Centrifugation for laboratory use

1. Principle

A body is rotated in a circular movement at speed. This creates a force that drives the body away from the centre of the circular movement (Fig. 3.35). To calculate the relative centrifugal force (rcf) for an individual centrifuge, measure the radius (r) of the rotor arm (in cm) and the number of revolu- tions per minute (rpm) and use the formula below: rcf = 1.118 x 10-6 x r x (rpm)2

For example, if the radius is 25 cm and the rpm is 1300 rev/min, the rcf is about 50g.

Weighing: use of laboratory balances

Balances may be either electrically or manually operated. All types should be posi- tioned on a firm level bench away from vibrations, draughts and direct sunlight. 

The balance is used to weigh chemicals for production of reagents, and cleanliness is essential if accurate results are to be obtained: 

● Remove dust by blowing or using a soft brush. 

● Remove stains or chemicals using a soft brush. 

● Use a plastic weigh boat or filter-paper to weigh chemicals on the balance; never place chemicals directly on to the pan.

Important: If water has been used to clean the balance, make sure that it is thoroughly dry before weighing. Always set the balance to zero before weighing. Check the accuracy of the balance regularly according to the manufacturer’s recommen- dations. Handle loose weights with forceps. 

Chronic Myeloid Leukaemia

Chronic myeloid leukaemia (CML) is a clonal malignant myeloproliferative disorder believed to originate in a single abnormal haemopoietic stem cell. The progeny of this abnormal stem cell proliferate over months or years, so that, by the time the leukaemia is diagnosed, the bone marrow is grossly hypercellular and the number of leucocytes is greatly increased in the peripheral blood. Normal blood cell production is almost completely replaced by leukaemia cells, which, however, still function almost normally.

Gram-Negative Cocci

For a general discussion on Gram-negative bacteria, readers are referred to the last part of this chapter. With reference to Gram-negative cocci, members of the follow- ing three genera are considered important pathogens:

• Neisseria spp.

 N. gonorrhoeae

 N. meningitidis

• Moraxella spp.

 M. catarrhalis

• Haemophilus (It is a coccobacillus and sometimes listed among the Gram- negative bacilli)

 H. influenzae

Gram-Positive Bacteria with Rudimentary Filaments

Taxonomically, bacteria with rudimentary filaments rank between classic bacilli and the Actinomycetes, bacteria with branching filaments. They represent an important evolutionary link in microbiology. Among the bacteria with rudimentary filaments, Corynebacterium and Mycobacterium are two important pathogenic genera.

CORYNEBACTERIUM  DIPHTHERIAE

Members of the genus Corynebacterium are aerobic to facultative anaerobic, catalase positive, and do not produce spores. They are common constituents of resident microbiota on human skin and in the mouth and upper respiratory tract. More than 16 species are recognized. Of these, C. diphtheriae is the most important pathogenic species. Like several other causal agents of diseases, asymptomatic carriers of C. diphtheriae are not uncommon.

Gram-Positive Bacilli

Important pathogens in this group of bacteria are restricted to three genera that include aerobes as well as anaerobes:

• Clostridium spp. (anaerobic)

• Bacillus spp. (aerobic)

• Listeria spp. (aerobic to facultative anaerobic)

Most Gram-positive rods grow well on blood agar. However, selective media are available in some cases, for example, cycloserine–cefoxitin–fructose–egg yolk agar (CCFA) for C. difficile.

Polycythaemia, Essential Thrombocythaemia and Myelofibrosis

Polycythaemia vera (PV), essential thrombocythaemia (ET) and idiopathic myelofibrosis (IMF), known collectively as the classic myeloproliferative disorders (MPDs), are clonal disorders originating from a neoplastic haemopoietic stem cell. They are most common in middle or older age, and share several features, including a potential to transform into acute leukaemia and into each other. Treatment of PV and ET can greatly influence prognosis, hence the importance

The Hereditary Anaemias

Hereditary anaemias include disorders of the structure or synthesis of haemoglobin (Hb), deficiencies of enzymes that provide the red cell with energy or protect it from chemical damage and abnormalities of the proteins of the red cell’s membrane. Inherited diseases of haemoglobin (haemoglobinopathies) are by far the most important. The structure of human Hb changes during development (Fig.3.1). By the 12th week of gestation, embryonic haemoglobin is replaced by fetal haemoglobin (Hb F), which is slowly replaced after birth by the adult haemoglobins, Hb A and Hb A2. Each type of haemoglobin consists of two different pairs of peptide chains; Hb A has the structure α2β2 (namely, two α chains plus two β chains), Hb A2 has the structure α 2δ 2 and Hb F, α 2γ2.

Macrocytic Anaemias

Macrocytosis is a rise in the mean cell volume (MCV) of red cells above the normal range (in adults 80–95 fl). It is detected using a blood count, in which the MCV and other red cell indices are meas- ured. The MCV is lower in children than in adults, with a normal mean of 70 fl at 1 year of age, rising by about 1 fl each year until it reaches the adult volume at puberty.

The causes of macrocytosis fall into two groups: (i) deficiency of vitamin B12 (cobalamin) or folate (or rarely abnormalities of their metabolism), in which the bone marrow is megaloblastic (Box 2.1) and (ii) other causes (Box 2.2), in which the bone marrow is usually normoblastic. In this chapter, the two groups are considered sepa- rately. The steps to diagnose the cause of macrocytosis and subse- quently to manage it are then considered.

Iron Deficiency Anaemia

OVERVIEW

• Iron deficiency is the commonest cause of anaemia worldwide 

• Iron deficiency is usually easily diagnosed from the red cell indices

• A drop in haemoglobin is generally a late feature of iron deficiency

• The serum ferritin is a reliable means of confirming the diagnosis but may be falsely normal or even elevated as a reactive phenomenon as ferritin is an acute phase protein

• Iron deficiency is not a diagnosis in itself and in males and postmenopasual women blood loss from the gastrointestinal tract must be excluded

• Oral iron is preferred for iron replacement therapy, but occasionally parenteral iron is required

Iron deficiency is the commonest cause of anaemia worldwide and is frequently seen in general practice. Iron deficiency anaemia is caused by defective synthesis of haemoglobin, resulting in red cells that are smaller than normal (microcytic) and contain reduced amounts of haemoglobin (hypochromic).

Iron metabolism

Iron has a pivotal role in many metabolic processes, and the average adult contains 3–5 g of iron, of which two-thirds is in the oxygen- carrying molecule haemoglobin.

A normal Western diet provides about 15 mg of iron daily, of which 5–10% is absorbed (~ 1 mg), principally in the duodenum and upper jejunum, where the acidic conditions help the absorption of iron in the ferrous form. Absorption is helped by the presence of other re- ducing substances, such as hydrochloric acid and ascorbic acid. The body has the capacity to increase its iron absorption in the face of in- creased demand, for example, in pregnancy, lactation, growth spurts and iron deficiency (Box 1.1).

Once absorbed from the bowel, iron is transported across the mucosal cell to the blood, where it is carried by the protein transferrin to developing red cells in the bone marrow. Iron stores comprise fer- ritin, a labile and readily accessible source of iron and haemosiderin, an insoluble form found predominantly in macrophages.

About 1 mg of iron a day is shed from the body in urine, faeces, sweat

Haematological Findings in Health and Disease

The blood film and count in healthy individuals

The microscopic features of normal blood cells and the normal range for the blood count have been discussed in Chapter 1. In assessing what is ‘normal’ it is necessary to consider the gender, age and ethnic origin of the person being investigated.

Gender

Adult men have a higher normal range for RBC, Hb and PCV/Hct than adult women but women tend to have a somewhat higher WBC and platelet count (see Table 1.2).

Neonates, infants, and children

The blood counts of healthy neonates, infants and children differ greatly from those of healthy adults (see Table 1.3). Neonates have a higher Hb, MCV, WBC, neutrophil count and lymphocyte count than adults. Children in general have a higher lymphocyte count than adults. They tend to have a slightly lower Hb and MCV.

Pregnancy

Physiological variation in the blood count occurs during preg- nancy. The Hb falls, the MCV rises slightly and the WBC and neutrophil count rise. Immature cells (myelocytes and occasion-al promyelocytes) appear in the blood and there may be ‘toxic’granulation and Döhle bodies. 

Ethnic variation 

The blood counts of healthy Africans and Afro-Caribbeans (see Table 1.3) often show a lower white cell and neutrophil count than is usual in Caucasians (see Table 1.2). There is also a tendency to a lower platelet count, particularly in Africans. 

Abnormalities of red cells 

Polycythaemia 

Polycythaemia is an increase in the Hb. It is usually accompa- nied by an increase in the RBC and PCV/Hct. It can be caused by a true increase in the total volume of red cells in the circulation (true polycythaemia) or by a decrease in the total plasma volume (apparent or relative or pseudo-polycythaemia). It is not possible to distinguish true from apparent polycythaemia by a blood film or count. True polycythaemia is caused by overproduction of red cells. Normally red cell production is driven by erythropoietin production in response to a diminished oxygen supply to the kidney. Overproduction of red cells may be an erythropoietin- mediated physiological response to hypoxia or it may be caused by inappropriate secretion of erythropoietin or by mechanisms independent of erythropoietin. Some of the important causes of polycythaemia, classified according to mechanism, are shown in Table 4.1. 

Polycythaemia vera 

Polycythaemia vera, also referred to as polycythaemia rubra vera or primary proliferative polycythaemia, is a myeloproliferative disorder characterized by overproduction of red cells. In many patients there is also overproduction of white cells and platelets. Clinical features are facial plethora and sometimes a moderate degree of splenomegaly. The disease may be complicated by arterial thrombosis and peripheral ischaemia.

Table 4.1  Some  important causes of polycythaemia, classified according to mechanism.

Use of a microscope in laboratory

The microscope is an essential instrument for the diagnosis of disease. It is a preci- sion instrument and requires careful maintenance to prevent damage to the me- chanical and ocular parts and also to stop fungi from obscuring the lenses. 

1. Components of a microscope 

The various components of the microscope can be classified into four systems: 

— the support system 

— the magnification system 

— the illumination system 

— the adjustment system. 

Support system (Fig. 3.1) 

This consists of: 

— the foot (1) 

— the limb (2) 

— the revolving nosepiece (objective changer) (3) 

— the stage (4) 

— the mechanical stage (5), which gives a slow con- trolled movement to the object slide. 

Magnification system (Fig. 3.2) 

This consists of a system of lenses. The lenses of the microscope are mounted in two groups, one at each end of the long tube — the body tube. 

● The first group of lenses is at the bottom of the tube, just above the preparation under examination (the object), and is called the objective. 

● The second group of lenses is at the top of the tube and is called the eyepiece. 

Objectives

Magnification

The magnifying power of each objective is shown by a figure engraved on the sleeve of the lens (Fig. 3.3):

— the x 10 objective magnifies 10 times;

— the x 40 objective magnifies 40 times;

— the x 100 objective magnifies 100 times.

Fig. 3.1 Components of the support system of a microscope
1: foot; 2: limb; 3: revolving nosepiece; 4: stage;
5: mechanical stage.

Equipments for laboratory use

The following is a list of the apparatus needed to equip a laboratory capable of carrying out all the examinations described in this manual. Such a laboratory would usually be located in a small rural hospital (district level) which might have be- tween 60 and 100 beds.

1. Essential laboratory instruments

Microscopes

The laboratory should be equipped with two microscopes.

● One microscope is for use in haematology. It should have an inclined binocular tube, a mechanical stage, three objectives (x 10, x 40, x 100), two eyepieces (x 5,x 10), a condenser and an electric lamp that can be connected to the mains electricity supply or a battery.

● The second microscope is for use in other laboratory procedures (parasitology, urine analysis, bacteriology, etc.) and should have an inclined binocular tube and accessories as listed above.

At the health centre level one binocular microscope is sufficient.

Gram-Positive Cocci

BACTERIAL TAXONOMY (AN OVERVIEW)

The classic approach to the classification of bacteria is based on size and shape, later aided by reaction to Gram stain. As advances in microscope-making were made and simple biochemical tests became available, gradually the system became more refined. The science of taxonomy matured with the availability of the electron microscope and advances in molecular biology. Muller may have been the first biologist to attempt to classify bacteria in late 18th century, but his attempts were limited by lack of understanding of bacteria and crudeness of microscopes. Cohn, in late 19th century, made further advances, but serious attempts to classify bacteria were first made only in 20th century. Bergey’s Manual of Determinative Bacteriol- ogy was first published in 1923 and it instantly became the foundation of and the most authoritative source for bacterial taxonomy. Aided by inputs from the American Society for Microbiology, international societies for bacterial taxonomy and nomen- clature, and the International Journal of Systemic Bacteriology, Bergey’s manual has been greatly refined and its scope enlarged. Its 9th edition was published in 1994. Another remarkable contribution to bacterial taxonomy was made by the publication of Bergey’s Manual of Systematic Bacteriology, a five-volume set that examines bacterial taxonomy in greater detail. Students interested in bacterial taxonomy are encouraged to consult these highly authoritative sources.

Water for laboratory use

The medical laboratory needs an adequate water supply for its work. It requires: 

— clean water 

— distilled water 

— demineralized water (if possible) 

— buffered water (if possible). 

1. Clean water 

To check whether the water supply is clean, fill a bottle with water and let it stand for 3 hours. Examine the bottom of the bottle. If there is a deposit, the water needs to be filtered. 

Filtering

Using a porous unglazed porcelain or sintered glass filter

This type of filter can be attached to a tap. Alternatively, it can be kept immersed in a container of the water to be filtered (Fig.2.28).

Important: Filters of this type must be dismantled once a month and washed in boiling filtered water.

Fig. 2.28 Filtering water using  a porous unglazed porcelain or sintered glass  filter

Antiseptics and Disinfectants

The terms “antiseptic” and “disinfectant” are often confused and misused in micro- biology and medicine. Typically, “antiseptic” refers to an agent used to minimize, destroy, or remove microbial population on a living surface, such as the skin of a person who needs to be prepared for injection or a surgical procedure. A disinfectant, on the other hand, is a substance used to eliminate or minimize microbial presence on an inanimate surface, such as a work bench, glassware, or surgical instruments. It is noteworthy that both antiseptics and disinfectants can either be a microbicide or a microbistatic. In this chapter, we will treat the two entities together under the banner of control of microbial population. The microbial population in or on a surface, material, or product can be controlled, minimized, or eradicated either by physical means or by chemical means, some of which are summarized below.

Hyponatraemia: pathophysiology

Hyponatraemia is defined as a serum sodium concentration below the reference interval of 133–146 mmol/ L. It is the electrolyte abnormality most frequently encountered in clinical biochemistry. 

Development of hyponatraemia 

The serum concentration of sodium is simply a ratio, of sodium (in millimoles) to water (in litres), and hyponatraemia can arise either because of loss of sodium ions or retention of water.

- Loss of sodium. Sodium is the main extracellular cation and plays a critical role in the maintenance of blood volume and pressure, by osmotically regulating the passive movement of water. Thus when significant sodium depletion occurs, water is lost with it, giving rise to the characteristic clinical signs associated with ECF compartment depletion. Primary sodium depletion should always be actively considered if only to be excluded; failure to do so can have fatal consequences. 

- Water retention. Retention of water in the body compartments dilutes the constituents of the extracellular space including sodium, causing hyponatraemia. Water retention occurs much more frequently than sodium loss, and where there is no evidence of fluid loss from history or examination, water retention as the mechanism becomes a near certainty. 

Water retention

The causes of hyponatraemia due to water retention are shown in Figure 8.1

Fig 8.1 The causes of hyponatraemia.

Water and sodium balance

Body water and the electrolytes it con- tains are in a state of constant flux. We drink, we eat, we pass urine and we sweat; during all this it is important that we maintain a steady state. A motor car’s petrol tank might hold about 42 L, similar to the total body water content of the average 70 kg male. If 2 L were lost quickly from the tank it would hardly register on the fuel indicator. However, if we were to lose the same volume from our intravascular compart- ment we would be in serious trouble. We are vulnerable to changes in our fluid compartments, and a number of important homeostatic mechanisms exist to prevent or minimize these. Changes to the electrolyte concentration are also kept to a minimum. 

To survive, multicellular organisms must maintain their ECF volume. Humans deprived of fluids die after a few days from circulatory collapse as a result of the reduction in the total body water. Failure to maintain ECF volume, with the consequence of impaired blood circulation, rapidly leads to tissue death due to lack of oxygen and nutrients, and failure to remove waste products.

Water 

Normal water balance is illustrated in Figure 7.1.

Water intake largely depends on social habits and is very variable. Some people drink less than half a litre each day, and others may imbibe more than 5 L in 24 hours without harm. Thirst is rarely an overriding factor in determining intake in Western societies.

Fig 7.1 Normal water balance.

Fluid and electrolyte balance: Concepts and vocabulary

Fluid and electrolyte balance is central to the management of any patient who is seriously ill. Measurement of serum sodium, potassium, urea and creatinine, frequently with bicarbonate, is the most commonly requested biochemical profile and yields a great deal of information about a patient’s fluid and electrolyte status and renal function. A typical report is shown in Figure 6.1

Reference intervalsme

Below, in Tables 5.1 and 5.2, is a list of reference intervals for a selection of tests that are performed in clinical biochem- istry laboratories. Where available, refer- ence intervals have been adopted from those suggested by Pathology Harmony, which is a U K-based project aiming to harmonize reference intervals for common analytes across the U K. In the absence of this approach, individual laboratories should use reference inter- vals that are based on values obtained from subjects appropriately selected from local populations, but this is not always feasible. For some analytes, e.g. glucose and cholesterol, conversion factors are supplied to allow different units to be compared. The list is not intended to be comprehensive; it is merely provided for guidance in answering the cases and examples in this book. Please note that age- and/or sex-specific reference intervals are avail- able for a range of analytes including alkaline phosphatase, creatinine, and urate. The sex-specific ranges for urate are shown in Table 5.1. Glucose, insulin and triglyceride all rise postprandially and should, where possible, be meas- ured in the fasting state.

Point of care testing

The methods for measuring some bio- logical compounds in blood and urine have become so robust and simple to use that measurements can be made away from the laboratory – by the patient’s bedside, in the ward sideroom, at the GP’s surgery, at the Pharmacy or even in the home. Convenience and the desire to know results quickly, as well as expectation of commercial profit by the manufacturers of the tests, have been the major stimuli for these developments. Experience has shown that motivated individuals, e.g. diabetic patients, fre- quently perform the tests as well as highly qualified professionals.

The immediate availability of results at the point of care can enable the appropriate treatment to be instituted quickly and patients’ fears can be allayed. However, it is important to ensure that the limitations of any test and the sig- nificance of the results are appreciated by the tester to avoid inappropriate intervention or unnecessary anxiety.

Outside the laboratory

Table 4.1 shows what can be commonly measured in a blood sample outside the normal laboratory setting. The most common blood test outside the labora- tory is the determination of glucose concentration, in a finger stab sample, at home or in the clinic. Diabetic patients who need to monitor their blood glucose on a regular basis can do so at home or at work using one of many commercially available pocket-sized instruments.

Figure 4.1 shows a portable bench analyser. These analysers may be used

Fig 4.1 A portable bench analyser.

Macroscopic and Microscopic Characteristics of Ocular Fungal Isolates (Part 3)

LASIODIPLODIA THEOBROMAE 

Ecology

Worldwide, well known plant pathogen.

Pathogenicity

Lasiodiplodia theobromae is a widespread saprophyte and wound-parasite on a considerable range of hosts in the tropics. It is an important parasite of bananas in storage, causing several forms of fruit-rot. Lasiodiplodia theobromae, a rare cause of mycotic keratitis and endophthalmitis.

Macroscopic Morphology

• Colonies on potato dextrose agar greyish sepia to mouse grey to black, fluffy with abundant aerial mycelium (Fig. 8.38).

• Colonies reverse fuscous black to black.

Microscopic Morphology (Fig. 8.39)

• Conidia initially unicellular, hyaline, granulose, subovoid to ellipsoide-oblong, thick- walled, base truncate; mature conidia 1-septate, cinnamon to fawn, often longitudinally striate, (18-) 20-30 × 10-15 μm.

• The pycnospores are elliptical, at first unicellular and hyaline, becoming brown and 1-septate, sometimes with longitudinal striations, 20-30 × 10-18 μm.

Fig. 8.38: Lasiodiplodia theobromae growth on potato dextrose agar (10 days)

Macroscopic and Microscopic Characteristics of Ocular Fungal Isolates (Part 2)

CANDIDA 

Ecology 

Candida is a genus of yeasts. Clinically, the most significant member of the genus is Candida albicans, which can cause numerous infections (called candidiasis or thrush) in humans and other animals, especially in immunocompromised patients. (Ryan KJ et al, 2004). Various Candida species are members of gut flora in animals, including C. albicans. 

Pathogenicity 

C. albicans is the most common cause of both superficial and systemic candidosis. It is also often present as part of the commensal flora of the mouth, vaginal mucosa and gastrointestinal tract, and may be isolated from these sites in the absence of disease. 

Most episodes of yeast infections in corneal ulcers and other ocular infections are due to various Candida species, predominantly Candida albicans and usually occur in the presence of systemic illness (diabetes mellitus or immunocompromise) or ocular diseases like lid abnormalities or dry eyes) or endogenous endophthalmitis and in patients receiving prolonged topical medications or topical corticosteroids. 

Macroscopic Morphology 

• Candida albicans grows well on potato dextrose agar (Fig. 8.19), Sabouraud’s agar and most routinely used bacteriological media. 

• Convex, entire margin, non-mucoid, smooth texture of cream colored pasty colonies usually appear after 24 - 48 hours incubation at 35-37°C. 

• The colonies have a distinctive yeast smell. 

Microscopic Morphology 

• The round—oval shaped budding cells can be easily seen by direct microscopy in stained or unstained preparations. 

• Candida albicans produces true germ tubes when incubated in serum for 2-3 hours at 37°C (Fig. 8.20). 

• These are parallel-sided tubes which are formed at right angles to the parent cell and are at least twice as long as the parent cell before cross walls are formed.

Fig. 8.19: Cream colored pasty colonies on potato dextrose agar (2 days)

Macroscopic and Microscopic Characteristics of Ocular Fungal Isolates (Part 1)

ACREMONIUM 

= Cephalosporium (Corda, 1839). 

Pathogenicity 

Acremonium 

Acremonium has been reported as a rare cause of keratitis and endophthalmitis. 

In the literature 17 cases of Acremonium keratitis have been reported between 1965 and  1991. Rosa et al (1994) found 3.2% of Acremonium keratitis in their series and Rodriguez-Areset al reported this as an extremely rare cause of suppurative corneal infection. 

Ecology 

Cosmopolitan, isolated from soil and plant debris. 

MACROSCOPIC MORPHOLOGY 

• The growth rate of Acremonium colonies is moderately rapid, maturing within 5 days. The diameter of the colony is 1-3 cm following incubation at 25°C for 7 days on potato glucose agar. 

• The texture of the colony is compact, flat or folded, and occasionally raised in the center. It is glabrous, velvety, and membrane-like at the beginning. Powdery texture may also be observed. By aging, the surface of the colony may become cottony due to the overgrowth of loose hyphae. 

• The color of the colony is white, pale grey or pale pink on the surface. The reverse side is either uncolored or a pink to rose-colored pigment production is observed (Fig. 8.1). 

MICROSCOPIC MORPHOLOGY

• Acremonium spp. possesses hyaline, septate hyphae which are typically very fine and narrow. Vegetative hyphae often form hyphal ropes. Unbranched, solitary, erect phialides are formed directly on the hyphal tips, the hyphal ropes, or both. The phialides are separated from hyphae by a septum and taper towards their apices. At the apices of the phialides is the hyaline conidia 2-3 × 4-8 μm in size. They usually appear in clusters, in balls or rarely as fragile chains.

• The conidia are bound by a gelatinous material. They may be single or multicellular, fusiform with a slight curve or resemble a shallow crescent. These structural properties of conidia vary depending on the species.

• Acremonium falciforme usually produces crescentic, nonseptate conidia. Sometimes, 2 or 3 celled conidia may also be observed. Acremonium kiliense, on the other hand, has short straight conidia and the conidia of Acremonium recifei are usually crescentic and nonseptate (Figs 8.1 and 8.2).

Fig. 8.1: Acremonium species on potato dextrose agar, 25°C, 7 days

Identification of Common Ocular Fungal Isolates

TECHNIQUES USED FOR MOULD IDENTIFICATION 

Colony Characteristics

• To evaluate colony characteristics of filamentous fungi, it is necessary to subculture the fungus to the same media that the original colony descriptions are based upon.

• Visual examination of the colony will rapidly reveal important data concerning color, texture, diffusible pigments, exudates, growth zones, aerial and submerged hyphae, growth rate, colony topography, and macroscopic structures such as ascocarps, pycnidia, sclerotia, sporodochia, and synnemata.

Lactophenol Mounts

Prepare a mount of the fungus in lactophenol within the biological safety cabinet.

Incubation and Processing of Cultures in the Laboratory

Once the specimen has been inoculated on the media by the ophthalmologist, and received in the microbiology laboratory, the plates and tubes or broths must be placed in the appropriate atmosphere and temperature for isolation. Occasionally it is necessary to process specimens that are not set up at the bedside but are submitted to the microbiology laboratory on swabs or in syringes. Given below is the description for processing of specimens for fungal cultures.

INCUBATION REQUIREMENTS 

Conditions (Fig. 6.1) 

Temperature: 25°C to 30°C 

Atmosphere: Sabouraud’s dextrose agar or potato dextrose agar for fungi is incubated in a regular non - CO2 incubator.

Fig. 6.1: Mycological incubator: Maintains temperature at 26°C

Recommendations for Isolation of Fungi from Ocular Specimens

RECOMMENDED MEDIA FOR FUNGI ISOLATION

Clinical specimens are processed promptly and plated to isolation media as a means to recover fungi that may be causing disease. Media and incubation temperatures are selected to allow for the growth of pathogenic and opportunistic yeasts and fungi.

Isolation Media

The following Table is intended as a guideline for media required for the primary isolation of common isolates in ocular infections. Because some of these sites may be sterile sources and others are nonsterile sources, isolates considered pathogenic differ by site.

Staining Procedure for Rapid Identification of Fungi

Scrapings from involved ocular site complemented with the appropriate staining method can offer the ophthalmologist circumstantial as well as definitive information concerning the identity of the invading organism.

STAINING METHODS 

Light Microscopy

• KOH

• Gram’s stain

• Giemsa stain

• Grocott-Gomori Methenamine Silver (GMS) stain.

Fluorescent Microscopy

• Calcofluor white staining

• KOH-Calcofluor white procedure.

KOH mount

Principle: Potassium hydroxide is used as a mounting fluid for visualization of fungal filaments as it helps in the clearance or lyses of all surrounding tissues. In addition, Acanthamoeba cysts and Nocardia filaments can also be visualized.

Preparation of 10% KOH

• Approximately 1 gm (8 pellets) of KOH is weighed

• It is dissolved in 10 ml of distilled water

• One drop of 10% glycerol is added

• Fresh stock should be prepared every week

Can be kept at room temperature in a dropper bottle like a penicillin bottle (Fig. 4.1).

Fig. 4.1: KOH pellets and preparation of 10% KOH

Ocular specimen collection

Standard procedures followed for ocular specimen collection, culture isolation and identification of the organisms are described below.

SPECIMEN COLLECTION KIT TO BE KEPT IN THE OPHTHALMOLOGIST OFFICE

The following are the items that must be available for collecting the corneal specimens.

• Culture plates (blood agar, potato dextrose agar)

• Sterile Kimura’s spatula or scraper

• Topical anesthetic agents

• Bunsen burner

• Clean glass slides

• Clean cover slips.

Corneal Scraping

Corneal scraping is performed under aseptic conditions by an ophthalmologist using a sterile Kimura spatula. The procedure is performed under magnification of a slit lamp or binocular loupe following instillation of topical anesthetic agents such as 0.5% proparacaine or 4% lignocaine.

Material obtained from scraping the leading edge and the base of each ulcer specimen are inoculated directly onto sheep blood agar, potato dextrose agar (PDA) and Brain Heart Infusion broth (BHI) without gentamycin sulphate. The material from the corneal scraping is also smeared and labeled onto slides in a thin, even manner to prepare a 10% KOH wet mount and Gram staining. In cases of suspected actinomycetes keratitis Kinyoun’s metho of acid fast staining is performed (Fig. 3.1). Corneal scrapings collected by an opthalmologist by using an ophthalmic microscope in shown in Figures 3.4A to F.

In some of the patients, the ulcer may be predominately in the deeper stromal lesion, with inflammatory outpourings in the anterior chamber. In such a case, a paracentesis is made using a 26 gauge needle mounted on a 2cc plastic disposable tuberculin syringe (Figs 3.2A and B).

Fig. 3.1: Materials used for collecting specimens from corneal ulcers

Antibiotics and Other Chemotherapeutic Agents

Technically, the antibiotic era began with the discovery of penicillin by Sir Alexan- der Fleming in 1929. However, its development could occur only during World War II. By that time, an energetic soil scientist, Dr. Selman Waksman, had established a school of soil microbiology in New Jersey’s Rutgers University. Focusing on soil- borne aerobic actinomycetes, his group started a systematic program that lead to the discovery of streptomycin, an antibiotic credited with saving lives of millions of tuberculosis patients all over the world. Since then, his group at Rutgers as well as his students in various educational and industrial research laboratories went on to discover thousands of antibiotics, which include almost all the powerful drugs, such as tetracycline, erythromycin, chloramphenicol, amphotericin B, and vancomycin. Traditionally, the term “antibiotics” has been used for the antimicrobial agents derived from microorganisms. Since a number of antibiotics currently in use are actually synthetic, the term antibiotics has become synonymous with antimicrobial agents used for the treatment of infectious diseases.

Host-Microbe Interactions

The human body reacts in many different ways to microorganisms. These interac- tions can be summarized in the following categories:

RESIDENT MICROBIOTA

All surfaces of the human body, including the skin as well as the mucous membranes that surround the inner parts of the mouth, nostrils, genitals, and gastrointestinal tract, are inhabited by a distinct set of microbial communities, which are specifically adapted to the local physical and chemical environment. Such normal microorgan- isms, called resident microbiota, perform extremely important roles.

1. Resident microbiota engage all available binding sites on the host cell sur- faces, thus invaders have a diminished possibility of attaching to the host cell surfaces.

2. Many microbes secrete vitamins that are absorbed by the host and serve important nutritional needs.

TRANSMISSION OF INFECTIOUS DISEASE (MODE OF DISSEMINATION)

Airborne (Inhalation of Bioaerosols) 

A bioaerosol contains bacteria in its center, surrounded by air and a small amount of liquid, generally saliva. Bioaerosols may be produced due to sneezing, coughing, or talking. Depending on the force of sneezing or coughing, the bioaerosol-borne microorganism can travel up to several meters in air. Almost all respiratory tract infections are airborne; some can also pass from person to person through the inhala- tion of bioaerosols. Some of the examples of airborne infections include tuberculo- sis, strep throat, diphtheria, pertussis, legionellosis, influenza, and chicken pox, and a wide range of mycotic diseases such as aspergillosis, zygomycosis, cryptococcosis, histoplasmosis, and coccidioidomycosis.

Assessing White Cells and Platelets

White cells and platelets may be increased or decreased in number. They may also show morphological abnormalities, either inherited or acquired. Assessing whether the numbers of individual types of white cell are increased or decreased requires a differential count. However, the differential count is of little importance in itself and should only be used to calculate the absolute numbers of each cell type. The absolute counts are then compared with those expected in healthy people of the same age, sex and ethnic group. The terms used in describing numerical abnormalities in white cells and platelets are defined in Table 3.1.

Table 3.1  Terminology used  for abnormalities of white cell  and  platelet numbers

Assessing Red Cells

Red cells should be assessed as to their:

• number

• size

• shape

• degree of haemoglobinization

• distribution in the blood film.

Their appearance should be described using a standard terminology.

Assessing red cell number and distribution (anaemia, polycythaemia, rouleaux formation, red cell agglutination)

The thickness of a film of blood spread on a glass slide is deter- mined by how thick the blood is, i.e. by its viscosity. This in turn is determined by the Hb. In a normal blood film it is possible to find a part of the film which is ideal for microscopic examination where the red cells are touching but not overlapping. If the Hb is abnormally high (a condition referred to as polycythaemia) the blood has a high viscosity and the film of blood on the glass slide is thick. The red cells therefore appear packed together through- out the whole length of the film. The term ‘packed film’ is often used. Conversely, when a patient is anaemic the viscosity of the blood is low, the blood film is very thin and there are large spaces between the red cells. The effect of Hb on the blood film can be seen by comparing Figs 2.1, 2.2 and 2.3.

Usually red cells are distributed fairly regularly on the slide. Two abnormalities of distribution may occur. When there is an

Fig. 2.1  Anaemia (caused by iron  deficiency).