Triple Sugar Iron Agar (TSI)

The TSI is a multiple test medium. Its a slanted medium with a deep butt that is used to further investigate Gram-negative microorganisms. It differentiates the microbes by their ability to ferment glucose, lactose and/or sucrose with or without the production of gas and production of hydrogen sulfide.

The TSI medium contains three carbohydrates; 1.0% lactose, 1.0% sucrose and 0.1% glucose. Phenol red is added as a pH indicator. Ferric ammonium citrate and sodium thiosulfate are added as indicators for the production hydrogen sulfide.

There are multiple reactions that can be observed;

A reaction of alkaline/no change is denoted K/NC and it means that the organism can only catabolize peptones aerobically, hence only the slant exhibited a color change, usually a red/orange color. This means that no carbohydrates were utilized.

When the slant is alkaline after 18-24 hours of incubation it means that there was rapid depletion of glucose and there is a subsequent reliance on peptides for nutrients. This occurs because the concentration glucose is so low and therefore it is consumed quickly. Catabolism of peptones results in the release of ammonia (NH3) which yields an alkaline pH. The butt of the medium remains acid because the degradation of peptones occurs aerobically (i.e. in the slant).

Some organisms have the ability to ferment lactose and/or sucrose with glucose for their nutrients. This results in an acid slant and acid butt reaction denoted A/A and a color change of yellow/yellow will appear. Because the concentrations of lactose and sucrose are 10x the amount of glucose, therefore a large amount of acid is produced.

A TSI medium also is used to determine whether or not a microorganism can produce carbon dioxide and hydrogen gases from the fermentation of the carbohydrates present. Gas production is seen when a bubble forms, which splits the medium. A clear disc shaped area is seen within the medium.

Ferric ammonium citrate and sodium thiosulfate are both indicators that are added to view the presence of H2S hydrogen sulfide. A microorganism in an acidic environment acts on the sodium thiosulfate to produce H2S gas. H2S reacts with ferric ions to produce ions that produce ferrous sulfide which is an insoluble black precipitate.

It should be noted that the black precipitate of ferrous sulfide that indicates H2S production may mask an acidic condition in the butt of the tube. Since H2S is only produced under acidic conditions, when the butt of the tube is black, an acid butt exists as well even without the presence of the yellow color.

TSI Results K/K K/A A/A +g A/A +g,+H2S A/K +g, +H2S

-Caleb

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The Gram Stain

Gram staining is the first step and common technique used to differentiate Gram-positive bacteria and Gram-negative bacteria. Bacterial cell walls contain the constituent peptidoglycan.

Gram_stain_01

In the photo above you’ll see Staphylococcus aureus gram positive stained purple and Escherichia coli gram-negative stained pink.

Bacterial cell walls lack membranes around their organelles. A major component of a prokaryotic cell wall is the structure of peptidoglycan. The peptidoglycan gives the cell shape and surrounds and surrounds the cytoplasmic membrane. Peptidoglycan consists of a polymer of disaccharides (glycan) which is cross-linked by short chains of amino acid monomers. The backbone of the peptidoglycan molecule consists of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) that are connected via peptide bridges. The NAM molecule varies slightly between bacterial species. The peptidoglycan molecules are transported across the cytoplasmic membrane by bactoprenol; a carrier molecule. The peptidoglycan provides receptor sites for viruses and antibiotics.

In Gram-Negative bacteria the cell wall is composed of a single layer of peptidoglycan surrounded by a membrane structure called an outer membrane. The outer membrane contains a unique lipopolysaccharide (LPS) component. The LPS is an endotoxin that generates a strong immune response when come into contact with. The gram-negative bacteria do not retain the crystal violet which stains the peptidoglycan, but are able to retain he safranin which is a counterstain added after the crystal violet. The safranin is responsible for the red or pink color that gram-negative bacteria appear under the microscope.

Gram-positive bacteria are stained dark blue or purple due to the thick layer of peptidoglycan which retains the crystal violet. Gram-positive bacteria lack the LPS that gram-negative have, but contain a group of molecules called teichoic acids. Teichoic acids give the bacteria an overall negative charge due to the presence of phosphodiester bonds between the monomers. The primary function is unknown, but they are believed to serve as a means of adherence for the bacteria.

The process of gram staining is quite simple;

First make a slide of cell sample that needs to be stained. Heat fix the slide by carefully passing it through a Bunsen burner a few times. Then add the primary stain crystal violet and let it sit for one minute. After one minute rinse the remaining stain off the slide. The next step is to grams iodine for one minute. The Gram’s iodine fixes the crystal violet to the bacterial cell wall. After the Gram’s iodine rinse the sample with acetone or alcohol and rinse with water. The alcohol acts to decolorize the sample in the case of a Gram-negative bacteria, however its important to be careful because if the alcohol remains on the sample for too long, it may decolorize Gram-positive cells. The last step is to add the secondary stain called the safranin and let sit for one minute. After one minute, wash the stain off and let dry.

Gram staining is an important step in identify cultures and often the first step in the process in differentiating whether the pathogen is gram-negative or gram-positive.

-Caleb

No Organisms Were Recovered On Buffered Charcoal Yeast Extract Medium After 2 Days of Incubation

Serum samples collected from a patient with pneumonia demonstrate a rising antibody titer to Legionella. A bronchoalveolar lavage (BAL) specimen from this patient had a positive antigen test for Legionella but no organisms were recovered on buffered charcoal yeast extract medium after 2 days of incubation. What is the best explanation?

Legionella BCYE

First of all, Legionella is a pathogenic group of gram-negative bacteria with the chief species being Legionella pneumophilia which is the causative agent of legionnaires disease. Legionella lives within amoeba in the natural environment. Transmission occurs via inhalation of water droplets from a contaminated source. Upon inhalation the bacteria infects alveolar macrophages where it begins to replicate. Legionella pneumophilia is a non-encapsulated aerobic bacillus with a single flagellum. It is unable to hydrolase gelatin or produce urease. Legionella is a non-pigment producer and does not auto fluoresce. It requires cysteine and iron to survive, thus media used to inoculate legionella must contain those two nutrients to grow.

The pathogenesis of legionella begins after an incubation period of up to two weeks. The initial prodromal symptoms are flu-like that include fever, chills and dry-cough. Through advancement of the disease the patient will experience gastrointestinal tract and the nervous system involvement. Pneumonia is also a common occurrence. The most at risk for infection with legionella are those that are immunocompromised, and the elderly. Legionella is also a prevalent nosocomial infection that has a fatality rate of 28%.

The medium of choice for when legionella is suspected is a buffered charcoal yeast extract agar (BCYE). The BCYE is a modified medium from the pre-existing F-G agar. Yeast serves as the protein source instead of casein. Beef extractives and starch are not added in the BCYE like they are in the F-G agar. Macroscopic colonies of legionella pneumophilia are visible on the BCYE after 3 days, but media should be incubated at 35-37 degrees Celsius for at least 7 days for a definitive answer. This is compared to the F-G agar where visible colonies were present after 4 days of incubation.

To answer the original question, the best explanation as to why colonies were present on the BCYE after 2 days is because the medium was simply not incubated long enough. It should be incubated for 7 days to make a definitive diagnosis even though colonies should start to appear after 3 days. The colonies on the BCYE should appear gray-white with a textured, cut glass appearance or opal.

-Caleb

Disseminated Intravascular Coagulation

Disseminated intravascular coagulation (DIC) is a generalized activation of homeostasis secondary to a systemic disease. There are multiple different diseases that can activate different homeostatic factors that can contribute to DIC. Conditions such as physical trauma and endothelial cell damage exposing tissue factor that finds its way into circulation; or being exposed to tissue factor through vasodilation from hypovolemic shock, malignant hypertension or even heat stroke. There is a long list of secondary conditions that can set off an array of events that can lead to DIC. DIC involves all aspects of homeostasis; the vascular intima, platelets, leukocytes, coagulation, coagulation regulation, and fibrinolysis. DIC is often called a consumptive coagulopathy as it is consuming platelets at a rapid rate that form fibrin microthrombin that partially occlude small vessels. These thrombi that form are small and ineffective so systemic hemorrhage occurs which is often the first sign of DIC or one of the first signs prevalent.

To understand the pathophysiology of DIC its important to have a handle on normal primary and secondary homeostasis in the body. Normal physiological coagulation initiation begins on tissue-bearing cells such as fibroblasts and the subendothelial cells. This is called the extrinsic tenase complex which is composed of tissue factor, factor VIIa, and calcium. When tissue factor that is released from damaged subendothelial cells comes into contact with coagulation factor VII it activates it and that complex produces factors Xa, and IX and miniscual amounts of thrombin. There is also a minute amount of factor VIIa that is circulating in the blood that is resistant to breakdown from tissue factor pathway inhibitor (TFPI) and can bind to tissue factor to start coagulation. The initiation phase and the small amount of thrombin produced starts the initial fibrin formation by splitting the fibrinogen peptides A and B from fibrinogen and activates platelets through cleavage of protease activated receptors PAR-1 and Par-4, cofactors, factor Va released from the platelet alpha granules, factor VIIIa to be released by vWF, and procoagulants such as factor IX to be used further in propagation.

Coagulation Cascade

Propagation is where more than 95% of the thrombin is generated and occurs on the surface of the platelets. Initiation attracts a copious amount of platelets to adhere to the site of the injury from both the low-level thrombin released and exposed collagen. These initial platelets are sometimes called COAT-platelets or platelets partially activated by collagen and thrombin. The COAT-platelets have a higher level of procoagulant activity than platelets activated by collagen alone. These platelets also provide a surface for the intrinsic and prothrombinase tenases to form. Factors Va and VIIIa that were activated by thrombin in initiation bind to platelet surfaces and become receptors for factors IXa and Xa. Factor IXa binds to VIIIa and forms in the intrinsic complex. The intrinsic complex then activates factor Xa, which binds to factor Va that forms the prothrombinase complex. The prothrombinase complex activates prothrombin which generates thrombin. Thrombin activates factor XIII to stabilize the fibrin clot by covalently cross-linking the fibrin polymers initiated by the extrinsic tenase, binds to its cofactor thrombomodulin to activate the protein C pathway and also activates thrombin activatable fibrinolysis inhibitor (TAFI) to inhibit fibrinolysis to protect the formation of the fibrin clot.

Platelets as mentioned above have an important role in homeostasis. Platelet activation occurs once the platelet binds to collagen or the vWF that is present on the surface of the damaged endothelial cells. Adhesion occurs through the integrin GP IX V platelet receptor. Upon binding they secrete their primary granules that secretes molecules such as ADP, epinephrine, serotonin, and calcium. Calcium and other molecules like ADP activate phospholipase A2 through GCPRs otherwise known as 7 transmembrane receptors. Thomboxane A2 (TXA2) is then synthesized by thromboxane synthase in multitude of events. TXA2 generates secondary messengers DAG and IP3. DAG helps mediate actin contraction for shape conformational changes and IP3 binds to the IP3 receptors in the dense tubular system that opens calcium channels to allow release of more calcium. The activation of DAG and IP3 induces a conformational change that activates the fibrinogen receptor GP IIb/IIIa which allows adjacent platelets to aggregate and form the initial platelet plug. Platelets are also important in that they allow a surface for propagation of coagulation to occur.

With a basic background of primary and secondary homeostasis it will now be easier to understand what actually occurs during DIC. Triggering events may activate coagulation at any point in its pathway. Circulating thrombin that is released activates platelets, activates coagulation proteins that have positive feedback loops within the coagulation cascade and catalyzes fibrin formation. The fibrinolytic system enzymes such as plasminogen and TPA may become active subsequent to fibrin clot formation. Monocytes may also be induced to released tissue factor caused by inflammation in DIC. Normally thrombin cleaves fibrinogen creating fibrin monomers which spontaneously polymerize to from this insoluble gel which is strengthened through factor XIII. In DIC, a high percentage of the fibrin monomers fail to polymerize and just circulate in plasma as soluble monomers. These circulating monomers coat platelets and coagulation proteins which doesn’t allow any binding creating an anticoagulant effect. Plasmin, which is the activated form of plasminogen is a part of the fibrinolytic system. In normal homeostasis plasmin only cleaves the solid fibrin clot formed. Although in DIC, plasmin circulates in the plasma and degrades all forms of fibrin. It is because of this that fibrin degradation products, otherwise known as D-dimers become detectable in the plasma in concentrations commonly exceeding 20,000 ng/mL. The normal range for the D-dimer is 0-240 ng/mL. At the same time coagulation pathway control is lost as protein C, protein S, and anti-thrombin are consumed by the plasmin. Plasmin also digests factors V, VIII, IX, and XI. The platelets become enmeshed within the fibrin monomers and become exposed to thrombin which triggers platelet further platelet activation and consumption. Plasmin can also trigger complement which causes hemolysis and the kinin system which triggers inflammation and hypotension and as an end result shock.

It is important to diagnose DIC early and as a physician be aware of the early signs. A lot of times the symptoms of DIC are masked by the underlying disease and may be chronic or acute. The initial laboratory testing includes a platelet count, blood film examination, PT, aPTT, D-dimer and fibrinogen assay. The PT time is usually >14 seconds (Ref. 11-14) aPTT is usually >35 seconds (Ref. 25-35). These time intervals clue in that there is a coagulation issue as the PT tests the function of the extrinsic pathway and the aPTT tests the function of the intrinsic pathway. The platelet count is lower than 150,000 uL (Ref. 150,000-450,000 uL). The D-dimer as noted previously is significantly elevated. Although a D-dimer alone can’t diagnose DIC because a D-dimer is elevated in other conditions such as inflammation, pulmonary embolism and deep vein thrombosis. The fibrinogen levels may drop below 220 mg/dL (Ref. 220-498), but that provides little diagnostic information because in a lot of cases the fibrinogen level may not rise or may become elevated because of the level of inflammation occurring while the patient is in DIC. A peripheral blood smear confirms thrombocytopenia as well as the presence of schistocytes. Schistocytes are broken red blood cells because the microvessel walls are occluded they get shredded while passing through the blood vessels. Although not one test result can rule in DIC or rule it out, a panel of specialized tests can help in the diagnosis. It’s important to get the whole picture.

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Treatment of chronic DIC is to diagnose and treat the underlying condition. This may include surgery, anti-inflammatory agents, or antibiotics to stabilize homeostasis. Supportive therapy to maintain fluid and electrolyte balance is important in the treatment of chronic DIC. In acute DIC where there is multi organ failure from microthrombi and hemorrhagic bleeding therapies are targeted at slowing the clotting process and to replace the consumed coagulation factors and proteins. Unfractionated heparin is commonly used for its anti-thrombotic properties. Normally the aPTT is used to monitor heparin therapy, but in the case of DIC other assays must be used so it’s important to pay close attention to the patient when administering heparin as it can aggravate bleeding tendencies. Physicians may also order fresh frozen plasma (FFP), platelets, and red cell transfusions as needed. FFP will replace the coagulation factors and proteins. The platelets will correct for the thrombocytopenia and the red cells are transfused because of the resulting anemia. Cryoprecipitate can also be administered to replace the low levels of fibrinogen. A physician may use an INR to figure out the best way to treat the DIC as well as monitor the therapy. The INR or international normalized ratio is a way of standardizing the PT results, regardless of test methods and where the testing occurred. A normal INR should be between 2-3. An INR too low puts the patient as risk for blood clots, on the contrary and INR too high puts the patient as too high of a risk for bleeding. As the underlying condition begins to stabilize the DIC will begin to subside and patient will slowly recover.

There will be more covered about DIC and how it relates to different leukemias and solid tumor cancers. This article provides an overview and how it affects normal homeostasis.

-Caleb

Hemoglobin, What is it?

Function

Hemoglobins main function is to carry oxygen to the tissues and cells of the body. Hemoglobin can bind and transport four molecules of oxygen. Hemoglobin is made up of 4 polypeptide globin chains, two alpha and two beta that forms a tetramer heme group with iron located within the tetramer. Globin is the protein part of hemoglobin. The different globin chains are typically 141-146 amino acids in length and each chain is designated by a greek letter. Each chain is subdivided into eight helices designated an alphabetical letter with each helice divided by seven non-helical segments. The globin chains loop to form a cleft pocket for heme which is suspended between the E and the F helices of the polypeptide chain. The ferrous iron in each heme molecule reversibly binds to one oxygen molecule.

Heme is a four ring consisting of carbon, hydrogen and nitrogen atoms called the protoporphyrin IX. The single carbon atoms act as connecting bridges. There are alternating double bonds where the electron resonation absorbs light which is the reason why heme is colored. There are two propionic acid side chains on end rendering it polar, with the rest of the heme molecule being non-polar and hydrophobic.

Globin Synthesis

Globin is a protein so naturally translation and synthesis occurs in the ribosomes while transcription occurs in the nucleus. Transcription of the alpha globin gene which occurs on chromosome 16 produces more mRNA than the beta globin genes which is transcribed on chromosome 11. There are four alpha genes, and only two beta genes. To make up for that discrepancy, translation of the alpha globin is less efficient than that of the beta globin so there are equal amounts of both produced in Hgb A.

Heme Synthesis

Heme is synthesized in the mitochondria and cytoplasm of the bone marrow erythrocyte precursor cells. Biosynthesis begins in the mitochondria with the condensation of glycine and succinyl CoA catalyzed by aminolevulinate synthase (ALA synthase) to form ALA in the cytoplasm. Porphobilinogen synthase converts ALA to porphobilinogen (PBG). Porphobilinogen synthase is the enzyme that is inhibited by lead. PBG is then converted to hydroxymethylbilane which is further converted to uroporphyrinogen III. Uroporphyrinogen III is converted to coproporphyrinogen III. Synthesis then continues back in the mitochondria by the conversion of coproporphyrinogen III to protoporphyrinogen IX. Protoporphyrinogen IX is then converted to protoporphyrin IX by protoporphyrinogen oxidase. From there protoporphyrin IX is converted to heme in the presence of ferrous iron and ferrochelatase. Heme has a negative feedback mechanism on ALA by inhibiting the transcription of the ALA synthase enzyme.

Hemoglobin Synthesis

With the synthesis of globin and heme covered the next step is the assembly of the hemoglobin molecule as one. After the globin is released by the ribosomes, each polypeptide chain binds to a single heme molecule. An alpha globin:heme complex and a beta globin:heme polypeptide then combine to form a heterodimer. Now remember that step is repeated as there are four polypeptide chains in a hemoglobin molecule. Two heterodimers then combine to form a tetramer to complete the assembly of the hemoglobin molecule.

hhemo_rib1

Oxygen Binding

When in the deoxygenated state, the iron within the heme molecule is pulled out of plane of the heme ring. When oxygen binds it pulls the iron back into the plane of the heme ring and also causes a shape change in the polypeptide chains which causes a ripple effect among all four polypeptide chains. This phenomenon is allosteric regulation. The allosteric effect is the conformational change in the entire hemoglobin molecule caused by the binding of one oxygen molecule to one ferrous iron molecule within the heme cleft. In the deoxygenated state ionic bridges form creating a stable and rigid configuration. A single molecule of 2,3-DPG binds adjacent polypeptides to further stabilize the hemoglobin molecule. In the oxygenated state there is that allosteric effect which alters the shape of the hemoglobin molecule enough so that ionic bridges are broken which causes the globin molecules to relax and heme cleft enlarges. This allows the remaining three oxygen molecules to bind readily to the ferrous iron.

Oxygen/Hemoglobin Dissociation Curve Effects

The oxyhemoglobin is formed when oxygen binds during physiological respiration within the pulmonary capillaries. Various factors such as pH, CO2 concentration, and 2,3-DPG concentration affect the way that oxygen binds. When talking in terms of the oxygen/hemoglobin dissociation curve it is generally said that curve is sigmoidal; meaning that there is low hemoglobin affinity for oxygen at low oxygen tension and high affinity for oxygen at high oxygen tension. There are values such as the P50 which is defined in terms of the amount of oxygen needed to saturate 50% of the hemoglobin molecule. The PO2 of the lungs are ~ 100 mm/Hg so that means that hemoglobin is 100% saturated when the RBC is in the lungs. Normally a P02 of 27 mm/Hg results in 50% hemoglobin saturation. You can also have shifts to the right or left of the curve. These shifts come from the various factors that were mentioned above. Hemoglobin exists as two forms, a taut (tense) phase and a (R) form or relaxed form. Low pH, high CO2 (such as in the tissues), and high 2,3-DPG initiate a shift to the right, meaning that there is less affinity for oxygen and the oxygen is released into the tissues resulting in the taut form of hemoglobin. Conversely a high pH, low CO2 (such as in the lung capillaries), and low 2,3-DPG results in the relaxed form and a shift to the left creating a higher affinity to oxygen. The partial pressure of the system also affects the affinity to oxygen. At high PO2 levels, such as those present in the lungs, a high affinity relaxed state is favored. In a low PO2 state, such as in the tissues, the low affinity, taut form is favored. Hemoglobin needs to be able to bind oxygen and release it. There is no point in the molecule binding if there is no chance at releasing it. The sigmoidal curve causes it to be efficient at taking up oxygen in the lungs and efficient at releasing it in the tissues

oxygen-dissociation curve

Hemoglobin is an essential aspect of homeostasis for the cells. It needs to functioning and be assembled correctly to efficiently do its job. This is just a brief overview of what it does and the structure of it. More will come on the different hemoglobinopathies (qualitative) and thalassemias (quanitative).

-Caleb