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.

Advertisements

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.

Streptococcus Identification and Work Up

Streptococcus is a genus of coccus gram positive bacteria. They typically grow in chains of pairs as cell division occurs along a single axis in this family of bacteria. Most species in the streptococcus genus are oxidase negative, catalase negative, and most are facultative anaerobes. Many streptococcal species are not intact pathogenic and are part of the commensal human microbiota of the skin, mouth, intestine and upper respiratory tract. However, certain streptococcus species are the pathogenic agent for many cases of pink eye, meningitis, bacterial pneumonia, endocarditis, erysipelas, and necrotizing fasciitis.

strep

Species of Streptococcus are classified based on their hemolytic properties. Alpha-hemolytic species cause oxidation of the iron within the red cells, giving a greenish color to appear on the blood agar. The bacteria produces hydrogen peroxide which oxidizes the hemoglobin to biliverdin. This is also referred to as incomplete hemolysis or partial hemolysis. Beta-hemolytic streptococci cause complete degradation of the red cells, appearing as wide clear areas surrounding the bacterial colonies. Gamma-hemolytic species cause no hemolysis.

The beta-hemolytic species are further classified by the Lancefield grouping. This is a serotype classification that describes the specific carbohydrate present on the bacterial cell wall. There are serotypes for Lancefield groups A-V. For times sake, the only ones that will be discussed are Group A, and Group B.

Alpha-Hemolytic Strep

Strep pneumoniae, often referred to as pneumococcus is the leading cause of bacterial pneumonia. It can also be the etiological agent for otitis media, sinusitis, meningitis and peritonitis. The viridian’s group of alpha-hemolytic streptococci are a large group of commensal organisms. They possess no Lancefield antigens (carbohydrates) and can or can not be hemolytic.

optochin_sensitive_strep_pneumo1362426051762-thumb400

Beta-Hemolytic Strep

Streptolysin which is an exotoxin is the enzyme produced by the strep species that causes complete hemolysis of the red cells in the media. There are two types of streptolysin; Streptolysin O (SLO) and Streptolysin S (SLS). Streptolysin O is an oxygen-sensitive (labile) cytotoxin which is secreted by most of the Group A Streptococcus (GAS) which interacts with cholesterol in the membrane of the red cells. Streptolysin S is an oxygen-stable cytotoxin also produced by GAS species that affects the innate immune system of the host. It works in preventing the host immune system from clearing the infection.

37444tn

Group A Strep

Group A strep, otherwise known as S. pyogenes is the causative agent for strep infections, invasive and non-invasive. The most common infection is pharyngitis, otherwise known as strep throat, impetigo, and scarlet fever. All these infections are non-invasive. Invasive GAS infections include necrotizing fasciitis, pneumonia, and bacteremia (bacteria present in the blood). Complications can arise from GAS infections. Rheumatic fever is a disease that affects the joints, kidneys, and the heart valves. It is the consequence of an untreated strep A infection. Antibodies created by the immune system cross-react with proteins in the body which causes an immune-mediated attack on the hosts own cells. Essentially an acquired auto-immune disease. GAS is also implicated in pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS). Autoimmune antibodies affect the basal ganglia causing rapid onset of psychiatric, motor, sleep and other neurological symptoms, primarily in the pediatric population.

GAS is diagnosed by either an rapid strep test or by culture.

Group B Strep

GBS, otherwise known as S. agalactiae, causes pneumonia and more importantly meningitis in neonates and the elderly. The American Congress of Obstetricians and Gynecologists now recommends that all women between 35 and 37 weeks gestation to be tested for group B strep. Those who test positive should be given prophylactic antibiotics during labor. The bacteria can cause premature rupture of the membranes during pregnancy and can colonize and cross the placenta to infect the fetus.

Laboratory diagnosis and Workup

After initial culture and preliminary identification of beta-hemolytic streptococcus is suspected there are further identification tests to make an accurate diagnosis. The first is by Lancefield antigen determination. Commercially available Lancefield antisera is used for the differentiation of beta-hemolytic species. These usually come as small kits and are directed to Lancefield groups A, B, C, F, and G. Antigen detection is demonstrated by agglutination by specific antibodies that are provided.

The PYR test is a rapid colormetric method most often used to distinguish S. pyogenes from other beta-hemolytic species. The PYR tests for the presence of the enzyme pyrrolidonyl aminopeptidase. This enzyme hydrolyzes L-pyrrolidonyl-b-naphthylamide (PYR) to B-naphthylamide, which produces a red color when a specific reagent is added. The test can be performed on paper strips that contain dried chromogenic substrates for the pyrrolidonyl aminopeptidase. S. pyogenes is PYR positive and in the case of an unknown organism displaying S. pyogenes morphology plus being PYR positive, it is acceptable to presumptively identify as S. pyogenes.

Bacitracin susceptibility is another test that can be used to differentiate S. pyogenes from other beta-hemolytic strep species. S. pyogenes has an increased susceptibility to bacitracin. A pure culture must be obtained and streaked on a sheep blood agar plate. A small disk containing 0.04 U of bacitracin is placed on the plate and incubated overnight at 35 degrees celsius in 5% CO2. A zone of inhibition surrounding the disk indicates susceptibility.

With the introduction and advancement of nucleic acid detection and serological methods in the laboratory it is now getting easier and faster to detect strep species without relying on culturing and further confirmation testing. It should be important to note that this does not replace the use of a culture or any of the further testing mentioned above. Serological testing relies on antibodies to anti-streptolysin O and anti-DNase B. The antibody levels against streptolysin O rise within one week of infection and peak around 3-6 weeks. DNase B is a nuclease among many that S. pyogenes uses to escape neutrophil extracellular nets. DNase B is specific for S. pyogenes. The antibody levels to DNase B rise post two weeks infection and reach maximum titers at around 6-8 weeks.

The Optochin test is used to differentiate alpha-hemolytic streptococci. This is either S. pneumoniae or strep viridians. S. pneumoniae species are sensitive to the chemical ethylhydrocupreine hydrochloride, otherwise known as optochin. Optochin disks, sometimes called P disks can be obtained from a commercial vendor. A pure culture is obtain and incubated to allow growth overnight. When growth has occurred, a subculture is plated and a P disk is placed on the agar and incubated overnight at 35 degrees celsius with 5% CO2. The zone of inhibition is then measured and a zone greater than 14 mm indicates sensitivity and allows for the presumptive diagnosis of pneumococci.

The bile solubility test is usually used as confirmatory test for S. pneumoniae that distinguishes it from other alpha-hemolytic species. Sodium deoxycholate (Bile) will lyse the cell wall of pneumococcal species.

-Caleb