DIPLOMA PAPER IIIDIAGNOSTIC MICROBIOLOGY –II

(VIROLOGY, MYCOLOGY AND PARASITOLOGY)

UNIT –ILaboratory methods in basic Mycology –Collection and transport of clinical specimens–Direct Microscopic examination, culture media and incubation, Serological tests forfungi – Antifungal susceptibility testingUNIT –IILaboratory methods for parasitic infections – Diagnostic techniques for faecal,gastrointestinal and urino-genital specimen.UNIT –IIIIdentification of Intestinal Protozoa –Amoeba, Blood protozoa – Malaria, IntestinalHelminthes and Blood Helminthes.UNIT –IVLaboratory methods in basic virology- detection of viral antigen (fluorescent antibodyand solid phase immunoassays). Viral Serology- Special consideration- Hepatitis andAIDS.UNIT –VViral culture- Media and cells used –Specimen processing – isolation and identificationof viruses.References1. Diagnostic Microbiology, Bailey and Scott’s., 1990. Eighth edition. The MosbyCompany.2. Medical laboratory techniques, Abdul Khader, 2003, First edition. FrontlinePublications, Hyderabad.3. Virology, Sawant, K.C., 2005, First edition, Dominant Publishers and distributors,Delhi.4. Medical Parasitology, Rajesh Karyrkarte, Ajit Damla, 2004. Books and alliedpublishers Ltd. Kolkata.5. Textbook of Medical Parasitology, Subash O. Barija , 1996. First edition. All IndiaPublishers and Distributors Regd. 920 Poonamallee High Road, Chennai.6. Rajesh Karyakarte and Ajith Damle (2005)Medical Parasitology, ooks and

Allied(P)Ltd.

UNIT-I

PART-A

1.Comment on CSF

Collection and 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.

2.Define 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.

3.Comment on 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.

      

4.Comment on 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.

5.Comment on Rectal swab.

     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.

       

PART-B

1.Briefly explain the 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.

2.Briefly explain the collection and processing of  Blood.

Whole blood is required for bacteriological examination. Serum separated from blood is used for serological techniques. Skin antisepsis is extremely important at the time of collection of the sample. Tincture of iodine (1-2%), povidone iodine (10%) and chlorhexidine (0.5% in 70% alcohol) are ideal agents. However, some individuals may be hypersensitive to iodine present in some of these. While collecting blood for culture,the following points must be remembered:

     Collect blood during the early stages of disease since the number of bacteria in blood is higher in the acute and early stages of disease.

     Collect blood during paroxysm of fever since the number of bacteria is higher at high temperatures in patients with fever.

     In the absence of antibiotic administration, 99% culture positivity can be seen with three blood cultures.

     Small children usually have higher number of bacteria in their blood as compared to adults and hence less quantity of blood needs to be collected from them (Table 2).

Table 2: Volume of blood to be collected at different ages

Age Volume in 2 bottles< 2 years 2 ml2-5 years 8 ml

6-10 years 12 ml>10 years 20 ml

 

3.Briefly explain the collection and processing of 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 NormalClear with Tyndall effect(sparkling appearance against incident light)

High protein content

Clear yellowish Old haemolysisClear red Fresh haemolysisTurbid blood-stained HaemorrhageTurbid white High cell or protein contentTurbid clot (after overnight storage) Fibrin clots

  4.Briefly explain the collection and processing of Sputum samples in detail

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.

PART-C

 1.Briefly explain the collection and processing of urine and stool sample in detail

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.

 2.Briefly explain the collection and processing of Throat swab, bone marrow and rectal swab sample in detail.

      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.

        3.Explain the Transportation of specimens in detail.

     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 laboratory’s 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.

UNIT -2

1.Define virology.

The study of virus is called as virology.

2. Define Bacteriology.

The study of virus is called as virology.

3. Define mycology.

The study of fungi is called as mycology

Part-B

1.Briefly explain the identification of amoebae in detail.

      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 Page▓s 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. Page▓s 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.

2. Briefly explain the Locomotive form of amoebae in detail.

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 two-three 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.

3. Briefly explain the floating form of amoebae in detail.

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.

Part-C

1.Explain the Nuclear structure and crystals of amoeba in detail

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.

UNIT-III

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 non-viable 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/µL 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 child’s 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 Newer Techniques1. Complement fixation tests (CFT) 1. Radioimmunoassay (RIA)2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA)3. Immunofluorescence techniques (IF) 3. Particle agglutination4. Neutralization tests 4. Western Blot (WB)5. Single Radial Haemolysis 5. Recombinant immunoblot assay (RIBA), line immunoassay

(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. real-time 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/re-activation.

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-EmbryoThe 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 PreparationThe 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 VirusThe 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 ml2  mM glutamine 5 ml100 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 days1:5 split should be 70-80% confluent and ready for an experiment in 2 to 4 days1:10 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 cm2 flasks

1 x 25 cm2 flask Split 1:3 Or

1 x 75 cm2 flask

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 37°C 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 37°C CO2 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 sub-culture

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 flask1:5 split from 100 ml take 20 ml into a new flask1:10 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 mediaeg. in 25 cm2 flask approx 5-10 ml75 cm2 flask approx 10-30 ml175 cm2 flask approx 40-150 ml

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 cm2 flask approx 1 ml75 cm2 flask approx 5 ml175 cm2 flask approx 10 ml

4. Roll flask gently to ensure trypsin contact with all cells. Place flask in 37°C 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 volumee.g. in 25 cm2 flask approx 5-10 ml75 cm2 flask approx 10-30 ml175 cm2 flask approx 40-150 mlLeave 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 mlFor 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 suspensionFor 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 37°C 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 37°C 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.