- All specimens should be immediately covered and taken to the laboratory.
Types of Urine Specimen
1. First Morning Specimen - a specimen obtained during the first urination of the
day. Most concentrated & bladder is incubated.
Best for: Chemical test and Microscopic examination
Required material:
 Clean, dry and leak proof container
 Container holder
 Test tube
 Test tube holder
 Disinfectant ( alcohol & bleach)
 Slide and cover slide
Procedure:
A. Inform the patient to collect the first morning urine in the container
B. Transfer the urine in to the test tube
C. Label the urine

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D. Report the appearance of urine

E. Prepare the urine for analysis. (eg. Prepare wet-mount)
2. Random Specimen - a specimen obtained at any time during examination. Most
convenient & most common.
Good for: Chemical Screen and Microscopic examination
Required material:
 Similar with first morning urine
Procedure:
 Inform the patient to collect the urine voiding at any time and follow the
same procedure with the first one.
3. Postprandial: a specimen obtained 2 hours after meal.
 It is good for glucose analysis.
4. 24- Hour specimen - a specimen obtained within 24 hours.
 Necessary for quantitative tests, especially for quantitative determination of
protein.
Procedure for Collection of 24 hour Urine Specimen
1. Inform or direct the patient to completely empty his bladder and discard his urine
exactly at the beginning of the 24 hour time collection (let say at 6:00 a.m.).
2. Collect all urine voided during the following 24 hours, including that voided exactly at
the end of the 24 hour period in a container (at 6:00 a.m.) of the following (second)
day.
3. All the urine collected must be preserved.
4. The container should be labeled with
 The test order
 The patient's name
 Time of collection
 The preservative added
5. Mid-stream Clean-Catch Urine Specimen
 Used for microbial culture and routine urinalysis.
 When specimens are collected for bacteriological examination they should be collected
by the clean catch method or by catheterization in to sterilized container.

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Catheterization is the process of passing a tube through the urethra to the bladder for
the withdrawal of urine (It may introduce urinary tract infection).
 Used as an alternative to the catheterized specimen as it provides a safer, less traumatic
method for obtaining urine for bacterial culture and routine urinalysis.
 The best method is properly collected mid-streamclean catch urine, which is collected
as follows:
 The genital area should be cleaned with soap and water and rinsed well. This is to
keep off bacteria on the skin from contaminating the urine specimen.
 The patient should urinate a small amount and this should be discarded.
 The urine that comes next, the mid-steam specimen, should be collected into a sterile
container of 30 to 50 ml.
 During collection and handling of urine sample, the inside of the container or the lid
should not be touched.
 After obtaining the specimen the patient continues to urinate and this is discarded.
6. Supra pubic – sacking urine by syringe directly from the bladder by skilled person.

Sources of Error in the Collection of Urine

 Bacteriologically or chemically contaminated specimen.
 Wrong type/ amount of preservative.
 Partial loss of specimen or inclusion of two-morning specimen in the 24 hour
collection.
 Inadequate mixing of specimen before examination.
 Careless measuring of the 24 hour volume.

Physical examination of urine

Color:
 Normally urine has pale yellow to yellow color depending on concentration.
 Deep Yellow: Mild to severe dehydration, Jaundice
 Red to brown: Haematuria, haemoglobinuria, myoglobinuria, porphyria
 Brown to black: Alkaptonuria
Appearance
 Normal urine is clear and transparent when freshly voided.

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 It may become turbid (cloudy) if exposed for long time due to the urea being acted upon
by bacteria and converted into ammonium carbonate.
Preservation of Urine Specimen:
 Urine should be examined immediately as much as possible after it is passed, because
some urinary components are unstable.
 If urine specimen cannot be examined immediately, it must be refrigerated or preserved
by using different chemical preservatives.
 The maximum time that urinary content to be maintained in urine specimen is 1 hour.
 Long standing of urine at room temperature can cause:
 Growth of bacteria.
 Break down of urea to ammonia by bacteria leading to an increase in the pH of
the urine and this may cause the precipitation of calcium and phosphates.
 Oxidation of urobilinogen to urobilin.
 Destruction of glucose by bacteria.
 Lysis of RBCs, WBCs and casts.

Method of Preservation of Urine Specimen

a.) Physical Method
1. Refrigeration
- Is a simple and reliable measure of retarding alternations including bacteriological action
and glycolysis.
- the refrigerator temperature is about 4 oC (2-6 oC)
- it prevents growth of bacteria, thus maintaining original PH with no growth alterations.
- Advantage: - no interference with chemical tests.
- Disadvantage: - used for short period of time.
2. Freezing
-it completely inhibits the growth of bacteria
-it uses a temperature of -5 oC or less. Mostly used for specimen transport.
b.) Chemical Method
- Chemical preservatives are usually reserved for 24hr urine collection, as they may
interfere with parts of the routine urinalysis. Include:

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1. Toluene
- is flammable liquid lighter than urine or water.
- Prevent growth of bacteria by excluding contact of urine with air.
- Toluene is added till it forms thin layer over the urine.
- During examination, the toluene should be skimmed of the urine pippeted from beneath
of it.
2. Formalin
- Excellent liquid preservative for formed elements.
- It interferes glucose determination.
- 1 drop/30ml urine
3. Thymol
- A crystalline substance used 5 mmdiameter/100ml urine
- Preserve most constituents
- Interfere with tests for protein and bilirubin.

4. Boric acid (1% w/v)

- At a concentration of 10 g/l (1% w/v), bacteria remain viable without multiplying.
- White cells, red cells, and casts are also well preserved, and there is no interference in
the measurement of urinary protein and glucose.
- Boric acid has been shown to be inhibitory to some enterococci and Pseudomonas
strains.
5. HCL
- It interferes with protein estimation, destroys formed elements & precipitate uric acid
- 1 drop/15 ml urine (15-20ml/24hr urine)
6. Chloroform
- An effective preservative for steroids
- Urine preserved with chloroform is unstable for glucose testing.
- 1 tablet/60 ml urine (10ml of chloroform/24hr urine)

2.4.2.2 Preparation of urine sample for testing

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Urine examination is one of the most helpful indicators of health and disease, especially, it is
useful as a screening test for the detection of various endocrine or metabolic abnormalities in
which the kidneys function properly but they will excrete abnormal amounts of metabolic end-
products specific of a particular disease. It is also used to detect intrinsic conditions that may
adversely affect the kidneys or urinary tract.
In order to make Urinalysis reliable the urine must be properly collected. Improper collection
may invalidate the results of the laboratory procedures, no matter how carefully and skillfully the
tests are performed.
Whenever possible, the first urine passed by the patient at the beginning of the day should be
sent for examination. This specimen is the most concentrated and therefore the most suitable for
culture, microscopy, and biochemical analysis.
Methods of Obtaining Specimens
A freshly voided urine specimen is adequate for most urinalysis except the microbiological
culture. The patient should be instructed to void directly into a clean, dry container, or a clean,
dry bedpan so that the specimen can be transferred to an appropriate container. Specimens
from infants and young children can be collected in a disposable collection apparatus. If a urine
specimenn is likely to be contaminated with vaginal discharge or menstrual blood, this period
has to be avoided and the patient must be informed to bring a clean-voided specimen. All
specimens should be immediately covered and taken to the laboratory.
Describe the appearance of the specimen
Report:
─ Colour of specimen
─ Whether it is clear or cloudy (turbid)
Normal freshly passed urine is clear and pale yellow to yellow depending on concentration.
Preparation and examination of a wet preparation
1. Aseptically transfer about 10 ml of well mixed urine to a labeled conical tube.
2. Centrifuge at 500–1000g (approximately 3000 RPM) for 5 minutes. Pour the supernatant
fluid (by completely inverting the tube) into a second container not the original one.
This can be used for biochemical tests to avoid contaminating the original urine which
may need to be cultured (depending on the findings of the microscopical examination).

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3. Remix the sediment by tapping the bottom of the tube. Transfer one drop of the well
mixed sediment to a slide and cover with cover glass.
Note: Do not discard the remaining sediment because this may be needed to prepare a
Gram smear if WBCs and, or, bacteria are seen in the wet preparation.
4. Examine the preparation microscopically using the 10xand 40x objective with the
condenser iris closed sufficiently to give good contrast.
5. Urine is examined microscopically as a wet preparation to detect:
 significant pyuria, i.e. WBCs in excess of 10 cells/µl of urine
 red cells
 casts
 yeast cell
 T. vaginalis motile trophozoite
 S. haematobium egg
 Bacteria (providing the urine is freshly collected) Examination of a Gram stained
smear Prepare and examine a Gram stained smear of the urine when bacteria and,
or white cells are seen in the wet preparation.
6. Transfer a drop of the urine sediment to a slide and spread it to make a thin smear. Allow
to air dry, protected from insects and dust. Heat fix or methanol fix the smear and stain it
by the Gram technique.
Examine the smear first with the 40× objective to see the distribution of material, and then with
the oil immersion objective.

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2.4.3. Collection and Process of blood sample

2.4.3.1. Collection of blood sample

 Blood must be collected with care and adequate safety precautions to ensure test results
are reliable,
 Contamination of the sample is avoided and infection from blood transmissible pathogens
is prevented. Protective gloves should be worn when collecting and handling blood
samples.
 Lancets, needles, and syringes must be sterile, and dry, and blood collecting materials
must be discarded safely to avoid injury from needles and lancets.
Method of blood collection
1. Capillary blood
 Capillary blood is mainly used when the patient is an infant or young child and the
volume of blood required is small.
o e.g. to measure haemoglobin, perform a WBC count, and to make thick and thin
blood film
Disadvantages in using capillary blood for blood tests include:
 Greater possibility of sampling errors particularly when the blood is not free-flowing,
e.g. dilution of the sample with tissue juice can occur if the puncture area is squeezed
excessively.
 Difficulty in obtaining sufficient blood particularly when it is required for more than
one test. Rapid clotting of blood in a pipette is common, particularly in tropical
temperatures.
 Tests cannot be repeated immediately or further tests performed when results are
unexpected or seriously abnormal. A patient may have also received treatment
following collection of the capillary sample, e.g. an antimalarial drug.
 It is not possible to estimate platelets in blood films made from capillary blood
(platelets clump).
 Site of collection
 Heel for less than one year age infant
 Ring finger for children and adult

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 Required material for capillary blood collection

- Glove
- Lancet
- Cotton and alcohol
- Labeling material
- Slide
- Capillary tube
 Procedure
1. Make sure the puncture area is warm to allow the blood to flow freely. On cold days
soak the hand or foot of an infant in warm water prior to collecting a sample.
2. Cleanse the puncture area with 70% ethanol.
3. Allow the area to dry.
4. Using a sterile pricker or lancet, make a rapid puncture, sufficiently deep to allow the
free flow of blood.
5. Wipe away the first drop of blood with a dry piece of cotton wool and use the next
few drops for the test. Do not squeeze too hard because this will result in an
unreliable test result.
6. When sufficient blood has been collected, press a piece of dry cotton wool over the
puncture area until bleeding stops.
2. Venous blood
 Anticoagulated venous blood is used when more than 100μl of whole blood is required or
when serum from a clotted blood sample is needed.
o e.g. for compatibility tests or antibody tests.
 Venous blood is preferable to capillary blood for the reasons previously described;
particularly when the patient is an adult and several tests are required.
 When venous blood is required from infants, this should be collected by a medical doctor.
 Do not collect blood for hematological tests from intravenous lines.
Site of collection
Adult: Forearm: include three type of vein
 Basilic vein, Median cubital vein, and Cephalic vein (mostly preferable site)
Infant: Jagular vein and femoral vein

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Required material
 Glove,
 Sterile Syringe and needle,
 Alcohol and cotton,
 Test tube,
 Tourniquet,
 Labeling material etc.
 Procedure
1. Select a sterile, dry, preferably plastic syringe of the capacity required, e.g. 2.5 ml, 5 ml, or
10 ml. Attach to it a 19 or 20 SWG needle (preferably a disposable one). If the patient is a
child or adult with small veins, use a 23 SWG needle.
2. Apply a soft tubing tourniquet or velcro fastening arm band to the upper arm of the patient
(to enable the veins to be seen and felt.
3. Using the index finger, feel for a suitable vein, selecting a sufficiently large straight vein that
does not roll and with a direction that can be felt.
4. Cleanse the puncture site with 70% ethanol and allow to dry. Do not re-touch the cleansed
area.
5. With the thumb of the left hand holding down the skin below the puncture site, make the vein
puncture with the bevel of the needle directed upwards in the line of the vein. Steadily
withdraw the plunger of the syringe at the speed it is taking the vein to fill. Avoid moving the
needle in the vein.
6. When sufficient blood has been collected, release the tourniquet and instruct the patient to
open his or her fist. Remove the needle and immediately press on the puncture site with a
piece of dry cotton wool. Remove the tourniquet completely. Instruct the patient to continue
pressing on the puncture site until the bleeding has stopped.
7. Remove the needle from the syringe and carefully fill the container(s) with the required
volume of blood. Discard the needle safely. Do not attempt to re-sheath it because this can
result in needle-stick injury.
8. Mix immediately the blood in an EDTA or citrate anticoagulated container. When required,
make a thick blood film from the blood remaining in the syringe. Immediately label carefully
all the blood samples.
9. Check that bleeding from the venepuncture site has stopped. Cover the area with a small
dressing.
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Evacuated tube collection systems:

 These disposable blood collecting containers are available from several manufacturers
but they are more expensive to use for collecting venous blood than a syringe and needle.
 The container has a vacuum which is used to draw the blood into the container. One end
of the needle is situated in the patient’s vein and the other end through the cap of the
container.
 Evacuated collection systems minimize contact with blood, help to ensure the correct
amount of blood is added to anticoagulant, and simplify multiple sample collection.
Note
 Do not apply the tourniquet too tightly or for longer than 2 minutes.
Ask the patient to make a tight fist which will make the veins more
prominent.
 If a vein cannot be felt, apply a pressure cuff above the elbow and
raise the pressure to 80 mm (deflate the cuff once the needle is in the
vein).
 Do not fill a container with the needle attached to the syringe. Forcing
the blood through the needle can cause haemolysis.

2.1.3.3 Blood Preservative

1. Physical (refrigeration)
2. Anticoagulants: serve as both preservative and prevent clotting of blood
Type of anticoagulants:
A. EDTA (Ethylenediamine tetra-acetic acid)
 also called sequestrene
 These chemicals prevent blood from clotting by removing calcium.
 Used for most tests, e.g. haemoglobin, PCV, WBC count, platelet count, reticulocyte
count, and reporting blood cell morphology. It is not suitable for coagulation tests.
 Recommended to use EDTA at a concentration of 1.5± 0.25 mg/ml of blood.

B. Tri-sodium citrate

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 These chemicals prevent blood from clotting by removing calcium.

 trisodium citrate, 32 g/l, is used to anticoagulate blood
 Usually recommended for
 The ESR, with 1.6 ml of venous blood being mixed with 0.4 ml of sodium
citrate anticoagulant.
 Coagulation tests, with 9 ml of venous blood being mixed with 1 ml of
sodium citrate anticoagulant.
C. Heparin anticoagulant:
 It is an excellent natural anticoagulant produced by liver or pancreatic cells.
 It is mainly used for clinical chemistry tests and immunophenotyping.
 It is not recommended for routine haematological tests because it causes cells to
clump and heparin gives a blue background to blood films

D. Balanced or Double Oxalate

 Salts of oxalic acid (combination of two salts: Ammonium oxalate & Potassium oxalate).
 have the ability to bind and precipitate Ca++ as calcium oxalate
 serve as suitable anticoagulants for many hematologic investigations.
 3 parts of ammonium oxalate is balanced with 2 parts of potassium oxalate
 neither salt is suitable by itself, i.e., ammonium oxalate causes cellular swelling and
potassium oxalate causes erythrocyte shrinkage
 It is used in the proportion of 1-2mg/ml of blood.
E. ACD (Acid Citrate Dextrose)
 Mainly used in blood bank to store whole blood for months
F. Sodium fluoride (NaF)
 Prevent haemolysis
 Used for the determination of glucose in whole blood in clinical chemistry laboratory.

2.4.2.2 Preparing blood sample for testing

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I. Preparation of blood films

Examination of the blood film is an important part of the hematologic evaluation and the validity
or reliability of the information obtained from blood film evaluation, the differential leucocyte
count in particular depends heavily on well-made and well- stained films. While blood film
preparation is a disarmingly simple straight – forward procedure, there is abundant and
continuing evidence that the quality of blood films in routine hematology practice leaves much
room for improvement. If not made from skin puncture, films should be prepared within 1 hour
of blood collection into EDTA. Adequate mixing is necessary prior to film preparation if the
blood has been standing for any appreciable period of time.
A. Thin blood films preparation
Three methods of making films are described: the two slides or wedge method, the cover glass
method, and the spinner method. Preparation of blood films on glass slides has the following
advantages:
• Slides are not easily broken
• Slides are easier to label
• When large numbers of films are to be dealt with, slides will be found much easier to handle.
Method
I. Wedge method (Two-slide method)
• A small drop of blood is placed in the center line of a slide about 1-2cm from one end. Another
slide, the spreading slide placed in front of the drop of blood at an angle of 300 to the slide and
then is moved back to make contact with the drop. The drop will spread out quickly along the
line of contact of the spreader with the slide.
• Once the blood has spread completely, the spreader is moved forward smoothly and with a
moderate speed. The drop should be of such size that the film is 3-4cm in length (approx. 3/4th
of the length of the slide). It is essential that the slide used as a spreader have a smooth edge and
should be narrower in breadth than the slide on which the film is prepared so that the edges of
the film can be readily examined.
• It can be prepared in the laboratory by breaking off 2mm from both corners so that its breadth
is 4mm less than the total slide breadth. If the edges of the Spreader is rough, films with ragged
tails will result and gross qualitative irregularity in the distribution of cells will be the rule.

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• The ideal thickness of the film is such that there is some overlap of the red cells throughout
much of the film’s length and separation and lack of distortion towards the tail of the film.
• Thickness and length of the film are affected by speed of spreading and the angle at which the
spreader slide is held. The faster the film is spread the thicker and shorter it will be. The bigger
the angle of spreading the thicker will be the film.
• Once the slide is dry, the name of the patient and date or a reference number is written on the
head of the film using a lead pencil or graphite. If these are not available, writing can be done by
scratching with the edge of a slide. A paper label should be affixed to the slide after staining.
B. Thick blood smears preparation
Thick blood smears are widely used in the diagnosis of blood parasites particularly malaria. It
gives a higher percentage of positive diagnosis in much less time since it has ten times the
thickness of normal smears. Five minutes spent in examining a thick blood film is equivalent to
one hour spent in traversing the whole length of a thin blood film.
Method
Place a small drop of blood on a clean slide and spread it with an applicator stick or the corner of
another slide until small prints are just visible through the blood smear. This corresponds to a
circle of approximately 2cm diameter.
Staining blood films
There are different types of staining methods, the following are the most commonly used
methods for staining of blood films.
A. Wright staining method
1. Place the air-dried smear film side up on a staining rack (two parallel glass or metal rods
kept 5cm apart).
2. Cover the smear with undiluted Wright stain and leave for 1 minute. The methyl alcohol
in the stain fixes the smear. When it is planned to use an aqueous or diluted stain, the air
dried smear must first be fixed by flooding for 3-5 minutes with absolute methanol. If
films are left unfixed for a day or more, it will be found that the background of dried
plasma stains pale blue and this is impossible to remove without spoiling the staining of
the blood cells.
3. Dilute with distilled water (approximately equal volume) until a metallic scum appears.
Mix by blowing. Allow this diluted stain to act for 3-5 minutes.

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4. Without disturbing the slide, flood with distilled water and wash until the thinner parts of
the film are pinkish red.
B. Giemsa staining method
Staining thin films using Giemsa stain
1. Dry the films in the air then fix by immersing in a jar containing methanol for 3 minutes.
2. Transfer the films to a staining jar containing Giemsa stain freshly diluted with 9 volumes
of buffered water pH 6.8. Allow to stain for 7-10 minutes.
3. Transfer the slides to a jar containing buffered water, pH 6.8; rapidly wash in 3 or 4
changes of water.
4. Place the slides on draining rack to dry.
Staining of thick smears using Giemsa stain
The stains used employ the principle of destroying the red cells and staining leucocytes and
parasites.
Method
1. Cover the air-dried smear with a 1:10 diluted Giemsa using buffered distilled water at pH
6.8 as a diluent. Leave the stain to act for 10 minutes. Do not fix the films before staining.
2. Wash with distilled water and air dry.
II. Preparing plasma and serum

 Specimens that are mostly used for serologic and clinical chemistry tests include serum
and plasma.

 The difference between plasma and serum is that the latter lacks fibrinogen and some of
the coagulation factors.

 To prepare serum or plasma sample, first 2-3ml venous blood is collected from a patent
using a sterile syringe and needle.

 If serum is required, allow the whole blood to clot at room temperature for at least one
hour, then centrifuge the clotted blood for 10 minutes at 2000 r.p.m. then transfer the
serum to a labeled tube with a Pasteur pipette and rubber bulb.

 Plasma samples are obtained by treating fresh blood with an anti-coagulant, then
centrifuge and separate the supernatant.

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 The specimen should be free from hemolyzed. Finally, the specimen containing tube
should be labeled with full patients identification (age, sex, code no, etc).

 The test should be performed within hours after sample collection. If this couldn`t be
done preserve the sample in a suitable preservative.

Anticoagulants

Anticoagulants are chemical substances that are added to blood to prevent coagulation. For
various purposes, a number of different anticoagulants are available such as EDTA, sodium
citrate, heparin and the like.
1. Ethylenediamine tetraacetic acid (EDTA)
It is the preferred anticoagulant for cell counts and morphological studies. It is especially the
anticoagulant of choice for platelet counts and platelet function tests since it prevents platelet
aggregation. The amount of EDTA necessary to prevent clotting of blood is 0.02ml of 10%
(W/V) solution of EDTA for 1ml of blood.
2. Trisodium Citrate
Nine volumes of blood are added to 1 volume of the sodium citrate solution and immediately
well mixed with it. Sodium citrate is also the anticoagulant for the erythrocyte sedimentation rate
(ESR); for this, 4 volumes of venous blood are diluted with 1 volume of the sodium citrate
solution.

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2.4.4. Collection and processing of Sputum

2.4.4.1 Collection of sputum
 When our body is infected by mycobacterium tuberculosis (TB) an inflammatory reaction
that leads to a liquefied destruction of lung tissue with caseation (breakdown of diseased
tissue into a cheese-like mass) will occur.
 Erosion through the wall of a bronchus leads to the discharge of the liquefied tissue and
the formation of a cavity.
 Bacilli multiply in the wall of the cavity and can be found in the sputum (‘open’
infectious stage).
Sputum for microbiological investigation is collected follows:
1. Give the patient a clean (need not be sterile), dry, wide-necked, leak-proof container (50
ml capacity), and request him or her to cough deeply to produce a sputum specimen.
Sputum Collection Container: Specifications
• Wide-mouthed containers desirable so that the patient can expectorate easily inside the
container without contaminating the outside
• Made of translucent or clear material in order to observe specimen volume and quality
without opening the container
• Single use containers which can be tightly sealed to prevent any leakage during
transport
•They should be made of unbreakable transparent plastic.
Specimen Collection: Safety
 The laboratory worker is at a much greater risk of infection from TB suspect who is
coughing than from a sputum specimen.
 Instruct patient to cover the mouth when coughing.
 Preparing smears presents a much lower risk to the lab worker than exposure to direct
coughing.
 Sputum specimens must never be collected inside the clinic or laboratory.
 Always collect sputum OUTSIDE, and away from other people.
 Do not stand near patient during specimen collection.
Advantages of Open Air Collection
 Adequate ventilation removes the droplet nuclei.

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 Direct sunlight (Ultraviolet rays) kills tubercle bacilli but they can survive in darkness or
in a humid environment for several hours and even days.
Caution: When a sputum specimen is being collected, adequate safety precautions must be taken
to prevent the spread of infectious organisms and to avoid contaminating the outside of the
container.
Important:
 The specimen must be sputum, not saliva.
 Sputum is best collected in the morning soon after the patient wakes and before any
mouth-wash is used. The following are sputum sampling strategies:
A. Spot - Morning - Spot strategies (previous one)
 When pulmonary tuberculosis is suspected, up to three specimens may need to be
examined to detect AFB.
 To ensure optimal recovery of TB bacilli from sputum, collect and process three
specimens.
 The first specimen is collected on the spot when the patient presents at the diagnostic
center. The patient is then given another sputum container and instructed to collect an
early morning specimen on the next day and return to the clinic. A third specimen (spot)
is collected when the early morning specimen is delivered to the laboratory.
 Early morning specimens have the highest yield of AFB.
 For hospitalized patients, collect early morning specimens on three successive days,
since such samples often contain more bacilli and thus are more likely to be positive by
microscopy.

B. Spot – Spot strategy

 WHO recommend Spot-Spot strategy in 2011
 Spot-spot compared with Spot - Morning - Spot
o 2.8% Positivity drop out
o 5.2% Patient dropout reduction
 Two consecutive sputum specimens collected on spot-spot schedule for AFB smear
microscopic examination
 Instruct on how to produce good quality of specimen on same day
o Give one labeled container to produce the first sample (Spot) immediately.

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 MODULE TITLE : Collecting and Processing Medical Samples

o Give the second container when the patient brings the first sample and
instruct to produce the second sample(Spot) after 30 minutes to 1 hour.
 One sputum smear positive result confirms the diagnosis of bacteriologically
confirmed Tuberculosis
 For patients whose sputum smears microscopy result is twice negative and in whom
clinical suspicion for TB remains high after administration of Broad spectrum
Antibiotic, GeneXpert MTB/RIF Assay is advised as follow on test.
 A single sputum specimen collected at spot for Xpert MTB/RIF assay
Advantages of Spot – Spot strategy

o Greatly reduces the workload of laboratories

o Has the potential for offering same-day diagnosis
o Is better for patients because it reduces the number of visits while largely
maintaining sensitivity
Disadvantages
o Very minor loss in the number of cases detected.
Sputum collection for follow-up of treatment:
 For patients on treatment, collect follow-up specimens at intervals specified by the NTP.
 Follow-up sputum examination is a tool for to monitor treatment effectiveness.
 Early morning sputum is the preferred specimen.
2.Clearly instruct the patient on:
 the importance of sputum examination for diagnosis or follow-up of TB;
 how to open and close the containers;
 the need for collecting real sputum, not saliva;
 how to produce good sputum (i.e., by repeated deep inhalation and exhalation of breath
followed by cough from as deep inside the chest as possible);
 how to avoid contamination of the exterior of the container (i.e., by carefully spitting and
closing the container);
 how to collect and safely deliver the morning sputum to the laboratory; and
 the need for multiple sputa to facilitate diagnosis.
Label the container, and complete a request form as follows:

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 MODULE TITLE : Collecting and Processing Medical Samples

 The specimen bottle must be labeled with the patient’s name or laboratory number, the
date and time the specimen was collected.
 The laboratory slip must be completed with the patient’s name, laboratory number, age,
sex, address and reason for examination.
Note that the amount of sputum collected should be 1-2 table spoons.
2.4.4.2 Specimen handling and referral
Specimen handling
 For optimum patient management, process the specimen as soon as possible (i.e., < 24
hours).
 For microscopic examination the interval between collection and staining matters little.
Acceptable results can be obtained even on delayed specimens.
 If the peripheral health center does not perform microscopy, there are several options.
Patient referral
 Ideally, you can refer a patient to the microscopy center so that a specimen can be
collected under monitored conditions.
 If an unsatisfactory specimen is submitted, then a repeat sample can be obtained
immediately.
 The disadvantage of this option is that the patient may find it expensive or impractical to
travel to the microscopy center.
Specimen referral
 Alternatively, the peripheral health center can supervise the patient in collecting an
appropriate specimen, which is then forwarded to a microscopy center.
 Transport specimens once or twice each week, although in some remote settings this may
not always be possible.
 Package specimens carefully to prevent leaks and breaks.
 Clearly label each specimen with the patient identification and include a completed
request for sputum examination.
Slide referral
 Time delays for slide referrals may occur.
 There are, however, several advantages. Fixed sputum smears are less infectious than
sputum specimens are and require less packaging for transport.

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 Current practice is patient referral

 Specimen referral will be a good option.

Describe the appearance of the specimen:

Describe whether the sputum is:
 Purulent:Green-looking, mostly pus
 Mucopurulent:Green-looking with pus and mucus
 Mucoid:Mostly mucus
 Mucosalivary:Mucus with a small amount of saliva
 When the sputum contains blood, this must also be reportedanother specimen needs to be
requested.

Unsuitable sputum specimens:

When the sputum is mostly saliva, report the specimen as ‘Unsuitable for
microbiological investigation’ and request another specimen.

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 MODULE TITLE : Collecting and Processing Medical Samples

2.4.4.3 Preservation of sputum

 It is better to process sputum samples as soon as possible.

 If a delay of a few days cannot be avoided, keep specimens cool (refrigerated but not
frozen) up to a week.
 This will not significantly affect the positivity rate of smear microscopy; however, the
additional growth of contaminants will result in an increased contamination rate on
culture media.
 If the delay exceeds 3 days, an equal volume of Cetyl Pyridinium Chloride (CPC;
solution of 1% CPC in 2% sodium chloride) should therefore be added to sputum.
 Sputum containing CPC can be kept for up to 7 days but must be kept at room
temperature (>20 °C since CPC crystallizes at lower temperatures).
 The addition of CPC must be indicated on the accompanying documents (see form
below) because CPC has to be removed before culturing.

2.4.4.4 Processing of sputum sample

Specimens for immediate attention: When pneumonia or bronchopneumonia is suspected, the
sputum must be cultured as soon as possible because organisms such as H. influenzae and S.
pneumoniae do not survive well in specimens.
Caution: Whenever possible, sputum specimens should be examined in a biological safety
cabinet
Note: Before culturing sputum, many laboratories examine a wet preparation or Field’s stained
smear microscopically for cells. When large numbers of squamous epithelial cells (often covered
with bacteria) are present and only a few or no pus or macrophage cells, this indicates that the
specimen is unsuitable for culturing.
Preparation of smear
If smears are to provide reliable information they must be prepared, labeled, and fixed correctly
prior to being stained.
Labeling slides
Every slide must be labelled clearly with the date and the patient’s name and number. Whenever
possible, smears should be spread on slides which have one end frosted for labeling. With the
increased use of such slides in recent years, their price is now little more than slides without a

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 MODULE TITLE : Collecting and Processing Medical Samples

frosted end. A lead pencil should be used for writing on the frosted area because pencil marks,
unlike biro and grease pencil marks, will not be washed off during the staining process.
Caution: Slides from positive AFB smears should always be discarded and never reused.
Scratched, chipped, and discolored slides should also be discarded.
Use a piece of clean stick to transfer and spread purulent and caseous material on a slide. Soak
the stick in a phenol or hypochlorite disinfectant before discarding it
Smears should be spread evenly covering an area of about 15–20 mm diameter on a slide.
The techniques used to make smears from different specimens are as follows:
● Purulent specimen: Using a sterile wire loop, make a thin preparation. Do not centrifuge a
purulent fluid, e.g. c.s.f. containing pus cells.
● Non-purulent fluid specimen: Centrifuge the fluid and make a smear from a drop of the well-
mixed sediment.
● Culture: Emulsify a colony in sterile distilled water and make a thin preparation on a slide.
When a broth culture, transfer a loopful to a slide and make a thin preparation.
● Sputum: Use a piece of clean stick to transfer and spread purulent and caseous material on a
slide. Soak the stick in a phenol or hypochlorite disinfectant before discarding it.
● Swabs: Roll the swab on a slide. This is particularly important when looking for intracellular
bacteria such as N. gonorrhoeae (urethral, cervical, or eye swab). Rolling the swab avoids
damaging the pus cells.
Drying and fixing smears
After making a smear, leave the slide in a safe place for the smear to air-dry, protected from dust,
flies, cockroaches, ants, and direct sunlight. When a smear requires urgent staining, it can be
dried quickly using lamp heat. Smears taken from in-patients and during out-patient clinics must
always be transported to the laboratory in a covered container. The purpose of fixation is to
preserve microorganisms and to prevent smears being washed from slides during staining.
Smears are fixed by heat, alcohol, or occasionally by other chemicals. Microorganisms are not
always killed by heat fixation, e.g. M. tuberculosis.
Heat fixation
This is widely used but can damage organisms and alter their staining reactions especially when
excessive heat is used. Heat fixation also damages leucocytes and is therefore unsuitable for
fixing smears which may contain intracellular organisms such as N. gonorrhoeae and N.

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meningitidis. When used, heat fixation must be carried out with care. The following technique is
recommended:
1. Allow the smear to air-dry completely.
2. Rapidly pass the slide, smear uppermost, three times through the flame of a spirit lamp or
pilot flame of a Bunsen burner.
Note: After passing the slide through the flame three times, it should be possible to lay the slide
on the back of the hand without the hand feeling uncomfortably hot. When this cannot be done,
too much heat has been used.
3. Allow the smear to cool before staining it.
Alcohol fixation
This form of fixation is far less damaging to microorganisms than heat. Cells, especially pus
cells, are also well preserved.
Alcohol fixation is therefore recommended for fixing smears when looking for Gram negative
intracellular diplococci.
Alcohol fixation is more bactericidal than heat (e.g. M. tuberculosis is rapidly killed in sputum
smears after applying 70% v/v alcohol). A method of alcohol fixing smears is as follows:
1. Allow the smear to air-dry completely.
2. Depending on the type of smear, alcohol-fix as follows:
– For the detection of intracellular Gram negative diplococci (N. gonorrhoeae or N.
meningitidis), fix with one or two drops of
absolute methanol or ethanol.
– For the detection of other organisms including M. tuberculosis, fix with one or two drops of
70% v/v methanol or ethanol (absolute
methanol can also be used but a 70% v/v solution is adequate).
3. Leave the alcohol on the smear for a minimum of 2 minutes or until the alcohol
evaporates.
Ziehl-Neelsen smear to detect AFB
Studies have shown that the chances of detecting AFB in sputum smears are significantly
increased when sputum is first treated with 5% v\v sodium hypochlorite (NaOC1), i.e. bleach,
followed by centrifugation or overnight sedimentation. Because NaOC1 kills M. tuberculosis, the

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NaOC1 concentration technique is also safer for laboratory staff. NaOC1 treated sputum cannot
be used for culture.
Sodium hypochlorite centrifugation technique to concentrate AFB
1. Transfer 1–2 ml of sputum (particularly that which contains any yellow caseous material)
to a screw-cap Universal bottle or other container of 15–20 ml capacity.
Caution: Open specimen containers with care and at arms length to avoid inhaling infectious
aerosols. When available, handle the specimen inside a safety cabinet.
2. Add an equal volume of concentrated sodium hypochlorite (bleach) solution and mix
well.
Sodium hypochlorite (NaOC1) solution: This is widely available as bleach for domestic and
laundering purposes. These domestic bleach solutions generally contain about 5% available
chlorine and should be used undiluted in the NaOC1 technique to concentrate AFB.
Caution: Bleach is corrosive and toxic when ingested or inhaled. It also has an irritating vapour
and therefore it should be used in a well-ventilated place. Store it out of direct sunlight in a cool
place, away from acids, methanol and oxidizing chemicals. When in contact with acids, sodium
hypochlorite liberates toxic gas.
3. Leave at room temperature for 10–15 minutes, shaking at intervals to break down the
mucus in the sputum.
4. Add about 8 ml of distilled water, or when unavailable use boiled filtered rain water. Mix
well.
5. Centrifuge at 3000 rpm for 20 minutes.
Note: When a centrifuge is not equipped to take Universal containers, divide the specimen
between two conical tubes (which can be capped).
When centrifugation is not possible, leave the NaOC1 treated sputum to sediment overnight.
6. Using a glass Pasteur pipette or plastic bulb pipette, remove and discard the supernatant
fluid. Mix the sediment. When two tubes have been used, combine the two sediments.
Transfer a drop of the well-mixed sediment to a clean scratch-free glass slide. Spread the
sediment to make a thin preparation and allow to air-dry.
7. Heat-fix the smear and stain it using the Ziehl- Neelsen technique.

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Ziehl-Neelsen staining technique for M. tuberculosis and M. ulcerans

Required
– Carbol fuchsin stain (filtered )
– Acid alcohol, 3% v/v
– Malachite green, 5 g/l (0.5% w/v)*
*If preferred, methylene blue, 5 g/l may be used instead of malachite green.
Method
1 Heat-fix the dried smear
Alcohol-fixation: This is recommended when the smear has not been prepared from sodium
hypochlorite (bleach) treated sputum and will not be stained immediately.
M. tuberculosis is killed by bleach and during the staining process. Heat-fixation of untreated
sputum will not kill M. tuberculosis whereas alcohol-fixation is bactericidal.
2 Cover the smear with carbol fuchsin stain.
3 Heat the stain until vapour just begins to rise (i.e. about 60 _C). Do not overheat. Allow the
heated stain to remain on the slide for 5 minutes.
Heating the stain: Great care must be taken when heating the carbol fuchsin especially if
staining is carried out over a tray or other container in which highly flammable chemicals have
collected from previous staining. Only a small flame should be applied under the slides using an
ignited swab previously dampened with a few drops of acid alcohol or 70% v/v ethanol or
methanol. Do not use a large ethanol soaked swab because this is a fire risk.
4 Wash off the stain with clean water.
Note: When the tap water is not clean, wash the smear with filtered water or clean boiled
rainwater.
5 Cover the smear with 3% v/v acid alcohol for 5 minutes or until the smear is sufficiently
decolorized, i.e. pale pink.
Caution: Acid alcohol is flammable; therefore use it with care well away from an open flame.
6 Wash well with clean water.
7 Cover the smear with malachite green stain for 1–2 minutes, using the longer time when the
smear is thin.
8 Wash off the stain with clean water.

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9 Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not
blot dry).
10 Examine the smear microscopically, using the 100_ oil immersion objective. When available,
use 7_eyepieces because these will give a brighter image. Scan the smear systematically
.Note: Do not touch the smear with the end of the oil dispenser because this could transfer AFB
from one preparation to another. After examining a positive smear, the oil must be wiped from
the objective
Auramine-phenol fluorochrome staining technique

The auramine-phenol fluorochrome staining technique can be used to detect M. tuberculosis in

sputum, cerebrospinal fluid, and other specimens when facilities for fluorescence microscopy are
available. Compared with the Ziehl-Neelsen technique, the auramine-phenol fluorochrome
technique enables a more rapid examination of smears because the 40_ objective can be used.
When tubercle bacilli are few they are more likely to be successfully detected in auramine-
phenol stained smears.
Auramine-phenol to demonstrate AFB
Auramine fluoresces when illuminated (excited) by blueviolet or ultra-violet (UV) light. It can be
used to demonstrate AFB because it binds to the mycolic acid in the mycobacterial cell wall. No
heating of the stain is required. After being stained with auramine, the smear is decolorized with
acid alcohol which removes the dye from the background. The smear is then washed with a weak
solution of potassium permanganate to darken the background. Tubercle bacilli fluoresce white-
yellow against a dark background
Required reagents

– Auramine-phenol stain (filtered)

– 1% acid alcohol Reagent No. 3

– Potassium permanganate, 1g/l (0.1% w/v)

Method

Whenever possible use a sodium hypochlorite technique to concentrate the bacilli prior to
staining

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1. Heat-fix the dried smear as described in subunit

2. Cover the fixed smear with the auramine-phenol stain for 10 minutes. Always include a
positive control smear.

3. Wash off the stain with clean water.

Note: When the tap water is not clean, wash the smear with filtered or clean boiled rainwater.

4. Decolorize the smear by covering it with 1% v/v acid alcohol for 5 minutes.

Caution: Acid alcohol is flammable; therefore use it with care well away from an open flame.

5. Wash off the acid alcohol with clean water.

6. Cover the smear with the potassium permanganate solution for about 10 seconds, followed

by several rinses with clean water.

7. Wipe the back of the slide clean and place it in a draining rack for the smear to dry. Do not
blot dry. To prevent fading of the fluorescence, protect the stained smear from sunlight and

bright light.
8. Systematically examine the smear for AFB by fluorescence microscopy using the
40_objective.
2.4.5. Collection and Processing of CSF sample
2.4.5.1 Collection of CSF
 CSF must be collected by a physician or a specially trained nurse.
 About 1-2ml of CSF is collected for examination.
 It must be collected aseptically to prevent organisms being introduced into the central
nervous system.
 The fluid is usually collected from the arachnoid space by inserting sterile lumbar
puncture needle between:
o 3rd and 4th lumbar vertebrae
o 4th and 5th lumbar vertebrae to a depth of 4–5cm.

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 IMPORTANT: Advise the laboratory before performing a lumbar puncture so that staff
are prepared to receive and examine the specimen immediately.
 Note: A delay in examining CSF reduces the chances of isolating a pathogen. It will also
result in a lower cell count due to WBCs being lyzed, and to a falsely low glucose value
due to glycolysis. When trypanosomes are present, they will be difficult to find because
they are rapidly lyzed once the CSF has been withdrawn.
 In practice, three sterile tubes containing about 5ml each are collected during spinal tap.
These tubes are numbered in sequence of collection and immediately brought to the
laboratory.
 The tubes that are sequentially collected and labeled in order of collection are generally
dispersed and utilized for analysis (after gross examination of all tubes) as follows:
Tube 1: for chemical and immunologic tests
Tube 2: For Microbiological tests
Tube 3: For Total cell counts and differential cell counts (for Hematology tests).
This is least likely to contain cells introduced by the puncture procedure
itself.
 Important: Cerebrospinal fluid must be examined without delay, and the results of tests
reported to the physician as soon as they become available, especially a Gram smear
report. The fluid should be handled with special care because a lumbar puncture is
required to collect the specimen.
 Cells must be counted within 1 hour of collection (cells disintegrate rapidly). If delay is
unavoidable store 2-8oC.
Gross Appearance
 As soon as the CSF reaches the laboratory, note its appearance. Report whether the fluid:
is clear, slightly turbid, cloudy or definitely purulent (looking like pus),
contains blood,
contains clots.
 Normal spinal fluid is crystal clear and has no clot. It looks like distilled water.
o Color and clarity are noted by holding the sample beside a tube of water against a
clean white paper or a printed page.

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 Purulent or cloudy CSF indicates presence of pus cells (Pleocytosis), suggestive of acute
pyogenic bacterial meningitis.

Blood in CSF
 Bloody CSF can result from a traumatic tap or from subarachnoid hemorrhage.
 If blood in a specimen results from a traumatic tap (inclusion of blood in the specimen
from the puncture itself), the successive collection tubes will show less bloody fluid,
eventually becoming clear.
 If blood in a specimen is caused by a subarachnoid hemorrhage, the color of the fluid
will look the same in all the collection tubes.
 If only one tube of CSF is available, wait for erythrocytes to settle (or centrifuge at
2000g for 5 min) and examine the supernatant fluid. Then:
o If the supernatant fluid is clear, the blood is there because of accidental injury to a blood
vessel.
o If the supernatant fluid is bloodstained, the blood is there because of a subarachnoid
hemorrhage
 In addition, subarachnoid bleeding is indicated by the presence of Xanthochromia.
o This is the presence of a yellow color in the supernatant CSF.
o It is the result of the release of hemoglobin from hemolyzed red blood cells,
which begins 1 to 4 hours after hemorrhage.
 Clots in CSF indicates a high protein concentration with increased fibrinogen, as can
occur with pyogenic meningitis, severe jaundice or when there is spinal constriction.
Note: tubes should be pre-numbered, labelled with the patient information and marked with the
specified time the sample was taken.

2.4.5.2. Processing CSF for testing)

Depending on the appearance of the c.s.f., proceed as follows:

Purulent or cloudy c.s.f. Suspect pyogenic meningitis and test the c.s.f. as follows:
_ Immediately make and examine a Gram stained smear for bacteria and polymorphonuclear
neutrophils (pus cells). Issue the report without delay.
_ Culture the c.s.f.

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 MODULE TITLE : Collecting and Processing Medical Samples

Slightly cloudy or clear c.s.f.

Test the c.s.f. as follows:
_ Perform a cell count and note whether there is an increase in white cells and whether the cells
are mainly pus cells or lymphocytes.
_ When cells predominantly pus cells:
– Examine a Gram stained smear for bacteria.
– Examine a wet preparation (sediment from centrifuged c.s.f.) for motile amoebae which could

be Naegleria (rare).
– Culture the c.s.f.
_ When cells predominantly lymphocytes: This could indicate viral meningitis, tuberculous
meningitis, cryptococcal meningitis, trypanosomiasis encephalitis, or other condition in which
lymphocyte numbers in the c.s.f. are increasedPerform the following tests:
– Measure the concentration of protein or perform a Pandy’s test. The c.s.f. protein is raised in

most forms of meningitis and meningoencephalitis.

– Measure the concentration of glucose. This is helpful in differentiating viral meningitis in
which the c.s.f. glucose is usually normal from tuberculous meningitis and other conditions
in which the c.s.f. glucose is reduced
– Examine a wet preparation for encapsulated yeast cells that could be C. neoformans.
– Examine a wet preparation for trypanosomes and a Giemsa stained smear for morula
(Mott) cells when late stage trypanosomiasis is suspected.
Gram smear
A Gram smear is required when the c.s.f. contains pus cells (neutrophils). It should be the first
investigation to be performed and reported when the c.s.f. appears purulent or cloudy (suggestive
of acute pyogenic meningitis).
Making a smear of c.s.f. for Gram staining
1. Mix the CSF sample and centrifuge most of it at approximately 3000 rpm for 5–10
minutes (leave a small amount of uncentrifuged CSF for a cell count should this be
required).

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 MODULE TITLE : Collecting and Processing Medical Samples

Purulent c.s.f. Do not centrifuge a purulent fluid. A smear for Gram staining is best
prepared from the uncentrifuged CSF.
2. Transfer the supernatant fluid to another tube (to be used for glucose and protein tests
should these be required).
3. Mix the sediment. Transfer several drops of the sediment to a slide, but do not make the
preparation too thick because this will make it difficult to decolorize adequately. Allow
the preparation to air-dry in a safe place.
4. Alcohol-fix the preparation and stain it by the Gram technique.
Gram stain Procedure
1. Prepare smears from specimen or culture.
2. Allow the smear to air-dry.
3. Rapidly pass slide three times through flame.
4. Cover fixed smear with crystal violet for one minute and wash with tap water.
5. Tip off the water and cover the smear with Gram’s iodine for one minute.
6. Wash off iodine solution with tap water.
7. Decolorize with acetone-alcohol for 30 seconds.
8. Wash off the acetone-alcohol with clean water.
9. Cover the smear with safranine for one minute.
10. Wash off the stain and wipe the back of the slide. Let the smear air-dry
11. Examine the stained smear with oil immersion objective to look for bacteria

Ziehl-Neelsen smear when tuberculous meningitis is suspected

1. Centrifuge the c.s.f. at high speed for 20–30 minutes. Remove the supernatant fluid and
mix the sediment. Transfer several drops of the sediment to a slide, allowing each drop to
dry before adding the next.
2. Fix the dry preparation with methanol and stain by the Ziehl-Neelsen technique.
Cell count
A white cell count with an indication whether the cells are pus cells or lymphocytes, is required
when the c.s.f. appears slightly cloudy or clear or when the Gram smear does not indicate
pyogenic bacterial meningitis.
Note: Samples that are heavily blood stained or contain clots are unsuitable for cell counting.
Method

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 MODULE TITLE : Collecting and Processing Medical Samples

1. Cover the counting chamber with the coverslip supplied.

2. Gently mix uncentrifuged CSF and fill the chamber with the fluid:
 undiluted, if the CSF appears clear;
 diluted, if the CSF appears cloudy.
Make a 1 in 20 dilution using 0.05 ml of the CSF and 0.95 ml of Türk solution.
Pipette into a small bottle and mix.
3. Leave the counting chamber on the bench for 5 minutes to allow the cells to settle. Place
the chamber on the microscope stage.
4. Count the cells
Differential white blood cell count in CSF
Method
1. Centrifuge the CSF sample at 3000 rpm for 5-10 minute.
2. Transfer the supernatant fluid in to another test tube (the supernatant can be used for
serological studies).
3. Put a small drop from the sediment on to a microscopic slide and make a thin smear.
4. Allow the smear to be air dried
5. Fix the dried smear using absolute methanol.
6. Stain the smear by Giemsa staining technique.
Biochemical testing of c.s.f
Biochemical c.s.f. tests which may be required include the measurement of protein and glucose.
Measurement of c.s.f. glucose
Glucose must be measured within 20 minutes of the CSF. being withdrawn otherwise a falsely
low result will be obtained due to glycolysis. Use the supernatant fluid from centrifuged CSF or
uncentrifuged CSF if the sample appears clear.
Measurement of c.s.f. total protein and globulin test
Use the supernatant fluid from centrifuged CSF or uncentrifuged CSF when the sample appears
clear.
Wet preparation to detect amoebae and trypanosome
Examine a wet preparation for motile amoebae or trypanosome as follows:
1. Transfer a drop of uncentrifuged purulent csf or a drop of sediment from a centrifuged
specimen to a slide and cover with a cover glass.

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2. Examine the preparation using the 10× and 40× objectives, with the condenser closed
sufficiently to give good contrast.

2.4.6. Serous fluids collection

Composition and formation
Serous fluid is the small amount of fluid that lies between the membranes lining the body
cavities (parietal) and those covering the organs within the cavities (visceral). It is considered an
“ultrafiltrate” of the plasma and closely resembles it. Production and reabsorption are normally at
a constant rate. They are influenced by:
 Changes in osmotic and hydrostatic pressure in the blood
 Concentration of chemical constituents in the plasma
 Permeability of blood vessels and the membranes
There are three types of serous fluids:
• Peritoneal fluid (ascitic fluid) - from the abdominal cavity - paracentesis
• Pleural fluid (thoracic fluid) - lung - thoracentesis
• Pericardial fluid - heart - pericardiocentesis
. Indications – infections, hemorrhages, malignancies, other disorders.
Collection
 Needle aspiration – paracentesis, thoracentesis, pericardiocentesis
3 sterile tubes, often in EDTA to prevent clotting.
 Lavage – peritoneal
Specimen: Transudates vs. exudates – A build up of serous fluid is called an effusion.
Classifying a cause can be aided by determining if it is a transudate or exudate.
Transudate
 Due to a systemic disorder, ex. congestive heart failure
 Disruption in the balanced regulation of fluid filtration (formation) and its reabsorption
 Thought of as a mechanical process
Exudate
• Produced by conditions that directly involve the membranes of the particular cavity, ex.
infections, inflammation, and malignancies
• Thought of as an inflammatory process
Transudate Exudate
Appearance Clear Cloudy
Specific Gravity <1.015 > 1.015
Total Protein <3.0 g/d >3.0 g/dl
Fluid Protein:Serum Protein Ratio <0.5 >0.5

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Lactic Dehydrogenase (LDH) <200 IU >200 IU

Fluid LDH:Serum LD Ratio <0.6 >0.6
WBC Count <1000/ul >1000/ul
RBC Count 0-low high
Spontaneous Clotting No Possible

Laboratory procedures performed on all serous fluids:

Hematology / Gross Examination
a. Color/clarity – Normally yellow and clear.Otherwise use the same descriptives as CSF (except
for the term Xanthochromic which is only used for CSF)
b. Cell count (performed same as for CSF)
Chemistry
a. Total protein, and ratio to serum protein
b. LDH, and ratio to serum LDH
c. Glucose
d. Amylase & Lipase evaluation for pancreatic disorders
e. Bilirubin and alkaline phosphatase – occasionally on peritoneal fluid
f. pH and ammonia
Microbiology
a. Gram stain and acid fast
b. Culture (aerobic and anaerobic)
4. Serology – rarely done
5. Cytology – fluid and/or biopsy
6. QC - No prepared/commercial controls available for serous fluids, usually run chemistry
serum controls.

2.4.7. Synovial fluid

Composition and formation
Secreted by the cells of the synovial membrane. Very viscous fluid containing Hyaluronic acid,
Mucopolysaccharides and Small amount of plasma protein. increased volume of synoval fluid
may be due to variety of pathological conditions. pathologically it can be classified in to four
groups
 Non inflammatory (osteoarthritis)
 Inflammatory(eg,rheumatoid arthritis,gout)
 Septic (eg bacterial infection,fungal infection)
 heamorrhagic (eg ,heamophilia ,trauma)
Functions
 Supplies nutrients to the cartilage
 Acts as a lubricant

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C. Indications – infections, hemorrhage, degenerative disorders (arthritis), inflammatory disease

(SLE)
Collection
 Arthrocentesis — needle aspiration of joint fluid
 Volume — normal knee = approximately 3.5 mL

Tubes
a. Heparin — chemical and immunological testing
b. Plain sterile tube — microbiological culturing and crystal examination
c. EDTA — cell counts and differential
Laboratory Procedures
1. Hematology / Gross Examination
a. Color/clarity
1) Normal = yellow and clear (tho viscous)
2) Abnormal = same descriptives as for other serous fluids
a) Hemarthrosis
b) Traumatic Tap - differentiate the same as for CSF
b. Viscosity - this test is unique to synovial fluid. It is essential for lubrication of joints, is the
result of polymerization of the hyaluronic acid

2.4.8 Collection and processing of of urogenital specimens

 Urogenital specimens should be collected by a medical officer or an experienced nurse.
 Amies medium is the most efficient medium for transporting urethral, cervical, and
vaginal swabs.
 Specimens should be transported in a cool box.

2.4.8.1 Collection of urethral discharge from male patients

1. Cleanse around the urethral opening using a swab moistened with sterile physiological
saline.
2. Gently massage the urethra from above downwards. Using a swab, collect a sample of
discharge. Make a smear of the discharge on a microscope slide by gently rolling the
swab on the slide. This will avoid damaging pus cells which contain the bacteria.
3. Label the specimens and deliver to the laboratory as soon as possible.
Note: Very few pus cells may be present if the patient has recently passed urine. Allow 2–4 hours
after urination before collecting a specimen.

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2.4.8.2 Collection of cervical specimens from female patients

A specimen collected from the endocervical canal is recommended for the isolation of N.
gonorrhoeae by culture. Use a sterile vaginal speculum to examine the cervix and collect the
specimen.
1. Moisten the speculum with sterile warm water, and insert it into the vagina.
Note: Do not lubricate the speculum with a gel that may be bactericidal.
2. Cleanse the cervix using a swab moistened with sterile physiological saline.
3. Pass a sterile cotton-wool swab 20–30 mm into the endocervical canal and gently rotate
the swab against the endocervical wall to obtain a specimen.
4. Label the specimens and deliver to the laboratory as soon as possible.

2.4.8.3 Collection of vaginal swab:

1. Wear glove
2. Open the swab packaging.
3. Remove the swab from the plastic wrapper.
4. Do NOT touch the cotton tip with your hands or lay the swab down.
5. Insert the swab into the vaginal orifice about 5 cms.
6. Gently rotate for 10 to 30 seconds or 3 times.
7. Withdraw the swab without touching the skin.
Appearance and pH of vaginal discharge in Candida, Trichomonas, and
Gardnerella infections:
 T. vaginalis: Yellow-green purulent discharge with pH over 5.
 C. albicans: White odourless discharge with pH below 5.
 G. vaginalis: Grey, offensive, smelling thin discharge with pH over 5.
*The normal reaction of vaginal discharge (puberty to menopause) is pH 3.0–3.5.

Collection of specimen to detect T. pallidum

To detect motile T. pallidum spirochaetes, a specimen must be collected before antibiotic
treatment.
1. Wearing protective rubber gloves, cleanse around the ulcer (chancre) using a swab
moistened with physiological saline. Remove any scab which may be present.

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Caution: T. pallidum spirochaetes are highly infectious.

2. Gently squeeze the lesion to obtain serous fluid. Collect a drop on a cover glass and
invert it on a microscope slide.
Note: The cover glass and slide must be completely clean.
3. Immediately deliver the preparation to the laboratory for examination by dark-field
microscopy.
2.4.8.4 Collection of semen sample
Semen is collected for investigation of infertility problem in men.
1. Give the person a clean, dry, leak-proof container, and request him to collect a specimen
of semen at home following 3–7 days of sexual abstinence.
 Condom: When a condom is used to collect the fluid, this must be well-washed to remove
the powder which coats the rubber. It must be dried completely before being used.
 Coitus interruptus: This method of collection should not be used because the first portion of
the ejaculate (often containing the highest concentration of spermatozoa) may be lost. Also
the acid pH of vaginal fluid can affect sperm motility and the semen may become
contaminated with cells and bacteria.
 Masturbation: can be performed at home or in a special room in the laboratory.
2. Ask the person to write his name on the container, date and time of collection, period of
abstinence, and to deliver the specimen to the laboratory within 1 hour after collection.
3. During transit to the laboratory, the fluid should be kept as near as possible to body
temperature. This is best achieved by placing the container inside a plastic bag and
transporting it in a pocket in the person’s clothing.
Note: Semen specimen should be tested within 30 minutes.
It should not be stayed more than a maximum of 2 hours.
2.4.9 Collection of pus, ulcer material, skin specimens
 Specimens should be collected by a medical officer or an experienced nurse.
 Pus from an abscess is best collected at the time the abscess is incised and drained, or
after it has ruptured naturally.
 When collecting pus from abscesses, wounds, or other sites, special care should be taken
to avoid contaminating the specimen with commensal organisms from the skin.
 As far as possible, a specimen from a wound should be collected before an antiseptic
dressing is applied.
In a hospital with a microbiology laboratory

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1. Using a sterile technique, aspirate or collect from a drainage tube up to 5 ml of pus.

Transfer to a leak-proof sterile container. When pus is not being discharged, use a sterile
cotton-wool swab to collect a sample from the infected site. Immerse the swab in a
container of Amies transport medium.
2. Label the specimen and as soon as possible deliver it with a completed request form to
the laboratory.
 When the tissue is deeply ulcerated and necrotic (full of dead cells): Aspirate a sample of
infected material from the side wall of the ulcer using a sterile needle and syringe.
Transfer to a sterile container.
 Fluid from pustules, buboes, and blisters: Aspirate a specimen using a sterile needle and
syringe. Transfer to a sterile container.
 Serous fluid from skin ulcers, papillomas, or papules, that may contain treponemes:
Collect a drop of the exudate directly on a clean cover glass and invert it on a clean slide.
Immediately deliver the specimen to the laboratory for examination by dark-field
microscopy.
Skin specimens for ringworm fungi: Collect and examine as follows:
1. Cleanse the affected area with 70% v/v ethanol.
2. Collect skin scales, crusts, pieces of nail or hairs on a clean piece of paper about 5 cm
square (use dark coloured paper so that the specimen is easy to see).
Skin scales: Collect by scraping the surface of the margin of the lesion using a sterile blunt
scalpel.
Crusts: Collect by removing part of the crust nearest to healthy skin using sterile scissors and
tweezers.
Nail pieces: Collect by taking snippings of the infected part of the nail using sterile scissors.
Where the nail is thickened, also collect scrapings from beneath the nail.
Hairs: Collect by removing dull broken hairs from the margin of the lesion using sterile tweezers
or scraping the scalp with a blunt scalpel.
3. Fold the paper to enclose the specimen and use a paper clip to close it. Label it with the
patient’s name and number, source of material, and the date.

Transporting fungal specimens to a mycology laboratory

 Ringworm specimens are best transported in paper packages (rather than screw-cap
containers) to reduce humidity and the multiplication of bacteria.

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 Spores of ringworm fungi resist drying and remain viable for several months when stored
in paper.
Caution: Ringworm specimens are not very infectious but the hands should always be washed
with soap and water after collecting specimens.

Method of collecting and examining a skin smear for M. leprae

A smear for the examination of M. leprae must be collected by a trained and experienced
observer using an aseptic and safe technique. The site sampled should be the edge of a leprosy
lesion.
1. Explain the procedure to the patient (or parent if the patient is a child). The patient
should sit with his or her back to the table on which the equipment for taking the smear is
placed.
2. Fit a new scalpel blade in its scalpel holder. Sterilize the blade by wiping it carefully
with a piece of absorbent cotton wool soaked in 70% v/v ethanol (alcohol) and flaming it
for 2–3 seconds in the flame of a spirit lamp. Allow the blade to cool; making sure it is
not touching any unsterile surface.
3. Wearing protective rubber gloves, cleanse the area from where the smear is to be taken,
using a cotton wool swab moistened with 70% v/v ethanol (alcohol). Allow the area to
dry.
4. Pinch the skin tightly between the thumb and index finger until it becomes pale due to
loss of blood.
Important: The area must be kept bloodless while the smear is collected because a smear which
contains red cells will be difficult to examine and report.
5. Using the sterile blade, make a small cut through the skin surface, about 5 mm long and
deep enough into the dermis (2–3 mm) where the bacteria will be found. Continue to hold
the skin tightly.
6. Using a dry piece of cotton wool, blot away any blood which appears at the site of the
cut.
Note: Providing the pressure is maintained between the thumb and index finger, little or no
bleeding should occur.
7. Turn the scalpel blade until it is at a right angle to the cut. Using the blunt edge of the
blade, scrape firmly two or three times along the edges and bottom of the cut to collect a
sample of tissue juice and cells.

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8. Transfer the sample to a slide. Make a small circular smear, covering evenly an area
measuring 5–7 mm in diameter.
9. Cover the cut with a small dressing. Instruct the patient to remove the dressing as soon as
the cut has healed.
10. Ensure the slide is clearly labelled with the patient’s name and identification number.
Caution: Specimens from patients with suspected plague or anthrax are highly infectious. Label
such specimens HIGH RISK and handle them with care.

In a health center for dispatch to a microbiology laboratory

1. Collect the specimen using a sterile cotton-wool swab. Insert it in a container of Amies
transport medium, breaking off the swab stick to allow the bottle top to be replaced
tightly.
When the material is aspirated fluid from a pustule, transfer the fluid to a sterile, leak-proof
container. Stopper, and seal in a leak-proof plastic or metal container.
Note: It is not possible to transport exudate from a suspected treponemal ulcer because the
treponemes remain motile for only a short time.
2. Make a smear of the material on a clean slide (for Gram staining) and allow to air-dry in
a safe place. Heat-fix the smear.
Caution: Do not make a smear for transporting when the specimen is from a patient with
suspected anthrax or bubonic plague.
3. Send the specimens with a completed request form to reach the microbiology laboratory
within 6 hours.

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Unit Three
Sample Transportation
1. Required materials for sample transport
1.1. Sample containers

 Hazardous materials, both chemical and biological hazards, MUST be transported in a

durable, leak-proof secondary container with a tight-fitting lid.

PRIMARY CONTAINER:

 A container that provides an immediate barrier between a hazardous agent and the
environment. Examples: sample tubes, blood tubes, syringes, vials, chemical bottles

SECONDARY CONTAINER:

 A container that a hazardous agent in a primary container can be placed inside of and
securely closed.

 To provide a second layer of protection between the hazardous agent, either chemical or
biological, and the surrounding environment if the primary container leaks or breaks.

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Requirements for a secondary container

Durable, leak-proof container with a tight fitting lid, preferably either a snap-top or

screwtop.

For biohazardous samples:

 The universal biohazard sign posted on the outside of the secondary container.
For large sample volumes or for multiple blood tubes:

 An absorbent need to be placed between the inner container and the secondary container.
This absorbent should be sufficient to absorb all of the liquid that is being transported.

 After a hazardous sample has been securely placed inside of secondary containment, all
personal protective equipment must be removed before you travel outside of the

laboratory!!!!

 This means NO gloves or lab coats outside of the laboratory

 if your secondary container hard to handle place the secondary container in a cooler with a
handle making it easy to carry. If the sample needs to remain cold, ice can be added to the

cooler.

 If your secondary container heavy, or you need transport your sample a long distance.

 for transporting heavy containers or transporting samples longer distances, the use of cart is
recommended

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Packaging, Labelling and Documentation for Transport

 Because of the distinction of risks between infectious substances and diagnostic

specimens, there are variations to the packaging, labelling and documentation requirements.
Basic triple packaging system
The system consists of three layers as follows.
1. Primary receptacle.
 A labelled primary watertight, leak-proof receptacle containing the specimen.
 The receptacle is wrapped in enough absorbent material to absorb all fluid in case of
breakage.
2. Secondary receptacle.
 A second durable, watertight, leak-proof receptacle to enclose and protect the primary
receptacle(s).
 Several wrapped primary receptacles may be placed in one secondary receptacle.
Sufficient additional absorbent material must be used to cushion multiple primary
receptacles.
3. Outer shipping package.
 The secondary receptacle is placed in an outer shipping package which protects it and its
contents from outside influences such as physical damage and water while in tran

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 Specimen data forms, letters and other types of information that identify or describe the
specimen and also identify the shipper and receiver should be taped to the outside of the
secondary receptacle.

Fig.Triple packaging system

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Dispatch of microbiological specimens collected in health centres or district hospitals

Without culture facilities

 Specimens for dispatch must be packed well and safely.

 When specimens are to be mailed, the regulations regarding the sending of ‘Pathological
Specimens’ through the post should be obtained from the Postal Service and followed
exactly.
 When dispatching microbiological specimens, the following apply:
● Keep a register of all specimens dispatched. Record the name, number, and ward or health
centre of the patient, type of specimen, investigation required, date of dispatch, and the method
of sending the specimen (e.g. mailing, hand-delivery, etc).

• When the report is received back from the microbiology laboratory, record the date of
receipt in the register.
● Check that the specimen container is free from cracks, and the cap is leak-proof. Seal around
the container cap with adhesive tape to prevent loosening and leakage during transit.

● Use sufficient packaging material to protect a specimen, especially when the container is a
glass tube or bottle (use a plastic container whenever possible). Place the packaged container in a
strong protective tin or box, and seal completely. When the specimen is fluid, use sufficient
absorbent material to absorb it should a leakage or breakage occur.

● Mark all specimens that may contain highly infectious organisms, ‘HIGH RISK’ (Do not mail
such specimens.

● Dispatch slides in a plastic slide container or use a strong slide carrying box or envelope.

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● Label specimens dispatched by mail, ‘ FRAGILE WITH CARE – PATHOLOGICAL

SPECIMEN’.

 When a specimen is likely to deteriorate unless kept cool, transport it in an insulated

container, such as a polystyrene box or thermos flask containing ice cubes.
 The specimen must be sealed inside a waterproof bag or tin to prevent the label being
washed off when the ice cubes melt.
 Precautions must also be taken to keep the request form dry.
 Postal transport regulations If using the postal system, laboratories must follow their
national postal regulations which apply to the mailing of biological infectious
substances. These will be available from the postal service.
 The postalsystem is appropriate for sending formol saline preserved biopsies or fixed
smears to a histopathological laboratory.

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Unit Four
Maintain a Safety work environment
 Everyone employed by the department has a personal responsibility to become involved in
solving health and safety problems by Follow standard laboratory safety practices.
 The department's goal is to have all employees working together to identify and control
situations that could cause harm and to integrate health and safety practices into their daily
activities.
 Worker participation is crucial to effective health and safety.
The department:
 recognizes that each employee has a right to a work environment which will not adversely
affect his or her health and safety;
 is committed to providing safe workplaces for all its employees;
 is committed to protecting the health and safety of its contracting parties and the public;
 will diligently carry out the employer duties contained in the Occupational Health and Safety
Act and regulations;
 The clinical laboratory contains a wide variety of safety hazards, many capable of producing
serious injury or life threatening disease.
 the chain of infection of microorganisms is necessary to prevent infection
 The chain of infection requires a continuous
 link between three elements:
1. A source
2. Method of transmission,
3. Susceptible host.
 Therefore, safety precautions are designed to protect health-care workers

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 Preventing the transmission of microorganisms from infected sources to susceptible hosts is

critical in controlling the spread of infection

Types of Safety Hazards

• Biologic: can be source of Infectious agents and cause bacterial, fungal, viral, or parasitic
infections.

• Sharp: Needles, lancets, and broken glass cause Cuts, punctures, or bloodborne pathogen
exposure.

• Chemical: Preservatives and reagent Exposure to toxic, carcinogenic,caustic agents

• Radioactive Equipment and radioisotopes causer adiation exposure

• Electrical: Burns or shock

• Fire/explosive: Bunsen burners and organic chemicals cause Burns

• Physical: Wet floors, heavy boxes, and patients Falls, sprains, or strains

 Procedures used to prevent microorganism transmission include:

F hand washing

F wearing personal protective equipment (PPE),

F Isolating highly infective or highly susceptible

patients

F properly disposing contaminated materials.

 Strict adherence to guidelines published by:

 Centers for Disease Control and Prevention (CDC)
 Occupational Safety and Health Administration (OSHA) is essential
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Laboratory Waste Management

 Laboratory personnel put all waste into a secured storage area where it is picked up for
disposal by specially designated transport.
 Labeling of Waste
-If the contents of the bottle are not known, the next person to use the bottle could All
bottles of chemical waste must have Label with date and contents.
-accidentally combine incompatible chemicals causing a fire and explosion.
- Store waste in a bottle with the words "Hazardous Waste".

 Do not use metal cans for waste. Do not store waste in a fume hood where reactions are
being carried out.
• If reaction gets out of control, the waste bottle could explode and lead to a fire or mixing of
incompatible chemicals.

• Remove waste bottles from hoods where reactions are being carried out.

• Use only glass or polyethylene containers for waste.

• Do not store flammable waste containers on a bench or floor. Store waste containers in an
explosion-resistant solvent cabinet.

• Do not store waste bottles in a sink or floor drain.

• Toxic chemicals can enter the drain, and emit toxic gas causing health hazard or explosion

• Try to have only ONE bottle of each kind of waste in the laboratory.

• If the organic waste bottle is full, take it to the waste storage area.

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