DIPLOMA PAPER III

DIAGNOSTIC MICROBIOLOGY II
(VIROLOGY, MYCOLOGY AND PARASITOLOGY)
UNIT I
Laboratory methods in basic Mycology Collection and transport of clinical specimens
Direct Microscopic examination, culture media and incubation, Serological tests for
fungi Antifungal susceptibility testing
UNIT II
Laboratory methods for parasitic infections Diagnostic techniques for faecal,
gastrointestinal and urino-genital specimen.
UNIT III
Identification of Intestinal Protozoa Amoeba, Blood protozoa Malaria, Intestinal
Helminthes and Blood Helminthes.
UNIT IV
Laboratory methods in basic virology- detection of viral antigen (fluorescent antibody
and solid phase immunoassays). Viral Serology- Special consideration- Hepatitis and
AIDS.
UNIT V
Viral culture- Media and cells used Specimen processing isolation and identification
of viruses.
References
1. Diagnostic Microbiology, Bailey and Scotts., 1990. Eighth edition. The Mosby
Company.
2. Medical laboratory techniques, Abdul Khader, 2003, First edition. Frontline
Publications, Hyderabad.
3. Virology, Sawant, K.C., 2005, First edition, Dominant Publishers and distributors,
Delhi.
4. Medical Parasitology, Rajesh Karyrkarte, Ajit Damla, 2004. Books and allied
publishers Ltd. Kolkata.
5. Textbook of Medical Parasitology, Subash O. Barija , 1996. First edition. All India
Publishers and Distributors Regd. 920 Poonamallee High Road, Chennai.
6. Rajesh Karyakarte and Ajith Damle (2005)Medical Parasitology, ooks and
Allied(P)Ltd.

Collection and Transportation of Clinical Specimens

The laboratory diagnosis of an infectious disease begins with the collection of a clinical specimen for
examination or processing in the laboratory (the right one, collected at the right time, transported in the
right way to the right laboratory). Proper collection of an appropriate clinical specimen is the first step in
obtaining an accurate laboratory diagnosis of an infectious disease. Guidelines for the collection and
transportation of specimens should be made available to clinicians in a lucidly written format. The guidelines
must emphasize two important aspects:
Collection of the specimen before the administration of antimicrobial agents.
Prevention of contamination of the specimen with externally present organisms or normal flora of the
body.
General rules for collection and transportation of specimens are summarized in Table 1.
Table1: Collection and transportation of specimens
Apply strict aseptic techniques throughout the procedure.
Wash hands before and after the collection.
Collect the specimen at the appropriate phase of disease.
Make certain that the specimen is representative of the infectious process (e.g. sputum is the specimen for
pneumonia and not saliva) and is adequate in quantity for the desired tests to be performed.
Collect or place the specimen aseptically in a sterile and/or appropriate container.
Ensure that the outside of the specimen container is clean and uncontaminated.
Close the container tightly so that its contents do not leak during transportation.
Label and date the container appropriately and complete the requisition form.
Arrange for immediate transportation of the specimen to the laboratory.
Criteria for rejection of specimens
Criteria should be developed by a laboratory on the basis of which the processing of a specimen may not be
done by the laboratory. The following are some examples:
Missing or inadequate identification.
Insufficient quantity.
Specimen collected in an inappropriate container.
Contamination suspected.
Inappropriate transport or storage.
Unknown time delay.
Haemolysed blood sample.
Collection of specimens
The clinical state of the patient will not necessarily be reflected by the result of laboratory investigation despite
correct laboratory performanceunless the specimen is in optimal condition required for the analysis.Some of
the important specimens and their proper collection and transportation methods are described here so as to
ensure quality.
Blood
Whole blood is required for bacteriological examination. Serum separated from blood is used for serological
techniques. Skin antisepsis is extremely important at the time of collection of the sample. Tincture of iodine (12%), povidone iodine (10%) and chlorhexidine (0.5% in 70% alcohol) are ideal agents. However, some
individuals may be hypersensitive to iodine present in some of these. While collecting blood for culture,the
following points must be remembered:

Collect blood during the early stages of disease since the number of bacteria in blood is higher in the
acute and early stages of disease.
Collect blood during paroxysm of fever since the number of bacteria is higher at high temperatures in
patients with fever.
In the absence of antibiotic administration, 99% culture positivity can be seen with three blood
cultures.
Small children usually have higher number of bacteria in their blood as compared to adults and hence
less quantity of blood needs to be collected from them (Table 2).
Table 2: Volume of blood to be collected at different ages

Age

Volume in 2 bottles

< 2 years

2 ml

2-5 years

8 ml

6-10 years

12 ml

>10 years

20 ml

Cerebrospinal fluid (CSF)

Examination of CSF is an essential step in the diagnosis of any patient with evidence of meningeal irritation or
affected cerebrum. Almost 3-10 ml of CSF is collected and part of it is used for biochemical, immunological and
microscopic examination and remaining for bacteriological or fungal examination. The following important
precautions need to be taken for CSF collection and transportation:
Collect CSF before antimicrobial therapy is started.
Collect CSF in a screw capped sterile container and not in an injection vial with cotton plug.
Do not delay transport and laboratory investigations.
Transport in a transport medium if delay in processing is unavoidable.
CSF is a precious specimen, handle it carefully and economically. It may not be possible to get a
repeat specimen.
Perform physical inspection immediately after collection and indicate findings on laboratory requisition
form.
Store at 37oC, if delay in processing is inevitable.
The characteristics of the appearance of CSF are outlined in Table 3.
Table 3: Appearance and interpretations of CSF
Clear and colourless

Normal

Clear with Tyndall effect

(sparkling appearance against incident light)

High protein content

Clear yellowish

Old haemolysis

Clear red

Fresh haemolysis

Turbid blood-stained

Haemorrhage

Turbid white

High cell or protein content

Turbid clot (after overnight storage)

Fibrin clots

Sputum
Sputum is processed in the laboratory for aetiological investigation of bacterial and fungal infections of the
lower respiratory tract. It is of utmost importance in the diagnosis of pulmonary tuberculosis.
Select a good wide-mouthed sputum container, which is preferably disposable, made of clear thin
plastic, unbreakable and leak proof material.
Give the patient a sputum container with the laboratory serial number written on it. Show the patient
how to open and close the container and explain the importance of not rubbing off the number written
on the side of the container.
Instruct the patient to inhale deeply 2-3 times, cough up deeply from the chest and spit in the sputum
container by bringing it closer to the mouth.
Make sure the sputum sample is of good quality. A good sputum sample is thick, purulent and
sufficient in amount (2-3 ml).
Give the patient an additional container with laboratory serial number written on it for an early morning
specimen. Explain to the patient to rinse his/her mouth with plain water before bringing up the sputum.
Urine
Under normal circumstances urine is sterile. The lower part of the urethra and the genitalia are normally
colonised by bacteria, many of which may also cause urinary tract infection. Since urine is a good growth
medium for all sorts of bacteria, proper and aseptic collection assumes greater importance for this specimen.
For microbiological examination urine must be collected as a "clean catch-mid-stream" specimen.
Urine specimens should be transported to the laboratory within one hour for bacteriological examination,
because of the continuous growth of bacteria in vitro thus altering the actual concentration of organisms.
Stool
Faecal specimens for the aetiological diagnosis of acute infectious diarrhoeas should be collected in the early
stage of illness and prior to treatment with antimicrobials. A stool specimen rather than a rectal swab is
preferred.
The faeces specimen should not be contaminated with urine.
Do not collect the specimen from bed pan.
Collect the specimen during the early phase of the disease and as far as possible before the
administration of antimicrobial agents.
1 to 2 gm quantity is sufficient.

If possible, submit more than one specimen on different days.

The fresh stool specimen must be received within 1-2 hours of passage.
Store at 2-8oC.
Modified Cary and Blair medium (see chapter 5) is recommended as a good transport medium. It is a
very stable medium and can be stored for use in screw capped containers. It is a semi-solid
transport medium. At least two swabs should be inoculated. Most pathogens will survive for up to 48
hours at room temperature. Specimens are unacceptable if the medium is held for more than one
week or if there is detectable drying of the specimen.
Alternative transport media are Venkataraman-Ramakrishnan medium (V-R fluid) or alkaline peptone water. VR
fluid should be prepared in 30 ml (1 oz) screw capped bottles (MacCartney bottles). It preserves vibrios for
more than six weeks and has also proved to be a very convenient medium for transportation as it can be kept
at room temperature after collection of the specimen.
Throat swab
Depress the tongue with a tongue blade.
Swab the inflammed area of the throat, pharynx or tonsils with a sterile swab taking care to collect the
pus or piece of membrane.
Transport in sterile transport tube.
Bone marrow
Bone marrow is collected by a doctor who is well trained in this procedure
Decontaminate the skin overlying the site from where specimen is to be collected with 70% alcohol
followed by 2% tincture of iodine.
Aspirate 1 ml or more of bone marrow by sterile percutaneous aspiration.
Collect in a sterile screw-cap tube.
Send to laboratory immediately.
Rectal swab
Insert swab at least 2.5 cm beyond the anal sphincter so that it enters the rectum.
Rotate it once before withdrawing.
Transport in Cary and Blair or other transport medium.
Transportation of specimens
Specimens to be sent to other laboratories require special attention for safe packing of the material. Guidelines
are usually issued by national authorities and the same should be strictly followed. For hand-carried
transportation over a short distance, the specimen should be placed upright in appropriate racks. For long
distance transportation, it should be placed in three containers, i.e:
A primary container which has the specimen and is leakproof with a screw-cap.
A secondary container which is durable, waterproof and made of metal or plastic with a screw-cap. It
should have enough absorptive material to absorb the contents of the primary container should the latter
break or leak. On its outside, the details of the specimen should be pasted.
A tertiary container is usually made of wood or cardbox. It should be capable of withstanding the shocks
and trauma of transportation. Dry ice can be kept between this and the secondary container along with
sufficient absorbents and provision for the escape of carbondioxide to prevent a pressure build-up inside
(Fig 1).
In general, most specimens should be processed in the laboratory within 1 to 2 hours after collection. In
practice, a 2-to 4-hour time limit is probably more practical during a normal working day. The laboratory must
be organized to permit processing of the specimens as soon as they arrive, and the collection of most
specimens should be limited to the working hours of the laboratory. However, some arrangements must be
made to allow for the initial handling of the few specimens that have to be collected outside of the laboratorys
working hours.
Figure 1: Transportation container

A continuous effort must be made in order to ensure proper collection and transportation of clinical specimens.
Full cooperation of nursing staff and others concerned with specimen collection is required and can be achieved
once they are made aware of the principles involved and the significance of what they are being asked to do.

Identification of amoebae
Identification of amoebae remains one of the most difficult problems, and a lot is written
about this. For correct generic and specific identification EM is obligate in the very most of
cases. Another problem is our relatively low level of knowledge about amoebae biodiversity the chances to find new species in any habitat are very high. For example, detailed faunistic
survey of a freshwater lake revealed 32 Gymnamoebia species, of which 15 were found to be
new for science (Smirnov & Goodkov 1995). You should always be ready to meet unknown
species in your cultures.
In order to identify an amoeba you need first to decide with the appropriate level of
detalisation of your identification. If your are satisfied with the level of a morphotype, you
have no need to clone amoebae, observation from initial cultures are sufficient. However, if
you are going to follow further in systematical identification you must fulfil all requirements,
for example, of Pages key (or other respective literature) concerning the set of necessary
species data. This may be rather laborious. It is strongly preferable to stop with the
reasonably identified morphotype or to identify genus using respective EM and respective
literature, rather than to make non-reliable suggestions about generic or specific position of
your strain, if you are not sure or unable to fulfil all requirements of systematic identification.
Identification itself consists of several distinct steps. We will consider them subsequently,
and this schedule should be used in real work as it is described. You are welcome to stop
either at the first step, if you are going to identify a morphotype only, or to follow them all for
systematic identification, using methods as required in respective literature (cited for each
morphotype).

If you are going to deal with systematic identification of amoebae first consult F.C.
Pages keys (1988 - in English, 1991 - in German). They allow you to have a good deal of
information and provide you with the basic steps for systematical identification. They may be
sufficient for species identification, however when you will decide with the species always
check the original description and latest papers dedicated to this species (if availiable).
Review the literature, dedicated to your species and relative taxa which was published after
1988. These papers may contain descriptions of new, post-Page genera and species which
have been found already in many taxa of amoebae. Consult the cheklist of valid amoebae
species at this homepage. It is difficult to give further advises, as here you will be already at
the expert level of identification, thus follow the recommendation of Page's keys and related
literature for further work.
Step 1. Locomotive form.
Locomotive form - the form of an amoeba in continuos, directed movement is a
background for any further speculations. If you are working with water-immersion or
inverted optics, find locomotive amoeba on the clean area of the bottom, free of sufficient
debris of bacteria and detritus (presence of a material on the bottom of the dish may influence
locomotive form). If you are working with agar culture, wash amoebae from the agar with a
drop of respective media, place this drop on the object slide and cover with a coverslip. The
drop should be of a size that the coverslip does not touch cells. If you are working with large
amoebae, scrape a piece of vax by each corner of a coverslip prior to use it. This will form
small legs on the coverslip which predicts contact with the cells. It is very important to
avoid depressement of amoebae with the coverslip, as this influence sufficiently on the
locomotive form and may result in misidentification. It may require some time for amoebae
to start movement and adopt locomotive forms, thus it is better to place ready preparations in
a wet camera for two-three hours and only than to observe them.
Choose actively moving cell and note the shape and characteristic details of the
locomotive form (uroid, hyaloplasm, ridges, lateral flatness, shape of subpseudipodia, lobs
and wrinkles, if present). Sketches, videoprints or photographs of moving amoeba may be
highly useful in further work. Measure the locomotive forms using micrometer. Preferably
several amoebae should be measured, but make sure that they all belong to the same species!
If you are going to identify species you should work with clones, and measure not less than
30 amoebae to have average measurements. Note the nuclear size and structure, shape and
size of crystals (if present), typical position of contractile vacuole (if any).
If amoebae were maintained on the agar without overlay, it may be extremely difficult to
observe moving cells. It seems that been cultivated under these conditions for several
generations, cells loose partly their locomotive capacities. To avoid this, prior to observe
locomotive forms cover the agar with the overlay of respective media and leave it for twothree days. In the very most of cases this is enough for cells to restore locomotion. In worst
case try several passages using agar with overlay.
Step 2. Floating form
Floating form is very important for species identification. Observation of the floating
forms should be done preferably in clonal cultures, unless the size difference of amoebae you
are interested in from any other existing in this dish is sufficient. In some cultures with
overlay or in liquid you may easily see floating forms at any time under dissection

microscope. If not, to observe floating form, shake a culture (if it has an overlay or is liquid
one) carefully and observe under the dissection microscope development of the floating
forms. Far not all amoebae form them readily, and you need to see the dynamic of the shape
changes in floating amoebae to make sure that you have seen developed floating forms. For
smaller amoebae and amoebae which are maintained on the agar without overlay, wash of
cells from the dish with the drop of respective media, place the drop on the object slide, close
with coverslip and observe immediately. Sometimes it is possible to see floating form and
than locomotive forms, subsequently, on the same object slide.
Note the appearance of the floating form, shape and number (min/max.) of pseudopodia,
the material of pseudopodia (hyaloplasm only, or with the granuloplasm), shape of the ends
of pseudopodia, their thickness. Note if amoeba has tendencies to form coiled or spiral
pseudopodia. Measure the length of pseudopodia comparing it with the size of a central mass
of the cytoplasm in radiate floating forms. Some amoebae species has a tendency to gradual
modification of the floating form with the increment of the time of cultivation, thus floatings
form of fresh isolates may differ slightly from the floating form of the same species in culture
collection.
There are amoebae species which do not adopt any specific floating forms. They flotate
remaining usual, locomotive-like. There are minor of them, and careful observations required
to make a conclusion that an amoeba do not have differentiated floating form.
Step 3. Nuclear structure and crystals
Nucleus and crystals are well visible with oil immersion optics, using 100x objective
lense. Amoeba for these observation preferably should be slightly pressed with the coverslip
in order to make nucleus and crystals better visible. Note nuclear structure, number and
position of nucleoli, shape and size of crystals, count approximate number of crystals.
However you should not measure nucleus under these conditions! It should be done in
locomotive form.
Step 4. Cysts
Cyst formation and cyst structure is a very important criteria in amoeba systematics.
However, far not all species form cysts in culture. Basically, to observe cysts you need to
have pure clonal culture. Cyst may be found after 7-15 days in agar cultures and after longer
periods (up to month) in liquid. Some species loose the capacities of encystment after some
time of cultivation, some do form cysts only in cultures with overlay. Different conditions
should be applied and all cultures should be traced for at least a month prior to conclude
about cyst formation. Cysts should be observed with LM under 100x oil immersion, shape,
structure and number of cyst walls, presence and disposition of cyst pores, size of cysts must
be noted. Cysts should be a subject of EM studies as well as trophozoites.

Diagnosis of malaria
Diagnosis of malaria involves identification of malaria parasite or its
antigens/products in the blood of the patient. Although this seems simple, the
efficacy of the diagnosis is subject to many factors. The different forms of the

four malaria species; the different stages of erythrocytic schizogony; the

endemicity of different species; the population movements; the inter-relation
between the levels of transmission, immunity, parasitemia, and the symptoms;
the problems of recurrent malaria, drug resistance, persisting viable or nonviable parasitemia, and sequestration of the parasites in the deeper tissues; and
the use of chemoprophylaxis or even presumptive treatment on the basis of
clinical diagnosis can all have a bearing on the identification and interpretation of
malaria parasitemia on a diagnostic test.
The diagnosis of malaria is confirmed by blood tests and can be divided
into microscopic and non-microscopic tests.
Microscopic Tests
For nearly a hundred years, the direct microscopic visualization of the parasite on
the thick and/or thin blood smears has been the accepted method for the
diagnosis of malaria in most settings, from the clinical laboratory to the field
surveys. The careful examination of a well-prepared and well-stained blood film
currently remains the "gold standard" for malaria diagnosis.
The microscopic tests involve staining and direct visualization of the parasite
under the microscope.
1. Peripheral smear study
2. Quantitative Buffy Coat (QBC) test
Peripheral smear examination for malarial parasite is the gold-standard in
confirming the diagnosis of malaria. Thick and thin smears prepared from the
peripheral blood are used for the purpose.
The peripheral blood smear provides comprehensive information on the species,
the stages, and the density of parasitemia with a sensitivity of 5 to 10
parasites/L of blood for an experienced laboratory professional. The efficiency of
the test depends on the quality of the equipment and reagents, the type and
quality of the smear, skill of the technician, the parasite density, and the time
spent on reading the smear. The test takes about 20 to 60 minutes depending on
the proximity of the laboratory and other factors mentioned above. It is
estimated to cost about 12 to 40 US cents per slide in the endemic countries.
Problems: The exacting needs of the blood smear examination are often not
met in certain remote and poor parts of the world. Detection of low levels of
parasitemia, sequestered parasites of P. falciparum and past infections in
aspiring blood donors; ascertaining viability of the detected parasites; difficulties
in maintaining the required technical skills and resultant misdiagnosis due to
poor familiarity and problems in accessing and activating the facility in
emergencies are some of the deficiencies with the blood smear examination.
Alternative microscopic methods have been tried, including faster methods
of preparation, dark-field microscopy, and stains like benzothiocarboxypurine,
acridine orange and Rhodamine-123. Acridine orange has been tried as a direct
staining technique, with concentration methods such as thick blood film or the
centrifugal Quantitative Buffy Coat system and with excitation filter in the
Kawamoto technique. Inability to easily differentiate the Plasmodium species,

requirements of expensive equipment, supplies and special training as well as

the high cost limit the use of these methods.
1. Preparation of the smear: Use universal precautions while preparing
the smears for malarial parasites - use gloves; use only disposable
needles/lancets; wash hands; handle and dispose the sharp instruments
and other materials contaminated with blood carefully to avoid injury
Intestinal Helminthis

Intestinal helminths include the following:

Ascaris lumbricoides

Strongyloids stercolaris

Enterobias vermicularis

Trichuris trichura

Ankylostoma duodenale

Necator americanus

Taenia saginata

Taenia solium

Ascaris lumbricoides is one of the most common of the intestinal worms. It is a roundworm
and infection with it is called Ascariasis. Children are more frequently and more heavily
infected than adults because of their habit of putting all kinds of things into their mouths. If
these objects are contaminated with ascaris eggs from human faeces the children swallow the
eggs and thus become infected.
The round worm lives in the small intestines. The female lays as many as 200,000 eggs a day.
These are passed in stool and develop in the soil. They are then transmitted as follows:

Eggs passed out in stool are embryonated in stool before they are infective.

The embryonated eggs are carried away from the contaminated place into houses by
feet, foot wear or in dust by wind. They also can reach vegetables and fruits

A child then eats and swallows food or fruits contaminated with eggs.

The eggs hatch into larva in the intestinal canal.

The larva penetrates the intestinal wall and reach the liver via the portal system.

The larva is then carried to the lungs.

In the lungs, they penetrate into the airway and pass via the bronchioli, bronchi, and
trachea to the pharynx.

They are coughed up and are swallowed a second time, thus returning to the intestinal
tract.

They then settle into the jejunum where they develop.

In two months, they mature as adult worms and can live for about a year.

Signs and Symptoms Usually, children with mild infestation are symptomless. They may,
however, present with symptoms indicative of larval migration like pneumonitis or urticarial
rash. There may be some vague abdominal discomfort. Sometimes a worm may leave the
body through vomitus or stool.
Complications In heavy infections, complications may occur. I will briefly describe them
here:

Intestinal obstruction: This is a serious complication of heavy roundworm infestation.

A ball of worms forms, usually at the narrowest part of the intestine (the ileocaccal
junction) where the small intestine enters into the large intestine. The child is ill with
abdominal pains, constipation, vomiting, abdominal distension and an abdominal
mass. If the obstruction is complete the child is not passing gas or stool at all, urgent
surgery is needed and you should refer the child to hospital urgently.

Wandering of the worms: Wandering ascaris may reach abnormal foci and cause acute
symptoms. Vomiting of the worm may course swelling of the glottis and larynx. This
results in difficulty in breathing. Blockage of the bile ducts may cause obstructive
jaundice and migration into the liver may result in liver abscess.

Malnutrition: Ascariasis contributes to serious malnutritional states such as stunting,

kwashiorkor and Vitamin A deficiency. The adult worms absorb the childs digested
food in small intestines. The worms also interfere with the absorption of nutrients in
the small intestine thus causing malnutrition.

Diagnosis If you work in a unit with laboratory facilities then you should request a
microscopic examination of the stool. A characteristic ovum is seen under microscope on
stool preparation.
You can also make a diagnosis of ascariasis when:

The caretaker tells you the child has passed the worm in stool or vomited it.

You are able to see the worm in a child's stool or vomitus.

Treatment A child with ascariasis should be treated as follows:

A child who is aged 2 years or more can be given albendazole 400 mg of

mebendazole 500 mg single dose.

A child aged less than 2 years should receive half that dose.

TAPEWORM (Taenia saginata and Taenia solium)

Tapeworm infestation is caused by two worms called Taenia saginata and Taenia solium. The
infestation is referred to as Taenasis. Tapeworm infestation is common in areas where beef
and pork is eaten raw or undercooked.
Life Cycle
Let us now learn about how one can get infested with tape worm. Study the illustration in
Fig. 15.4 and the descriptive notes below. The life cycle of the tapeworm begins with an
infected person and is transmitted as follows:

Stool containing grand segment of the worm or eggs is passed;

Cattle or pigs ingest the eggs or segments;

In the gastrointestinal tract of the animals, the embryos hatch and penetrate the bowel
wall;

The larvae are then carried via the blood stream to striated muscles;

In the muscle, the larvae grow and form the infective cysts called cysticerci. Pork or
beef containing these cysts is called "mealy" pork or beef;

When a child ingests lightly cooked meat containing cysticerca, the cysts are
dissolved by the gastric acid in the stomach and the embryo is release;

Taenia saginata embryo attaches itself to the wall of the small bowel by its head and
grows into an adult worm;

Taenia solium behaves differently by penetrating the intestinal wall. It is then carried
by the blood stream to striated muscles or the brain.

Signs and Symptoms

Most infections with Taenia saginata cause no signs or symptoms. In some children
there is a loss of weight, abdominal discomfort and pruritus ani.
A child with Taenia solum infection may present with neurological signs like epilepsy,
or muscular pains. This is because the cysticerca invade the brain or muscle
respectively.
Diagnosis

You can diagnose tapeworm infestation when you are given a history of the presence
of segments in the stool. The segments can migrate out through the anus and be found
on the buttocks or they are passed with the faeces.
Treatment
The drug of choice in the treatment of tapeworm infestation is Niclosamide. You
should give it according to the schedule in Table 15.1.
Table 15.1: Niclosamide Treatment for Tapeworm
Age

Dose

Duration

< 2 years

500 mg as a single dose

1 day

2-6 years

1 gm as a single dose

1 day

> 6 years

2 gm as a single dose

1 day

When administering the drug Niclosamide, you should take note of the following:

It is better to give Niclosamide at breakfast and the tablets should be chewed.

Two hours after administering Niclosamide, give a purgative like Bisacodyl

(5mg/tablet) to the child.

An alternative treatment is mebendazole tablets as described in the treatment for Ascaris.

Laboratory methods in virology

Overview of diagnostic methods
In general, diagnostic tests can be grouped into 3 categories.: (1) direct detection, (2) indirect
examination (virus isolation), and (3) serology. In direct examination, the clinical specimen
is examined directly for the presence of virus particles, virus antigen or viral nucleic acids.
In indirect examination, the specimen into cell culture, eggs or animals in an attempt to grow
the virus: this is called virus isolation. Serology actually constitute by far the bulk of the
work of any virology laboratory. A serological diagnosis can be made by the detection of
rising titres of antibody between acute and convalescent stages of infection, or the detection
of IgM. In general, the majority of common viral infections can be diagnosed by serology.
The specimen used for direction detection and virus isolation is very important. A positive
result from the site of disease would be of much greater diagnostic significance than those

from other sites. For example, in the case of herpes simplex encephalitis, a positive result
from the CSF or the brain would be much greater significance than a positive result from an
oral ulcer, since reactivation of oral herpes is common during times of stress.
1. Direct Examination of Specimen
1. Electron Microscopy morphology / immune electron microscopy
2. Light microscopy histological appearance - e.g. inclusion bodies
3. Antigen detection immunofluorescence, ELISA etc.
4. Molecular techniques for the direct detection of viral genomes
2. Indirect Examination
1. Cell Culture - cytopathic effect, haemadsorption, confirmation by neutralization,
interference, immunofluorescence etc.
2. Eggs pocks on CAM - haemagglutination, inclusion bodies
3. Animals disease or death confirmation by neutralization
3. Serology
Detection of rising titres of antibody between acute and convalescent stages of infection, or
the detection of IgM in primary infection.
Classical Techniques
1. Complement fixation tests (CFT)
2. Haemagglutination inhibition tests
3. Immunofluorescence techniques (IF)
4. Neutralization tests
5. Single Radial Haemolysis

Newer Techniques
1. Radioimmunoassay (RIA)
2. Enzyme linked immunosorbent assay (EIA)
3. Particle agglutination
4. Western Blot (WB)
5. Recombinant immunoblot assay (RIBA), line immun
(Liatek) etc.

1. Direct Examination
Direct examination methods are often also called rapid diagnostic methods because they can
usually give a result either within the same or the next day. This is extremely useful in cases
when the clinical management of the patient depends greatly on the rapid availability of
laboratory results e.g. diagnosis of RSV infection in neonates, or severe CMV infections in
immunocompromised patients. However, it is important to realize that not all direct
examination methods are rapid, and conversely, virus isolation and serological methods may
sometimes give a rapid result. With the advent of effective antiviral chemotherapy, rapid
diagnostic methods are expected to play an increasingly important role in the diagnosis of
viral infections.

1.1. Antigen Detection

Examples of antigen detection include immunofluorescence testing of nasopharyngeal
aspirates for respiratory viruses e.g.. RSV, flu A, flu B, and adenoviruses, detection of
rotavirus antigen in faeces, the pp65 CMV antigenaemia test, the detection of HSV and VZV
in skin scrappings, and the detection of HBsAg in serum. (However, the latter is usually
considered as a serological test). The main advantage of these assays is that they are rapid to
perform with the result being available within a few hours. However, the technique is often
tedious and time consuming, the result difficult to read and interpret, and the sensitivity and
specificity poor. The quality of the specimen obtained is of utmost importance in order for the
test to work properly.

1.2. Electron Microscopy (EM)

Virus particles are detected and identified on the basis of morphology. A magnification of
around 50,000 is normally used. EM is now mainly used for the diagnosis of viral
gastroenteritis by detecting viruses in faeces e.g. rotavirus, adenovirus, astrovirus, calicivirus
and Norwalk-like viruses. Occasionally it may be used for the detection of viruses in vesicles
and other skin lesions, such as herpesviruses and papillomaviruses. The sensitivity and
specificity of EM may be enhanced by immune electron microscopy, whereby virus specific
antibody is used to agglutinate virus particles together and thus making them easier to
recognize, or to capture virus particles onto the EM grid. The main problem with EM is the
expense involved in purchasing and maintaining the facility. In addition, the sensitivity of
EM is often poor, with at least 105 to 106 virus particles per ml in the sample required for
visualisation. Therefore the observer must be highly skilled. With the availability of reliable
antigen detection and molecular methods for the detection of viruses associated with viral
gastroenteritis, EM is becoming less and less widely used.
1.3. Light Microscopy
Replicating virus often produce histological changes in infected cells. These changes may be
characteristic or non-specific. Viral inclusion bodies are basically collections of replicating
virus particles either in the nucleus or cytoplasm. Examples of inclusion bodies include the
negri bodies and cytomegalic inclusion bodies found in rabies and CMV infections
respectively. Although not sensitive or specific, histology nevertheless serves as a useful
adjunct in the diagnosis of certain viral infections.
1.4.Viral Genome Detection
Methods based on the detection of viral genome are also commonly known as molecular
methods. It is often said that molecular methods is the future direction of viral diagnosis.
However in practice, although the use of these methods is indeed increasing, the role played
by molecular methods in a routine diagnostic virus laboratory is still small compared to
conventional methods. It is certain though that the role of molecular methods will increase
rapidly in the near future.Classical molecular techniques such as dot-blot and Southern-blot
depend on the use of specific DNA/RNA probes for hybridization. The specificity of the
reaction depends on the conditions used for hybridization. These techniques may allow for

the quantification of DNA/RNA present in the specimen. However, it is often found that the
sensitivity of these techniques is not better than conventional viral diagnostic methods.
Newer molecular techniques such as the polymerase chain reaction (PCR), ligase chain
reaction (LCR), nucleic acid based amplification (NASBA), and branched DNA (bDNA)
depend on some form of amplification, either the target nucleic acid, or the signal itself.
bDNA is essentially a conventional hybridization technique with increased sensitivity.
However, it is not as sensitive as PCR and other amplification techniques. PCR is the only
amplification technique which is in common use. PCR is an extremely sensitive technique: it
is possible to achieve a sensitivity of down to 1 DNA molecule in a clinical specimen.
However, PCR has many problems, the chief among which is contamination, since only a
minute amount of contamination is needed to give a false positive result. In addition, because
PCR is so sensitive compared to other techniques, a positive PCR result is often very
difficult to interpret as it does not necessarily indicate the presence of disease. This problem
is particular great in the case of latent viruses such as CMV, since latent CMV genomes may
be amplified from the blood of healthy individuals. Despite all this, PCR is being increasingly
used for viral diagnosis, especially as the cost of the assay come down and the availability of
closed automated systems that could also perform quantification (Quantitative PCR) e.g. realtime PCR and Cobas Amplicor.systems. Other amplification techniques such as LCR and
NASBA are just as susceptible to contamination as PCR but that is ameliorated to a great
extent by the use of propriatory closed systems. It is unlikely though that other amplification
techniques will challenge the dominance of PCR since it is much easier to set up an house
PCR assay than other assays.
2. Virus Isolation
Cell cultures, eggs, and animals may be used for isolation. However eggs and animals are
difficult to handle and most viral diagnostic laboratories depend on cell culture only. There
are 3 types of cell cultures:
2.1. Types of cell cultures
1. Primary cells - e.g. Monkey Kidney. These are essentially normal cells obtained from
freshly killed adult animals. These cells can only be passaged once or twice.
2. Semi-continuous cells - e.g. Human embryonic kidney and skin fibroblasts. These are
cells taken from embryonic tissue, and may be passaged up to 50 times.
3. Continuous cells - e.g. HeLa, Vero, Hep2, LLC-MK2, BGM. These are immortalized
cells i.e. tumour cell lines and may be passaged indefinitely.
Primary cell culture are widely acknowledged as the best cell culture systems available since
they support the widest range of viruses. However, they are very expensive and it is often
difficult to obtain a reliable supply. Continuous cells are the most easy to handle but the range
of viruses supported is often limited.
2.2. Identification of growing virus
The presence of growing virus is usually detected by:

1. Cytopathic Effect (CPE) - may be specific or non-specific e.g. HSV and CMV
produces a specific CPE, whereas enteroviruses do not.
2. Haemadsorption - cells acquire the ability to stick to mammalian red blood cells.
Haemadsorption is mainly used for the detection of influenza and
parainfluenzaviruses.
Confirmation of the identity of the virus may be carried out using neutralization,
haemadsorption- inhibition, immunofluorescence, or molecular tests.
2.3 Problems with cell culture
The main problem with cell culture is the long period (up to 4 weeks) required for a result to
be available. Also, the sensitivity is often poor and depends on many factors, such as the
condition of the specimen, and the condition of the cell sheet. Cell cultures are also very
susceptible to bacterial contamination and toxic substances in the specimen. Lastly, many
viruses will not grow in cell culture at all e.g. Hepatitis B and C, Diarrhoeal viruses,
parvovirus etc.
2.4 Rapid Culture Techniques
Rapid culture techniques are available whereby viral antigens are detected 2 to 4 days after
inoculation. Examples of rapid culture techniques include shell vial cultures and the CMV
DEAFF test. In the CMV DEAFF test, the cell sheet is grown on individual cover slips in a
plastic bottle. After inoculation, the bottle then is spun at a low speed for one hour (to speed
up the adsorption of the virus) and then incubated for 2 to 4 days. The cover slip is then taken
out and examined for the presence of CMV early antigens by immunofluorescence.
The role of cell culture (both conventional and rapid techniques) in the diagnosis of viral
infections is being increasingly challenged by rapid diagnostic methods i.e. antigen detection
and molecular methods. Therefore, the role of cell culture is expected to decline in future and
is likely to be restricted to large central laboratories.
3. Serology
Serology forms the mainstay of viral diagnosis. This is what happens in a primary humoral
immune response to antigen. Following exposure, the first antibody to appear is IgM, which
is followed by a much higher titre of IgG. In cases of reinfection, the level of specific IgM
either remain the same or rises slightly. But IgG shoots up rapidly and far more earlier than in
a primary infection. Many different types of serological tests are available. With some assays
such as EIA and RIA, one can look specifically for IgM or IgG, whereas with other assays
such as CFT and HAI, one can only detect total antibody, which comprises mainly IgG. Some
of these tests are much more sensitive than others: EIAs and radioimmunoassays are the most
sensitive tests available, whereas CFT and HAI tests are not so sensitive. Newer techniques
such as EIAs offer better sensitivity, specificity and reproducibility than classical techniques
such as CFT and HAI. The sensitivity and specificity of the assays depend greatly on the
antigen used. Assays that use recombinant protein or synthetic peptide antigens tend to be
more specific than those using whole or disrupted virus particles.
3.1. Criteria for diagnosing Primary Infection

1. A significant rise in titre of IgG/total antibody between acute and convalescent

sera - however, a significant rise is very difficult to define and depends greatly on the
assay used. In the case of CFT and HAI, it is normally taken as a four-fold or greater
increase in titre. The main problem is that diagnosis is usually retrospective because
by the time the convalescent serum is taken, the patient had probably recovered.
2. Presence of IgM - EIA, RIA, and IF may be are used for the detection of IgM. This
offers a rapid means of diagnosis. However, there are many problems with IgM
assays, such as interference by rheumatoid factor, re-infection by the virus, and
unexplained persistence of IgM years after the primary infection.
3. Seroconversion - this is defined as changing from a previously antibody negative
state to a positive state e.g. seroconversion against HIV following a needle-stick
injury, or against rubella following contact with a known case.
4. A single high titre of IgG (or total antibody) - this is a very unreliable means of
serological diagnosis since the cut-off is very difficult to define.
3.2. Criteria for diagnosing re-infection/re-activation
It is often very difficult to differentiate re-infection/re-activation from a primary infection.
Under most circumstances, it is not important to differentiate between a primary infection
and re-infection. However, it is very important under certain situations, such as rubella
infection in the first trimester of pregnancy: primary infection is associated with a high risk
of fetal damage whereas re-infection is not. In general, a sharp large rise in antibody titres is
found in re-infection whereas IgM is usually low or absent in cases of re-infection/reactivation.
3.3. Limitations of serological diagnosis
How useful a serological result is depends on the individual virus.
1. For viruses such as rubella and hepatitis A, the onset of clinical symptoms coincide
with the development of antibodies. The detection of IgM or rising titres of IgG in the
serum of the patient would indicate active disease.
2. However, many viruses often produce clinical disease before the appearance of
antibodies such as respiratory and diarrhoeal viruses. So in this case, any serological
diagnosis would be retrospective and therefore will not be that useful.
3. There are also viruses which produce clinical disease months or years after
seroconversion e.g. HIV and rabies. In the case of these viruses, the mere presence of
antibody is sufficient to make a definitive diagnosis.
There are a number of problems associated with serology:1. long length of time required for diagnosis for paired acute and convalescent sera
2. mild local infections such as HSV genitalis may not produce a detectable humoral
immune response

3. Extensive antigenic cross-reactivity between related viruses e.g. HSV and VZV,
Japanese B encephalitis and Dengue, may lead to false positive results
4. immunocompromised patients often give a reduced or absent humoral immune
response.
5. Patients with infectious mononucleosis and those with connective tissue diseases such
as SLE may react non-specifically giving a false positive result
6. Patients given blood or blood products may give a false positive result due to the
transfer of antibody.

3.4. Antibody in the CSF

In a healthy person, there should be little or no antibodies in the CSF. Where there is a viral
meningitis or encephalitis, antibodies may be produced against the virus by lymphocytes in
the CSF. The finding of antibodies in the CSF is said to be significant when ratio between the
titre of antibody in the serum and that in the CSF is less than 100. But this does depend on an
intact blood-brain barrier. The problem is that in many cases of meningitis and encephalitis,
the blood-brain barrier is damaged, so that antibodies in the serum can actually leak across
into the CSF. This also happens where the lumbar puncture was traumatic in which case the
spinal fluid would be bloodstained. So really, one should really check the integrity of the
blood-brain barrier before making a definite diagnosis. One way to check the integrity of the
blood brain barrier is to use a surrogate antibody that most individuals would have, such as
measles virus, since most people would have been vaccinated. So the patient's serum and
CSF for measles antibody. If the blood-brain barrier is intact, there should be little or no
measles antibodies in the CSF.

Cultivation
of
Animal
Viruses
(i)
In
Animal
Cells
Suitable living mammals (such as sheep or calves or rabbits) are selected for cultivation of viruses. The
selected animals should be healthy and free from any communicable diseases. The specific virus is
introduced into the healthy animals. The site of administration varies according to the type of virus is
allowed to grow in the living animal. At the end of incubation period, the animals are slaughtered and
washed
thoroughly
and
viruses
are
obtained
from
them.
(ii)
Im
Chick-Embryo
The animal viruses can be successfully cultivated using chick-embryo technique. In this method fertile
hen eggs are selected. Eggs must not be more then 12 days old. To prepared the egg for virus
cultivation, the shell surface is first disinfected with iodine and penetrated with a small sterile drill. After
inoculation, the drill hole is sealed with gelatin and the egg is then incubated. Viruses may be able to
region. For convenience, the mayxoma virus grows well on the chorioallantoic membrane, whereas the
mumps virus prefers the allantoic cavity., The infection may produce a local tissue lesion known as pock,
whose appearance often is characteristic of the virus.

(iii)
In
Vitro
Culture
(Tissue
Culture
Technique)
More recently developed in vitro cultivation of animal viruses has eliminated the need to kill the
animals. This technique has become possible by the development of growth media for animal cells and
by the availability of antibiotics which prevent bacterial and fungal contaminations in cultures.
Cultivating animal viruses using tissue culture technique involves following three main step:

Monolayer Preparation. Live tissues of vital organs (e.g., heart or kidney) are taken and the
cells are separated from the tissue by digesting the intracellular cement substance with
dispersing agents such as trypsins or collagenase or ethylenediaminetetraacetic acid (EDTA).
The cell suspension is passed through screen filters so that the coarse particles are removed
from the separated cells. The cells are washed free of dispersing agents. The cells are
centrifuged if required and resuspended in nutrient medium contained in glass or plastic
vessels. The composition of medium and other conditions of incubation depends on the type of
cells used. Upon incubation the cells quickly settle and attach firmly to the bottom of the flask.
If undisturbed, these cells grow and spread to form monolayers.

Clonal
Cell
Line
Preparation
The monolayer cells are first removed and washed with saline solution devoid of calcium and
magnesium ions and then added to the dilute solution of EDTA (1 : 3000) to chelate intracellular
magnesium or calcium ions. After sometime, the loosened cells are shaken and resuspended in growth
medium in fresh culture vessels and incubated. The cells are cultivated under 5% CO2 condition. The
cultures of cell obtained so are called diploid cell strain. It is extremely difficult to distinguish primary
cell and the diploid cell strain. On repeated subculturing, each cell starts multiplying to form separate
colony. If each colony is removed and cultivated separately, it forms pure culture. These bunch of cells
from single cell is called clonal cell lines.
Infection with Virus
The clonal cell lines suspended in suitable media are infected with any desired virus which replicates
inside the multiplying cells. If the virus is virulent, they cause lysis of cells and virus particles are
released in the surrounding medium. These newly produced virus particles (virions) infect the adjacent
cells. As a result localized areas of cellular destruction and lysis (called plaques) often are formed.
Plaques may be detected if stained with dyes, such as neutral red or trypan blue, that can distinguish
living from dead cells. Viral growth does not always result in the lysis of cells to form a plaque. Animal
viruses, in particular, can cause microscopic or macroscopic degenerative changes or abnormalities in
host cells and in tissues called cytopathic effects, cytopathic effects may be lethal, but plaque
formation from cell lysis does not always occur.

Cell Culture

Preparing an aseptic environment

1. Hood regulations
(a) Close hood sash to proper position to maintain laminar air flow
(b) Avoid cluttering
2. Autoclaving
(a) Pipette tips (or can be purchases pre-autoclaved, DNAse/RNAse free)
3. (b) Glass 9 Pasteur pipettes
4. (c) 70% ethanol (Be sure to spray all surface areas)

All media, supplement and reagents must be sterile to prevent microbial growth in the
cell culture. Some reagents and supplements will require filter sterilization if they are not
provided sterile.

2. Preparation of cell growth medium

Before starting work check the information given with the cell line to identify what media type, additives and
recommendations should be used.
Most cell lines can be grown using DMEM culture media or RPMI culture media with 10% Foetal Bovine
Serum (FBS), 2 mM glutamine and antibiotics can be added if required (see table below).

Check which culture media and culture supplements the cell line you are using requires
before starting cultures. Culture media and supplements should be sterile. Purchase
sterile reagents when possible, only use unders aseptic conditions in a culture hood to
ensure they remain sterile.

General example using DMEM media

DMEM - Remove 50 ml from 500 ml bottle then add the other constituents.

450 ml

10% FBS

50 ml

2 mM glutamine

5 ml

100 U penicillin / 0.1 mg/ml streptomycin

5 ml

3. Creating the correct culturing environment

Most cell lines will grow on culture flasks without the need for special matrixes etc. However, some cells,
particularly primary cells, will require growth on special matrixes such as collagen to promote cell
attachment, differentiation or cell growth. We recommend reviewing the relevant literature for further
information on the cells you are culturing.
The following is an example for endothelial and epithelial cells:
For human cells, coat flasks with 1% gelatin. Alternatively, for other cell types such as BAEC, flasks can be
coated with 1% fibronectin.
1. Prepare 10mL of coating solution composed of 1% gelatin or 1% fibronectin by diluting with
distilled water, followed by filtration. This is efficient to coat about 5 flasks.
2. Pipette coating solution into flask. Rock back and forth to evenly distribute the bottom of the flask.
Let sit in an incubator for 15-30 minutes.
3. Completely remove coating solution by aspirating before seeding.

4. Checking cells
1. Cells should be checked microscopically daily to ensure they are healthy and growing as
expected.Attached cells should be mainly attached to the bottom of the flask, round and plump or
elongated in shape and refracting light around their membrane.Suspension cells should look
round and plump and refracting light around their membrane. Some suspension cells may
clump.Media should be pinky-orange in colour.
2. Discard cells if:
They are detaching in large numbers (attached lines) and/or look shrivelled and grainy/dark in color.
They are in quiesence (do not appear to be growing at all).

5. Sub-culturing
1. Split ratios can be used to ensure cells should be ready for an experiment on a particular day, or
just to keep the cell culture running for future use or as a backup. Suspension cell lines often
have a recommended subculture seeding density. Always check the guidelines for the cell line in

use. Some slow growing cells may not grow if a high split ratio is used. Some fast growing cells
may require a high split ratio to make sure they do not overgrow. Note that most cells must not be
split more than 1:10 as the seeding density will be too low for the cells to survive.
As a general guide, from a confluent flask of cells:
1:2 split should be 70-80% confluent and ready for an experiment in 1 to 2 days
1:5 split should be 70-80% confluent and ready for an experiment in 2 to 4 days
1:10 split should be 70-80% confluent and ready for sub-culturing or plating in 4 to 6 days.
Attached cell line split ratios are done on volume of flask surface area:

3 x 25 cm flasks
2

1 x 25 cm flask
2

Split 1:3

Or

1 x 75 cm flask
2

Suspension cell line split ratios are done on volume of culture cell suspension:

25ml cell suspension +

75 ml fresh media in 5 seperate new flasks

100 ml cell suspension

Split 1:4

Or

50 ml cell suspension +
150 ml fresh media in 2 larger flasks

2. If cells are less then 70-80% confluent but you wish to subculture them on (eg Friday before the
weekend) then they should be split at a lower split ratio in order to seed the cells at a high enough
density to survive e.g. use 1:2 or 1:5 split.

6. Splitting

1. When the cells are approximately 80% confluent (80% of surface of flask covered by cell
monolayer) they should still be in the log phase of growth and will require sub-culturing. (Do not
let cells become over confluent as they will start to die off and may not be recoverable).
2. To sub-culture, first warm the fresh culture medium at 37C water bath or incubator for at least 30
min. Then carry out one of the appropriate following procedures:
Make sure flasks are labelled with the cell line, passage number, split ratio, date, operator initials
and the vial number of the cells. Place flask(s) straight into 37C CO 2 incubator. Write down the
details of the sub-culturing in the culture record log sheet. There should be a separate log sheet
for each vial of cells resuscitated and in use.

7. Sub-culturing loosely attached cell lines requiring cell scraping for subculture
1. When ready, carefully pour off media from flask of the required cells into waste pot (containing
approximately 100 ml of 10% sodium hypochlorite) taking care not to increase contamination risk
with any drips.
2. Replace this immediately by carefully pouring an equal volume of pre-warmed fresh culture media
into the flask.
3. Using cell scraper, gently scrape the cells off the bottom of the flask into the media. Check all the
cells have come off by inspecting the base of the flask before moving on.
4. Take out required amount of cell suspension for required split ratio using a serological pipette.
e.g. for 1:2 split from 100 ml take 50 ml into a new flask
1:5 split from 100 ml take 20 ml into a new flask
1:10 split from 100 ml take 10 ml into a new flask
5. Top the new flasks up to required volume (taking into account split ratio) with pre-warmed fresh
culture media
eg. in 25 cm flask approx 5-10 ml
2

75 cm flask approx 10-30 ml

2

175 cm flask approx 40-150 ml

2

8. Sub-culturing attached cell lines requiring trypsin

Note not all cells will require trypsinization, and to some cells it can be toxic. It can also induce temporary
internalization of some membrane proteins, which should be taken into consideration when planning
experiments. Other methods such as gentle cell scraping, or using very mild detergent can often be used
as a substitute in these circumstances.
1. When ready, carefully pour off media from flask of the required cells into waste pot (containing
approximately 100 ml 10% sodium hypochlorite) taking care not to increase contamination risk
with any drips.

2. Using aseptic technique, pour/pipette enough sterile PBS into the flask to give cells a wash and
get rid of any FBS in the residual culture media. Tip flask gently a few times to rinse the cells and
carefully pour/pipette the PBS back out into waste pot.
This may be repeated another one or two times if necessary (some cell lines take a long time to
trypsinize and these will need more washes to get rid of any residual FBS to help trypsinization)
3. Using pipette, add enough trypsin EDTA to cover the cells at the bottom of the flask.
e.g. in 25 cm flask approx 1 ml
2

75 cm flask approx 5 ml
2

175 cm flask approx 10 ml

2

4. Roll flask gently to ensure trypsin contact with all cells. Place flask in 37C incubator. Different cell
lines require different trypsinization times. To avoid over-trypsinization which can severely
damage the cells, it is essential to check them every few minutes.
5. As soon as cells have detached (the flask may require a few gentle taps) add some culture media
to the flask (the FBS in this will inactivate the trypsin)
6. Using this cell suspension, pipette required volume of cells into new flasks at required split ratio.
These flasks should then be topped up with culture media to required volume
e.g. in 25 cm flask approx 5-10 ml
2

75 cm flask approx 10-30 ml

2

175 cm flask approx 40-150 ml

2

Leave cells overnight to recover and settle. Change media to get rid of any residual trypsin.

9. Sub-culturing of suspension cell lines

1. Check guidelines for the cell line for recommended split ratio or sub-culturing cell densities.
2. Take out required amount of cell suspension from the flask using pipette and place into new flask.
e.g. For 1:2 split from 100 ml of cell suspension take out 50 ml
For 1:5 split from 100 ml of cell suspension take out 20 ml
3. Add required amount of pre-warmed cell culture media to fresh flask.
e.g. For 1:2 split from 100 ml add 50 mls fresh media to 50 ml cell suspension
For 1:5 split from 100 ml add 80mls fresh media to 20 ml cell suspension

10. Changing media

1. If cells have been growing well for a few days but are not yet confluent (eg if they have been split
1:10) then they will require media changing to replenish nutrients and keep correct pH. If there
are a lot of cells in suspension (attached cell lines) or the media is staring to go orange rather
than pinky-orange then media change them as soon as possible.
2. To change media, warm up fresh culture media (section 5.1) at 37C in water bath or incubator for
at least 30 min. Carefully pour of the media from the flask into a waste pot containing some

disinfectant. Immediately replace the media with 100 ml of fresh pre-warmed culture media and
return to CO2 37C incubator.

11. Passage number

The passage number is the number of sub-cultures the cells have gone through. Passage number should
be recorded and not get too high. This is to prevent use of cells undergoing genetic drift and other
variations.