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Biochemistry

Amylase kit

Amylase Kit(Direct Substrate Method):- INTENDED USE:- This diagnostic reagent kit is intended for in-vitro quantitative determination of amylase activity in human serum or plasma. PRINCIPLE Amylase catalyzes the hydrolysis of a 2-chloro-4-nitrophenyl linked substrate. The rate of hydrolysis is proportional to amylase activity in the sample. REACTION CNPG₃ + Amylase → CNP + G₃ + G₄ CONTENT Reagent-1 / Amylase Reagent MATERIALS REQUIRED BUT NOT PROVIDED Clean & dry glassware Laboratory glass pipettes / micropipettes & tips Bio-Chemistry Analyzer SAMPLES Serum free from hemolysis, heparinized plasma, or EDTA plasma. Specimen should be tested as fresh as possible. PROCEDURE Pipette into clean labeled test tubes: Component Blank Sample Working Reagent 1000 µl 1000 µl Sample — 20 µl Mix well and read the absorbance after 1, 2 & 3 minutes.Calculate the mean absorbance per minute (ΔA/min). CALCULATION OF RESULTS Amylase Activity (U/L) = ΔA/min × 3178 NORMAL VALUE Serum Amylase: Up to 100 U/L at 37°C CLINICAL SIGNIFICANCE Amylase is secreted by the pancreas into the duodenum. It catalyzes the hydrolysis of starch into sugars. Measurement of amylase activity is useful in the diagnosis of pancreatic disorders such as acute pancreatitis, pancreatic cancer, and pancreatic duct obstruction. GENERAL SYSTEM PARAMETERS Reaction Type Kinetic Wavelength 405 nm Temperature 37°C Delay 60 sec Incubation 60 sec Reaction 60 sec Sample Vol. 20 µl Reagent Vol. 1000 µl LIMITATIONS AND PRECAUTIONS   Use non-hemolyzed samples Avoid contamination Do not use turbid or lipemic samples Do not freeze reagents QUALITY CONTROL Run normal and pathological controls with each assay.

Biochemistry

SGPT

SGPT INTENDED USE:This reagent kit is intended for in-vitro quantitative determination of SGPT (ALT) activity in serum. PRINCIPLE:SGPT (ALT) catalyzes the transfer of amino group between L-alanine and α-ketoglutarate to form pyruvate and glutamate. The pyruvate formed reacts with NADH in the presence of Lactate Dehydrogenase (LDH) to form lactate and NAD. The rate of oxidation of NADH to NAD is measured as a decrease in absorbance which is proportional to the SGPT (ALT) activity in the sample. REACTION:Alanine aminotransferaseL-Alanine + α-Ketoglutarate → Pyruvate + L-Glutamate Lactate DehydrogenasePyruvate + NADH + H⁺ → Lactate + NAD⁺ CONTENTS:Reagent 1: SGPT Enzyme ReagentReagent 2: SGPT Substrate Reagent MATERIALS REQUIRED BUT NOT PROVIDED:• Clean & Dry Glassware• Micropipettes & Tips• Bio-Chemistry Analyzer SAMPLES:Serum free of hemolysis. SGPT (ALT) is reported to be stable in serum for 3 days at 2–8°C. PROCEDURE:Pipette into clean dry test tubes as (T): Addition sequence Blank Test Working Reagent 1000 µl 1000 µl Sample — 100 µl Mix well and read the initial absorbance after 1 min and repeat the absorbance reading after every 1 min. Calculate the mean absorbance change per minute (ΔA/min). CALCULATION:SGPT activity (U/L) = ΔA/min × 1746

Biochemistry

POTASSIUM

POTASSIUM (Monotest) BEACON INTENDED USE: This reagent kit is intended for the in-vitro quantitative determination of Potassium in Serum. PRINCIPLE: Potassium reacts with sodium tetraphenyl boron in a specially prepared buffer to form a colloidal suspension. The amount of turbidity produced is directly proportional to the concentration of potassium in the sample. CONTENTS: Reagent 1: Potassium Reagent Reagent 2: Potassium Standard 5 mEq/L SAMPLE COLLECTION AND PRESERVATION: Separate serum from the clot as soon as possible as potassium may leach from red blood cells which can elevate results. REAGENT PREPARATION AND STORAGE: All reagents are ready to use. Temperature: 25–30°C. PROCEDURE: Pipette into cuvettes labeled as Blank (B), Standard (S), and Test (T). Addition Sequence B S T Potassium Reagent 1.0 ml 1.0 ml 1.0 ml Standard — 20 µl — Sample — — 20 µl Mix well and incubate at RT for 5 mins. Measure the absorbance of Standard and Test against reagent blank at 630 nm. CALCULATION: Concentration of Potassium (mEq/L) = (Abs. T / Abs. S) × 5 NORMAL VALUES: 3.5 – 5.5 mEq/L Each laboratory should establish its own normal range. GENERAL SYSTEM PARAMETERS: Reaction type: End point Wavelength: 630 nm Cuvette: 1 cm Reaction temperature: Room temperature Zero setting: Reagent blank Sample volume: 10 µl Reagent volume: 1.0 ml Incubation time: 5 mins Standard concentration: 5 mEq/L REAGENT SYSTEM STABILITY: Reagents are stable until the expiration date mentioned on the label. LINEARITY: The procedure is linear up to 7 mEq/L.If values exceed this limit, dilute the sample with distilled water and multiply results with dilution factor. NOTES: As potassium is widely distributed, care should be taken to avoid contamination. All glassware should be free from alkali to avoid falsely high values. BIBLIOGRAPHY: Tietz NW, Fundamentals of Clinical Chemistry, W.B. Saunders Co., Philadelphia Henry RF et al., Clinical Chemistry Principles and Techniques David A. Sacks et al., Tietz Textbook of Clinical Chemistry Trinder, P., Ann. Clin. Biochem., 1969 (6: 159) QUALITY CONTROL: It is recommended that controls be included in each set of assays.

Biochemistry

LDL CHOLESTROL

LDL CHOLESTEROL DIRECT REAGENT KITBEACON INTENDED USE: The reagent kit is intended for the in-vitro quantitative determination of LDL Cholesterol in Serum / Plasma. PRINCIPLE: Cholesterol is released from LDL particles and enzymatically determined. Cholesterol Ester + H₂O(Cholesterol esterase) → Cholesterol + Fatty acids Cholesterol + O₂(Cholesterol oxidase) → Cholest-4-en-3-one + H₂O₂ H₂O₂ + 4-Aminoantipyrine + Phenol(Peroxidase) → Quinoneimine dye + H₂O The intensity of the colored complex formed is proportional to the LDL cholesterol concentration and is measured at 575 nm. REAGENTS: Reagent 1 (R1): Buffer Reagent 2 (R2): Enzymes Standard: LDL Cholesterol MATERIALS REQUIRED BUT NOT PROVIDED: Semi auto / Fully auto analyzer Micropipettes & Tips Incubator Distilled water SAMPLE: Serum or plasma. Avoid hemolysis. PROCEDURE (37°C): Blank Standard Sample Reagent 375 µl 375 µl 375 µl Standard — 25 µl — Sample — — 25 µl Mix and incubate for 5 minutes at 37°C.Measure absorbance at 575 nm against reagent blank. CALCULATION: LDL-C (mg/dl) = (Abs. of Test / Abs. of Standard) × Standard concentration NORMAL VALUES: < 130 mg/dl : Desirable 130–159 mg/dl : Borderline high ≥ 160 mg/dl : High risk of CHD Each laboratory should establish its own reference range. CLINICAL SIGNIFICANCE: LDL particles are lipoproteins that transport cholesterol to tissues. LDL cholesterol is considered a major risk factor for coronary heart disease and atherosclerosis. Elevated LDL levels are associated with increased risk of cardiovascular disease. PREPARATION OF REAGENT & STABILITY: Mix R1 and R2 as per instructions. Working reagent is stable for 7 days at 2–8°C. LINEARITY: The procedure is linear up to 1000 mg/dl.Samples exceeding this value should be diluted with normal saline (NaCl 0.9%) and retested. INTERFERENCES: No interference observed up to: Hemoglobin: 500 mg/dl Bilirubin (conjugated): 40 mg/dl Bilirubin (unconjugated): 40 mg/dl Ascorbic Acid: 5 mg/dl Triglycerides: 1000 mg/dl                 BIBLIOGRAPHY:- NCEP ATP III Guidelines Burtis & Ashwood, Tietz Fundamentals of Clinical Chemistry Other standard clinical chemistry references

Biochemistry

SGOT

SGOT(SERUM GLUTAMIC OXALOACITIC TRANSAMINASE) INTENDED USE:This reagent kit is intended for in vitro quantitative determination of SGOT (AST) activity in serum. PRINCIPLE:SGOT (AST) catalyzes the transfer of amino group between L-Aspartate and α-ketoglutarate to form oxaloacetate and glutamate. The oxaloacetate formed reacts with NADH in the presence of Malate Dehydrogenase to form NAD. The rate of oxidation of NADH to NAD is measured as a decrease in absorbance which is proportional to the SGOT (AST) activity in the sample. REACTION: L-Aspartate + α-Ketoglutarate—(Aspartate aminotransferase)→Oxaloacetate + Glutamate Oxaloacetate + NADH—(Malate Dehydrogenase)→L-Malate + NAD⁺ CONTENTS:Reagent 1: SGOT Enzyme ReagentReagent 2: SGOT Substrate Reagent MATERIALS REQUIRED BUT NOT PROVIDED: Clean & Dry Glassware Laboratory Glass Pipettes or Micropipettes & Tips Bio-Chemistry Analyzer SAMPLES:Serum free of hemolysis. SGOT (AST) is reported to be stable in serum for 3 days at 2–8°C. PROCEDURE:Pipette into clean dry test tube labeled as (T): Addition sequence (T) Working Reagent 1 ml Sample 100 µl Mix well and read the initial absorbance A₁ at 1 min and repeat the absorbance reading after every 1 & 2 mins. Calculate the mean absorbance change per minute (ΔA/min.). CALCULATION:SGOT activity (U/L) = ΔA/min × 1746 NORMAL VALUE:Serum: < 40 U/LEach laboratory should establish its own normal range representing patient population. CLINICAL SIGNIFICANCE:SGOT is an enzyme found mainly in heart muscle, liver cells, skeletal muscle and kidneys. Injury to these tissues results in the release of the enzyme into blood. Elevated levels are found in myocardial infarction, cardiac operations, hepatitis, cirrhosis, acute renal diseases, primary muscle diseases. Decreased levels may be found in pregnancy, beri beri and diabetic ketoacidosis. GENERAL SYSTEM PARAMETERS: Reaction Type: Kinetic (Decreasing) Wavelength: 340 nm Cuvette Temp: 37°C Delay Time: 60 sec Interval Time: 60 sec No. of Reading: 2 Reagent Volume: 1 ml Sample Volume: 100 µl Zero Setting: Deionised Water Light Path: 1 cm Factor: 1746 LINEARITY:The procedure is linear up to 300 U/L. If the activity exceeds this limit, dilute the sample with normal saline (NaCl 0.9%) and multiply result by dilution factor. QUALITY CONTROL:For accuracy it is necessary to run known controls with every assay. LIMITATION & PRECAUTIONS: Storage conditions as mentioned on the kit to be adhered. Do not freeze or expose the reagents to higher temperature as it may affect the performance of the kit. Before the assay bring all the reagents to room temperature. Avoid contamination of the reagent during assay process. Use clean glassware free from dust or debris.                          PREPARATION OF REAGENT & STABILITY: Working reagent:Mix 4 parts of Reagent 1 with 1 part of Reagent 2. Working solution is stable for 4 days at 2–8°C.

Microbiology

GRAM STAIN

GRAM STAIN :- Objective :- To differentiate bacteria into Gram-positive and Gram-negative based on the ability of their cell wall to retain the primary stain (crystal violet) after decolorization. Principle Gram-positive bacteria: Thick peptidoglycan layer → retain crystal violet–iodine complex → appear purple. Gram-negative bacteria: Thin peptidoglycan, high lipid content → lose crystal violet on decolorization → take up safranin → appear pink/red. Materials Required Clean glass slides Inoculating loop / needle Bunsen burner Staining rack Wash bottle with water Blotting paper Reagents Crystal Violet (Primary stain) Gram’s Iodine (Mordant) Decolorizer (Acetone–alcohol or 95% ethanol) Safranin (Counterstain) Procedure 1. Preparation of Smear Clean the slide and label it. Place a small drop of water on the slide. Pick a small amount of culture and spread to form a thin smear. Air dry completely. Heat fix by passing the slide over flame 2–3 times (do not overheat). ·         2. Gram Staining Steps Step Reagent Time   1 Crystal Violet 1 minute 2 Wash gently — 3 Gram’s Iodine 1 minute 4 Wash gently — 5 Decolorizer (Alcohol/Acetone) Few seconds (5–15 sec) 6 Wash immediately — 7 Safranin 30–60 seconds 8 Final wash — 9 Blot dry — Microscopic Examination Observe under oil immersion (100x) objective. Findings Gram-positive → Purple / violet Gram-negative → Pink / red Note shape: cocci, bacilli, spirilla, clusters, chains, pairs.

Biochemistry, Uncategorized

HDL CHOLESTROL

HDL CHOLESTEROL DIRECT REAGENT KIT BEACON INTENDED USE: The reagent is intended for the direct in-vitro quantitative determination of HDL cholesterol in human serum. PRINCIPLE: The direct HDL cholesterol assay is a homogeneous method for directly measuring serum HDL. Using selective detergents and enzymatic reaction, HDL cholesterol is measured without interference from LDL, VLDL and chylomicrons. The cholesterol esterase and cholesterol oxidase react with HDL cholesterol to form a colored quinoneimine dye whose intensity is proportional to the HDL cholesterol concentration. Reaction: HDL-C + Esterase → Cholesterol + Fatty acidsCholesterol + Oxidase → Cholestenone + H₂O₂H₂O₂ + Chromogen → Colored quinoneimine CONTENTS: Reagent 1: R1 Reagent 2: R2 Standard: HDL Cholesterol Calibrator MATERIALS REQUIRED BUT NOT PROVIDED: Laboratory glassware Micropipettes & tips Autoanalyzer / Semi-auto analyzer STORAGE AND STABILITY: The reagents are stable up to the expiry date stated on the label when stored at 2–8°C. Do not freeze. SAMPLES: Serum, Plasma (Heparin) PREPARATION OF REAGENTS & STABILITY: The reagent is ready to use. Calibrator: Reconstitute with distilled water. Let stand for 10 minutes, mix gently. Working reagent is stable for 7 days at 2–8°C. PROCEDURE: Blank Calibrator Sample R1 Reagent 450 µl 450 µl 450 µl Calibrator — 5 µl — Sample — — 5 µl R2 Reagent 150 µl 150 µl 150 µl Mix and incubate at 37°C for 5 minutes. Measure absorbance at 578 nm against reagent blank. CALCULATION: HDL-C    =  Abs of Sample/ Abs of Calibrator      ×Calibrator concentration NORMAL VALUE: Male: ≥ 40 mg/dL Female: ≥ 50 mg/dL (Values may vary depending on population and laboratory.) CLINICAL SIGNIFICANCE: Lipoproteins are particles with many transport fats in blood plasma. There are two groups: low density lipoprotein (LDL) cholesterol and high density lipoprotein (HDL) cholesterol. LDL cholesterol is mainly triglycerides, though LDL is also transport some amount of cholesterol. LDL causes cholesterol to be deposited in blood vessels and cause atherosclerosis. HDL cholesterol removes cholesterol from blood and transports it back to the liver for excretion. HDL is considered “good cholesterol” as higher levels are associated with lower risk of heart disease. Estimation of HDL cholesterol is useful for the risk assessment of coronary heart disease. GENERAL SYSTEM PARAMETERS: Reaction Type: End point Wavelength: 578 nm (540–620 nm) Temperature: 37°C Reaction Volume: R1 450 µl + R2 150 µl Sample Volume: 5 µl Measuring: Against blank Linearity: 5–150 mg/dL Calibration: Single point Reaction Time: 5 minutes LINEARITY: This procedure is linear up to 150 mg/dL. If values exceed this limit, dilute the sample with saline and repeat the assay. QUALITY CONTROL: Use recommended commercial quality control sera with each run. Results should fall within acceptable limits.

Microbiology

CULTURE MEDIA INTRODUCTION &PREPARATION

INTRODUCTIONOF CULTURE MEDIA Most bacteria can be cultured artificially on culture media containing required nutrients, pH and osmotic pressure. The microorganisms grow in an atmosphere and temperature most suited to their metabolic reactions. The pathogens are isolated in pure culture so that they can be identified and tested for their sensitivity to antimicrobials. The specimens are cultured in known volumes, and the number of bacterial colonies appearing after incubation can be counted. Operation room requirements and blood from blood bank are frequently checked for sterility by using pure culture methods. Vaccines and antitoxins require the growing of bacteria under controlled conditions. Stock cultures are also useful for the teaching institutes for practical training purposes. COMPOSITION OF CULTURE MEDIA The basic ingredients, which are common to the many of the frequently used media are as follows: 1.  Water: It allows fluids to enter and leave cells more readily and to enhance the chemical reactions. 2.Sodium chloride: Presence of sodium chloride maintains the isotonicity  of bacterial cells. 3.Peptones: It is a source of readily available nitrogen. 4.Buffers: They maintain a constant pH in culture media. 5.Indicators:  In culture media the indicators are useful in the detection of acid or alkali production by microorganisms. Indicators such as phenol red, methyl red and Bromocresol purple are used to adjust pH of the culture media. 6.  Solidifying agents: Agar, gelatin, egg yolks and serum are used as solidifying agents of the culture media. 7.Selective agents: These are special chemicals introduced into the culture media for inhibiting some types of bacteria, while allowing other bacteria to grow. Examples: (a) Crystal violet inhibits Staphylococci but not tubercle bacilli (b) 6.5% (w/ v) sodium chloride is inhibitory to most Streptococci but not S. faecalis. 8.   Additive for enrichment: The substances such as sheep blood, horse blood, rabbit serum or calf’s ground hearts allow fastidious organisms to grow since these organisms, may not survive in ordinary culture media. 9.Reducing substances: The substances such as thioglycollate are used to remove free oxygen from the medium for the growth of anaerobic bacteria. THE DIFFERENT TYPES OF CULTURE MEDIA Basic Media These support the growth of microorganisms that do not have special nutritional requirements. They are often used (a) To maintain stock cultures of control strains of bacteria and (b) For subculturing pathogens from selective media prior to performing biochemical and serological dentification tests. Examples: (1) Nutrient agar (2) Nutrient broth. Enriched Media These are enriched with (a) Whole blood (b) Lysed blood (c) Serum (d) Extra peptones and (e) Vitamins to support the growth of particular pathogens such as Hemophilus influenzae, Neisseria and Streptococcus species. Examples: (I) Blood agar (II) Tryptone soya media. Selective Media These media contain substances that accelerate the growth of required pathogens only and prevent or slow down the growth of other microorganisms. Example: XLD agar: It is used for the growth of Salmonellae and Shigellae. The bile salts present in this media inhibit the growth of many fecal commensals Differential (Indicator) Media These contain indicators, dyes or other substances which help to differentiate microorganisms. Example: TCBS agar contains the indicator bromothymol blue which differentiates sucrose fermenting from non-sucrose fermenting vibrio species. Transport Media When specimens are not cultured soon after collection, to prevent overgrowth and also to ensure survival of pathogens, transport media are used. These are mainly used to transport microbiological specimens from health centers to the district pathological laboratories. Example: (1) Amies transport medium (2) Cary Blair medium. DIFFERENT FORMS OF CULTURE MEDIA:- 1.      Solid Culture Media This is used in petri dishes and in test tubes (slope cultures) and prepared by adding Solidifying agent  (1.0 – 1.5%) (w/v). Microorganisms grow on this and form colonies after multiplication. This helps to identify the organism. 2.    Semisolid Culture Media This is prepared by adding Solidifying agent (0.4-0.5%) (w/v) to a fluid medium. These are used mainly as transport media and for the testing of motility of the organisms. 3.   Fluid Culture Media These media are mainly used as biochemical testing media, blood culture media or the enrichment media. PREPARATION OF CULTURE MEDIA Most culture media are available commercially in readymade dehydrated form. It is less costly to use readymade media, since the ingredients are often required in small amounts but available in large quantities if purchased. Some of the chemicals are also difficult to obtain. To ensure good performance and reproducibility in the results the following must be  performed correctly- 1.     Weighing and dissolving of the ingredients 2.     Addition of heat sensitive material 3.     pH testing 4.     Dispensing and sterilization 5.     Sterility testing and quality control 6. Storage Note 1.      The heat sensitive ingredients such as blood or serum should be brought to room temperature and added when the medium has cooled to about 50°C. 2.      A fluid medium should be tested for accurate pH by using a narrow, range pH paper. 3.      For sterilizing culture media, it is necessary to use manufacturer’s instructions. The commonly used methods for sterilization are- a) Autoclaving (b) Steaming at 100°C and (c) Filtration. It is necessary to use correct temperature and correct length of time. Precautions:- 1.  Autoclaving is used to sterilize most agar and fluid media. 2. Steaming at 100°C: Media such as Cary Blair transport medium contain ingredients that would break down above 100°C. Steaming can be performed in an autoclave with a loose lid. 3.    Filtration: Serum and solutions containing carbohydrates, urea, etc. are heat sensitive and hence cannot be autoclaved. Hence, the media containing such substances are filtered to remove bacteria. 4. Sterility testing: Media in tubes and bottles: Incubate the entire batch at 37°C overnight. Contamination is indicated by appearance of turbidity in a fluid medium and growth on a solid medium. 5.  Control of media: Appropriate control species are used to inoculate slants or plates of the medium (quarter part). After overnight incubation, the cultures are examined for (a) Degree of growth (b) Size of colonies and (c) Other characteristics. 6.Storage of culture media: Dehydrated culture media and dry ingredients (agars, peptones, bile salts, etc.) can be stored at room temperature (25°C ± 5°C) in a cool and dry place, away from

Microbiology, Uncategorized

INTRODUCTION OF MICROBIOLOGY

“Microbiology Worlds”  Microbiology is the scientific study of microorganisms, which are microscopic living organisms that include bacteria, viruses, fungi, and protozoa. These microorganisms play a crucial role in various biological processes and have a significant impact on human health, agriculture, industry, and the environment. French chemist and Microbiologist Louis Pasteur (1822-1895) coined the term “microbiology”. He used the term to describe his work with organisms at the microscopic level. The field of microbiology has a rich history that spans several centuries, with important discoveries and developments contributing to our understanding of the microbial world. Historical Background of Microbiology:- 1.0Aristotle (384-322 BC):-  The concept of spontaneous generation was proposed by various ancient civilizations and philosophers, including the ancient Egyptians and Greeks. One of the most notable proponents of spontaneous generation in ancient times was the Greek philosopher Aristotle. He proposed the idea that certain animals, insects, and even mice could arise spontaneously from decaying organic matter. In the Middle Ages, spontaneous generation was further supported by prominent figures such as Avicenna and Albertus Magnus. 2.0 Anton van Leeuwenhoek (1632 – 1723):- Anton van Leeuwenhoek made significant contributions to the field of microbiology, earning him the title of the  “father of microbiology & Protozoology.” Here are some of his key contributions: 1.  Discovery of Microorganisms:  In the 17th century, Leeuwenhoek designed powerful single-lens microscopes, enabling him to observe tiny organisms that were previously invisible to the human eye. He was the first to describe and document various microorganisms, including bacteria, protozoa, and other microscopic life forms.  In 1676 Observations of bacteria, which he called “animalcules,” provided crucial evidence against the theory of spontaneous generation. 2..  Microscopic Studies of Biological Samples: Leeuwenhoek examined a wide range of biological samples, including water, dental plaque, and even his own feces. 3. Advancements in Microscopy: Leeuwenhoek’s innovative improvements to microscope design, including using high-quality lenses and precise grinding techniques, significantly enhanced the magnification and clarity of his observations.  4..       The term “microbe” was first used by Sedillot in 1878. The word comes from the Greek words “mikros” meaning “small” and “bios” meaning “life”. It literally means “small life” or “microscopic life”. 3.0 Francesco Redi (1626-1697):- Francesco Redi was an Italian physician, naturalist, and poet who made significant contributions to the field of microbiology, particularly in the context of spontaneous generation, which was the widely held belief at the time, that living organisms could arise from non-living matter. Redi conducted a series of experiments that challenged this idea, providing evidence against spontaneous generation Francesco Redi experiments:- In 1668, Redi conducted a famous experiment to test the hypothesis that maggots (the larvae of flies) spontaneously generated from decaying meat. He set up three groups of jars, each containing decaying meat. One group was left open, allowing flies to access the meat and lay eggs, another group was covered with gauze, preventing flies from touching the meat but allowing air to pass, and the third group was completely sealed. Redi observed that maggots only appeared in the open jars, where flies could access the meat, and not in the covered or sealed jars. This experiment provided strong evidence against spontaneous generation, demonstrating that maggots only appeared when flies were able to lay their eggs on the decaying meat. 4.0 John Needham (1713 – 1781): In 1745, Needham conducted experiments where he heated nutrient broths and then sealed them in flasks. Afterward, he observed the growth of microorganisms in the sealed flasks and concluded that life could arise spontaneously from non-living matter. 5.0 Lazzaro Spallanzani (1729 – 1799):- Lazzaro Spallanzani Disproving Spontaneous Generation Theory and support Biogenesis Theory. In 1765, Lazzaro Spallanzani conducted experiments where he boiled nutrient-rich broth in sealed containers, effectively sterilizing the broth. His experiments showed that no microorganisms grew in the sealed flasks, even after long periods, unless they were exposed to air. This demonstrated that living organisms did not arise spontaneously but were introduced to the broth from the external environment. 6.0 Louis Pasteur (1822 – 1895): Louis Pasteur made several significant contributions to the field of microbiology, , earning him the title of the “father of Medical microbiology.” He coined the term “microbiology”, Aerobic and Anaerobic.      1.  Disproving Spontaneous Generation:  Pasteur conducted experiments that disproved the prevailing notion of spontaneous generation      2. Germ Theory of Disease:  Germ theory states that microorganisms are the cause of many diseases      3.  Pasteurization: Pasteurization is a heat treatment process developed by Louis Pasteur to kill or deactivate harmful microorganisms in food and beverages, such as milk and wine. 4.     Vaccination: He developed vaccines against several diseases, including rabies and anthrax.   5.Fermentation: Chemical process by which molecules such as glucose are broken down anaerobically 7.0 John Tyndall:- John Tyndall was a prominent 19th-century Irish physicist and naturalist who made significant contributions to the field of microbiology, particularly in the areas of sterilization.. 1.Tantalization:  Tyndall developed a process known as Tantalization, which involves intermittent sterilization through boiling, incubation, and e-boiling. This method was used to kill heat-resistant bacterial spores and became an important technique in microbiology for ensuring the elimination of  spore-forming bacteria in culture media and other substances.       2.  He Discovered highly resistant bacterial structure, later known as endospore. 8.0 Lord Joseph Lister:- Lord Joseph Lister, a British surgeon, is widely regarded as the pioneer of antiseptic surgery. He is also known as father of Antiseptic Surgery. His contributions to microbiology and healthcare have had a profound impact on the field, here are some key contributions of Lord Joseph Lister in microbiology. 1.       Introduction of Antiseptic Techniques 2….Impact on Infection Control 9.0 Robert Koch (1843 – 1910): Robert Koch, a German physician and microbiologist, made significant contributions to the field of microbiology, particularly in the areas of medical microbiology and infectious diseases. Koch’s Postulates: Koch formulated a set of postulates in the late 19th century that are used to demonstrate the association between a specific microorganism and a particular disease. These postulates are still considered fundamental in the field of microbiology and are

Microbiology

Bacteria

Bacteria:- Bacteria are single-celled microorganisms that have a relatively simple structure compared to other living organisms. They are prokaryotic organisms, which means they lack a true nucleus and membrane-bound organelles. Bacteria are some of the most numerous and diverse organisms on Earth, and they can be found in various environments, including soil, water, air, and inside the bodies of other organisms. Here is an overview of the basic structure of bacteria: 1. Cell wall bacterial cell wall is a rigid structure that surrounds the cell membrane of most bacteria. It provides structural support and protection to the cell, helping it maintain its shape and resist changes in the surrounding environment. The composition and structure of bacterial cell walls can vary widely among different types of bacteria, and these differences are used to classify bacteria into two main groups: Gram-positive and Gram-negative. Gram-Positive Bacteria cell wall:-  Gram-positive bacteria have a thick layer of peptidoglycan, a polymer made up of sugars and amino acid, in their cell walls. This layer is located outside the cell membrane and provides strength and rigidity to the cell wall. In addition to peptidoglycan, Gram-positive cell walls often contain teichoic acids, which are polymers of glycerol or ribitol phosphate. These acids contribute to the overall negative charge of the cell wall. Gram-Negative Bacteria cell wall:  Gram-negative bacteria have a thinner layer of peptidoglycan in their cell walls, located between the inner and outer membranes. Outside the peptidoglycan layer, there is an outer membrane made up of lipopolysaccharides (LPS) and proteins. LPS molecules are composed of lipid A (which anchors the LPS molecule in the outer membrane), a core polysaccharide, and an O antigen (which varies among different bacterial species). The outer membrane acts as a barrier against certain chemicals, including antibiotics, making Gram-negative bacteria often more resistant to these substances than Gram-positive bacteria. Function of bacterial cell wall:- Structural Support:  The cell wall maintains the shape of the bacterium and prevents it from bursting or collapsing due to changes in osmotic pressure. Protection:  The cell wall provides protection against physical damage and helps the bacterium resist the effects of harmful substances in the environment. Barrier:  In Gram-negative bacteria, the outer membrane acts as a barrier that can exclude or allow the passage of specific molecules into the cell. Virulence Factor:  Some components of the cell wall, such as lipopolysaccharides in Gram-negative bacteria, can trigger immune responses in the host organism, contributing to the bacterium’s virulence. 2.    Cell Membrane (Plasma Membrane):  The bacterial cell membrane, also known as the plasma membrane or cytoplasmic membrane, is a vital structure that surrounds the bacterial cell. It serves as a selectively permeable barrier, separating the interior of the cell from its external environment. The cell membrane is a phospholipid bilayer embedded with proteins and other molecules, and it performs several essential Functions of cell membrane in bacterial cells: 1.      Selective Permeability:   The phospholipid bilayer of the cell membrane acts as a barrier that prevents the passage of most substances, including ions and large molecules, into and out of the cell. Only specific molecules, such as gases (like oxygen and carbon dioxide) and small hydrophobic molecules, can freely diffuse through the lipid bilayer. Other substances, such as nutrients and waste products, require specialized transport proteins to facilitate their movement across the membrane. 2.      Nutrient Uptake:  Bacteria need various nutrients to survive, including sugars, amino acids, and ions. Specialized membrane proteins, such as transporters and permeases, help actively or passively transport these nutrients into the cell, allowing the bacterium to obtain the necessary building blocks and energy for its metabolic processes. 3.      Waste Elimination:  Similarly, the cell membrane aids in the removal of waste products and metabolic byproducts from the bacterial cell. These waste products are expelled from the cell through specific transport mechanisms 4.      Energy Production:    Bacterial cell membranes are also the sites of electron transport chains and ATP synthesis in many bacterial species. These processes are essential for the generation of energy in the form of ATP (adenosine triphosphate), which powers various cellular activities. 5.      Maintaining Cell Shape and Integrity:       The cell membrane plays a role imaintainingthe shape and integrity of the bacterial cell. It provides structural support and helps the cell maintain its specific shape, especially in the absence of a rigid cell wall. 1.      Sensory Functions: The cell membrane contains proteins and receptors that allow bacteria to sense changes in their environment. These proteins can detect environmental signals, such as changes in temperature, pH, or the presence of specific molecules, triggering appropriate cellular responses. 3. Cytoplasm:  The bacterial cytoplasm is the semi-fluid substance inside a bacterial cell, enclosed by the cell membrane. A complex, gel-like matrix contains various cellular components and is the site of numerous biochemical reactions essential for the bacterium’s survival and growth. Here are some key aspects of bacterial cytoplasm:   Composition: The cytoplasm is primarily composed of water, making up the majority of its volume. In addition to water, the cytoplasm contains ions, enzymes, nucleotides, amino acids, sugars, and other small molecules that are crucial for the bacterium’s metabolism. Genetic Material: The bacterial chromosome, a single circular DNA molecule, is located in the cytoplasm. Unlike eukaryotic cells, bacteria do not have a membrane-bound nucleus, so their genetic material is dispersed in the nucleoid region within the cytoplasm. The nucleoid lacks a membrane and is simply an area where the genetic material is concentrated. Ribosomes: Bacterial cytoplasm contains ribosomes, which are the cellular structures responsible for protein synthesis. These ribosomes read the genetic information encoded in mRNA (messenger RNA) and use it to assemble amino acids into proteins. Metabolic Reactions: Various metabolic pathways occur within the bacterial cytoplasm. These include processes such as glycolysis (the breakdown of glucose), the citric acid cycle (Krebs cycle), and the synthesis of essential molecules like nucleotides, amino acids, and fatty acids. Enzymes catalyzing these reactions are found in the cytoplasm. Storage Granules: Some bacteria store reserve materials such as glycogen, polyhydroxyalkanoates (PHAs), or sulfur granules in the cytoplasm. These storage compounds serve as a source of energy

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