Ppt gram stain procedure powerpoint presentation free. Perform gram stain procedure quality control per standard laboratory protocol. Specimen collection label 2 clean slides with the scharp label. Gram himself never used the red counterstaining in order to visualize the gram negative bacteria. Crystal violet primary stain for 1 minrinse gram s iodine mordant for 1 minrinse acetonealcohol decolorizer for 5 seconds safranin counterstain for 1 minrinse gram negative red and gram positive blue assess gram reaction a. The technician decides to make a gram stain of the specimen. The 100x oil immersion lens is essential for viewing individual bacteria. The ions will interact with negatively charged bacterial cells and stain them purple. Sep 05, 2014 this general microbiology practical lecture explains the gram staining procedure and the principle behind it. Gently rinse the slide with water no longer than 5 seconds to remove excess stain. Transfer a loopful of culture or the specimen by sterile loop and make a smear at the center.
Prepare the smear of suspension on the clean slide with a loopful of sample. The crystal violet dye is washed by acetone and counter stain stick on its cell wall. Flood the surface of the slide with crystal violet stain and let sit for one minute. Just like last time, when gram staining an unknown, the best method is to make three bacterial smears on the same slide.
The gram stain is the most important staining procedure in microbiology. Make sure you air dry the bacteria before heat fixing. Gram stain rare epithelial cell, few gram positive cocci 3. Macconkey agar mac a selective and differential media used to. It is the responsibility of the course instructorlab assistant to ensure that this sop is performed as described and to update the procedure when. It is mainly responsible for myonecrosis gas gangrene, food poisoning, and gangrenous cholecystitis.
Gram staining procedure animation microbiology principle. Beyer individual supplies microscope slide clothes pin inoculating loop or wood splint paper towels shared supplies crystal violet stain safranin red stain grams iodine ethyl alcohol denatured water bottle rinsing basin alcohol burner wax pencil matches microscope create a staining area on your microscope slide obtain a clean microscope slide draw a. Grams iodine grams iodine acts as a mordant and it forms a complex with crystal violet that is cvi complex. If the slide is heat fixed, allow it to cool to the touch before applying the stain. Flood the gram s iodine for 1 minute and wash with water. It tries to decolourise the cell by removing the cvi complex from the cell. Air dry and then heat fix by passing it through a flame a few times. The method for mounting bacteria on a microscope slide using aseptic technique is shown. Planted digital learning library gram stain procedure duration.
Clostridium perfringens is a gram positive, anaerobic, endosporeforming bacilli, often appear as boxcar shaped. The most widely used staining procedure in microbiology is the gram stain, discovered by the danish scientist and physician hans christian. In this staining, method bacteria are classified into two groups that are. It is the reason why gram staining is not that reliable in identifying organisms.
We perform gram in both cytological and histological samples either as first stain or secondary after destaining by hydrochloric acid. Gram stain procedural highlights preparing the smear concentrating fluids using a cytospin gram stain procedural highlights gram stain procedural highlights low power 10x examination 1020 fields, quantitate cells select area for high power examination gram stain procedural highlights high power 100x examination 2040 fields, quantitate. The cell walls of gram positive bacteria have a thick layer of proteinsugar complexes called peptidoglycan and lipid content is low. This donation link uses paypal on a secure connection. Which staining method has been opted for staining ofthis. The other should be a known gram negative organism like escherichia coli. Twice a year one set of slides will be sent to the central lab at the pittsburgh location.
Gram negative bacteria will be pink and gram positive bacteria will be purple. Remel qcslide gram stain control is a heatfixed quality control slide used for gram stain procedures. Gram staining involves a fourpart process, which includes. Bacteria are commonly diagnosed using the gram stain procedure when washed after staining.
Dilute your organism in a tube of sterile water to obtain a turbidity equivalent to a 0. The filter gram stain detected 2 to 100 bacteria, whereas the slide gram stain. Mycobacterium avium complex mac is a group of bacteria related to tuberculosis. Tf view the figure for the procedure of experiment 33. Remove excess water and flood smear with gram s iodine. Sign in to view the content amrita vishwa vidyapeetham. Hi i had a afb culture, gram stain and sputum culture done final test revealed 1. Gram staining is a universal staining technique used for identification and classification of organisms.
Then, flood the sample with several drops of crystal violet dye. Gently flood the smear with gram s iodine and let stand for 1 minute. After completing the gram stain procedure, the technician views the slide under the brightfield microscope and sees purple, grapelike clusters of spherical cells figure 4. Gram negative bacteria have a thin layer of peptidoglycan and stain red. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. It includes introduction, history,principle, method of gram staining procedure. Gram staining or gram s method is a way of classifying bacteria into two large groups. Add about 5 drops of huckers crystal violet to the culture. The gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain. When the bacteria is stained with primary stain crystal violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. This is because the cell walls of gram negative cells contain more lipids fatty substances. Unknown identification microbiology resource center.
Learn what it involves and when your doctor might order it. This complex increases affinity between cell and stain. Acharya tankeshwar, author at learn microbiology online. Use a dropper to apply the primary stain crystal violet to the slide and allow it to sit for 1 minute. Place slide on staining tray or hold with forceps above the sink. It was a few years later, that the german pathologist carl weigert18451904 from frankfurt, added a final step of staining with safranin.
It is used to differentiate between gram positive organisms and gram negative organisms. Gram positive bacteria have a thicker peptidoglycan layer and stain purple. Enter into ldms but do not useldms labels on the slides. Applying a primary stain crystal violet to a heatfixed smear of a bacterial culture. Examine the slide under themicroscope andclassify the organism on the basis of gram staining. Sputum culture heavy growth normal respiratory flora 2.
Wash slide in a gentle and indirect stream of tap water for 2 seconds. Ppt staining of bacteria powerpoint presentation free to download id. This will allow you to focus quickly and to look for areas of purulence. Flood the slide with grams iodine and time for one. Gram stain was described by danish bacteriologist hans christian gram in 1884 to differentiate streptococcus pneumoniae from klebsiella pneumoniae in lung tissue. Learn vocabulary, terms, and more with flashcards, games, and other study tools. The name comes from its inventor, hans christian gram. How to do a grams stain test grams stain is one of the most frequently used processes in identifying bacteria and is used daily in hospitals. Rinsing too long can remove too much color, while not rinsing long enough may allow too much stain to remain on gramnegative cells. The relation of the gram stain to the cell wall and the ribonucleic acid content of the cell. Gram stain procedural highlights preparing the smear concentrating fluids using a cytospin gram stain procedural highlights gram stain procedural highlights low power 10x examination 1020 fields, quantitate cells select area for high power examination gram stain procedural highlights high power 100x examination 20. Gram staining gram staining procedure prepare a heat fixed smear of the culture you wish to examine flood the smear with crystal violet 30 sec. Overheating of smears when heat fixation is used d.
Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. It is one of the differential stains that are used to characterize bacteria in one of two groups. Donations are key in helping us provide this resource with fewer ads. Wash briefly in water, do not let the section dry out. Smears for staining may be prepared in order of likely positive results i.
Heat fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they dont rinse out during the staining procedure. Gram staining procedure was discovered by hans christian gram in 1884. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. For both settings conventional xylenebased mounting media. The primary stain crystal violet binds to peptidoglycan, coloring cells purple.
Stain procedure follow the manufacturers instructions for performing the stain. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. Applies to gram staining samples using the 3step method to detect the presence of gram positive and gram negative microorganisms. Occurrence of a phosphoric ester in certain bacteria.
Purpose the gram stain is the most commonly used differential stain for determining cell morphology. To prepare a gram stain, fix the bacteria to the slide by passing it through a bunsen burner flame. Mycobacteria are called acidfast bacilli because they are a group of rodshaped bacteria bacilli that can be seen under the microscope following a staining procedure where the bacteria retain the color of the stain after an acid wash acidfast. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. Gram stain procedure 1 heatfix the glass slide containing the specimen. Here is the list of countries and us states that have the top 4 number of spo visitors. The slide is then washed in a solvent to decolorize the first stain. Sentinel laboratory guidelines for suspected agents of. This protocol is taken from bensons microbiological applications. Gram s method stains bacteria according to the chemical and physical properties of their cell walls. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. Additional test should be done to confirm the strain of bacteria in the medium and the macconkey agar test is a reliable test. Gram positive cells will retain the stain even after being washed in the solvent, but the stain will be removed from the gram negative cells.
Endospores resist adverse environmental conditions such as dryness, heat and poor nutrient supply. Ppt staining of bacteria powerpoint presentation free. Gram staining is a quick procedure used to look for the presence of bacteria in tissue samples and to characterise bacteria as gram positive or gram negative, based on the chemical and physical properties of their cell. Drain off crystal violet and rinse off with distilled water. Sterilize the inoculating loop on a flame of a bunsen burner. Gram stain is probably one of the most commonly used staining procedures used in the field of microbiology. Gram was studying lung tissue sections from patients who had died of pneumonia.
The gram stain divides most bacteria into two main groups based on the amount of the sugar peptidoglycan present in the bacteria cell wall. One should be a known gram positive organism like staphylococcus aureus. The cells appear pink because of the color of the counterstain safranin levinson, 2014. It is a primary test which quickly and cost effectively divides bacteria into one of two types. Steps in gram staining procedure follow the clock 1 on a rack, flood with filtered crystal violet 10 sec 2 wash briefly in water to remove excess crystal violet 3. In brief, the procedure involves the application of a solution of iodine potassium iodide to cells previously stained with crystal violet or gentian violet. Gram staining gram staining is the single most important and universally used staining technique in the laboratory. Rinse the crystal violet off and flood the smear with iodine.
This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Ppt microbiology lab tests powerpoint presentation free. Gram variable observations in gram staining the gram staining procedure does not always give clearcut results. The name comes from its inventor hans christian gram who developed the technique in 1882 published 1884. A gram stain is a test used to learn if bacteria are present in your body fluids or tissue. Hans christian joachim gram the gram stain was devised by the danish physician, hans christian joachim gram, while working in berlin in 1883. Gram stain gonorrhea culture nonulture nonc amplified tests commercially available naats include.
Student procedures illinois institute of technology. The cell wall can stain either positive or negative, depending on its chemistry. These germs are very common in food, water, and soil. Flood slide with crystal violet and let stain for 1 minute. Qslide gram a microscope slide for the quality control of. Gram negative bacteria are bacteria that do not retain the crystal violet dye in the gram stain procedure. Different bacteria stain differently to a common staining procedure. Laboratory manual in general microbiology eleventh edition. Gram staining is a method commonly used to determine the chemical make up of the cell wall of bacteria. A method of gram staining a specimen containing bacteria, which method comprises. Apr 04, 2016 corynebacterium diptheria seen in gram staining 35. The macconkey agar test was the alteration of the neutral red bile salt agar. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
Gram positive bacteria retain the crystal violetiodine complex after decolorization, are not counterstained, and appear purple gram negative bacteria have a different cell wall structure, dont retain the crystal violetiodine complex after decolorization, and so are counterstained by safraninfuchsin and appear pinkred paraffin sections. The procedure is based on the reaction between peptidoglycan in the cell walls of some bacteria. The gram stain procedure was originally developed by the danish physician hans christian gram to differentiate pneumococci from klebsiella pneumonia. Gram stain or gram staining, also called gram s method, is a method of staining used to distinguish and classify bacterial species into two large groups. Flood the grams iodine for 1 minute and wash with water. Oct 24, 2019 the procedure is based on the reaction between peptidoglycan in the cell walls of some bacteria. This video demonstrates the gram staining method for identifying unknown bacter. Jun 10, 2006 such staining methods are called differential staining methods, these include gram staining and acid fast staining. The name comes from the danish bacteriologist hans christian gram, who developed the technique. Afb laboratory tests detect the bacteria in a persons sample and help identify an infection. Drain crystal violet and gently rinse with tap water. The adobe flash plugin is needed to view this content. Swab of the vaginal fluid is rolled onto the slide. Macconkey agar purpose, test and results 2020 updated.
Beyer individual supplies microscope slide clothes pin inoculating loop or wood splint paper towels shared supplies crystal violet stain safranin red stain gram s iodine ethyl alcohol denatured water bottle rinsing basin alcohol burner wax pencil matches microscope create a staining area on your microscope slide obtain a clean. Crystal violet was poured and kept for about 30 seconds to 1 minutes and rinse with water. This technique is commonly used as an early step in identifying pathogenic bacteria. The filter gram stain was approximately 100fold more sensitive than the slide gram stain. Knowing the chemical make up makes it easier to manipulate the bacteria for. Flood slide with crystal or gentian violet 60 seconds.
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