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User talk:Tlsramek

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Talk:GAMSAT Guide/Experiences

GAMSAT UK 2008

The exam will be 21 September 2007 with registration closing mid-summer 2007. This is a big change in the dates so please spread the message far and wide

Caroline St George's

license: Creative Commons - Attribution Share-Alike - https://creativecommons.org/licenses/by-sa/4.0/

title: Talk:GAMSAT Guide/Experiences


text:
Chinese (Mandarin)/Traditional/Lesson 8

=Lesson 8= = 我叫王明. My name is Wang Ming. =

Dialogues
You can check out the translation here

title: Chinese (Mandarin)/Traditional/Lesson 8


text:
Basic Physics of Nuclear Medicine/X-Ray CT in Nuclear Medicine

The need for attenuation compensation in SPECT imaging has been understood for many years but it has only been in recent times that effective techniques have been applied in commercial systems. Gamma rays get attenuated by the medium through which they pass, as we've seen earlier, via photoelectric absorption and Compton scattering. The projections measured in SPECT imaging therefore contain impressions of this attenuation which reduce their fidelity for tomographic reconstruction, as illustrated in the following figure:

Patient specific attenuation maps can be used to correct the measured projections. These can be produced using the gamma camera to generate a transmission scan of the patient using a radioisotope source – see the following figure for an example:

The transmission scan contains the attenuation information for the volume of interest within the patient and a map of this attenuation pattern is subsequently applied to each SPECT projection prior to filtered back projection. A more powerful approach is to use an X-ray CT scanner to generate the attenuation maps – see the following figure. Numerous hybrid scanners have been produced as a result which combine nuclear medicine with CT imaging, as in SPECT/CT and PET/CT – see the following figure for an example:

An additional benefit of these developments is the simultaneous capability, with sufficient computing power, of blending the nuclear medicine and CT images so as to generate physiological images colocalized with images of patient anatomy – as shown in the following figure – and hence improve the diagnostic utility of nuclear medicine procedures.

Hybrid scanners generally have a single patient couch with the gamma camera gantry moulded to the front of a CT unit so that the patient can be transported into the X-ray beam before or after the SPECT acquisition. Some CT units are of a low resolution design and generate crude, single slice CT images which are sufficiently accurate for SPECT attenuation correction but are of insufficient quality on their own for diagnostic purposes – see the example below. There are also higher resolution CT units in use for hybrid scanning with capabilities of acquiring, for instance, 16-slice helical scans which can compete directly with the diagnostic capabilities of dedicated CT scanners.

This chapter provides an overview of the physical aspects of X-ray CT scanning from a nuclear medicine perspective. We begin with various scanner designs and will explore a number of computational techniques relevant to this form of imaging. The intention is to provide a foundation for understanding the application of X-ray CT in nuclear medicine.

Conventional Radiography
The attenuation of an X-ray beam in the body is used in conventional radiography to project a shadow onto an image receptor (as shown in the figure below). These shadowgraphs record a two-dimensional representation of a three-dimensional object. Small lesions are therefore not readily identified because of overlapping and underlying anatomy, image distortion occurs because of unequal magnification effects and low contrast masses are poorly delineated since scatter contributes substantially to the image data.

We can consider that there is a degeneracy introduced into such images, i.e. two anatomically quite different objects may produce the same film density because the effective attenuation they each produce is identical, as illustrated in the following figure:

Panel (a) shows a pencil beam of X-radiation of incident intensity, Io, attenuated by the body, represented here for simplicity by four volume elements each of thickness, Δx, and of different linear attenuation coefficients, μ1 thru μ4. The transmitted intensity, when no scattered radiation is detected, is given by I, which is dependent on the sum of these linear attenuation coefficients, according to the exponential attenuation equation. Panel (b) illustrates that two adjacent columns of volume elements can generate identical values of the transmitted intensity, I, even though the two columns contain volume elements with different linear attenuation coefficients. In other words, what is detected in the integrated contribution of the linear attenuation coefficients without any information about the contribution from any individual volume element.

One way to improve this situation is to use X-Ray Computed Tomography (CT). Note that the image reconstruction algorithms developed for X-ray CT can also be applied to gamma camera images (as in SPECT) to overcome limitations of planar nuclear imaging and to images generated using positron-emitting radioisotopes (as in PET).

Back Projection
We have considered this method of computerized image reconstruction in a previous chapter in the context of SPECT. Our intention here is to consider this reconstruction method in the context of X-Ray CT.

The following figure is a representation of how back projection works. The basic premise is that any attenuation of the X-ray beam is assumed to have occurred uniformly along the entire ray path.

Thus, the result of the back projection of the first profile, P1 is to put the values 7 and 9 in both elements of the first and second rows, respectively. The second profile, P2 adds a 4 to the top right element, 1 to the bottom left element and 11 to the other two elements when back projected. The other profiles are treated in similar fashion. Following subtraction of an offset (16 in this case) and renormalisation (division by 3 in this case) of the data set, the final image is obtained. You should confirm for yourself that this final matrix satisfies all projections.

The principle is demonstrated in more detail in the following figure:

A single profile is back projected to give a dark stripe across the entire image plane [panel (a)]. As we scan the phantom from many directions and back project the ray profiles onto the image plane [panel (b)], an image of the radio-dense dot, albeit a poor one, begins to resolve [panel (c)]. As the number of projections increases, the quality improves but some blurring will always remain in the image.

This blurring can be removed using what is known as Filtered Back Projection.

Filtered Back Projection
The blurring inherent in the simple back projection process can be suppressed mathematically using filtering techniques and the overall reconstruction process is then called Filtered Back Projection. In this technique (see the following figure), the attenuation profile is first filtered to counteract the effect of sudden density changes which cause the blurring in simple back projection. Note that the graphical representation in the figure is very crude as the filtered profile may be a quite complicated function of distance as illustrated in panel (c). The filter is also referred to as a convolution filter, or convolution kernel. After completion of the filtration process, the Inverse Fourier Transform is applied to the data for each profile before the back projection process is undertaken. Notice that the reconstructed image [panel (d)] is free of the star artefact generated in unfiltered back projection.

Most CT scanners offer a choice of filters which may be selected by the operator to enhance either soft tissue features or bone detail in images. Indeed, the image can be post-processed using a different filter after the scan has been completed if so desired. Images can be generated to enhance bone detail on the one hand and to observe subtle low contrast masses on the other, without the need to re-scan the patient. Thus, the filter choice will have a major impact on image quality. Two common filters used in X-ray CT are those due to Ramachandran & Lakshminarayanan (Ram-Lak) and to Shepp & Logan. In SPECT, Butterworth, Hanning and Metz are used, as you will remember. The ramp filter on its own compensates for artefacts introduced by the simple back projection process but does not compensate for the increasing noise content of the data with increasing spatial frequency. The Ram-Lak is a ramp filter which leaves all frequencies unchanged except those above a cut-off, which are eliminated. Most of the other filters suppress higher frequencies to varying degrees. Sometimes these filters are referred to as algorithms. The term kernel is also used. Images of an axial tomogram reconstructed with a soft tissue and with a bone algorithm are shown below as examples:

There's an excellent online CT reconstruction simulator available via the Biomedical Imaging Group at the EPFL. As an exercise, you might like to explore your understanding of filtered back projection by varying the type of filter used prior to reconstruction. You might also like to explore the influence of the number of angles used for profile acquisition on the quality of the reconstructed image.

CT Scanning Geometries

Most CT scanner designs use some form of mechanical gantry which allows movement of the X-ray tube and the detector around the patient. The X-ray beam was collimated to a pencil beam in early designs and consists of a wide fan beam in modern systems. The thickness of the slice, typically 1 to 10 mm, is generally defined by pre-patient collimation using motor driven adjustable wedges external to the X-ray tube. Even with relatively large fan beams the volume of tissue irradiated is still small quite compared with projection radiography and scatter is a less serious problem. The detectors have collimators placed in front of them to minimise the impact of out of slice scatter. To minimise the effects of beam hardening CT scanners use a heavily filtered beam (the Siemens Somatom Plus uses 2.7 mm Al plus 0.2 mm Cu, for instance) operating at 120 – 140 kVp. Note that even with the best radiographic geometry, such as in the third generation scanners, object magnification is substantial and the finite size of the focal spot, typically 1 mm, may well limit the achievable spatial resolution.

The first generation of CT scanner used what is referred to as Translate-Rotate geometry. The original EMI Mk 1 scanner (see the figure on the right), for instance, used a pencil X-ray beam and a single detector (a NaI:Tl scintillator coupled to a photomultiplier tube). During the translational motion of the gantry, the transmitted X-ray beam was sampled 160 times to produce a single profile. A rotation through 1 degree with the X-ray beam off then followed before a new profile was obtained. This procedure was completed until the patient's head was scanned from 180 different angles.

The whole procedure took about 5 minutes and depended on the patient remaining completely still. The patient's head was usually clamped to minimise patient motion. In addition, their head was surrounded by a water bag which helped to overcome problems associated with afterglow in the NaI(Tl) scintillator and the very substantial signal dynamic range that would otherwise be present at the detectors. Subsequent generations of scanners have sought to decrease the scan time to a few seconds to minimise the movement artifact.

The second generation of scanner addressed this issue by using a small fan beam (see the next figure) with multiple detectors – up to 30 in some designs. The detectors collect multiple data readings during a translation so that fewer translations and rotations are required. Each of the detectors collects its own limited profile during each translation. Typically, a rotation of 30 degrees was employed between translational movements so that only six rotational movements were required to obtain the data for one slice.

Scan times of between 5 to 90 seconds were achieved with this design. The detector elements were scintillators but the water bag was dispensed with and body scans could be performed for the first time. The use of multiple detectors gave a quantum leap in performance.

The third generation of CT scanner decreased scan times even further by using a Rotate-Rotate geometry (see the figure on the right). Most scanners today are of the third generation type. A typical machine employs a large fan beam such that the patient is completely encompassed by the fan. The number of detector elements is typically in the hundreds, e.g.
 * the GE Hispeed Advantage has 852 elements 1.0 mm apart;
 * the Siemens Somatom Plus 4 has 768 elements 1.1 mm apart; and
 * the Toshiba Xpress/SX has 896 elements 1.03 mm apart.

The detector elements are aligned along the arc of a circle centred on the focus of the X-ray tube. The X-ray tube and detector array rotate as one through 360 degrees during which time several hundred discrete profiles are obtained. Both solid state detectors and pressurised xenon gas detectors are used for data acquisition.

Because the fan beam totally encompasses the patient, translational motion is not needed and the tube and detector array rotate as one about the patient. The number of profiles recorded depends on the chosen scan parameters but is at least a few hundred and can be in excess of a thousand. Scan times can be as low as a second. Although the dynamic range of the detectors is very high, some manufacturers control excessive variations in signal strength by using bow-tie shaped filters chosen to suit the body or head shape. Such filters generally attenuate the peripheral part of the fan beam to a greater extent than the central part. It also helps overcome the effects of beam hardening and to minimise patient skin dose in the peripheral part of the field of view.

A number of variants on this geometry have been developed, which include those based on offsetting the centre of rotation and the use of a flying focus X-ray tube.

The fourth generation of CT scanner uses a Rotate-Fixed Ring geometry where a ring of fixed detectors completely surrounds the patient. The X-ray tube rotates inside the detector ring through a full 360 degrees with a wide fan beam. Only those detectors which see the beam are activated at any one time which means that only a small fraction of thousands of detector elements (e.g. the Picker PQ-5000V has 4800 detector elements 1.1 mm apart) are in use at any given time. Scan time is comparable with third generation scanners but the radiographic geometry is poor because the X-ray tube must be closer to the patient than the detectors, i.e. the geometric magnification is large.

The disadvantages of poor geometry noted above has been alleviated very neatly by the so called nutating geometry. The X-ray tube is external to the detector ring but slightly out of the detector plane. Scan times as low as 0.6 s can be achieved using machines of this type with interscan delays of as little as 100 ms. Excellent resolution (0.35 mm) can be obtained by using as many several thousand detector elements.

Helical Scanning
Innovations in slip-ring technology, beginning with the Siemens Somatom Plus and the Toshiba TCT 900S, have enabled the X-ray tube to rotate continuously in the same direction which overcomes problems of interscan delays. When the continuous motion of the X-ray tube is combined with a continuous advance of the patient table along the axis of the scanner we have helical (also called spiral) scanning, as illustrated the following figure. Typical table velocities are 1 – 10 mm/s; a complete 360 degree rotation can be achieved in 0.5 s and the nominal fan beam thickness is 1 mm or greater.

In the context of helical scanning a parameter called pitch is defined as the:

ratio of the distance that the patient couch moves in one rotation to the slice thickness.

In other words, for a couch advance of 10 mm and a nominal slice width of 10 mm, the pitch is 1. Pitch values are typically in the range of 1 to 2 depending on the required spatial resolution in the direction of the couch motion. Its a coverage indicator, in other words.

A significant reduction in scan times results from helical scanning because large volumes can be scanned contiguously without any gaps and with little influence from patient motion. The Somatom Plus-4A, for instance, allows up to 80 slices to be scanned contiguously in 60 seconds of non-stop scanning. Single breathhold scanning of the thorax is therefore possible and reconstruction, with minimal loss of spatial resolution, of sagittal and coronal planes is feasible.

Axial reconstruction in helical scanning is illustrated in the following figure. It is seen that no axial slices are actually irradiated during the scan and that any transaxial image data needs to be estimated from the acquired helical record. One estimation technique is referred to as z-interpolation, because it applies data interpolation techniques along the z-dimension of the acquired data, i.e. the patient's head-to-foot axis.

The filtered back projection reconstruction process is therefore modified to include an z-interpolation step prior to Fourier filtration, as illustrated in the following flow diagram:

Interpolation & Extrapolation
Before proceeding, let's briefly review the topic of interpolation and extrapolation to clarify our basic understanding and to put the subsequent discussion in context. Let's start with a simple one-dimensional case, as illustrated in the following graph. Suppose we've taken measurements A and B and plotted them on the graph, and wish to estimate the value at an in-between point, C. Such an estimation process is called interpolation, and if we assume that there's a linear relationship between variables X and Y as illustrated in our graph, the process of estimating point C is called linear interpolation. Other functions can also be used as interpolatants for more complex data than we're considering here.

If we also require knowledge about point D on our graph, i.e. a point outside the measured range, we refer to the estimation process as extrapolation, and linear extrapolation is illustrated in our graph above.

Mathematically we can write that given:

$$A \Rightarrow (x_1, y_1)\ \ B \Rightarrow (x_2, y_2)\ \ C \Rightarrow (x_3, y_3)$$

then the Y-value of point C is given by:

$$y_3 = y_1 + \frac{(x_3 - x_1)(y_2 - y_1)}{(x_2 - x_1)}$$

using linear interpolation. You might like to develop a similar equation as an exercise which can be used to determine the Y-value of point, D.

Since helical scanning is a three-dimensional phenomenon, we need to develop some two-dimensional perspectives on the situation before proceeding with our discussion – as illustrated in the following figure:

We'll use the side view in the following discussion.

Interpolation in Helical CT
A large number of computational methods have been developed for helical scanning. The 360o linear interpolation algorithm is illustrated in the following figure as an example:

The following equation can be developed on the basis of our earlier discussion for linear interpolation of the transaxial data of interest:

$$P_z(i, \alpha) = (1-w)P_j(i, \alpha) + wP_{j+1}(i, \alpha)\,\!$$

where:

 * Pz(i, α) is the interpolated projection for an angle, α, at position Zref;
 * Pj(i, α) is the helical projection measured in rotation j at position Zj and projection angle α;
 * Pj+1(i, α) is the helical projection measured in rotation j+1 at position Zj+1 and projection angle α; and

$$w = \frac{Z_{\text{ref}} - Z_j}{d}$$

This interpolation process is applied throughout all data points in the helix to generate estimates of the axial data, prior to filtered backprojection. Higher order interpolation is also possible where data points from, say, four rays are used in the estimation. In addition, sophisticated z-axis filters can be applied for these estimations in multi-slice CT.

Volume Imaging
It is possible to produce a number of slices per rotation when the fan beam of a CT scanner is broadened along the z-axis and two-dimensional detectors are used. The fan beam can be formed into the shape of a cone on this basis and concepts such as volume imaging and cone beam CT can be applied. Terms such as multi-slice and multi-detector CT can also be used in this context, although as you will see the second of these two terms is probably the more appropriate.

The 2-D detectors are solid-state devices and the simplest example is the Matrix Array, as illustrated below:

The array can consist of (say) 912 columns by 16 rows of identical detectors, each 1.25 mm square, curved to fit an arc of the x-ray tube rotation. Eight of these columns are shown in panel (a) above being irradiated by an X-ray beam of width 6 mm, with the implication that data for four 1.25 mm axial slices can be acquired simultaneously. More rows, up to sixteen in this case, can be irradiated simultaneously with this arrangement when the x-ray beam is broadened to encompass the width of the detector array.

Flexibility can be built into this design by coupling the outputs of adjacent detector rows, as illustrated in panels (b), (c) and (d), where the outputs of 2, 3 and 4 rows, respectively, are summed to simultaneously generate four 2.5 mm thick slices, or four 3.75 mm slices, or (you guessed it!) four 5 mm slices.

A more flexible design is provided by the Adaptive Array detector, as illustrated in the next figure:

Rather than columns of square detectors, this array uses columns of detectors of variable width, such that the width in the two central columns is relatively narrow, e.g. 1 mm as in the figure above, with the column width increasing towards the periphery, e.g. from 1.5 mm, through 2.5 mm to 5 mm as in our figure. The reason for this pattern is explained below.

Four modes of operation of this detector array are shown in the following figure:

We can see in panel (a) that two 0.5 mm slices can be acquired when a 1 mm thick fan beam is aligned with the central columns of the adaptive array. In panel (b) we can see that four 1 mm slices can be acquired using a fan beam of thickness 4 mm. Detector coupling is illustrated in panels (c) and (d), where the outputs of detectors in the four central columns are coupled so as to simulate two 2.5 mm wide detection columns and hence acquire data for four 2.5 mm slices. How do you think this coupling process is applied in panel (d) to generate four 5 mm slices?

A second advantage of the adaptive array is that the number of individual detector elements on each row can be considerably reduced, from 16 to 8 in the case we've just discussed. This adds greatly to the speed with which data can be generated by the array and reduces the number of computations necessary for uniformity and other corrections that must also be applied to the measured data.

The interpolation process in multi-slice helical CT is illustrated in our final figure where the situation in a 4-slice system using a 360o multi-slice filtered interpolation algorithm is shown:

The number of detector rows in Multi-Detector CT (MDCT) in 2010 is typically 8, 16 or 64, or greater – even a 320-slice scanner has been developed.

CT Image Display
After the computer calculates the linear attenuation coefficient for each pixel via filtered back projection, the values are normalised to the value for water as a reference, scaled and presented as a Hounsfield or CT-number which is defined as:

$$H = 1000 \frac{\mu_m - \mu_{\text{water}}}{\mu_{\text{water}}}$$

where μm and μwater are the linear attenuation coefficients for the tissue material and for water, respectively. The CT-number of water is therefore zero. CT-numbers for a number of tissues are shown in the following table:

The image is usually presented on a computer monitor using a grey scale. The grey scale is chosen to encompass all or some part of the entire range of CT-numbers by selecting a suitable window level and window width. The window width is the range of CT-numbers we select for display and the window level is, usually but not always, the central CT-number about which the window is chosen. Typically, the highest number is assigned to white and the lowest number to black with all intervening numbers assigned intensities on a linear scale. Thus, air will be displayed as black and cortical bone will appear relatively bright.

Examples of image display manipulation are shown in the following two figures. In the first, the same image of a slice through a patient's liver is displayed using a relatively narrow window (high contrast) and also with a wide window. The image with the narrower window appears noisier, but this is merely a reflection of the fact that the gray scale spreads over a narrow range of CT-numbers.

In the second figure, a relatively narrow window has been used to highlight pathology:

Scanned Projection Radiography
Most CT systems use scanned projection radiography (SPR), also known as a scout view and scanogram, to establish a consistent set of anatomical markers for all subsequent procedures. In SPR, the fan beam is not rotated about the patient but is fixed, and the patient couch is moved slowly through the beam. Each detector element measures a varying amount of radiation intensity as the anatomy changes between the focus and the detector. These discrete detector signals are digitised but not processed to any great extent. The computer stores the signals from all the detectors as a series of line images. The width of each line is small, typically 1.5 – 2 mm, corresponding to the finite width of the fan beam. Subsequently, a projection radiograph is synthesized by making a composite of these line scan images with the grey scale suitably chosen to display the anatomy with an adequate contrast – see an example image below:

The volume of tissue to be examined in CT mode can be defined once the SPR image has been generated. The CT procedure can then be automatically controlled by the computer once the appropriate scanning factors have been selected.


text:
User:Elizej

hi, i'm fluent in Afrikaans and English, can converse on a intermediate level of French (read and write on advanced level, i hope) and can speak and understand basic cantonese (can only read about 20 characters, though!). Love languages and anything chinese or french. Simone de Beauvoir and Frida Kahlo are my ultimate heroes and i hope to be just like them one day!

title: User:Elizej


text:
A-level Applied Science/The Role of the Pathology Service/Haematology

Haematology (US English: hematology) is the branch of medicine that is concerned with blood, the blood-forming organs and blood diseases. Haematology includes the study of etiology, diagnosis, treatment, prognosis, and prevention of blood diseases.

Blood diseases affect the production of blood and its components, such as blood cells, haemoglobin, blood proteins, the mechanism of coagulation, etc.

Tests that require examination and measurement of the cells of blood, as well as blood clotting studies, are grouped under haematology. apart from clotting studies, tests on serum or plasma come under biochemistry.

(Serum is the yellow watery part of blood that is left after blood has been allowed to clot and all blood cells have been removed. This is most easily done by centrifugation which packs the more dense blood cells and platelets to the bottom of the centrifuge tube, leaving the liquid serum fraction resting above the packed cells. Plasma is essentially the same as serum, but is obtained by centrifuging the blood without clotting. Plasma therefore contains all of the clotting factors, including fibrinogen.)

= Haematologists = Medical doctors who work in haematology are known as haematologists. Their routine work may range from the management of the haematology laboratory, work at the microscope viewing blood films and bone marrow slides, interpretation of various haematological test results, care of in-patients and care of out-patients.

Haematologists may specialise further or have special interests, for example in:
 * treating bleeding disorders such as haemophilia.
 * treating haematological malignacies such as lymphoma and leukaemia (onco-haematology).
 * treating haemoglobinopathies.
 * in the science of blood transfusion and the work of a blood bank.

Haematologists receive whole blood and citrated plasma. They do full blood counts, blood films and coagulation investigations. This would identify conditions such as leukaemia and haemophilia.

= Classification of haematology diseases =
 * Haemoglobinopathies (congenital abnormality of the haemoglobin molecule or of the rate of haemoglobin synthesis)
 * e.g. Sickle-cell disease
 * Anaemias (lack of red blood cells or haemoglobin)
 * Decreased numbers of cells
 * Myeloproliferative disorders (Increased numbers of cells)
 * Haematological malignancies
 * Lymphomas
 * Hodgkin's disease
 * Non-Hodgkin's lymphoma
 * Myelomas
 * Multiple myeloma
 * Leukemias
 * Coagulopathies (disorders of bleeding and coagulation)
 * Disorders of clotting proteins
 * Haemophilia
 * Disorders of platelets
 * Miscellaneous
 * HIV/AIDS
 * Malaria
 * Leishmaniasis

= Tests = Tests used in the investigation of haematological problems include:
 * Full blood count
 * Erythrocyte sedimentation rate (ESR)
 * Blood film
 * Bone marrow biopsy
 * Coombs test
 * Serum ferritin level
 * Vitamin B12 and folate levels
 * Prothrombin time
 * Partial thromboplastin time
 * Protein electrophoresis
 * Haemoglobin electrophoresis
 * D-dimer

= Treatments = Treatments include:
 * Diet advice.
 * Oral medication - tablets or liquid medicines.
 * Anticoagulation therapy.
 * Intramuscular injections (for example, Vitamin B12 injections).
 * Blood transfusion (for anaemia).
 * Venesection (for iron overload or polycythaemia).
 * Bone marrow transplant (for example, for leukaemia).
 * Chemotherapy (for example, for leukaemia).
 * Radiotherapy (in decline, for example, for leukaemia).

= Type of work =

Much work is carried out by technicians using microscopes. Automation is less widespread than in biochemistry because in many tests the cell types need to be identified visually.

Health and safety
Safe handling of blood products is extremely important. There is a high risk of infection if the blood is infected with pathogens such as HIV or a hepatitis virus.

Blood culture
Blood culture is microbiological culture of blood. It is employed to detect infections that are spreading through the bloodstream (bacteraemia, septicaemia).

Method
About 30 cm3 of blood are taken through venipuncture and injected into two "blood bottles" with specific media for aerobic and anaerobic organisms.

Care needs to be taken that the bottles are not contaminated with bacteria from staff members or other patients. To that end, the patient's skin and the top of the blood bottles are rubbed or sprayed with denatured alcohol.

To maximise the diagnostic yield of blood cultures, multiple samples are sometimes taken from different veins.

Uses
Any infection causing fever may prompt hospital physicians to request blood cultures. Identifying the agent may aid in choosing the correct antibiotic and addressing particular risk factors.

Blood culture is essential in the diagnosis of infective endocarditis. In this elusive disease, blood cultures may have to be repeatedly taken during febrile episodes, when bacteria are shed from the heart valves into the bloodstream (bacteraemia).

Haemocytometry

The haemocytometer is a device originally used to count blood cells (as the name suggests). It is now used to count other cells and many types of microscopic particles. It consists of a thick glass microscope slide with a rectangular indentation that creates a chamber of certain dimensions. This chamber is etched with a grid of perpendicular lines.

The device is carefully crafted so that the area bounded by the lines is known, and the depth of the chamber is also known. Therefore it is possible to count the number of cells in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall.

Principles

The ruled area of the haemocytometer consists of one large square known as a type A square. Within the type A square there are 16 smaller squares known as type B squares. These are further divided into 16 smaller squares known as type C. Each type C square has an area of 1/256 mm2 and each type B square has an area of 1/16 mm2. There are three lines around each side of the type B squares. The cell-sized structures to be counted are those which lie between the middle of the three lines on the top and right of the square and the inner of the three lines on the bottom and left of the square.

Some designs have 25 type B squares in each type a square. In this design, each type C square has an area of 1/400 mm2 and each type B square has an area of 1/25 mm2.

Usage
When a liquid sample containing immobilised cells is placed on the chamber, it is covered with a cover glass, and capillary action completely fills the chamber with the sample. Looking at the chamber through a microscope, the number of cells in the chamber can be determined by counting. Different kinds of cells can be counted separately as long as they are visually distinguishable. The number of cells in the chamber is used to calculate the concentration or density of the cells in the mixture from which the sample was taken: it is the number of cells in the chamber divided by the chamber's volume (the chamber's volume is known from the start), taking account of any dilutions and counting shortcuts:

$$\mbox{concentration of cells in original mixture} = $$

$$\left (\frac{\mbox{number of cells counted}}{(\mbox{proportion of chamber counted})(\mbox{volume of chamber})} \right ) \left (\frac{\mbox{volume of sample dilution}}{\mbox{volume of original mixture in sample}} \right )$$

Haemocytometers are often used to count blood corpuscles, organelles within cells, blood cells in cerebrospinal fluid after performing a lumbar puncture, or other cell types in suspension. Using a haemocytometer to count bacteria results in a 'total count' as it is difficult to distinguish between living and dead organisms.

Usage tricks

 * Mix your original mixture thoroughly before taking a sample. This ensures that your sample is representative, and not just an artefact of the particular region of your original mixture from which you drew it.
 * Use an appropriate dilution of your mixture with regard to the number of cells you hope to count. If your sample is not diluted enough, the cells will be too crowded and difficult to count. If it is too dilute, your sample size will not be enough to make strong inferences about the concentration in the original mixture. Naturally, you must have a rough idea of the concentration before you begin in order to guess an appropriate dilution. If your mixture is coloured, it may be helpful to memorise a particular intensity of that colour at which the mixture tends to be easy to analyse.
 * Analyse multiple chambers. By performing a redundant test on a second chamber, you can compare the results. If they differ greatly, your method of taking the sample may be unreliable (e.g. the original mixture is not mixed thoroughly). You can use the average of your results for a more accurate calculation.
 * Make sure to put enough liquid on the instrument that some leaks out of the cover glass when it is placed over the chamber. Otherwise, it is uncertain whether the space under the cover glass is completely filled with liquid. This volume should be the same every time you use the instrument.
 * Do not use a paper wipe to dry the excess liquid. The same capillary action that filled the chamber will then dry it out.
 * Watch out for the objective lens. Remember that the haemocytometer is thicker than a normal microscope slide. If you focus too closely, your objective lens may contact the instrument. This may affect your choice of which objective lens you use - carefully figure out what will fit, before you start.
 * Count across the rows or down the columns. Use the gridlines to help you remember which areas' cells have already been counted.
 * You don't have to count the whole chamber. If there are lot of cells, you can just perform your count in a section of the chamber and use the grid to determine what proportion of the chamber that is. You can then extrapolate to estimate how many cells are in the chamber, and use that figure in your final calculation. This gives you speed at the expense of potential accuracy; if possible, using a more appropriate dilution is better.
 * Are the lines in or out? Some cells inevitably fall smack on top of the outside gridlines that mark the edges of the chamber. The usual practice is to include cells overlapping the top and left lines, but not those overlapping the bottom or right lines - this has the advantage of eliminating redundant counting if you count adjacent regions.


text:
XForms/Address

Here is a simple address form. Note that HTML is the default namespace. We interleave both XForms and HTML tags but the XForms tags have xf as a prefix. We use the HTML fieldset and legend tags to put related form elements in a group. We use the XForms group element to tell us where in the model to get our instance data.

Link to XForms Application
Load XForms Application

Discussion
The form labels are not lined up very nicely. This can be done by adding a few lines of cascading style sheet to the program.

Note that the data uses an upper camel case naming convention used in ebXML, ACORD, UBL, GMXDM, NIEM and other US federal naming and design conventions.

title: XForms/Address


text:
XForms/Repeat

Here is a simple example of how to get a list of repeating data elements out to your screen. This is done using the repeat tag and the nodeset attribute. You use the nodeset to specify where you want to begin your listing in the model. In this case we are just using a model embedded into the page.

Output
Here is the output for this program under the FireFox browser:

Note that all of the names are displayed, not just the first name. Note how we are mixing both HTML tags and XForms tags together in the body.

Discussion
The key line here is the  statement:

The  element has an attribute named. This tells you where in your model to get the data. In this case we will be iterating over all Person records and outputting the Name and Phone for each Person.

You can also use an absolute path reference:

As an alternative you can also use an instance reference:

Note that each  starts a new line for each person record. This can be turned into a clean tabular layout using a simple style sheet. You can do this by having the second output be associated with a class called "column2" and then adding a column2 formatting rule to the style sheet.

title: XForms/Repeat


text:
Horticulture/Conyza canadensis

Conyza canadensis (formerly Erigeron canadensis L.) is an annual plant native throughout most of North America and Central America. Common names include Horseweed, Canadian Horseweed, Canadian Fleabane, Coltstail, and Butterweed. Horseweed is a common weed of fields, meadows, wastes, and gardens throughout its native range, colonizing areas where perennials are not established. It often grows in cracks of hardscape surfaces as well, and can cause problems with heaving in thin materials such as flagstone.

It is an invasive species in Britain, found from northern Scotland to Cornwall. It is the only one of the British Conyza species that grows as a weed of arable land: the others are casuals of waste and disturbed ground in towns and by roads and railways. It is not invasive of any natural or semi-natural habitats.

Description
Horseweed is an annual plant growing to 1.5 m (5 ft) tall, with sparsely hairy stems. The leaves are slender, 2-10 (0.5" to 4") cm long and up to 1 cm (0.5") broad, with a coarsely toothed margin. The flowers are produced in dense inflorescences 1 cm (0.5") in diameter, with a ring of white or pale purple ray florets and a centre of yellow disc florets.

Control
In summer, patrol upwind meadows and remove plants if possible.

title: Horticulture/Conyza canadensis


text:
User:Spikespeigel42~enwikibooks

I mainly edit Blender3d: Noob to pro, but i also clean up and wikify some other stuff.

title: User:Spikespeigel42~enwikibooks


text:
Talk:Horticulture/Conyza canadensis

Transwiki notes
Copied from Conyza canadensis 15:40, 20 September 2006 (UTC) using this version].

title: Talk:Horticulture/Conyza canadensis


text:
Engineering Analysis

Preface
This book is about the topic of mathematical analysis, particularly in the field of engineering. It attempts to be a companion piece to high-level engineering texts that will rely on a certain fundamental mathematical background among readers.

This will build on topics covered in Probability, Algebra, Linear Algebra, Calculus, and Ordinary Differential Equations. Readers of this book are expected to have background knowledge in all those topics. Topics covered will be inter-disciplinary engineering topics, and will be highly mathematical. However, overlap between this book and other mathematics books, except where necessary, will be minimized.

This book is intended to accompany a graduate course of study in engineering analysis.

Linear Algebra

 * Vector Spaces
 * Vector Basics
 * Linear Independence and Basis
 * Linear Transformations
 * Minimization
 * Projections
 * Linear Spaces
 * Matrices
 * Matrix Forms
 * Quadratic Forms
 * Eigenvalues and Eigenvectors
 * Diagonalization
 * Spectral Decomposition
 * Error Estimation

Matrix Calculus

 * Matrix Functions
 * Cayley-Hamilton Theorem
 * Matrix Exponentials
 * Lyapunov Equation
 * Function Spaces
 * L2 Space
 * Banach and Hilbert Spaces
 * Fourier Series
 * Arbitrary Basis Expansion
 * Bessel Equation and Parseval Theorem
 * Multi-Dimensional Fourier Series

Algorithms Design And Analysis

 * Wavelets
 * /Wavelet Transforms/

Stochastic Processes

 * Random Variables
 * Probability Functions
 * Distributions
 * Expectation and Entropy
 * SISO Transformations
 * MISO Transformations
 * Correlation
 * Random Vectors

Analysis

 * Leibnitz' Rule
 * /Differential Operator Theory/
 * /Linear Operators/
 * /Complex Analysis/

Optimization and Minimization

 * Optimization
 * /Unconstrained Optimizations/
 * /Constrained Optimizations/

Appendices

 * /Resources/

title: Engineering Analysis


text:
A-level Applied Science/The Role of the Pathology Service/Biochemistry

Clinical biochemistry (also known as chemical pathology or clinical chemistry) is the area of pathology that is generally concerned with analysis of bodily fluids.

The discipline originated in the late 19th century with the use of simple chemical tests for various components of blood and urine. Subsequently other techniques were applied including the use and measurement of enzyme activities, spectrophotometry, electrophoresis and immunoassay.

Most current laboratories are now highly automated and use assays that are closely monitored and quality controlled.

Tests that require examination and measurement of the cells of blood, as well as blood clotting studies, are not included as these are usually grouped under haematology.

=Type of work= Biochemical tests are also known as 'chemical pathology'. The tests are performed on any kind of body fluid, but mostly on serum or plasma. Serum is the yellow watery part of blood that is left after blood has been allowed to clot and all blood cells have been removed. This is most easily done by centrifugation which packs the more dense blood cells and platelets to the bottom of the centrifuge tube, leaving the liquid serum fraction resting above the packed cells. Plasma is essentially the same as serum, but is obtained by centrifuging the blood without clotting. Plasma therefore contains all of the clotting factors, including fibrinogen. Biochemists test samples for different biochemical components. A large laboratory will accept samples for up to about 700 different kinds of tests. Even the largest of laboratories rarely does all these tests themselves and some need to be referred to other labs. This large array of tests can be further sub-categorised into sub-specialities of:
 * Immunology test for antibodies.
 * DNA analysis is also done in large medical laboratories.
 * Hormones
 * Minerals
 * Vitamins
 * Metabolites
 * Enzymes
 * General or routine chemistry
 * Endocrinology - the study of hormones
 * Immunology - the study of the immune system and antibodies
 * Pharmacology or Toxicology - the study of drugs

Chemical pathology tests
Common chemical pathology tests include:
 * Sodium;
 * Potassium;
 * Chloride;
 * Bicarbonate;
 * Urea;
 * Creatinine;
 * Calcium;
 * Phosphate;
 * Albumin;
 * Bilirubin;
 * AST;
 * ALT;
 * GGT;
 * Alkaline phosphatase;
 * Magnesium;
 * Osmolality;
 * Urate;
 * Iron;
 * Transferrin;
 * Total protein;
 * Globulins;
 * Glucose;
 * C-reactive protein;
 * HbA1c.
 * Blood gases ( $$ [H^+], P_{{\mathrm{CO}}_2}, P_{{\mathrm{O}}_2} $$)

=Automation=

The biochemistry department uses more automated analysis than other departments. This is due to the:
 * reliability and validity of testing procedures.
 * volumes of samples required.
 * numbers samples tested in one day.

=Gel electrophoresis=

Gel electrophoresis is a group of techniques used by scientists to separate molecules based on physical characteristics such as size, shape, or isoelectric point. Gel electrophoresis is usually performed for analytical purposes, but may be used as a preparative technique to partially purify molecules prior to use of other methods such as mass spectrometry, PCR, cloning, DNA sequencing, or Western immuno-blotting for further characterisation.

Separation
"Gel", refers to the matrix used to separate the molecules. In most cases the gel is a crosslinked polymer whose composition and porosity is chosen based on the weight and composition of the target of the analysis. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually made with different concentrations of acrylamide and a cross-linker, producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is purified agarose (a component of agar which is a red seaweed extract). In both cases, the gel forms a solid but porous matrix that looks and feels like clear jelly. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and needs to be handled using Good Laboratory Practices (GLP) to avoid poisoning.

"Electrophoresis", refers to the electromotive force (EMF) that is used to push or pull the molecules through the gel matrix; by placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, towards the anode if negatively charged or towards the cathode if positively charged (note that gel electrophoresis operates as an electrolytic cell; the anode is positive and the cathode is negative).

Visualisation
After the electrophoresis runs, when the smallest molecules have almost reached the anode, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie blue dye can be used. Other methods can also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules luminesce under ultraviolet light, a photograph can be taken of the gel under ultraviolet light. If the molecules to be separated contain radioactive atoms, an autoradiogram can be recorded of the gel (as in the example shown here).

If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components.

Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are special markers available - ladders - which contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule.

Applications
Gel electrophoresis is used in molecular biology, genetics, microbiology and biochemistry. The results can be analysed quantitatively by visualising the gel with UV light, or by staining the separated molecules. A gel imaging device records the image with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel. The measurement and analysis are mostly done with specialized software.

Nucleic acids
In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the natural negative charge carried on their sugar-phosphate backbone. Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their radius of gyration, or, for non-cyclic fragments, roughly size. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to renature the nucleic acids and cause them to behave as long rods again.

Gel electrophoresis of large DNA] or RNA molecules is usually done by agarose gel electrophoresis. See the "Chain termination method" page for an example of a polyacrylamide DNA sequencing gel.

Proteins
Proteins, on the other hand, can have different charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are usually denatured in the presence of a detergent such as sodium dodecyl sulphate/sodium dodecyl phosphate (SDS/SDP) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the size of the protein (usually 1.4 g SDS per gram of protein), so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like they were long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape.

Proteins are usually analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), by native gel electrophoresis], by quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE), or by 2-D electrophoresis.

=Class experiments=

Using simple clinical dipsticks, a mock urine sample can be analysed. Glucose, ketone and protein levels can be established to identify possible diseases.

Thin layer chromatography separations of carbohydrates, proteins and amino acids or lipids are simple to perform and introduce candidates to the concepts of separation of molecules being related to the chemical properties.

''If access to electrophoresis equipment is not possible, teachers could approach the hospital biochemistry department or a local university biological sciences department and request a demonstration day, or arrange for someone to visit who can demonstrate the technique with samples gathered from the candidates. Protein electrophoresis equipment is available from a range of companies and the National Centre for Biotechnology Education offers a range of practical activities and resources for electrophoresis.''

=Paper chromatography=

Paper chromatography is an analytical technique for separating and identifying compounds that are 'or can be coloured, especially pigments. This method has been largely replaced by thin layer chromatography, however it is still a powerful teaching tool. Two-way paper chromatography, also called two-dimensional chromatography, involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of similar compounds, for example, amino acids.

Technique
A small spot of solution containing the sample is applied to a strip of chromatography paper about 1 cm from the base. This sample is adsorbed onto the paper and may form interactions with it. Any substance that reacts or bonds with the paper cannot be measured using this technique. The paper is then dipped in to a suitable solvent such as ethanol or water, and placed in a sealed container.

The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper and to one another. As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the fibres in the paper. Paper chromatography takes anywhere from several minutes to several hours.

In some cases, paper chromatography does not separate pigments completely; this occurs when two substances appear to have the same Rf values in a particular solvent. In these cases, two-way chromatography is used to separate the multiple-pigment spots. The chromatogram is turned by ninety degrees, and placed in a different solvent in the same way as before; some spots separate into multiple spots, showing the presence of more than one pigment. As before the Rf value is calculated, and the two pigments identified.

=Thin-layer chromatography=

Thin layer chromatography (TLC) is a widely-used chromatography technique used to separate chemical compounds. It involves a stationary phase consisting of a thin layer of adsorbent material, usually silica gel, alumina, or cellulose immobilised onto a flat, inert carrier sheet. A liquid phase consisting of the solution to be separated dissolved in a solvent is drawn through the plate via capillary action, separating the experimental solution. It can be used to determine the pigments a plant contains, to detect pesticides or insecticides in food, in forensics to analyse the dye composition of fibres, or to identify compounds present in a given substance, among other uses.

Plate preparation
TLC plates are made by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulphate (gypsum) and water. This mixture is spread as a thick slurry on an inert carrier sheet, usually glass, thick aluminium foil, or plastic, and the resultant plate is dried and activated by heating in an oven. The thickness of the adsorbent layer is typically around 0.1–0.25 mm for analytical purposes and around 1–2 mm for preparative TLC.

Technique
The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products.

A small spot of solution containing the sample is applied to a plate, about one centimeter from the base. The plate is then dipped in to a suitable solvent, such as ethanol or water, and placed in a sealed container. The solvent moves up the plate by capillary action and meets the sample mixture, which is dissolved and is carried up the plate by the solvent. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the stationary phase.

=Analysis of chromatograms= After development, the spots corresponding to different compounds may be located by their colour, by ultraviolet light, ninhydrin or by treatment with iodine vapours. The final chromatogram can be compared with other known mixture chromatograms to identify sample mixes using the Rf value.

As in most other forms of chromatography, paper chromatography uses Rf values to help identify compounds. Rf values are calculated by dividing the distance the pigment travels up the paper by the distance the solvent travels (the solvent front). Because Rf values are standard for a given compound, known Rf values can be used to aid in the identification an unknown substance in an experiment.

As the chemicals being separated may be colourless, several methods exist to visualise the spots:
 * Often a small amount of a fluorescent compound, usually Manganese-activated Zinc Silicate, is added to the adsorbent that allows the visualisation of spots under a blacklight(UV254). The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.
 * Iodine vapours are a general unspecific colour reagent
 * Specific colour reagents exist into which the TLC plate is dipped or which are sprayed onto the plate

Once visible, the Rf value of each spot can be determined by dividing the distance travelled by the product by the total distance travelled by the solvent (the solvent front). These values depend on the solvent used, and the type of TLC plate, and are not physical constants.


text:
A-level Applied Science/The Role of the Pathology Service/Microbiology

Microbiology laboratories receive swabs, faeces, urine, blood, sputum, medical equipment, as well as possible infected tissue. They culture this to check for any pathogenic microbes.

Pathogenic microbes may be bacteria, fungi, viruses or other parasites.

Viruses are difficult to culture because they require living cells to reproduce. Virology is therefore done in larger medical laboratories.

Bacteria, fungi and other non-viral parasites can grow independently of other living organisms if they are provided with the correct nutrients and growth conditions. Culturing the organism in this way will assist in identifying it and studying possible therapies against it.

=Infectious diseases=

An infectious disease is a clinically evident disease of humans or animals that damages or injures the host so as to impair host function, and results from the presence and activity of a pathogenic microbial agent, including viruses, bacteria, fungi, protozoa, multicellular parasites, and aberrant proteins known as prions. Transmission of an infectious disease may occur through several pathways; including through contact with infected individuals, by water, food, airborne inhalation, or through vector-borne spread.

A contagious disease (also called a communicable disease) is an infectious disease that is capable of being transmitted from one person or species to another. Contagious diseases are often spread through direct contact with an individual, contact with the bodily fluids of infected individuals, or with objects that the infected individual has contaminated.

The term infectivity describes the ability of an organism to enter, survive and multiply in the host, while the infectiousness of a disease indicates the comparative ease with which the disease is transmitted to other hosts. An infection however, is not synonymous with an infectious disease; as an infection may not cause clinical symptoms or impair host function.

Overview
Among the almost infinite varieties of microorganisms, relatively few cause a disease in an otherwise normal or healthy individual. These highly virulent microorganisms are classified as primary pathogens. Organisms, which cause an infectious disease in a host with depressed resistance, are classified as opportunistic pathogens. Opportunistic infectious disease may be caused by organisms that are ordinarily in contact with the host, such as bacteria or fungi in the colon or in the upper respiratory tract; following an injury, whether mechanical (such as a open Bone fracture|fracture); or by a disease with immunosuppressive activity (such as measles or malaria, or one induced by cytotoxic chemotherapy). Primary pathogens may also cause more explosive disease in a host with depressed resistance.

Most primary pathogens are inhabitants of, and pathogens for, humans only. Opportunistic pathogens, in contrast, may cause disease in many species of mammals. Some exceptions are tetanus, anthrax and rabies which may inhabit and cause disease in many animal species including humans.

Agents and vectors
Infectious disease requires an agent and a mode of transmission (or vector). A good example is malaria, which is caused by Plasmodial parasites, chiefly Plasmodium falciparum but does not affect humans unless the vector, the Anopheles mosquito, is around to introduce the parasite into the human bloodstream.

The vector does not have to be biological. Many infectious diseases are transmitted by droplets which enter the airway (e.g. common cold and tuberculosis).

Clearance and immunity
Infection with most pathogens does not result in death of the host and the offending organism is ultimately cleared after the symptoms of the disease have waned. This process requires immune mechanisms to kill or inactivate the inoculum of the pathogen. Specific acquired immunity against infectious diseases may be mediated by antibodies and/or T lymphocytes. Immunity mediated by these two factors may be manifested by:
 * a direct effect upon a pathogen, such as antibody-initiated complement-dependent bacteriolysis, opsonoisation, phagocytosis and killing, as occurs for some bacteria,
 * neutralization of viruses so that these organisms cannot enter cells,
 * or by T lymphocytes which will kill a cell parasitized by a micro organism.

The immune response to a micro organism often causes symptoms such as a high fever and inflammation, and has the potential to be more devastating than direct damage caused by a microbe.

Resistance to infection (immunity) may be acquired following a disease, by asymptomatic carriage of the pathogen, by harbouring an organism with a similar structure (cross reacting), or by vaccination. Knowledge of the protective antigens and specific acquired host immune factors is more complete for primary pathogens than for opportunistic pathogens. Immune resistance to an infectious disease requires a critical level of either antigen-specific antibodies and/or T cells when the host encounters the pathogen. Some individuals develop natural serum antibodies to the surface polysaccharides of some agents although they have had little or no contact with the agent, these natural antibodies confer specific protection to adults and are transmitted to newborns.

The work of an infectiologist
Doctors who specialise in the medical treatment of infectious disease are called infectiologists or infectious disease specialists. Generally, infections are initially diagnosed by primary care physicians or internal medicine specialists. For example, an "uncomplicated" pneumonia will generally be treated by the internist or the pulmonologist (lung physician).

The services of the infectious disease team are called for when:
 * The disease has not been definitively diagnosed after an initial workup
 * The patient is immunocompromised (for example, in AIDS or after chemotherapy);
 * The infectious agent is of an uncommon nature (e.g. tropical diseases);
 * The disease has not responded to first line antibiotics;
 * The disease might be dangerous to other patients, and the patient might have to be isolated.

The work of the infectiologist therefore entails working with patients and doctors on one hand and laboratory scientists and immunologists on the other hand.

Mortality from infectious diseases
The relationship between virulence and transmission is complex, and has important consequences for the long term evolution of a pathogen.

If a disease is rapidly fatal, the host may die before the microbe can get passed along to another host. However, this cost may be overwhelmed by the short term benefit of higher infectiousness if transmission is linked to virulence, as it is for instance in the case of cholera (the explosive diarrhoea aids the bacterium in finding new hosts) or many respiratory infections (sneezing, coughing etc. create infectious aerosols).

The classification of infectious disease
Epidemiology is another important tool used to study disease in a population. For infectious diseases it helps to determine if a disease outbreak is sporadic (occasional occurrence), endemic (regular cases often occurring in a region), epidemic (an unusually high number of cases in a region), or pandemic (a global epidemic).

=Collecting samples=

Before starting treatment, the physician will collect a sample from a suspected infected compartment: a blood sample when bacteria possibly have invaded the bloodstream, a sputum sample, a urine sample, etc. These samples are transferred to the microbiology lab, which looks at the sample under the microscope, and tries to culture the bacteria. This can help in the diagnosis.

=Serology=

Serum is the yellow watery part of blood that is left after blood has been allowed to clot and all blood cells have been removed. This is most easily done by centrifugation which packs the more dense blood cells and platelets to the bottom of the centrifuge tube, leaving the liquid serum fraction resting above the packed cells. Serologists receive serum samples to look for evidence of diseases such as hepatitis or HIV.

Certain agents cannot be microbiologically cultured, for example Treponema pallidum and most viruses. The first serological markers were developed to diagnose syphilis (the Wassermann test, later replaced by the VDRL and TPHA tests). Serology involves detecting the antibodies against an infectious agent in the patient's blood. In immunocompromised patients (e.g. AIDS), serology can be troublesome, because the antibody reaction is blunted.

A more recent development is direct detection of viral proteins and/or DNA in blood or secretions. This can be done by PCR (polymerase chain reaction), involving the amplification of viral DNA and its subsequent detection with anti-DNA probes.

=Aseptic technique=

Aseptic technique is the name given to the procedures used by microbiologists to prevent microbial contamination of themselves, which may result in infection, contamination of the environment they are working in, and contamination of the specimen they are working on, which is especially important when a pure culture is desired. It is used whenever specimens are to be transferred between media, for example, when subculturing. Such a procedure, using a flame sterilisation method, might occur as follows:
 * 1) A person would assemble the closed tube or flask from which — and the closed tube or flask to which  — the specimen is to be transferred, an inoculating loop, and a flame source, all on a clean, preferably microbe-free surface with some overhead protection from airborne microbes.
 * 2) The person would start the flame, and move the end of the inoculating loop, in a slow back-and-forth motion, through the top of the blue part of the flame. The person would not allow the loop to touch anything except the specimen itself, until the entire procedure is finished.
 * 3) Preparing to execute the specimen transfer, the person would hold both of the tubes or flasks in one hand, probably the opposite of the writing hand. The person would then open the tube or flask containing the specimen source and briefly hold the top of it in the flame, to kill unwanted microbes.
 * 4) Quickly, so as to minimize the possible time for contamination of the specimen in the source tube or flask, the person would use the inoculating loop with their writing hand to retrieve the specimen, and then sterilize the top of the tube or flask again before immediately closing it.
 * 5) Keeping in mind that the specimen on the inoculating loop could be contaminated every of time it is exposed, the person would repeat the previous step identically with the tube or flask in which the specimen is to be deposited; however, the person would be depositing the sample into the tube or flask.

Students of microbiology are taught the principles of aseptic technique by means of hands-on laboratory experience. Practice is critical to learning how to handle the laboratory tools without contaminating them.

=Microbiological cultures=

Diagnosis is initially by medical history and physical examination, and imaging (such as X-rays), but the principal tool in infectious disease is the microbiological culture. In a culture, a growth medium is provided for a particular agent. After inoculation of a specimen of diseased fluid or tissue onto the medium, it is determined whether bacterial growth occurs. This works for a number of bacteria, for example Staphylococcus or Streptococcus.

A microbiological culture, or microbial culture, is a method of growing a microbial organism to determine what it is, its abundance in the sample being tested, or both. It is one of the primary diagnostic methods of microbiology. It is often used a tool to determine the cause of infectious disease by letting the agent multiply (reproduce) in predetermined media in laboratory.

The most common method of microbiological culture uses Petri dishes with a layer of agar-based growth medium in them to grow bacterial cultures. This is generally done inside of an incubator. Another method is liquid culture, where the bacteria are grown suspended in a liquid nutrient medium. Bottles of liquid culture are often placed in shakers in order to introduce oxygen to the liquid and maintaining the uniformity of the culture.

The term culture can also, though infrequently and informally, be used as a synonym for tissue culture, which involves the growth of cells or tissues explanted from a multi-cellular organism.

A sputum culture is a test to detect and identify bacteria or fungi that are infecting the lungs or breathing passages. Sputum is a thick fluid produced in the lungs and in the airways leading to the lungs. A sample of sputum is placed in a container with substances that promote the growth of bacteria or fungi. If no bacteria or fungi grow, the culture is negative. If organisms that can cause infection (pathogenic organisms) grow, the culture is positive. The type of bacterium or fungus will be identified with a microscope or by chemical tests.

If bacteria or fungi that can cause infection grow in the culture, other tests may be done to determine which antibiotic will be most effective in treating the infection. This is called susceptibility or sensitivity testing.

This test is done on a sample of sputum that is usually collected by coughing. For people who cannot cough deeply enough to produce a sample, a suction tube or needle may be inserted in the airway to collect the sputum.

In a hospital setting, a sputum culture is most commonly ordered if a patient has a pneumonia. The Infectious Disease Society of America recommends that sputum cultures be done in pneumonia requiring hospitalization, while the American College of Chest Physicians does not. The reasons for the discrepancy is this: normal, healthy lungs have bacteria, and sputum cultures collect both normal bacteria and the bacteria causing disease. It is difficult to tell which bacteria is causing the disease in this setting.

=Antibiograms=

The role of antibiograms
In clinical practice, antibiotics are most frequently prescribed on the basis of general guidelines and knowledge about sensitivity: e.g. uncomplicated urinary tract infections can be treated with a first generation quinolone, etc. This is because Escherichia coli is the most likely causative pathogen, and it is known to be sensitive to quinolone treatment. Infections that are not acquired in the hospital, are called "community acquired" infections.

However, many bacteria are known to be resistant to several classes of antibiotics, and treatment is not so straightforward. This is especially the case in vulnerable patients, such as patients in the intensive care unit. When these patients develop a "hospital-acquired" (or "nosocomial") pneumonia, more hardy bacteria like Pseudomonas aeruginosa are potentially involved. Treatment is then generally started on the basis of surveillance data about the local pathogens probably involved. This first treatment, based on statistical information about former patients, and aimed at a large group of potentially involved microbes, is called "empirical treatment".

When a culture has proven to be positive, the sensitivity (or, conversely, the antibiotic resistance) of an agent can be determined by exposing it to test doses of antibiotic. This way, the microbiologist determines how sensitive the target bacterium is to a certain antibiotic. This is usually reported as being: Sensitive, Intermediate or Resistant. The antibiogram can then be used to determine optimal therapy for the patient. This can reduce the use of broad-spectrum antibiotics and lead to a decrease in antibiotic resistance.

An antibiogram is the result of a laboratory testing for the sensitivity of an isolated bacterial strain to different antibiotics. It is by definition an in vitro-sensitivity test.

Antibiogram methods
Once a culture is established, there are two possible ways to get an antibiogram:
 * a semi-quantitative way based on diffusion (Kirby-Bauer method); small discs containing different antibiotics, or impregnated paper discs, are dropped in different zones of the culture in the Petri dish. The antibiotic will diffuse in the area surrounding each tablet, and a disc of bacterial lysis will become visible. Since the concentration of the antibiotic was the highest at the centre, and the lowest at the edge of this zone, the diameter is suggestive for the Minimum Inhibitory Concentration (conversion of the diameter in millimeter to the MIC, in µg/ml, is based on known linear regression curves).
 * a quantitative way based on dilution: a dilution series of antibiotics is established (this is a series of reaction vials with progressively lower concentrations of antibiotic substance). The last vial in which no bacteria grow contains the antibiotic at the Minimal Inhibiting Concentration.

Once the MIC is calculated, it can be compared to know values for a given bacterium and antibiotic: e.g. a MIC > 0,06 µg/ml may be interpreted as a penicillin-resistant Streptococcus pneumoniae. Such information may be useful to the clinician, who can change the empirical treatment, to a more custom-tailored treatment that is directed only at the causative bacterium.

=Health and safety=

Waste materials from this department are likely to be highly infectious. Their safe disposal is very important and microbiology laboratories will have rigorous disposal procedures.

=Practical work=

Candidates could be given a range of ‘unknown’ cultures which they need to characterise by looking at colony morphologies, growth characteristics, media requirements, and further characterise by performing Gram staining.

Candidates could investigate the antimicrobial activity of liquid soaps, toothpastes or similar household products which make antibacterial claims.

If possible a range of different bacteria or fungi could be investigated and links made to reasons behind the different efficacies of antibiotics.

Bacterial colony shape
The shape (morphology) of bacterial colonies on an agar plate is a valuable aid to identifying the bacteria.

Bacterial cell shape
The shape (morphology) of bacterial cells under the microscope is a valuable aid to identifying the bacteria.

Gram staining

Gram staining (or Gram's method) is an empirical method of differentiating bacterial species into two large groups based on the chemical and physical properties of their cell walls.

The method is named after the inventor, the Danish scientist Hans Christian Gram (1853–1938), who developed the technique in 1884.

Uses
Gram stains are performed on body fluid or biopsy when infection is suspected. It yields results much quicker than culture, and is especially important when infection would make an important difference in the patient's treatment and prognosis; examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis. It necessitates the 24 hour staffing of microbiological laboratories in hospitals.

Mechanism
Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan which is capable of retaining the violet dye/iodine complex. Gram-negative bacteria have a thin cell wall made of a layer of peptidoglycan. In addition to an inner membrane, they also have an outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space.

The decolourising mixture causes dehydration of the multilayered peptidoglycan in the Gram-positive cell wall, thus decreasing the space between the molecules and causing the cell wall to trap the crystal violet-iodine complex within the cell. But in Gram-negative bacteria, the decolourising mixture acts as a lipid solvent and dissolves the outer membrane of the Gram-negative cell wall. The thin layer of peptidoglycan is unable to retain the crystal violet-iodine complex and the Gram-negative cell is decolourised. The decolourisation step is the crucial one, and requires some degree of skill, as Gram-positivity is not an all-or-none phenomenon.

As a rule of thumb (which has exceptions), Gram-negative bacteria are more dangerous as disease organisms, because their outer membrane is often hidden by a capsule or slime layer which hides the antigens of the cell and so acts as "camouflage" - the human body recognises a foreign body by its antigens; if they are hidden, it becomes harder for the body to detect the invader. Often the presence of a capsule will increase the virulence of a pathogen. Additionally, Gram-negative bacteria have lipopolysaccharide in their outer membrane. Lipopolysaccharide is an endotoxin which increases the severity of inflammation. This inflammation may be so severe that septic shock may occur. Gram-positive infections are generally less severe because the human body does not contain peptidoglycan, and in fact the human body produces an enzyme called lysozyme which attacks the open peptidoglycan layer of Gram-positive bacteria. Gram-positive bacteria are also much more susceptible to beta-lactam antibiotics, such as penicillin.

Procedure

 * 1) First, an inoculum is taken from a culture using an inoculation loop and put on a slide and then allowed to air dry. If the culture is solid, it is diluted by adding a drop of water or sterile saline on the slide and mixing with the loop. It is important here to take a very small inoculum so that the end result is a sparse single layer of bacteria. It is a common mistake for beginners to put far too much inoculum at this step.
 * 2) The specimen is heat-fixed by passing the slide, inoculum side up, through a Bunsen flame 1-2 times, without allowing the slide to become hot to the touch.  This prevents the bacteria from being washed away later and it also kills the bacteria
 * 3) A basic dye, crystal violet or gentian violet, is used to stain the slide. This dye is taken up by both Gram-positive and Gram-negative bacteria. Allow to stain for 1 minute. The slide should look purple to the unaided eye, and if examined microscopically at this point both Gram-positive and Gram-negative bacteria are purple. Lugol can also be used instead of crystal violet.
 * 4) Rinse off with water for a maximum of 5 seconds.
 * 5) Add iodine (Gram's iodine) solution (1% iodine, 2% potassium iodide in water) for 1 minute. This acts as a mordant and fixes the dye.
 * 6) Rinse with water.
 * 7) Apply 95% ethanol or a mixture of propanone and alcohol several times until no more colour appears to come from the sample. This washes away all the unbound basic dye, (usually crystal violet) and leaves Gram-positive organisms stained purple and Gram-negative organisms unstained (colourless).
 * 8) Rinse with water immediately to prevent over-decolourisation.
 * 9) Apply a suitable counterstain. Opinions vary as to the best choice but suitable stains include safranin or fuchsin. This stain is taken up by both Gram-positive and Gram-negative organisms, but does not alter the colour of Gram-positive organism much, as they are already purple. It does, however, make the Gram-negative organisms pinkish-red.
 * 10) Blot gently and allow to dry. Do not smear.

Interpretation
Inspect the slide under a microscope
 * Gram-positive organisms will appear blue-black or purple.
 * Gram-negative organisms will appear red or pink.

Organisms that cannot reliably be differentiated by this staining technique are said to be Gram-variable

=See also=
 * Infection
 * Microbiology
 * List of infectious diseases
 * Copenhagen Consensus
 * Important publications in infectious disease
 * Big killer
 * Staining

=References=
 * "MICROBIOLOGY: Classroom Assignment #2", a Portable Document Format computer file.
 * H. Krauss, A. Weber, M. Appel, B. Enders, A. v. Graevenitz, H. D. Isenberg, H. G. Schiefer, W. Slenczka, H. Zahner: Zoonoses. Infectious Diseases Transmissible from Animals to Humans. 3rd Edition, 456 pages. ASM Press. American Society for Microbiology, Washington DC., USA. 2003. ISBN 1-55581-236-8
 * Gram HC. Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpreparäten. Fortschr Med 1884;2:185-89.