Gram stain

The Gram staining method is named after the Danish bacteriologist Hans Christian Gram (1853 –1938) who originally devised it in 1882 (but published in 1884), to discriminate between pneumococci and Klebsiella pneumoniae bacteria in lung tissue. It is a differential staining method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. This reaction divides the eubacteria into two fundamental groups according to their stainability and is one of the basic foundations on which bacterial identification is built. Gram staining is not used to classify archaea, since these microorganisms give very variable responses.

Gram staining consists of four components:
Primary stain (Crystal violet, methyl violet or Gentian violet)
Mordant (Gram's Iodine)
Decolourizer (ethyl alcohol, acetone or 1:1 ethanol-acetone mixture)
Counterstain (Dilute carbol fuchsin, safranin or neutral red)

The original description of staining technique by Christian Gram in a publication titled "The differential staining of Schizomycetes in tissue sections and in dried preparations" in Fortschitte der Medicin; 1884, Vol. 2, pages 185-189 was slightly different from what we use today. The primary stain used was aniline gentian violet, mordant was Lugol's iodine (iodine-potassium iodide in water), decolorizer was absolute alcohol and bismark brown was the counterstain.

Procedure:

The smear on a glass slide is covered with few drops of one of the primary stains. Gentian violet is a mixture of methyl violet and crystal violet. The primary stain renders all the bacteria uniformly violet. After a minute of exposure to the staining solution, the slide is washed in water.

The smear is treated with few drop of Gram's Iodine and allowed to act for a minute. This results in formation of a dye-iodine complex in the cytoplasm. Gram's iodine serves as a mordant.

The slide is again washed in water and then decolorized in absolute ethyl alcohol or acetone. A mixture of ecetone-ethyl alcohol (1:1) can also be used for decolorization. The process of decolorization is fairly quick and should not exceed 30 seconds for thin smears. Acetone is a potent decolorizer and when used alone can decolorize the smear in 2-3 seconds. A mixture of ethanol and acetone acts more slowly than pure acetone. Decolorization is the most crucial part of Gram staining and errors can occur here. Prolonged decolorization can lead to over-decolorized smear and a very short decolorization period may lead to under-decolorized smear.

After the smear is decolorized, it is washed in water without any delay. The smear is finally treated with few drops of counterstain such as dilute carbol fuchsin, neutral red or safranin.

The slide is washed in water; excess water is removed using a blotting paper, dried in air and heat fixed before observing under microscope.

From bacteriology

Those bacteria that hold on to primary dye-iodine complex and remain violet are called Gram positive and those which get decolorized and subsequently take up counterstain (pink/red) are called Gram negative.

Basic fuchsin (present in dilute carbol fuchsin) stains many Gram negative bacteria more intensely than does safranin, making them easier to see. Some bacteria which are poorly stained by safranin, such as Haemophilus spp., Legionella spp., and some anaerobic bacteria, are readily stained by basic fuchsin.

In order to ascertain if the staining procedure was satisfactorily conducted, a control smear of known Gram positive organism (e.g., Staphylococcus aureus) and a known gram negative organism (Escherichia coli) must be stained simultaneously. While the fibrin in a clinical specimen may appear gram positive, the pus cells and epithelial cells are always gram negative.

Mechanism of Gram reaction:

Various theories have been proposed to explain why some bacteria retain the dye and some don't. Theories such as differences in cytoplasmic pH (2 in case of Gram positive bacteria and 3 in case of Gram negative bacteria), and presence of Magnesium ribonucleate in Gram positive bacteria and its absence in Gram negative bacteria have not received widespread acceptance. The thickness of Gram positive cell wall and presence of more lipids in Gram negative cell walls have been more acceptable reasons for Gram stain reactions.

It is believed that the positively charged crystal violet pass through the cell wall and cell membrane and binds to negatively charged components inside the cell. Addition of negatively charged iodine (in the mordant) binds to the positively charged dye and forms a large dye-iodine complex within the cell. Crystal violet (hexamethyl-para-rosaniline chloride) interacts with aqueous KI-I2 via a simple anion exchange to produce a chemical precipitate. The small chloride anion is replaced by the bulkier iodide, and the complex thus formed becomes insoluble in water. During decolorization, alcohol dissolves the lipid present in the outer membrane of Gram negative bacteria and it leaches the dye-iodine complex out of the cell. A thin layer of peptidoglycan does not offer much resistance either. The dye-iodine complexes are washed from the Gram negative cell along with the outer membrane. Hence Gram negative cells readily get decolorized. On the other hand Gram positive cells become dehydrated from the ethanol treatment, closing the pores as the cell wall shrinks during dehydration. The dye-iodine complex gets trapped inside the thick peptidoglycan layer and does not get decolorized.

Limitations of Gram staining:

Some Gram-positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-variable). When over-decolorized, even Gram positive bacteria may appear pink and when under-decolorized gram negative bacteria may appear Gram positive.

The Gram reaction also depends on the age of the cell. Old cultures of Gram positive bacteria (where cell walls may be weakened) may readily get decolorized. Gram positive cells affected by cell wall active agents such as lysozyme or antibiotics may become Gram negative. Gram-positive bacteria such Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these cells. In cultures of Bacillus, and Clostridium a decrease in peptidoglycan thickness during cell growth may cause some of them to appear Gram negative.

Certain group of bacteria can display variable response to the stain, which can be due to growth stress (e.g., unsuitable nutrients, temperatures, pHs, or electrolytes) that results in a number of nonviable, gram-negative cells in a gram positive culture, but certain bacterial species are known for their gram variability even under optimal growth conditions. Some bacteria tend to appear Gram negative when grown in acidic medium.

Loss of cell walls in Gram positive bacteria may render them Gram negative (L-forms). Bacteria totally devoid of cell wall (Mycoplasma) are always Gram negative. Bacteria such as Mycobacterium that have extra waxy content in their cell wall are difficult to stain. Small and slender bacteria such as Treponema, Chlamydia, Rickettsia are often difficult to stain by Gram's method. Gram positive bacteria that have been phagocytosed by polymorphs may also appear Gram negative.

Modifications of Gram stain:

There have been several modifications of Gram's stain. These are:
1. Kopeloff and Beerman's modification: Primary stain solution consists of freshly constituted methyl violet with sodium bicarbonate in distilled water. Mordant consists of iodine dissolved in 4% NaOH solution. Decolorization is either using acetone alone or a mixture of acetone and ethanol. Basic fuchsin is used to counterstain the smear. This method may be modified to stain tissue sections.

2. Jensen's modification: This method involves use to methyl violet as primary stain, iodine and potassium iodide in water as mordant, absolute alcohol as decolorizer and neutral red as counterstain. For Neisseria spp, Sandiford's counterstain is useful.

3. Weigert's modification: This modification is particularly useful for staining tissue sections. The primary stain carbol gentian violet is prepared using saturate alcoholic solution of gentian violet and 5% phenol solution. Gram's iodine is used as a mordant and aniline-xylol is used as a decolorizer. The counterstain carmalum (carminic acid and potassium alum in water), however is used ahead of primary stain. This method may be used to stain Pneumocystis cysts.

4) Preston and Morrell's modification: The primary stain used in this modification is ammonium oxalate-crystal violet. The smear is washed in Lugol's iodine and further treated with iodine solution. The smear is decolorized using iodine-acetone decolorizer and counterstained using dilute carbol fuchsin solution. This method has been further modified to overcome the irritating iodine in aerosols by reducing the iodine concentration to one-tenth and shortening the duration of decolorization to ten seconds.

Applications of Gram staining:

Differentiation of bacteria into Gram positive and Gram negative is the first step towards classification of bacteria.
It also the first step towards identification of bacteria in cultures.
Observation of bacteria in clinical specimens provides a vital clue in the diagnosis of infectious diseases.
Useful in estimation of total count of bacteria.
Empirical choice of antibiotics can be made on the basis of Gram stain’s report.
Choice of culture media for inoculation can be made empirically based on Gram’s stain report.

Miscellanea:

Although Gram stain is useful in staining bacteria, certain fungi such as Candida and Cryptococcus are observed as Gram positive yeasts.
Half-Gram stain refers to modified staining technique, where the smear is neither decolorized nor counterstained. It is useful to stain a known Gram positive bacterium.
Rapid Gram stain refers to quickened technique where the smear is exposed to only 30 seconds instead of one minute.
In specimen such as sputum, capsulated bacteria may stand out as clear spaces between the bacterium and the pink (mucus) background.
The spores may stand out as clear, unstained region in sporing bacteria.

Bacterial culture media

Introduction

There are various reasons why bacteria have to be grown (cultured) in the laboratory on artificial culture media. One of the most important reasons being its utility in diagnosing infectious diseases. Isolating a bacterium from sites in body normally known to be sterile is an indication of its role in the disease process. Indeed, isolating an organism from the clinical specimen is the first step in proving its role as an etiologic agent. Culturing bacteria is also the initial step in studying its morphology and its identification. Bacteria have to be cultured in order to obtain antigens from developing serological assays or vaccines. Certain genetic studies and manipulations of the cells also need that bacteria be cultured in vitro. Culturing bacteria also provide a reliable way estimating their numbers (viable count). Culturing on solid media is another convenient way of separating bacteria in mixtures.

Bacteria infecting humans (commensals or pathogens) are chemoorganoheterotrophs. When culturing bacteria, it is very important to provide similar environmental and nutritional conditions that exist in its natural habitat. Hence, an artificial culture medium must provide all the nutritional components that a bacterium gets in its natural habitat. Most often, a culture medium contains water, a source of carbon & energy, source of nitrogen, trace elements and some growth factors. Besides these, optimum pH, oxygen tension and osmolarity too have to be taken into consideration.

Ingredients
Some of the ingredients of culture media include water, agar, peptone, casein hydrolysate, meat extract, yeast extract and malt extract. While tap water is suitable for culture media, it must not be used if it contains high amount of minerals. In such situations, distilled or demineralised water should be used. Peptone is a byproduct of protein (plant or animal) digestion. Proteins are often obtained from heart muscle, casein, fibrin or soya flour and is digested using proteolytic enzymes such as pepsin, trypsin or papain. The final product contains peptones, proteoses and amino acids besides a variety of inorganic salts including phosphates, potassium and magnesium. Casein hydrolysate is obtained from hydrolysis of milk protein casein using HCl or trypsin. Meat extract is obtained by hot water extraction of lean beef and then concentrated by evaporation. Meat extract contains gelatin, albumoses, peptrones, proteoses, amino acids, creatinine, purines, and accessory growth factors. Yeast extract is prepared from washed cells of bakers’ yeast and contains wide range of amino acids, growth factors and inorganic salts. Malt extract is prepared by extracting soluble materials from sprouted barley in water at 55oC and concentrated by evaporation. It contains maltose, starch, dextrin, glucose and small amounts of protein and protein breakdown products and growth factors.

Brief history
Initially, culture media were very simple; Louis Pasteur used simple broths made up of urine or meat extracts. Robert Koch realized the importance of solid media and used potato pieces to grow bacteria. It was on the suggestion of Fannie Eilshemius, wife of Walther Hesse (who was an assistant to Robert Koch) that agar was used to solidify culture media. Before the use of agar, attempts were made to use gelatin as solidifying agent. Gelatin had some inherent problems; it existed as liquid at normal incubating temperatures (35-37oC) and was digested by certain bacteria.

Classification
Bacterial culture media can be classified in at least three ways; Based on consistency, based on nutritional component and based on its functional use.
Classification based on consistency:
Culture media are liquid, semi-solid or solid. Liquid media are sometimes referred as “broths” (e.g nutrient broth).
Liquid media are available for use in test-tubes, bottles or flasks. In liquid medium, bacteria grow uniformly producing general turbidity. Certain aerobic bacteria and those containing fimbriae (Vibrio & Bacillus) are known to grow as a thin film called ‘surface pellicle’ on the surface of undisturbed broth. Bacillus anthracis is known to produce stalactite growth on ghee containing broth. Sometimes the initial turbidity may be followed by clearing due to autolysis, which is seen in penumococci. Long chains of Streptococci when grown in liquid media tend to entangle and settle to the bottom forming granular deposits but with a clear medium. Culturing bacteria in liquid media has some drawbacks. Properties of bacteria are not visible in liquid media and presence of more than one type of bacteria can not be detected. Liquid media tend to be used when a large number of bacteria have to be grown. Culture media are suitable to grow bacteria when the numbers in the inoculum is suspected to be low. Inoculating in the liquid medium also helps to dilute any inhibitors of bacterial growth. This is the practical approach in blood cultures. Culturing in liquid medium can be used to obtain viable count (dilution methods).

Solid media:
Any liquid medium can be rendered by the addition of certain solidifying agents. Agar agar (simply called agar) is the most commonly used solidifying agent. The word "agar" comes from the Malay word agar agar (meaning jelly). It is also known as kanten, China grass, or Japanese isinglass. Agar is chiefly used as an ingredient in desserts throughout Japan. It is an unbranched polysaccharide obtained from the cell membranes of some species of red algae such as the genera Gelidium and Gracilaria, or seaweed (Sphaerococcus euchema). Commercially it is derived primarily from Gelidium amansii. Agar is composed of two long-chain polysaccharides (70% agarose and 30% agarapectin). It melts at 95oC (sol) and solidifies at 42oC (gel), doesn’t contribute any nutritive property, it is not hydrolysed by most bacteria and is usually free from growth promoting or growth retarding substances. However, it may be a source of calcium & organic ions. Most commonly, it is used at concentration of 1-3% to make a solid agar medium. New Zealand agar has more gelling capacity than the Japanese agar. Agar is available as fibres (shreds) or as powders.

For preparing agar in Petri plates, 3% agar (by weight) is added to the broth and autoclaved, when the medium is at ~50oC, it is poured on to sterile Petri plates and allowed to set. For preparing agar containing media in test-tubes, the culture medium is mixed with 3% agar and heated with stirring to melt. This ensures that all the tubes get equal amounts of agar. These tubes can then be sterilized by autoclaving.

Semi-solid media
Reducing the amount of agar to 0.2-0.5% renders a medium semi-solid. Such media are fairly soft and are useful in demonstrating bacterial motility and separating motile from non-motile strains (U-tube and Cragie’s tube). Certain transport media such as Stuart’s and Amies media are semi-solid in consistency. Hugh & Leifson’s oxidation fermentation test medium as well as mannitol motility medium are also semi-solid.

Biphasic media
Sometimes, a culture system comprises of both liquid and solid medium in the same bottle. This is known as biphasic medium (Castaneda system for blood culture). The inoculum is added to the liquid medium and when subcultures are to be made, the bottle is simply tilted to allow the liquid to flow over the solid medium. This obviates the need for frequent opening of the culture bottle to subculture.
Biphasic medium

Other solidifying agents
Besides agar, egg yolk and serum too can be used to solidify culture media. While serum and egg yolk are normally liquid, they can be rendered solid by coagulation using heat. Serum containing medium such as Loeffler’s serum slope and egg containing media such as Lowenstein Jensen medium and Dorset egg medium are solidified as well as disinfected by a process of inspissation.

Classification based on nutritional component:
Media can be classified as simple, complex and synthetic (or defined). While most of the nutritional components are constant across various media, some bacteria need extra nutrients. Those bacteria that are able to grow with minimal requirements are said to non-fastidious and those that require extra nutrients are said to be fastidious. Simple media such as peptone water, nutrient agar can support most non-fastidious bacteria. Complex media such as blood agar have ingredients whose exact components are difficult to estimate. Synthetic or defined media such as Davis & Mingioli medium are specially prepared media for research purposes where the composition of every component is well known.

Classification based on functional use or application:
These include basal media, enriched media, selective/enrichment media, indicator/differential media, transport media and holding media.
Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient broth and nutrient agar considered basal medium
Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched media. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Blood agar, chocolate agar, Loeffler’s serum slope etc are few of the enriched media.
Blood agar is preparing by adding 5-10% (by volume) to a basal medium such as nutrient agar or other blood agar bases. Since blood can not be sterilized, it has to be collected aseptically from the animal. Animals have to be bled and the blood is collected in sterile containers with anticoagulant or glass beads. While sheep blood is preferred, blood from rabbit, horse and ox can also be collected. Human blood must be avoided since it may contain inhibitory substances including antibiotics. After the blood agar base is autoclaved, blood is added to the medium at temperature just above the solidifying point of agar. The mixture is then poured on to the plates and allowed to solidify. Blood agar is useful in demonstrating hemolytic properties of certain bacteria. Two major types of hemolysis are often seen on blood agar; beta and alpha hemolysis. Beta hemolysis is the complete lysis of RBC resulting in clearing around the colonies whereas alpha hemolysis is the partial lysis of RBC resulting in greenish discolouration around the colonies. Gamma hemolysis is a misnomer and it indicates non-hemolytic colonies. Chocolate agar is also known as heated blood agar or lysed blood agar. The procedure is similar to that of blood agar preparation except that the blood is added while the molten blood agar base is still hot. This lyses the blood cells and releases their contents into the medium. This process turns the medium brown, hence the name. This medium is especially useful in growing Hemophilus and Neisseria.
Serum for medium can be obtained from animal blood but must be filtered through membrane or seitz filter before use.

Selective and enrichment media are designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogen from a mixture of bacteria. While selective media are agar based, enrichment media are liquid in consistency. Both these media serve the same purpose. Any agar media can be made selective by addition of certain inhibitory agents that don’t affect the pathogen. Various approaches to make a medium selective include addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these. Thayer Martin Agar used to recover N.gonorrhoeae contains Vancomycin, Colistin and Nystatin. Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contain 10% NaCl. Potassium tellurite medium used to recover C.diphtheriae contains 0.04% Potassium tellurite. McConkey’s Agar used for Enterobacteriaceae members contains Bile salt that inhibits most gram positive bacteria. Pseudosel Agar (Cetrimide Agar) used to recover P.aeruginosa contains cetrimide. Crystal Violet Blood Agar used to recover S.pyogenes contains 0.0002% crystal violet. Lowenstein Jensen Medium used to recover M.tuberculosis is made selective by incorporating Malachite green. Wilson & Blair’s Agar for recovering S.typhi is rendered selective by the addition of dye Brilliant green. Selective media such as TCBS Agar and Monsur’s Tellurite Taurocholate Gelatin Agar used for isolating V. cholerae from fecal specimens have elevated pH (8.5-5.6), which inhibits most other bacteria.

Enrichment media are liquid media that also serves to inhibit commensals in the clinical specimen. Selenite F broth, tetrathionate broth and alkaline peptone water are used to recover pathogens from fecal specimens.

Differential/Indicator media:
Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony colour. Various approaches include incorporation of dyes, metabolic substrates etc, so that those bacteria that utilize them appear as differently coloured colonies. Such media are called differential media or indicator media. When a particular carbohydrate is incorporated into a medium and a mixture of bacteria inoculated on it, only that bacterium that can ferment it produces acid. This change in pH is detected by using a pH indicator incorporated in the medium and the bacterium that can ferment the sugar appears in a different colour. This approach is used in MacConkey’s agar, CLED agar, TCBS agar, XLD agar etc. MacConkey’s agar is the most commonly used media to culture and identify gram negative bacilli (especially enterobacteriaceae members). It contains bile salts (selective agent), lactose (sugar), peptone and neutral red (pH indicator), agar and water. Those bacteria that can ferment lactose produce pink coloured colonies where non-lactose fermenting colonies produce colourless colonies. Similarly, Vibrio cholerae produces yellow coloured colonies on sucrose containing TCBS medium.
Reduction of potassium tellurite to metallic tellurium by Corynebacterium diphtheriae results in production of black coloured colonies on PT agar. Production of H2S by Salmonella typhi results in production of black coloured colonies on Wilson & Blair’s medium. Enterococcus fecalis produces black coloured colonies on bile esculin agar due to reduction of esculin to esculetin. Detection of hemolysis on blood agar can be considered as an indicator property of Blood agar.

Transport media:
Clinical specimens must be transported to the laboratory immediately after collection to prevent overgrowth of contaminating organisms or commensals. This can be achieved by using transport media. Such media prevent drying (desiccation) of specimen, maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria. Some of these media (Stuart’s & Amie’s) are semi-solid in consistency. Addition of charcoal serves to neutralize inhibitory factors. Cary Blair medium and Venkatraman Ramakrishnan medium are used to transport feces from suspected cholera patients. Sach’s buffered glycerol saline is used to transport feces from patients suspected to be suffering from bacillary dysentery. Pike’s medium is used to transport streptococci from throat specimens.

Anaerobic media:
Anaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation –reduction potential and extra nutrients.
Media for anaerobes may have to be supplemented with nutrients like hemin and vitamin K. Such media may also have to be reduced by physical or chemical means. Boiling the medium serves to expel any dissolved oxygen. Addition of 1% glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine or red hot iron filings can render a medium reduced. Robertson cooked meat that is commonly used to grow Clostridium spps medium contain a 2.5 cm column of bullock heart meat and 15 ml of nutrient broth. Before use the medium must be boiled in water bath to expel any dissolved oxygen and then sealed with sterile liquid paraffin. Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast extract and casein hydrolysate. Methylene blue or resazurin is an oxidation-reduction potential indicator that is incorporated in the medium. Under reduced condition, methylene blue is colourless.

Preparation and storage:
Care must be taken to adjust the pH of the medium before autoclaving. Various pH indicators that are in use include phenol red, neutral red, bromothymol blue, bromocresol purple etc. Dehydrated media are commercially available and must be reconstituted as per manufacturers’ recommendation. Most culture media are sterililized by autoclaving. Certain media that contain heat labile components like glucose, antibiotics, urea, serum, blood are not autoclaved. These components are filtered and may be added separately after the medium is autoclaved. Certain highly selective media such as Wilson and Blair’s medium and TCBS agar need not be sterilized. It is imperative that a representation from each lot be tested for performance and contamination before use. Once prepared, media may be held at 4-5oC in the refrigerator for 1-2 weeks. Certain liquid media in screw capped bottles or tubes or cotton plugged can be held at room temperature for weeks.

Monoclonal antibody production by hybridoma technique explained

I shall explain the hybridoma technique used in production of monoclonal antibodies in a simplified way.

An antigen (unless it is a small peptide) is a complex molecule with several antigenic determinants (or epitopes). When the immune system encounters such an antigen, it is usually processed to result in several fragments. Humoral (antibody-mediated) response may occur against some of these fragments. There are multiple clones of B cell, each against a specific epitope; resulting in production of antibodies against several epitopes. Such a response is said to be polyclonal. This is what that happens when our body encounters a microbial antigen following infection or immunization.

There are situations when it becomes necessary to have antibodies against a single antigenic determinant produced by a single clone of B cell. Such a response is said to be monoclonal. In order to produce monoclonal antibody, it is necessary to possess a purified antigen.

Hybridoma technique was developed by Georges Kolher and Cesar Milstein in 1975, for which they were awarded the Nobel Prize. The chief participants of this technique are the B cells and the myeloma cells. The B cells are obtained from the mouse which has been immunized with the antigen of choice. Myeloma cells are malignant B cells that are immortal and multiply continuously. Myeloma cells that have lost the ability to produce antibodies are chosen for this technique. In addition, these cells lack the ability to produce hypoxanthine-guanine phosphoribosyl transferase (HGPRT-) and thymidine kinase (TK-) enzymes through an induced mutation. Normal B cells have functional enzymes (HGPRT+ and TK+) and are able to produce antibodies. However, their life span is not beyond two weeks. Hybridoma technique involves physical fusion of both these cells so that the resulting hybrid (called hybridoma) has the features of both these cell types.

The first step towards the production of monoclonal antibodies is the immunization of the animal by antigen of choice. Mouse is the commonly used animal, but rat or hamster too can be used. Following repeated immunization (booster doses) the blood of the animal is tested for antibodies against the immunized antigen. Once it is determined that the animal has produced sufficient antibodies, it is killed and its spleen removed. The spleen is rich in B cells and would contain B cells specific to the immunized antigen among B cells of other specificities. The B-cells are separated from other cells and cultured. They are then mixed with cultured myeloma cells and allowed to fuse. Fusions of the cells are aided by polyethylene glycol (PEG). Not all cells fuse; present in the reaction mixture are unfused B cells, unfused myeloma cells and fused hybridoma cells. The next step involves separation of hybridoma cells from the unfused cells using a special selective medium.

In order to understand the functioning of selective medium, one must be aware of the following facts. Multiplying cells need to produce their DNA. Most cells produce their purines nucleotides and thymidylate (both precursors of DNA) utilizing tetrahydrofolates by a De-Novo pathway. This can be blocked using anti-folate drugs such as Aminopterin. The cells can then adopt Salvage pathway to synthesize DNA if hypoxanthine and thymidine are exogenously supplied. Purine nucleotides are produced from hypoxanthine using hypoxanthine-guanine phosphoribosyl transferase enzyme and thymidylate is produced from thymidine using thymidine kinase. The selective medium (HAT medium) used to select the hybridoma contains aminopterin, hypoxanthine and thymidine. Amiopterin inhibits the De-Novo pathway and presence of hypoxanthine and thymidine facilitates salvage pathway.

Normal unfused B cells can’t produce DNA by De-Novo pathway because of aminopterin but are able to undertake salvage pathway. This is because they contain functional enzymes (HGPRT+ and TK+). However, since they are mortal, they die after few multiplications. Unfused myeloma cells too can’t use De-Novo pathway because of aminopterin. They are unable to utilize the salvage pathway either because of deficient enzymes (HGPRT- and TK-). These cells die despite being immortal. However, fused hybridoma cells receive (HGPRT+ and TK+) trait from normal B cells and immortality from myeloma cells. These cells can utilize salvage pathway for DNA synthesis and yet be immortal. After two weeks, only the hybridoma cells survive in the selective medium.

The surviving hybridoma cells would have formed against different epitopes. The next step is to select the hybridoma produced against the desired antigen. The cultures are diluted to such an extent that only a single cell gets transferred to the wells of microtitre plate. The cells are allowed to multiply. These cells produce antibodies that can be readily detected in their supernatant fluids. Supernatant fluids from all the wells are tested for antibodies against the antigen of choice and the well that contains desired antibodies is selected and the rest may be discarded. Finally, a hybridoma cell producing antibodies against the epitope of choice is available.

These hybridoma cells may be lyophilized, cultured in vitro or injected intra-peritoneally into a mouse and monoclonal antibodies raised whenever required.

Humoral immunity

Humoral immunity: How does the antibody production occur?

Depending on the nature of the antigen and the physiology of the individual, the immune response of the body to a foreign antigen may include only antibody production or a cellular response (T cell) or even both. It is still not very clear how the body makes this choice. Some antigens induce only an antibody response while others induce both humoral and cell mediated immunity. Sometimes, there are no apparent responses at all, yet in some cases these responses become exaggerated and harmful.

I shall concentrate only on humoral (antibody-mediated) response. Body responds to different kinds of antigens differently even with antibody production. Antibody production may be quick in some cases and delayed in other cases. Immune system may retain memory cells towards some antigen and none in case of some other antigens. Although the primary antibody response is IgM, some people produce IgE or IgG class of antibodies. It all depends on the body’s constitution and the immune response genes.

The cells that actually produce are the B cells and their derivatives, the plasma cells. In fact, most of the antibody production is by plasma cells and B cells produce only little. Plasma cells are derived from B-cell only after appropriate stimulation and activation of B cells. B cells are predominantly located in lymphoid organs such as bone marrow, spleen and lymph nodes. They may also be found in circulation, but their numbers are less compared to T cells.

Antigens can be classified into two types (T-independent antigens and T-dependent antigens) based on their ability to induce B-cell activation.

Some antigens have multiple repeating identical units; such antigens can bind to several receptors on the B-cell surface and effectively cross link them. B lymphocytes are not perfectly spherical cells as is often depicted; in fact their surfaces have long projections and have 106 numbers of receptors on their surface. These surface receptors are either monomeric IgM or IgG immunoglobulins. Their role is to bind with the antigenic epitopes. When an antigen such as flagellin, which has multiple repeating identical units, binds to many of the surface receptors, the receptors are said to be cross-linked. This is the first signal in B-cell activation. Apart from antigens cross linking the surface receptors, B cells have another mechanism of cross-linking surface receptors that involves immunoglobulin receptor and complement receptor. If an antigen that has C3d bound to it binds to surface immunoglobulin, another receptor (CR2) can bind with the deposited C3d on the antigen. This process too can cross link the surface receptors and activate the B cell. Upon activation by these signals, B cells undergo proliferation and start producing antibodies. Since there is no involvement of T cells in antibody production, these antigens are said to be T-independent. Antibody response to non-protein antigens, such as polysaccharides and lipids do not need participation of antigen-specific helper T cells. Since T cells have role here, the antibody production is typically quicker. An unfortunate effect of this response is that the immune system does not retain any memory of antibody production. Antibodies to T-independent antigens are mainly of low-affinity and responses are simple and mainly consist of IgG and IgM.

Humoral immune response to protein antigens is more complex; these antigens are said to be T-dependent as it requires participation of T cells for B-cell activation. Since the CD4 T lymphocytes stimulate B cells, they are called helper T cells. Antigen-specific CD4+ T cells recognize a protein antigen only when it is presented by antigen presenting cell (B cell, macrophage, dendritic cell etc) along with MHC class II molecules. B cells are also capable of antigen presentation to T cells. When an antigen binds to a specific B-cell through the surface immunoglobulin receptor, it is endocytosed by a receptor mediated endocytosis process. The processed antigen is then presented on its surface coupled with MHC II molecules for recognition by specific T cell. The initial encounter between the antigen-specific T cell and B-cell occur at the interface of the primary follicles and T cell area. Following this initial binding and the binding of other co-receptors (such as B7 of B-cell and CD28 of T cell, CD40 of B-cell and CD40L of T cell), the T cell also gets activated. Cytokines such as IL-2, IL4 and IL-5, which are secreted by activated helper T cell acts on B-cell to induce B-cell proliferation. Many of these B cell clones transform into effector cells called plasma cells and start producing antibodies. Within the lymphoid tissue, antibody secreting cells are found mainly in extrafollicular sites, such as red pulp of spleen and medulla of lymph node. These cells also migrate to bone marrow at 2-3 weeks after antigen exposure, and bone marrow becomes the principal site of antibody production. Antibody secreting cells do not circulate actively. Some of the Antibody producing cells that migrate to the bone marrow and live for several years, where they continue to produce antibodies even when antigen has been eliminated
The secreted antibodies have same specificity to the surface Ig receptor that captured the antigen. The antibodies that are secreted initially are predominantly of the heavy chain µ (IgM) isotype. In response to CD40 engagement and cytokines, some of the progeny of activated B cells undergo the process of heavy chain isotype switch. This leads to production of antibodies with heavy chains of different classes such as γ (IgG), α (IgA) and ε (IgE).
Some of the antigen-activated B cells do not develop into antibody secretors. Instead, they acquire the ability to survive for long periods without antigenic stimulation. These memory cells are capable of mounting rapid antibody responses to subsequent introduction of antigen.

General Description of the Clinical Diagnosis

Basically there are three different types of clinical diagnosis identified among the five patients and they are: Urinary Tract Infection, Enterocolitis and Food Poisoning.

1) Urinary Tract Infection

Urinary tract infection (UTI) is a condition where one or more structures in the urinary tract become infected after bacteria overcome its strong natural defenses.

Among the 3 patients diagnosed with urinary tract infection, 2 were women and 1 was man.

Normally, only the lower part of the urethra is usually colonized by bacteria as the flushing action of the urinary flow protects against ascending infection. As the female urethra is short, urinary tract infection is more common in women.

Epidemiology and Pathogenesis

Dehydration, obstruction, disturbance of the smooth urinary flow or the presence of a foreign body example stone or urinary catheter, may predispose an individual to urinary tract infection. Trauma during sexual intercourse may precipitate infection in women whereas paediatric infection especially in boys are often associated with congenital abnormality e.g. ureteric reflux or urethral valves.

Possible Causes of Urinary Tract Infection

The most commonly isolated pathogens are Escherichia coli and Enterococcus spp.E.coli uses fimbriae to adhere to the urinary epithelium, thereby reducing the risk of being washed away. Infections caused by Proteus spp. Are more likely in patients who have stones as Proteus spp. have urease activity that raises urinary pH, thus encouraging stone formation. Staphylococcus saprophyticus is a common isolate from sexually active females. Many different Gram-negative organisms colonize urinary catheters, often becoming invasive infections.

Clinical Features / Signs and Symptoms

Lower urinary tract infections are characterized initially by urinary frequency, dysuria (dysuria refers to any difficulty in urination and is sometimes accompanied by pain) and suprapubic (above the pubic bone) discomfort, fever may be absent. In pyelonephritis (an ascending UTI that has reached the pyelum (pelvis) of the kidney), fever, loin (part of the body on either side of the backbone, between the ribs and pelvis) pain, renal angle tenderness and signs of septicaemia (sepsis of the bloodstream caused by bacteremia, which is the presence of bacteria in the bloodstream) may be present. In children, elderly and prenatal patients, UTI may be clinically silent. Recurrent infections can result in scarring and renal failure.

2) Enterocolitis

Enterocolitis is the inflammation of the large and small intestines.

Signs and Symptoms

Fever, abdominal swelling, nausea, vomiting and diarrhea.

There are different types of Enterocolitis and they are: salmonella enterocolitis, antibiotic-associated enterocolitis, hemorrhagic enterocolitis, pseudomembraneous enterocolitis, necrotizing enterocolitis (mostly premature babies), neutropenic enterocolitis, etc.

Since the patient is 28 years old, then necrotizing enterocolitis would be the least possible kind.

Antibiotic-associated enterocolitis is developed when treatment with antibiotics alters the bowel flora and results in diarrhea.

Hemorrhagic enterocolitis is an inflammation of the small intestine and colon, characterized by hemorrhagic breakdown of the intestinal mucosa with inflammatory-cell infiltration.

Pseudomembranous enterocolitis is an acute inflammation of the bowel mucosa with the formation of pseudomembranous plaques overlying an area of superficial ulceration, and the passage of the pseudomembranous material in the feces.

Neutropenic enterocolitis: viral diarrhea and yersinia enterocolitis are commonly found in children.

Possible causes of Enterocolitis
- Due to usage of antibiotics
Examples: Chloramphenicol, AK-Chlor, Chloroptic, Ophthochlor, Pentamycetin, Diochloram, Sopamycetin, Cetina, Clorafen, Paraxin, Quemicetina.

- Possible virus or bacteria infection
Examples: Clostridial organisms, fungi organism, campylobacter jejuni, shigella, Enteropathogenic P E.coli.

3) Food poisoning
Food poisoning is the result of eating organisms or toxins in contaminated food.
Food is an important mode of transmission of infectious diarrhea. Bacterial enters the food chain from animal infections, from poor hygiene during butchering, improper cleaning of storage and preparation areas and unclean utensils cause contamination of raw and cooked foods. Hens that are chronically colonized with salmonella produce eggs that may allow the multiplication of bacteria. Transmission of food poisoning is also facilitated where there is poor sanitation. In these situations, infections spread rapidly through the community, causing significant mortality. Cholera is capable of spreading world-wide. The temperature range in which most bacteria grow is between 40 degrees F (5 degrees C) and 140 degrees F (60 degrees C). Undercooking or improper processing of home-canned foods can cause very serious food poisoning.
Signs and Symptoms
Nausea and vomiting, diarrhea, bloody diarrhea, profuse watery diarrhea with consequent risk of dehydration, severe abdominal pain and cramps, fever, neurologic involvement such as paresthesias (a sensation of tingling, pricking, or numbness of a person's skin with no apparent long-term physical effect), motor weakness, visual disturbances, and cranial nerve palsies, autonomic symptoms such as flushing, hypotension, and anaphylaxis (a severe and rapid multi-system allergic reaction), Headache, dizziness, respiratory failure, and urticaria (a relatively common form of allergic reaction that causes raised red skin welts), myalgias (muscle pain), lymphadenopathy (swelling of one or more lymph nodes), appendicitis like presentation, oliguria (decrease production of urine), neck stiffness and meningeal signs.
Possible causes of food poisoning

- Possible bacteria infection
Examples: Staphylococcus aureus, Salmonella, Clostridium perfringens, Clostridium botulinum, Vibrio parahaemolyticus, Bacillus cereus, Listeria, Yersinia enterocolitica, Campylobacter jejuni, Enteropathogenic Escherichia coli.

Below is a link describing the different types of bacteria that might cause food poisoning.
http://aggie-horticulture.tamu.edu

PICTURES OF THE SUSPECTED MICROORGRANISMS

This picture shows Clostridium perfringens after Gram staining. It indicates that they are Gram positive bacilli.
Source
http://phil.cdc.gov/phil_images/20030124/13/PHIL_2995_lores.jpg
Staphylococcus aureus

Patient 2 - Posted by Hui Yan.

Name: Kwan Siew Lan (outpatient)
Age: 28 Gender: Female
Complaints: Diarrhoea
Diagnosis: Enterocolitis
Antibiotic treatment: Nil
Stool specimen is collected from the patient.

Possible Organisms that causes enterocolitis
1. Salmonella sp.
It is a type of enterobacteriaceae. This type of organism causes bloody diarrhea with mucus.
Key Characteristics of Salmonella sp.
- Gram-negative, motile rods
- facultative anaerobes
- Non-lactose fermenters
- Produces H2S
This organism causes Salmonella enterocolitis. Salmonella enterocolitis is an infection in the lining of the small intestine. Salmonella enterocolitis can range from mild to severe diarrheal illness. The infection is acquired through ingestion of contaminated food or water. Any food can become contaminated during preparation if conditions and equipment for food preparation are unsanitary.

2. Shigella sp.
It is a type of enterobacteriaceae. This type of organism causes watery diarrhea in later stage of disease, the stool contains blood, mucus or pus. The most common symptoms are diarrhea, fever, nausea, vomiting, stomach cramps, and straining to have a bowel movement.
Key Characteristics of Shigella sp.
- Gram-negative, non-motile and non-spore forming rods
- Does not produce H2S
- Aerobic microbe
This organism cause Shigellosis. Shigellosis or known as Shigella enterocolitis is a common cause of acute diarrhea in adults.

3. Campylobacter jejuni
It is gram-negative, curved, rod-shaped bacteria. It is motile with a single polar flagellum and it is micro-aerophillic type of microorganism.

Infection with C. jejuni usually results in enteritis (inflammation of small intestines), which is characterised by abdominal pain, diarrhea, fever, and malaise. Diarrhea can vary in severity from loose stools (watery stools) to bloody stools.

4. Entameba histolytica
Entamoeba histolytica is an anaerobic parasitic eukaryote protozoan. It infects predominantly humans and other primates. The active (trophozoite) stage exists only in the host and in fresh feces; cysts survive outside the host in water and soils and on foods, especially under moist conditions on the latter. When swallowed they cause infections by excysting (to the trophozoite stage) in the digestive tract.

5. Clostridial organisms- Clostridium difficile
Clostridial difficile is a species of bacteria of the genus Clostridium which are gram-positive, anaerobic, spore-forming rods. C. difficile is the most significant cause of pseudomembranous colitis, a severe infection of the colon, often after normal gut flora is eradicated by the use of antibiotics.

Since the patient is an outpatient and she does not have any antibiotic treatment, thus Clostridium difficile is not the cause of enterocolitis as it often is caused by antibiotic treatment and commonly, this type of infection is acquired in the hospital.

6. Giardia lamblia
Giardia lamblia (formerly also Lamblia intestinalis and also known as Giardia duodenalis and Giardia intestinalis) is a flagellated protozoan parasite that infects the gastrointestinal tract and causes giardiasis. Infection causes giardiasis, a type of gastroenteritis that manifests itself with severe diarrhea and abdominal cramps. Other symptoms can include bloating, flatulence, fatigue, nausea, vomiting and weight loss. Giardia is a major cause of intestinal disease worldwide.

7. Enteropathogenic E. Coli (EPEC)
EPEC is a gram-negative bacillus (rod-shaped organism). EPEC causes a profuse watery diarrheal disease and it is a leading cause of diarrhea in developing countries for infants.

The patient is 28 years old, thus EPEC is not the cause of enterocolitis.

Thus, possible organisms that causes enterocolitis in Patient 2 are:
- Salmonella sp.
- Shigella sp.
- Campylobacter jejuni
- Entameba Histolytica
- Giardia lamblia

Investigational tests
1. Microscopy test
A. Gram staining
This test is to find out whether the microorganism is gram positive or gram negative and the shape of the organism (coccus or bacillus). After that, relevant biochemical tests can be done to find out the identity of the suspected organisms.

B. Wet mount
This is used for checking for the presence of pus, blood and any parasites in the stool sample and motility of the microbes.

C. Stool ova and cyst
This test is to check for the presence of cyst and/or ova in the stool.

D. Parasite
This test is to check whether there is any presence of parasite in the stool.

2. Culture (Fecal) - allow the microbes to become enriched in numbers – e.g. using peptone and selenite broth.

3. Serology tests such as slide agglutination tests and Widal tests (tube agglutination) – this is to test whether the suspected microorganism reacts to certain antigens such as O, K, H and Vi antigens.

4. Kirby-Bauer test (using antibiotics discs) or known as Antibiotics Susceptibility test – this is to test whether the cause of enterocolitis is caused by the abnormal flora of certain microorganisms that are resistant to antibiotics

5. Other possible tests
A. Using Salmonella-Shigella agar – to find out whether the suspected microorganism is either Salmonella or Shigella sp.

B. Triple-Sugar Iron test – to find out whether the suspected microorganism ferments any of the 3 sugars (lactose, glucose, fructose) and whether it produce gas or not.

C. Using Campylobacter selective media at 42oC, 10% carbon dioxide, 3-4 days incubation – this is to find out whether the Campylobacter sp. is the cause of enterocolitis. This selective media only allows Campylobacter sp. to grow.

D. MacConkey agar – a selective media to grow Salmonella strains.
It is a selective and differential media used to differentiate between Gram negative bacteria while inhibiting the growth of Gram positive bacteria. The addition of bile salts and crystal violet to the agar inhibits the growth of most Gram positive bacteria, making MacConkey agar selective.

E. Xylose lysine deosycholate (XLD)agar – a selective growth media used in the isolation of Salmonella and Shigella species from clinical samples.
XLD also can be used for the culture of stool samples, and contains two indicators. It is formulated to inhibit Gram-positive bacteria, while the growth of Gram-negative bacilli is encouraged. The colonies of lactose fermenters appear yellow.

F. Blood Agar plate (BAP) - Contains mammalian blood (usually sheep), typically at a concentration of 5–10%. BAP are an enriched, differential media used to isolate fastidious organisms and detect hemolytic activity.

G. Hektoen Enteric (HE) - HE agar is designed to isolate and recover fecal bacteria belonging to the Enterobacteriaceae family. HE is particularly useful in isolating Salmonella and Shigella.

Reference
Brooks, G. F., Butel, J. S. & Ornston, L. N.; “Jawetz, Melnick & Adeberg’s Medical Microbiology”, 23rd edition, Appleton & Lange, 2004.

http://www.nlm.nih.gov