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Histopathology

TISSUE FIXATION IN HISTOLOGY

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INTRODUCTION:- It is a process by which the cells or tissues are fixed in chemical and partlyphysical state so that they can withstand subsequent treatment with variousreagents, with minimal distortion of morphology and no decomposition  AIMS OF FIXATION:- (a) To preserve the tissues as close to their living state as possible  (b) To prevent autolysis and bacterial attack  (c) To prevent tissues from changing their shape and size during processing  (d) To harden the tissues  (e) To allow clear staining of sections subsequently  (f) To improve the optical differentiation of cells & tissue  PRINCIPLE OF FIXATION:- Fixation results in denaturation and coagulation of protein in the tissues. Thefixatives have a property of forming cross links between proteins, therebyforming a gel, keeping everything in their in vivo relation to each other. PROPERTIES OF FIXATIVES AND FACTORS AFFECTING FIXATION:- 1. Coagulation and precipitation of proteins in tissues.  2. Penetration rate differs with different fixatives depending on the molecular weight of the fixative  3. pH of fixatives – Satisfactory fixation occurs between pH 6 and 8. Outside this range, alteration in structure of cell may take place.  4. Temperature – Room temperature is alright for fixation. At high temperature there may be distortion of tissues.  5. Volume changes – Cell volume changes because of the membrane permeability and inhibition of respiration.  6. An ideal fixative should be cheap, nontoxic and non-inflammable. The tissues may be kept in the fixative for a long time TYPE OF FIXATION:- Immersion fixationPerfusion fixationVapour fixationCoating/Spray fixationFreeze dryingMicrowave fixation/StabilizationThe most commonly used technique is simple immersion of tissues/smears in an excess of fixative. For all practical purposes immersion fixatives are most useful. These may be divided into routine and special SIMPLE FIXATIVES :-

Microbiology

Biochemical Reactions on Triple Sugar Iron Agar (TSI) Slants.

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TSI slants are useful in the identification of Enterobacteria by their specific reactions on the slants. v  Principle:- 1.     Alkaline reaction (red color) is shown by the organisms, who fail toferment any one of the sugars. 2.     Fermentation of the sugars is indicated by yellow color since pHrange of phenol red is 6.8 to 8.4 and color change from yellow to red. Sincethe glucose (dextrose) present on the surface of the medium is used up andsince the surface of the slant is exposed to atmosphere, under aerobicconditions, the acid reaction on the surface reverts to alkaline (red color) in18 to 24 hours. (which is a critical duration for this observation). In thebutt, since anaerobic condition exists, the color of the butt remains yellow. 3.     Gas production (carbon dioxide) is indicated by splitting of theagar 4.     Production of hydrogen sulfide imparts black shade to the slant byreacting with ferrous ions. It is an indication of H,S producing organisms. v  Procedure:- 1.      Streak the TSI slant with a loop and stab with a straight needle. 2.      Incubate at 37°C for 18-24 hours.  3.      The various reactions obsrved on the slants are as follows. v Quality control:- 1.      Use the following microorganisms to confirm the reliability of TSIslants. 2.      Positive control:   Acid slantand butt with gas: E. coli. 3.      Negative control:   Alkalineslant and acid butt and no gas: S. paratyphi A.

Microbiology

O-Nitrophenyl-B-D-Galactopyranoside (ONPG) test

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O-Nitrophenyl-B-D-Galactopyranoside (ONPG) test:-  v   Principle:- This test is used to detect an organism which produces B-galactosidase enzyme, thathydrolyses ONGP to produce the yellow colored end product orthonitrophenol(ONP). v Requirements:- 1.      ONPG disk 2.      Pure culture of suspected organism 3.      Normal saline v  Procedure:- 1.      Suspend loopful of the organisms in normal saline. 2.      Place an ONPG disk in the tube 3.      Incubate for 4 hours at 37°C. 4.      Observe the tubes for color change  v Observations:- 1             Yellow color of disk: Organism like E. coli 2              No change of color of disk: Negative test v Quality control:- 1.       Use following microorganisms to confirm the reliability of reagents: 2.       Positive test: E. coli 3.       Negative test: S. typhimurium

Microbiology

Optochin Test for pneumococci bacteria

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Optochin test:-     vPrinciple:-                This test is used to identify pneumococci. Optochin lyses pneumococci.        However, alpha-streptococci are resistant to optochin.     v   Requirements:-  1    Optochin disks        2   Pure culture of suspected organisms on 5% sheep blood agar vProcedure:- 1      Streak 2-3 suspect colonies of a pure culture onto half of a 5% sheep blood                agar.       2    Aseptically place an optochin disk on upper third of the streaked area.       3    Incubate the plate for 18-24 hours.       4    Observe and measure the zone inhibition including the diameter of the disk. v Observations:- 1     Positive: Zone of inhibition more than 14 mm in diameter with 6 mm disk: pneumococci        2      Negative: No zone of inhibition        3      Equivocal: Zone of inhibition less than 14 mm  v  Quality control:-          Use following microorganisms to confirm the reliability of reagents:          Positive test: Pneumococci          Negative test: alpha-streptococci

Uncategorized

RAITMAN- FRANKEL CALORIMETRIC (SGOT)

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SERUM SGOT SUMMERY:-     Serum Glutamate Oxaloacetate Transaminase (SGOT), also called as Aspartate aminotransferase (AST), belongs to the transferase class of enzymes. This enzyme shows high levels of activity in the heart, liver, skeletal muscles and kidneys. Since its level seems to be increasing enormously following Myocardial Infarction (MI), it can be used as supporting evidence in the diagnosis of MI (Especially 20-36 hrs after MI). Elevated levels are also seen in Viral / Toxic Hepatitis, Hepatic and Cardiac Necrosis, Muscular Dystrophy and Pulmonary Embolism. AIM:- Determination of Serum Glutamate Pyruvate Transaminase (SGPT or ALT) by Reifman & Frankel’s method  PRINCIPLE:- Alanine aminotransferase (GPT) catalyzes the transfer of the amino group from alanine to oxoglutarate with the formation of glutamate and pyruvate.   L-Aspartate + 2-Oxogluatarate                      GPT            Oxaloacetate +L-Glutamate        Oxaloacetate + 2,4 DNPH                                              Brownish red colored complex SGOT (AST) catalyses the transfer of amino group from Aspartic acid to 2- Oxoglutarate to form Oxaloacetate and L-Glutamate. The Oxaloacetate thus formed reacts with 2,4 Dinitrophenyl Hydrazine (2,4 DNPH) to form a corresponding Hydrazone, a brownish red colored complex in an alkaline medium.The color intensity is directly proportional to the SGOT concentration in the serum and is measured photometerically at 505 nm (490- 546).    REQUIREMENTS:-     ·        Four test-tube, ·        Colorimeter, ·        SGOT Reagent Kit, ·        Incubator, ·        Cuvette, ·        Pipette etc. ·        Fresh Serum PROCEDURE:-   1.      Pipette into test tubes labeled as Blank, Calibrator, Control, Test and proceed as per given below       Reagent Blank Calibrator Control Test Substrate Reagent 0.25 ml 0.25 ml 0.25 ml 0.25 ml Deionized Water 50µl       Serum Sample       50µl Calibrator (Conc. 170 U/L)   50µl      1.      Mix and incubate at 37ºC for 60 minutes. Color Reagent 0.25 ml 0.25 ml 0.25 ml 0.25 ml Serum Sample (Same Serum Sample Which isused above)     0.05 ml   1.      Mix and incubate at 37ºC for 20 minutes. Alkaline Reagent (Prediluted) 1.5 ml 1.5 ml 1.5 ml 1.5 ml 1.      Read absorbance of all the tubes against distilled water at 505 nm(490-546).   Calculation:-:- SGOT (AST) activity in U/L =                 Abs of Test – Abs of Control  x Conc of Calibrator (Conc :160 U/L)                              Abs of Calibrator– Abs of Blank Then,    If Absorption of,        Test                       =             0.35                                      Control                 =             0.30                                      Calibrator            =             0.36                                     Blank                     =             0.05 Then,  SGOT (AST) activity in U/L =            Abs of Test – Abs of Control      xConc of Calibrator (Conc :160 U/L)                                                       Abs of Calibrator– Abs of Blank Then,       SGOT (AST) activity in U/L =            0.35 –0.30  x 160 U                                                                 0.36 – 0.5          SGOT(AST) activity in U/L =          25.8   Result (U/L)                        =             25.8 NORMAL VALUE:-        =          0-35 U/L                    CLINICAL SIGNFINANCE The group of enzymes called transaminase exist in tissues of many organs. Necrotic activity in these organs causes a release of measured. Since heart tissue is rich in AST increased serum levels appear in patients after myocardial infraction, as well as in patients with muscle disease. Muscular dystrophy and dermatomyositis . The liver is especially rich in ALT, being this enzyme measurement used primarily as a test for infectious and toxic hepatitis, although high levels of both ALT and AST may also be found in cases of liver cell damage and acute pancreatitis, suggesting that the obstruction of billary tree by the adematous pancreas and the presence of associate hepatic disease may contribute to elevated AST levels these patients. Slight or moderate elevations of AST and ALT activities may be observed after intake of alcohol and after administration of various drugs, such as salicylates, opiates, and ampicilin

Histopathology

Component of a microscope

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Light source:- Light source can be external or inbuilt. Dispersal of heat, collection of greatest amout of light, direction and distance are carefully calculated by the designers of microscope for greatest efficiency Condensers:- Body Tube Objectives Stage Clamping Screw Condenser Mirror (Light Source) Fig. 2.3: Microscope Condensor Adjustment The purpose of condenser is to concentrate the light into the plane of the object. The more the light at the specimen, better is its resolution. All condensers have aperture diaphragm with which the diameter of the light beam can be controlled Object stage:- It is a rigid platform with an aperture through which the light can pass. It supports the glass slide. It allows controlled movement in two directions.  Objectives:- They are the most important parts of microscope. The main task of objective is to collect the maximum amount of light from the object, unite it and form a high quality magnified real image. Magnifying powers of objectives are from 1:1 to 100:1. Body tube:- Body tube can be monocular, binocular and the combine photo-binocular (also called trinocular). Binocular tubes have provision for inter-pupillary distanceadjustment, enabling each observer to adjust for his eyes  Eyepiece :- The final stage in optical path, the eyepiece’s function is to magnify the image formed by the objective within the body tube, and present the eye with a virtual image.  Use of the Microscope :-  * Illumination should be centered.  *The condenser should be centered and in proper position.   *Objectives should be properly screwed.   *Optical parts should be clean and free from dust.   *Use oil only for oil immersion objective.  After use, clean the oil objective with lens tissue.  Avoid use of xylene, alcohol or acetone.  Eyepieces get dirty by grease from eyelashes, clean them with lens paper.   *When changing slide, always lower the stage before removing the slide or change objective lens to scanner view.   *Make sure the slide is the right way up

Microbiology

Decarboxylase Tests (Moeller’s Method)

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Decarboxylase test:- v Principle:- This test is used to detect the enzymatic ability of an organism  to decarboxylase (hydrolyse) an amino acid to form an amine. Hydrolysis of an amino acid results in an alkaline pH change leading  to formation of pink color.   v Requirements:- 1.      Glucose non-fermenting organisms 2.      Glucose fermenting organisms 3.      Decarboxylase broaths (lysine, arginine, ornithine) 4.      Sterile mineral oil 5.      Suspensions of suspected organisms grown on 5% sheep blood agar (18-24 hours) in brain- heart infusion broth (BHIB).   v Procedure:- A.    Glucose non-fermenting organisms 1.      Prepare a heavy suspension of the organisms in brain-heart infusion broth. 2.      Inoculate each of the three decarbxylase broths and one control broth  (without amino   acid). 3.      Add a 4 mm layer of sterile mineral oil in each tube. 4.      Incubate at 37° up to seven days. 5.      Observe color change.   B.  Glucose fermenting organisms 1.      Inoculate each of the decarboxylase tubes with 1 drop of brain-heart infusion broth   culture. 2.      Add 4 mm layer of sterile mineral oil in each tube. 3.      Incubate at 37° up to seven days. 4.      Observe color change. vObservations: By comparing with the color of the control tube        1   Positive:   Purple color (alkaline color change)            Lysine:        Klebsiella pneumonia           Arginine:     Enterobacteria cloacae          Ornithine:    Enterobacteria cloacae        Negative:     No color change or yellow color          (Due to fermentation of glucose in BHIB). v  Use following microorganisms to confirm the reliability of reagents: 1   Positive test:       Lysine:      Klebsiella pneumonia                                   Arginine:   Enterobacter cloacae                                  Ornithine:  Enterobacter cloacae  2  Negative test:      Lysine:       Enterobacter cloaeae                                    Arginine:    Klebsiella pneumonia                                   Ornithine:   Klebsiella pneumonia v Note 1         Reference Book Text book of medical laboratory Technology by Dr. Praful B. Godkar                 

Microbiology

Acetamide Utilization

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Acetamide utilization test:- v  Principle This test is performed to identify nonfermentative Gram negative bacteria, which use acetamide. These bacteria (such as Pseudomonas aeruginosa) deaminate acetamide in the medium to release ammonia, leading to increase in pH of the medium. Due to increase in pH, the original blue color of the medium changes to blue. v Requirements:- 1.     Acetamide slants 2.     Culture (18-24 hour) v  Procedure:- 1     Inoculate acetamide slant using a needle using growth from 18-24 hour culture. 2      Incubate at 35° for up to 7 days. v  Result:- 1.      Color of medium blue:  Pseudomonas aeruginosa 2.      Color of medium green: Negative test v  Quality control:- Use following microorganisms to confirm the reliability of reagents: Positive test        : Pseudomonas aeruginosa Negative test      : E. coli 

Microbiology

. Phenylalanine Deamination Test

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This test is used mainly to assist in the identification of Proteus sp:- v Principle:- It is based on the ability of bacteria such as Proteus species to convert phenylalanine in the medium to phenylpyruvic acid by deamination. Phenylpyruvic acid is detected by the addition of ferric chloride ions, which give green color on the surface of the culturez. v Requirements:- 1.      Phenylalanine agar 2.      10 g/dl ferric chloride (freshly prepared) v Procedure:- 1.      Inoculate a slope of the phenylalanine agar with test organisms. 2.      Incubate at 35-37°C overnight. 3.      Add 3 to 4 drops of ferric chloride reagent and observe the color. v Observations:- 1.      Green color                     : Positive test 2.      No green color              : Negative test v  Quality control:- Use following microorganisms to confirm the reliability of reagents: Positive control         : Proteus sp. Negative control         : E. coli

Microbiology

Gelatin Liquefaction Test

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Gelatin Liquefaction test:- This test is performed in the identification of organisms such as Pseudomonas and Vibrio cholera vPrinciple:- Gelatinase enzyme is secreted by the organisms such as Pseudomonas and Vibrio cholerae which act on gelatin which is liquefied and it is confirmed by placing the test at 4°C for 30 minutes. v Requirements:-  Nutrient gelatin medium 1.      Peptone                     : 10.0 g 2.      Beef extract               : 3.0 g 3.      Gelatin                         : 120.0 g 4.      Distilled water            : 1000 ml Dissolve the solid ingredients in 100 ml of distilled water by heating. Dispense in 7 ml amounts in screw cap test tubes (15 x 125 mm). Sterilize by autoclaving at 121°C for 15 minutes v Procedure:-          1      Inoculate the medium with test organisms.                2.     Incubate at 35-37°C for 72 hours.     3.     Chill the tubes at 4°C for 30 minutes.     4      Observe for positive gelatin liquefaction v Observations:- 1. Medium in fluid state: Positive test 2.  Medium in solid state: Negative test v  Quality control:-  Use  following microorganisms to confirm the reliability of reagents:  Positive control        : Pseudomonas aeruginosa  Negative control      : E. coli

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