Practical Biochemistry - Third Stage PDF

Class: Third Stage Subject: Practical Biochemistry Lecturers: Asst. Lect. Lina Abbas Hasan Asst. Lect. Doaa Nasser Wahhab Micropipettes: are essential tools in laboratories, used to measure and transfer small volumes of liquids, typically in microliters....

Class: Third Stage Subject: Practical Biochemistry Lecturers: Asst. Lect. Lina Abbas Hasan Asst. Lect. Doaa Nasser Wahhab Micropipettes: are essential tools in laboratories, used to measure and transfer small volumes of liquids, typically in microliters. Components and how they work: Components of a Micropipette 1. Plunger: The top part you press to aspirate (draw up) and dispense (release) liquid. 2. Volume Adjustment Dial: A knob or digital display that allows you to set the desired volume. 3. Tip Ejector Button: A button that expels the attached tip. 4. Tip Holder: The part where the disposable tip is attached. How Micropipettes Work 1. Set the Volume: Adjust the volume dial to the desired amount. 2. Attach the Tip: Insert a sterile tip into the tip holder. 3. Aspirate: Press the plunger down to the first stop, then slowly release it to draw up the liquid. 4. Dispense: Press the plunger down to the first stop to expel the liquid, then press further to the second stop to ensure complete delivery. 5. Eject the Tip: Press the tip ejector button to safely dispose of the tip. Types of Micropipettes  Single-Channel: Used for transferring one sample at a time. 1   Multi-Channel: Used for transferring multiple samples simultaneously (e.g., 8-, 12-, or 16-channel).  Variable Volume: Allows you to adjust the volume within a specific range.  Fixed Volume: Has a set volume that cannot be changed. How to use micropipette? 1. Select the Correct Micropipette:  Choose a micropipette with a volume range that encompasses the desired volume.  Common ranges include: o 0.1-1 µL o 1-10 µL o 10-100 µL o 100-1000 µL 2. Set the Desired Volume:  Piston Adjustment: Turn the plunger (the top part) to set the desired volume. The volume is usually displayed in a digital readout.  Dial Adjustment: For older models, rotate the dial to set the volume. 3. Attach the Tip: 2   Press the Tip Ejector: Gently press the tip ejector button (usually located at the top or side) to create a space for the tip.  Insert the Tip: Carefully insert the tip into the pipette shaft. Ensure it's securely attached. 4. Aspirate the Liquid:  Immerse the Tip: Submerge the tip into the liquid, ensuring it's below the liquid surface.  Press the First Stop: Slowly press the plunger down to the first stop (usually around the bottom 80-90% of the total stroke). This creates a vacuum, drawing liquid into the tip.  Avoid Air Bubbles: Ensure no air bubbles are present in the tip, as this can affect the accuracy of the volume. 5. Dispense the Liquid:  Position the Tip: Position the tip over the desired target.  Press the Second Stop: Slowly press the plunger down to the second stop (usually at the bottom of the stroke). This expels the liquid from the tip.  Avoid Touching Surfaces: Avoid touching the tip to any surfaces during dispensing, as this can cause liquid to adhere and affect the accuracy. 6. Eject the Tip:  Press the Ejector: After dispensing, press the tip ejector button to safely dispose of the tip 3 4 Class: Third Stage Subject: Practical Biochemistry Lecturer: Dr. Safa Wahhab Asst. Lect. Doaa Nasser Wahhab Asst. Lect. Lina Abbas Hasan The Beer law When the light of an appropriate wavelength strikes a cuvette that contains a colored sample, some of the light is absorbed and the rest is transmitted through the sample to the detector. % percent transmittance which represents the proportion of light reaches the detector. Where: lo: is the intensity of light striking the sample. It : is the intensity of transmitted light. The Beer law If the concentration of a solution is increased, the It will decrease and then % T is decreased. - The relationship between the concentration and %T is not linear, but if the logarithm of the %T is plotted against the concentration, a straight line is obtained - The term absorbance is used to represent (-log % T). A=a Beer Lambert Law A=a b c Which states that "the absorbance of a solution is directly proportional with the concentration of the dissolved substance" Where: - A is the absorbance - a is the molar absorptivity coefficient. - b is the light bath through a solution. For x substance: (1) Abs(x) = a b Conc.(x) For standard substance: (2) Abs(st) = a b Conc.(st) From 1, 2 Then we can determine the concentration of x substance by measuring both sample and standard absorbance, which can be made by spectrophotometers. A spectrophotometer: is used to measure the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution. Components of spectrophotometer Light Source Monochromators Sample Cell (cuvette) Photodetectors Meter  Class: Third Stage Subject: Practical Biochemistry Lecturer: Asst. Lect. Lina Abbas Hasan Asst. Lect. Doaa Nasser Wahhab Drawing blood and sampling: is a medical procedure that requires proper training. Performing it without proper knowledge can lead to serious complications, including infections and tissue damage. Blood samples Blood samples can be collected from blood vessels (capillaries, veins, and sometimes arteries) by trained phlebotomists or medical personnel. The sample is obtained by needle puncture and withdrawn by suction through the needle into a special collection tube. Some specimens may be obtained by a finger puncture that produces a drop of blood, such as that used for PCV. Equipment: Tourniquet Alcohol swabs Gloves Needles Blood collection tubes Bandage 1   Steps: 1. Prepare the patient: o Have the patient sit comfortably with their arm extended. o Ensure the patient is well-hydrated. 2. Gather supplies: o Assemble all necessary equipment. 3. Apply the tourniquet: o Place the tourniquet around the upper arm, about 3-4 inches above the venipuncture site. o Tighten it enough to restrict blood flow without causing pain. 4. Locate the vein: o Palpate the vein using your index and middle fingers. o Look for a prominent, rolling vein. 5. Clean the site: o Use an alcohol swab to clean the venipuncture site in a circular motion. o Allow the area to air dry. 6. Insert the needle: o Angle the needle at a 45 degree angle to the skin. o Gently insert the needle into the vein. o You should feel a "pop" as the needle enters the vein. 7. Collect the blood: o Attach the blood collection tube to the needle. o Allow the blood to fill the tube. 8. Remove the needle: o Once the tube is full, gently remove the needle. 9. Apply pressure: o Apply pressure to the venipuncture site with a cotton ball or gauze pad until bleeding stops. 10.Secure the site: Place a bandage over the site. 2    3 Class: Third Stage Subject: Practical Biochemistry Lecturers: Asst. Lect. Lina Abbas Hasan Asst. Lect. Doaa Nasser Wahhab Glucose: Glucose is the main source of energy for most living organisms, including humans. It is belongs to a category of foods called carbohydrates, which are interconnected chains of glucose and other sugars. Glucose has a simple chemical structure and is therefore absorbed by the body directly through the intestines. Glucose is a sugar with the molecular formula C6H12O6. The name "glucose. The suffix "-ose" is a chemical classifier, denoting a carbohydrate. It is also known as dextrose or grape sugar, it contains 6 carbon atoms and it is classed as a hexose, a sub-category of monosaccharides. Hypoglycemia hypoglycemia is defined as a low blood sugar (glucose) level. This reaction happens when there is not enough glucose in blood. Causes The most common causes of hypoglycemia are: 1. too much insulin 2. too much exercise 3. not enough food 1 Symptoms of Hypoglycemia: 1- sweating 2- weakness 3- hunger 4- headache Hyperglycemia: Hyperglycemia, or high blood glucose, is the condition found in individuals with diabetes, either insulin-dependent or non-insulin-dependent. The most common causes of hyperglycemia are: caused by: 1-not taking enough insulin 2. illness (such as a cold or flu) 3. eating too much 4. certain medications Symptoms of Hyperglycemia: 1- blood glucose over 240 mg/dl 2- more urine output than usual 3- increased thirst 4- dry skin and mouth 2  Diabetes mellitus Is actually a group of metabolic diseases characterized by hyperglycemia resulting from Defects in insulin secretion, insulin action, or both. Diabetes mellitus divided into two broad categories: Type 1, insulin-dependent diabetes mellitus (IDDM); Type 2, non–insulin-dependent diabetes mellitus (NIDDM) The third is gestational diabetes. The different types of glucose blood tests include: 1- fasting blood glucose. Before the test, you will need to fast (not eat or drink) for eight hours before the test. 2-Oral glucose tolerance test (OGTT). 3- Random blood sugar. 4-Hemoglobin A1c (HbA1c). 3 Practical part: Calculation: Calculate the concentration of BL in the provided fasting blood samples using the absorbance reading of standard glucose and applying the following equation: Glucose (mg/dl) = (Abs. sample/Abs. standard)* standard conc. Normal Range : 1- (fasting = 70 – 110 mg/dl 2- (random = 125 mg/dL). 3-(hemoglobin A1c level is between 4% and 5.6%.). 4 Class: Third Stage Subject: Practical Biochemistry Lecturers: Asst. Lect. Lina Abbas Hasan Asst. Lect. Doaa Nasser Wahhab Protein Proteins are large biological molecules, or macromolecules, consisting of one or more long chains of amino acid residues. Proteins perform a vast array of functions within living organisms, including catalyzing metabolic reactions, replicating DNA, responding to transporting molecules from one location to another. A total serum protein test measures the total amount of protein in the blood. It also measures the amounts of two major groups of proteins in the blood: albumin and globulin. 1-Albumin is made mainly in the liver. It helps keep the blood from leaking out of blood vessels. Albumin also helps carry some medicines and other substances through the blood and is important for tissue growth and healing. 2-Globulin is made up of different proteins called alpha, beta, and gamma types. Some globulins are made by the liver, while others are made by the immune system. Certain globulins bind with hemoglobin. Other globulins transport metals. Some types of globulin (such as alpha-1 globulin) also may be measured. 1 Structure: Biochemists often refer to four distinct aspects of a protein's structure: polyamide. by hydrogen bonds. The most common examples are the alpha helix, beta sheet and turns. tiary structure: the overall shape of a single protein molecule; the spatial relationship of the secondary structures to one another. molecules (polypeptide chains), usually called protein subunits in this context, which function as a single protein complex. High values : High albumin levels may be caused by: - Severe dehydration. High globulin levels may be caused by: -Diseases of the blood, such as multiple myeloma, lymphoma, leukemia, or hemolytic anemia. - An autoimmune disease, such as rheumatoid , autoimmune hepatitis. -Kidney disease. -Liver disease. 2  Low values: Low albumin levels may be caused by: - A poor diet (malnutrition). - Kidney disease. - Liver disease. - An autoimmune disease - Uncontrolled diabetes. - Hyperthyroidism. - Heart failure. Total serum protein level: Total protein: 6.4–8.3 (g/dL) or 64–83 grams per liter (g/L) Albumin: 3.5–5.0 g/dL or 35–50 g/L Alpha-1 globulin: 0.1–0.3 g/dL or 1–3 g/L Alpha-2 globulin: 0.6–1.0 g/dL or 6–10 g/L Beta globulin: 0.7–1.1 g/dL or 7–11 g/L 3