Use of Selective and Differential Media

There are many different types of media that are used in a microbiology laboratory. Generally speaking there are three types of media used; selective, differential, and supportive or nutritive.

Selective media are manufactured to support the growth of one type of microorganism while inhibiting the growth of another, in other words, it selectively grows one type of microorganism. It is not uncommon or this media to contain antimicrobials, dyes such as crystal violet, or even alcohol. Some of the more routine selective media types are EMB agar, mannitol salt agar, MAC, and a PEA agar.

Differential media is used to distinguish microorganisms from one another based on growth characteristics that are evident when growth is obtained. There are visible differences between microorganisms when growth is achieved. One fo the more common differential media used is the MacConkey agar. This differentiates between lactose fermenters and non-lactose fermenters.

Supportive media is used to support the growth of a wide range of microorganisms. They are typically non-selective because they want to achieve growth of a wide array of microorganisms. Some more common ones include the sheeps blood agar, as well as the chocolate agar. It has the added X and V factors to support the growth of Haemophilus influenzae.

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Cells Cells Cells!

The immune system is the host defense system against foreign pathogens. It is an extremely adept system comprised of the innate immune system and the adaptive immune system, as well as complement. For more information on those two systems as a whole, review part one of the immune system.

This part of the immune system overview will focus on the leukocytes or granulocytes of the innate immune system.

The most abundant leukocyte is the neutrophil. It comprises about 40-70% of the white cells an individual has. The maturation of the neutrophil is myeloblast, promyelocyte, myelocyte, metamyelocyte, band neutrophil, segmented neutrophil. The cytokine responsible for stimulating neutrophil production in the bone marrow is G-CSF (granulocyte colony stimulating factor). There are three pools in the bone marrow, the stem cell pool, consisting of HSCs, the proliferative pool, full of mitotic cells, and the maturation (storage) pool. Full of metamyelocytes, bands, and PMNs. During the proliferative pool stage GM-CSF, G-CSF and IL-3 are all used as growth factors to help the neutrophil differentiate and mature. Granulocyte release from the bone marrow is stimulated by G-CSF. Once in circulation neutrophils are divided randomly into either a circulating pool and a marginated pool. The neutrophils in the marginated pool are loosely localized to the walls of capillaries in tissues. Neutrophils can move freely between the two pools. Integrins and selectins are important as they allow neutrophils to marginate and allow them to move into the tissues by using diapedesis. Diapedesis is the extravasation of blood cells through intact vessel walls.

In response to inflammatory mediators and chemoattractants the neutrophil is activated and it results in reorganization of the actin cytoskeleton, membrane ruffling, adhesion and motility. In basic terms, when an infection is ongoing, the surrounding cells and tissue release cytokines and inflammatory mediators that attract neutrophils specifically to come to the site and help control the infection. Chemotaxis is the term for this. The neutrophil attaches to the substratum (endothelial surface) which allows extensions of pseudopods to attach through integrins. Contraction allows the cell body to be pulled forward (still attached). Release of the neutrophil at the back allows the cell to move forward.

Once the neutrophil has reached the site of infection it aids in fighting the infection or pathogen by phagocytosis. Phagocytosis occurs when a neutrophil surface receptor recognizes an antigen either through direct recognition, or to recognize an opsonized antigen. An opsonized antigen remember is when particular cellular processes, such as complement, present pathogenic antigens to these neutrophils to aid in phagocytosis so the neutrophil doesn’t have to search for the pathogen. With recognition comes attachment and engulfment. Cytoplasmic pseudopodia surround the particle forming a phagosome within the neutrophil cytoplasm. The formation of the phagosome allows the NADPH oxidase complex to form which leads to the generation of reactive oxygen species (ROS) such as hydrogen peroxide which is converted to hypochlorite by myeloperoxidase. (O2 dependent). A series of metabolic changes can occur like the changing of the pH and that allows primary or secondary granules within the neutrophil to release numerous bactericidal molecules into the phagosome. (O2-Independent). Bactericidal molecules aid in the killing of foreign pathogens. There is a third mechanism to which neutrophils are able to fight off foreign invader and its by using NETS. Neutrophils can generate an extracellular net that consists of chains of nucleosomes from unfolded nuclear chromatin. These structures have enzymes from neutrophil granules and can trap and kill some gram positive and gram negative bacteria, and fungi. NETs are generated at the time neutrophils die.

Monocyte development is similar to that of neutrophilic maturation because they are both derived from the granulocyte monocyte progenitor. M-CSF (macrophage colony stimulating factor) is the major cytokine responsible for the growth and differentiation of monocytes. Once in the tissue, monocytes differentiate into macrophages, depending on the tissue that the monocytes migrate too, for example, in the lymph nodes they differentiate into dendritic cells, and in the liver, they differentiate into Kupffer cells.

Eosinophils make up 1-3% of the cells in the bone marrow. Eosinophil granules are full of synthesized proteins, cytokines, chemokines, growth factors and cationic proteins. Degranulation can occur in multiple ways; by classic exocytosis, granules move to and fuse with the plasma membrane and the granules secrete into the ECS (Extracellular Space). By compound exocytosis, the granules fuse in the cytoplasm before moving to the plasma membrane. Piecemeal degranulation is when vesicles remove specific proteins from the secondary granules and then migrate to the plasma membrane and then emptying into the ECS. Eosinophils play a role in immune regulation. Eosinophils secrete major basic protein (MBP) which is the cause of mast cell degranulation and cytokine production. Eosinophils are implicated in both type 1 and type 2 immune response, primarily being infectious diseases. Eosinophils are primarily implicated in parasitic infections and are the hallmark characteristic of helminth infections. They help drive antibody production and suppress phagocytosis by secreting arylsufatase which inactivated leukotrienes and secrete antihistamine which counteracts the action of mast cells and basophils.

Basophils and Mast cells are usually grouped together, although basophils are a true WBC because they mature in the bone marrow and circulate in the blood with granules. Mast cell precursors leave the bone marrow and migrate to a tissue where they mature. Basophils and mast cells have membrane bound IgE on their surface. When activated by an antigen causes degranulation (histamine and heparin, which leads to an inflammation causing vasodilation and edema. They also secrete cytokines that activate B and T cells. Basophils are capable of releasing large quantities of subtype 2 helper T cell cytokines such as IL-4, and IL-13 that regulate the TH2 immune response. Mast cells function in chronic allergic reactions, Basophils are the initiators of allergic inflammation through the release of preformed cytokines. Basophils can play a rule in angiogenesis through the release of VEGF and its receptors.

To recap everything that has been learned with this article; neutrophils are the most abundant leukocyte encountered and play a huge role in the innate immune system. They are often the first to a site of infection or inflammation and use multiple mechanics of phagocytosis to control the situation. Monocytes circulate and settle into a tissue where they become resident macrophages. Macrophages also phagocytize foreign antigens when it comes into contact with the tissue they reside in. Eosinophils are active in infections, particularly of parasitic origin. Eosinophilia is a common finding in helminth infections. Basophils and mast cells work in conjunction with IgE to mediate hypersensitivity allergic reactions. They are the ones to thank for making your nose run.

The Pseudohyponatremia Debacle

Measurement of electrolytes in a sample of plasma is probably one of the most common tests that is performed. Every patient who gets blood drawn will also get a Chem 7 or CP13 which will include an analysis of electrolytes. There are two different technological approaches to measuring electrolytes, depending on what kind of chemistry analyzer is employed. Some use an indirect ISE and some are centered on direct ISE.

When a whole blood sample is used it is typically centrifuged beforehand to separate the sample into its plasma and red cell layers. Measurement of the electrolytes will involve using the plasma portion of the sample. Plasma consists of 93% water and approximately 7% of solid constituents, mainly proteins and lipids. The electrolytes being measured are somewhere in the water portion the plasma.

The two different methods; indirect and direct ISE differ in that direct ISE is able to respond to the electrolyte concentration within the plasma water while the indirect measures the electrolyte concentration by volume of total plasma. Total plasma including the 93% water content and 7% protein and lipid portion. That is an important distinction because the distribution of the water and protein content of plasma is important. The ratio of water/protein content is not always going to be 93/7. Depending on the patient that ratio can be skewed and inaccurate results can be reported.

So lets recap, the direct ISE measures the electrolytes using the water content of plasma, while the indirect ISE measures the electrolytes using the ratio of water/protein in whole plasma.

Indirect ISE

Indirect ISE measures electrolytes using a total plasma sample that has been diluted with diluent. This requires that the whole blood sample has been centrifuged and separated. The method measures the mean concentration in the entire plasma; water and protein content of plasma. The concentration is then multiplied by the dilution factor.

Pseudohyponatremia

Variation of the content of proteins and lipids from a normal with cause an error in the reported electrolyte results when using an indirect ISE. In particular, when measuring sodium (Na+). Dilution, as needed in the indirect ISE involves taking a pre-determined volume of the “total” sample, not only the water content of the plasma, and adding to a diluent. The total number of measurable ions in the sample is expressed as the average concentration in the total volume of the original sample, because of this the reported values depend on the protein and lipid content of the samples. A patient with hyperlipidemia will skew the 93/7 ratio which will cause falsely decreased levels of electrolytes, most often sodium. If the low sodium level is due to lipids, its possible to centrifuge to clarify the sample to get a more accurate result. Sometime the best thing to do is to rerun the sample using direct ISE. If its possible to get a whole blood sample then use point-of-care analyzers, like an i-STAT, or some blood gas analyzers (ABG) which can give a more accurate result.

Direct ISE

Direct ISE measures electrolytes using non-diluted whole blood or a plasma sample. The actual measurement that is gained is based on the water content of the plasma. It measures the electrolyte activity in the plasma water. The electrochemical activity of the ions in the water is converted to the readout concentration by a fixed multiplier that is ion-specific. The use of the fixed factor reflects the actual, clinically significant result, irrespective of the level of proteins or lipids within the solid phase of plasma.