What is Gram staining and how does it work?
Under a Gram stain, different kinds of bacteria change one of two sets of colors (pink to red or purple to blue) under a special series of stains and are categorized as “gram-negative” or “gram-positive,” accordingly. Gram staining works by differentiating bacteria by the chemical and physical properties of their cell walls.
What are the procedures used for indirect or negative staining?
The procedures used for indirect or negative staining do not require smears to be heat-fixed prior to stain application, so cells retain their actual size and shape. Nigrosin, an aqueous solution containing carbon particles, and Congo red,an acidic stain, can both be used for indirect or negative staining procedures as follows:
What is the advantage of indirect staining over direct staining?
Although indirect or negative staining is not used extensively in this laboratory, it does have an advantage over direct staining because it causes less cellular distortion. The procedures used for indirect or negative staining do not require smears to be heat-fixed prior to stain application, so cells retain their actual size and shape.
What is an example of a direct stain?
For example, the Gram stain, acid-fast stain, and Shaeffer-Fulton endospore stain are all direct stains. The capsule stains and Dorner method endospore stain are a combination of direct and indirect stains. Stains or dyes are generally salts with one ion being colored and the other not.
Is gram staining direct?
In the era of rapid development of molecular and other diagnostic methods, direct Gram staining (DGS) tends to remain in the background, although it can provide both microbiologists and clinicians numerous benefits.
What type of staining technique is the Gram stain?
differential staining procedureThe Gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. Through a series of staining and decolorization steps, organisms in the Domain Bacteria are differentiated according to cell wall composition.
Why can bacteria be directly or indirectly stained?
The cell wall of most bacteria has an overall net negative charge and thus can be stained directly with a single basic (positively charged) stain or dye. This type of stain allows us to observe the shape, size and arrangement of bacteria.
What is the example of direct stain?
For example, the Gram stain, acid-fast stain, and Shaeffer-Fulton endospore stain are all direct stains. The capsule stains and Dorner method endospore stain are a combination of direct and indirect stains. Stains or dyes are generally salts with one ion being colored and the other not.
What type of stain is a Gram stain quizlet?
Terms in this set (14) What is a gram stain? It's a DIFFERENTIAL stain that allows for classification of a bacteria as either gram-positive or gram-negative. Discovered by Hans Christian Gram.
What is the principle and procedure of Gram staining?
The basic principle of gram staining involves the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment. Gram-positive microorganisms have higher peptidoglycan content, whereas gram-negative organisms have higher lipid content.
Which staining of the following is an indirect staining?
The Gram stain is a direct method, since the cells themselves retain dye. In indirect, or negative, staining, smears are produced by mixing material with India ink or acidic dyes such as nigrosine. Acidic dyes have a negative charge and are repelled by the negatively charged cell walls.
Is safranin a direct stain?
In Gram's staining, the safranin directly stains the bacteria that has been decolorized. With safranin staining, gram-negative bacteria can be easily distinguished from gram-positive bacteria.
Why is negative staining called indirect staining?
Why is negative staining also called either indirect or background staining? Negative sating is also known as indirect or background staining bc it ors not directly stain the bacterial cells rather it indirectly stains them by coloring the background making the cells more easily viewable.
Why do we use indirect staining?
The indirect method is used to enhance the fluorescence signal and also to facilitate multicolor staining of human cells when direct conjugated reagents are not available. 1,2 BD offers two methods for indirect staining: biotin-avidin (or streptavidin)
What is the difference between simple staining and gram staining?
The Gram stain is a differential stain, as opposed to the simple stain which uses 1 dye. As a result of the use of 2 dyes, making this procedure a differential stain, bacteria will either become purple/blue or pink during the procedure.
What is gram staining in microbiology?
A Gram stain is a test that checks for bacteria at the site of a suspected infection such as the throat, lungs, genitals, or in skin wounds. Gram stains may also be used to check for bacteria in certain body fluids, such as blood or urine.
Which staphylococcus is gram-positive?
Staphylococcus aureus is a gram-positive, catalase-positive, coagulase-positive cocci in clusters. S. aureus can cause inflammatory diseases, including skin infections, pneumonia, endocarditis, septic arthritis, osteomyelitis, and abscesses.
What is differential staining technique?
Differential staining is a staining process which uses more than one chemical stain. Using multiple stains can better differentiate between different microorganisms or structures/cellular components of a single organism.
What is the Gram stain method quizlet?
Gram stain technique. A staining procedure used to identify bacterial cells as gram-positive or gram-negative. developed by christian gram in the 1800s. -Cells are stained with crystal violet and Gram iodine solution and washed with a decolorizer. -Safranin is applied as a counterstain.
Why is Gram stain considered a differential stain?
The Gram stain is a differential stain because it will differentiate between Gram(+) (Gram positive) and Gram(-) (Gram negative) cells based on their different cell wall compositions.
What is indirect staining?
Indirect Stain using an Acidic Dye. In negative staining, the negatively charged color portion of the acidic dye is repelled by the negatively charged bacterial cell. Therefore, the background will be stained and the cell will remain colorless. Organism.
When to prepare a direct stain?
Prepare a direct stain when given all the necessary materials.
How to heat fix a smear of Escherichia coli?
Heat-fix a smear of either Escherichia coli or Enterobacter aerogenes as follows: Using the dropper bottle of distilled water found in your staining rack, place a small drop of water on a clean slide by touching the dropper to the slide. Aseptically remove a small amount of the culture from the agar surface.
How to remove agar culture from a slide?
Using the dropper bottle of distilled water found in your staining rack, place ½ drop of water on a clean slide by touching the dropper to the slide. Aseptically remove a small amount of the culture from the agar surface and touch it several times to the drop of water until it turns cloudy.
What happens if you dye a fabric too thick?
If the dye is too thick, not enough light will pass through; if the dye is too thin, the background will be too light for sufficient contrast. Results. Make drawings of your 3 direct stain preparations and your indirect stain preparation. Performance Objectives.
Why does acidic dye not react with bacterial cytoplasm?
An acidic dye, due to its chemical nature, reacts differently. Since the color portion of the dye is on the negative ion , it will not readily combine with the negatively charged bacterial cytoplasm (like charges repel). Instead, it forms a deposit around the organism, leaving the organism itself colorless.
Why do bacteria have negative cytoplasm?
Because of their chemical nature, the cytoplasm of all bacterial cells have a slight negative charge when growing in a medium of near-neutral pH. Therefore, when using a basic dye, the positively charged color portion of the stain combines with the negatively charged bacterial cytoplasm (opposite charges attract) and the organism becomes directly stained.
What is a stain called?
Stains or dyes are generally salts with one ion being colored and the other not. The colored ion is called a chromophore. If color is associated with the positive ion (cation), the stain is called a basic stain; if color is associated with the negative ion (anion), the stain is said to be acidic. Since cell membranes typically carry a slight negative charge, cells will readily attract and be colored by basic stains or dyes. Methylene blue, crystal violetand safranin are examples of basic stains.
What is it called when a stain leaves the background colorless?
When a staining procedure colors the cells present in a preparation, but leaves the background colorless (appearing as white), it is called a direct stain.
How to remove a smear from a slide?
2. Remove the slide from the stain rack, hold it at an angle toward the bottom of the sink and rinse the stained smear thoroughly with tap water. 3. Remove excess water from the slide bottom with paper towel and air-dry the smear surface.
How long to wait to stain a slide?
Cover each smear with one or more drops of the appropriate stain reagent and allow the stain to act for the required time (60 seconds for methylene blue and safranin; 10 seconds for crystal violet). DO NOT OVER STAIN!
How to direct straining?
1. Use a clean glass slide and a sterile loop, and if transferring organisms from a solid surface (bacteria from agar or cheek epithelium) place a loopful of water on the slide first. Aseptically transfer the desired organisms into the water and mix to form the smear.
What are the three shapes of bacteria?
Individual bacteria typically have one of three shapes; spherical (cocci- singular coccus), rod-like or cylindrical (bacilli- singular bacillus), or spiral (spirilla- singular spirillum). Depending upon the manner in which they divide, bacteria may occur as single cells or as multiple cells in specific arrangements as shown.
Can you stain after smears?
After smears have been prepared and heat fixed, they are ready for staining as indicated below. Since stain reagents are potentially messy and some can cause skin irritation, students are encouraged to use caution and to avoid unnecessary contact between stain reagents and skin surfaces. 1.
