Etiket Arşivleri: Gram Stain

‎Laboratory‎ > Stain Protocols

Stain Protocols – BIOL 2420 Simple Stain Simple stains provide a quick and easy way to determine cell shape, size, and arrangement. 1. Perform a bacterial smear, as discussed in Figure 3-52 on page 150 of your lab manual. 2. Saturate the smear with basic dye for approximately 1 minute. You may use crystal violet, safranin, or methylene blue. 3. Rinse the slide gently with water. 4. Carefully blot dry with bibulous paper. 5. Observe the slide under the microscope, using proper microscope technique. Negative Stain Negative staining is an excellent way to determine an organism’s cellular morphology. Since the cells themselves are not stained, their morphology is not distorted in any way. The nigrosin provides a dark background against which the shapes of the unstained cells are clearly visible. This method provides a high degree of contrast not available in most other staining procedures. 1. Place a single drop of nigrosin on a clean microscope slide, adjacent to the frosted edge. 2. Using a flamed loop and sterile technique, remove some organism from your tube or plate and mix it into the drop of nigrosin. Be sure there are no large clumps of organism, but try to avoid spreading the drop. 3. Place the end of another clean microscope slide at an angle to the end of the slide containing the organism and spread the drop out into a film. This is done by contacting the drop of nigrosin with the clean microscope slide and using the capillary action of the dye/microscope slide to spread the nigrosin across the smear: 4. Allow the film to air dry. 5. Observe the slide under the microscope, using proper microscope technique.

Stain Protocols – BIOL 2420 Gram Stain 1. Perform a bacterial smear, as discussed in Figure 3-52 on page 150 of your lab manual. 2. Saturate the smear with crystal violet for 1 minute. 3. Rinse the slide gently with water. 4. Saturate the smear with iodine for 1 minute. 5. Rinse the slide gently with water. 6. Decolorize with Gram decolorizer (acetone/alcohol) for 3-5 seconds ONLY; if you leave the decolorizer on too long, it will bleach the crystal violet out of a gram positive cell! 7. Rinse the slide gently with water. 8. Counterstain with safranin for 1 minute. 9. Rinse the slide gently with water. 10. Carefully blot the slide dry with bibulous paper. 11. Observe the slide under the microscope, using proper microscope technique. With proper staining technique… Gram positive bacteria will stain purple. Gram negative bacteria will stain red/pink. TIP: When making a smear of an unknown organism to Gram stain, place three organisms on your slide: a known gram positive, a known gram negative, and your unknown (and LABEL which is which!). That way, if there’s a problem with your staining technique, it will be reflected in the known organisms, and you’ll know you need to do the stain again to get accurate results.

Stain Protocols – BIOL 2420 Capsule Stain 1. Place a single drop of India ink on a clean microscope slide, adjacent to the frosted adge. 2. Using a flamed loop and sterile technique, remove some K. pneumoniae (or the organism you want to stain) from your tube or plate and mix it into the drop of India ink. Be sure there are no large clumps of organism, but try to avoid spreading the drop. 3. Place the end of another clean microscope slide at an angle to the end of the slide containing the organism. Spread out the drop out into a film. This is done by contacting the drop of India ink with the clean microscope slide and using the capillary action of the dye/ slide to spread the India ink across the smear. Refer to the Negative Stain portion of this handout for a diagram. 4. Allow the film to air dry. DO NOT heat or blot dry!!!! Heat will melt the capsule! 5. Saturate the slide with crystal violet for 1 minute. 6. Rinse the slide gently with water. 7. Allow the slide to air dry. DO NOT heat or blot dry!!!! Heat will melt the capsule! 8. Observe the slide under the microscope, using proper microscope technique. The background will be dark. The bacterial cells will be stained purple. The capsule (if present) will appear clear against the dark background. background capsule bacterium TIP: If you are staining an unknown organism, be sure your culture is several days old. Young, fresh cultures won’t have developed capsules yet. Smith

Stain Protocols – BIOL 2420 Acid-Fast Stain 1. Perform a bacterial smear, as discussed in Figure 3-52 on page 150 of your lab manual. 2. Place a small piece of bibulous paper over the smear and saturate the paper with carbolfuchsin. 3. Heat the slide gently over the Bunsen burner for 5 minutes. Be sure to keep the bibulous paper saturated with carbolfuchsin during heating. If the slide is steaming, you’re okay; if it stops steaming, add more carbolfuchsin! 4. Remove the bibulous paper from the slide, and rinse the slide gently with water. Dispose of the used bibulous paper in the trash. DO NOT leave the bibulous paper in the sink or drain! 5. Decolorize the slide with acid-alcohol until the rinsate runs clear. 6. Rinse the slide gently with water. 7. Counterstain with methylene blue for 2 minutes. 8. Rinse the slide gently with water. 9. Carefully blot the slide dry with bibulous paper. 10. Observe the slide under the microscope, using proper microscope technique. Acid-fast cells will stain fuchsia. Non-acid-fast cells will stain blue. Endospore Stain 1. Perform a bacterial smear of Bacillus or the organism you want to stain, as discussed in Figure 3-52 on page 150 of your lab manual. 2. Place a small piece of bibulous paper over the smear. Saturate the paper with malachite green. 3. Heat the slide gently over the Bunsen burner for 5 minutes. Be sure to keep the bibulous paper saturated with malachite green during heating. If the slide is steaming, you’re okay; if it stops steaming, add more malachite green! 4. Remove the bibulous paper from the slide, and rinse the slide gently with water. Dispose of the used bibulous paper in the trash. DO NOT leave the bibulous paper in the sink or drain! Parent cell or 5. Counterstain with safranin for 2 minutes. bacterium (red) 6. Rinse the slide gently with water. 7. Carefully blot the slide dry with bibulous paper. Endospore (green) 8. Observe the slide under the microscope, using proper microscope technique. Endospores will stain green. Parent cells will stain red. Smith

Laboratory‎ > The Gram Stain and Simple Stain Smear

FE204 Microbiology

Lab report

The Gram stain and simple stain Smear 

Purpose

       Preparation of smear and application of simple stain to observe size,shape, arrangement, color of the microorganisms using oil immersion objective.And application of differential staining method and classify microorganism as Gram positive, Gram negative.

Theory

 

Material

•                    Crystal violet

•                    Safranine

•                    Distilled Water

•                    Iodine

•                    Alcohol

•                    Bibulous paper

•                    Microscope

•                    E.coli in Nutrient broth culture

•                    Sterile Glass slide

•                    İnoculating loop

•                    Forcep

•                    İmmersion oil

•                    Bunsen burner

•                    Xylol

•                    Methylene blue

Procedure

       To preparation of smear, firstly in aseptic conditions a sterile microscopic slide was taken.It was flamed through a bunsen burner.After that, a few loops of E.coli broth culture was taken. The inoculum was putted on the slide.Then was waited for air drying.Next, heat fixation was applied and smear was prepared.

      In Gram staining process, firstly the ready smear was placed to staining pool.Then, a few drops of Crytal violet was added and was waited for 1 minute.After waiting, it was washed with water. Next, a few drops of Iodine in order to form CV-I complex was added and was waited for 1 minute.Then,it was washed with water again. After the this step, the smear was decolorized by using alcohol and it was washed with water.After that, 1 drop of Safranine was added and was waited for 1 minute.Then, it was washed with water and was dried with a paper.Finally, before it was examined under microscope at 100x, a few drops of oil added on the slide.And was decided the bacterial smear is G(+) or G(-).

Result

Discussion

       Bacteria are too small to see without the aid of a microscobe. Even with a microscope, bacteria cannot be seen easily. Because microbial cytoplasm is usually transparent, it is necessary to stain microorganisms before they can be viewed with the light microscope.There are many different ways to stain bacteria so that they can be more easily visualized under the microscope. Some stains can also be used to identify and classify bacteria. The Gram stain is a differential stain that allows you to classify bacteria as either Gram-positive or Gram-negative.

      Using a single stain to color a bacterial cell is commonly referred to as a simple stain.  The most common dyes for this type of stain are methylene blue, fuchsin, and crystal violet.    Staining times for most simple stains are relatively short, usually from 30 seconds to 2 minutes, depending on the affinity of the dye.  After a smear has been stained for the required time, it is washed off gently, blotted dry, and examined directly under oil immersion.  This type of slide is useful in determining basic morphology and the presence or absence of certain kinds of granules.

      Bacteria that decolorize easily are called Gram-negative (red) and those that retain the primary stain are called Gram-positive (purple). Bacteria stain differently because of differences in their cell walls. Gram-positive cell walls consist of many layers of peptidoglycan. The crystal violet-iodine complex is larger than either the crystal violet or iodine molecules that entered the cell and the complex cannot pass through this thick cell wall. Gram-negative bacteria have a thin layer of peptidoglycan and an outer lipopolysaccharide layer. The alcohol dissolves the lipopolysaccharides so that the crystal violet-iodine complex can wash out of the cell.

      Before bacteria can be stained, a smear of bacteria must be made on a slide and heat fixed. A smear is made by spreading a bacterial suspension on a clean slide and letting it air dry. The dry smear is heated on a hot plate or passed through a flame several times to heat fix it. Heat fixing denatures bacterial enzymes, preventing them from digesting cell parts, which causes the cell to break, a process called autolysis. The heat also enhances the adherence of bacterial cells to the slide. Finally, it is very important to prepare thin smears.  If a smear is too thick one will not be able to see individual cells, their arrangement, or the details of their microstructures.  Thick smears with large clumps of cells can entrap the stain preventing it from being washed.  The first step in preparing a smear depends on  whether the organism has been growing in liquid or solid media.  Two or more loopfuls of liquid media containing the organism can be placed on the slide.  One can use an inoculating loop to disperse organisms from solid media into a drop of water on the slide.

Laboratory‎ > ‎Gram Stain

PURPOSE:

The purpose of this experiment was to learn the gram staining method and to observe the characteristics of gram negative and gram positive microorganisms.

THEORY:

The gram stain is the most widely used staining procedure in bacteriology. It is called a differential stain since it differentiates between gram-positive and gram negative bacteria. Bacteria that stain purple with the gram staining procedure are termed gram-positivethose that stain pink are said to be gram-negative.
The terms positive and negative have nothing to do with electrical charge, but simply designate two distinct morphological groups of bacteria. Gram-positive and gram negative bacteria stain differently because of fundamental differences in the structure of their cell walls. The bacterial cell wall serves to give the organism its size and shape as well as to prevent osmotic lysis. The material in the bacterial cell wall that confers rigidity is peptidoglycan. The gram-positive cell wall appears thick and consists of numerous interconnecting layers of peptidoglycan. Also interwoven in the cell wall of gram-positive bacteria are teichoic acids. Generally, 60% -90% of the gram-positive cell wall is peptidoglycan. The gram-negative cell wall, on the other hand, contains a much thinner, single layer of peptidoglycan only two or three layers thick. This is surrounded by an outer membrane composed of phospholipids, lipopolysaccharide, and lipoprotein. Only 10% – 20% of the gram-negative cell wall is peptidoglycan.

The gram staining procedure involves four basic steps:

1. The bacteria are first stained with the basic dye crystal violet. Both gram positive and gram-negative bacteria become directly stained and appear purple after this step.

2. The bacteria are then treated with gram’s iodine solution. This allows the stain to be retained better by forming an insoluble crystal violet-iodine complex. Both gram-positive and gram-negative bacteria remain purple after this step.

3. Gram’s decolorizer, a mixture of ethyl alcohol and acetone, is then added. This is the differential step. Gram-positive bacteria retain the crystal violet-iodine complex while gram-negative are decolorized.

4. Finally, the counterstain safranin (also a basic dye) is applied. Since the gram-positive bacteria are already stained purple, they are not affected by the counterstain. Gram-negative bacteria, that are now colorless, become directly stained by the safranin. Thus, gram-positive appear purple, and gram-negative appear pink.

MATERIALS:

– Microscope                                                              – Bunsen burner

– Inoculating loop                                                        – Test tube rack

– Nutrient agar of Escherichia coli                              – Pisette

– Nutrient agar of Bacillus subtilus                             – Distilled water

– Crystal violet                                                            – Alcohol

– Iodine                                                                      – Safranine

– Oil immersion                                                           – Microscope slide

– Holder                                                                     – A piece of napkin

PROCEDURE:

In here, firstly, immersed to alcohol microscope slide was taken and was held to flame very short time, then plates of Escherichia coli and Bacillus subtilis were taken and in the meantime inoculating loop was held to flame as far as redness. Afterwards; one loop E. coli and B. subtilis were taken and spread out on microscope slide and a drop of water on a slide was added, meanwhile loop was held to re-flame, then microscope slide was waited in air for drying. After that; microscope slide of bacteria were passed through the flame of the bunsen burner 3 or 4 times to heat-fixed and thus heat-fixed smear of bacteria was prepared. Afterwards; crystal violet was added to microscope slide and was waited for one minute. Gently it was washed with distilled water. Then, gram’s iodine solution was added and was waited for one minute and then gently it was washed with distilled water. Then, on microscope slide, stained bacteria were decolorized immediately with alcohol for 30 seconds and again bacteria were washed with distilled water. Then, safranine was added on microscope slide, bacteria were stained with safranine and waited for one minute and rinsed with distilled water. After that; excess water was taken with a piece of napkin, then it was dried in air. Next, a drop of oil immersion was added and finally, slide was placed on light microscope and bacteria were observed under oil-immersion objective (100X) and colour, shape, size of bacteria was recorded.

RESULTS:

                    Bacillus subtilis                                            Escherichia coli

Colour : purple or dark pink                                        Colour : red

Shape  : rod                                                                Shape  : rod

Size      : long                                                               Size      : short

 

Because of these results, B. subtilis is gram positive bacteria and E. coli is gram negative bacteria

DISCUSSION:

In this experiment, gram staining technique was tried to apply. Firstly; Bacillus subtilis and Escherichia coli cultures were used. These were put on the microscope slide. The microscope slide was sterilized previously. Then, microscope slide was passed through flame, that is; heat fixation was taken place, because of this B. subtilis and E. coli cultures were to fix on the microscope slide and to prevent their motile, thus microorganisms were non-motile. After that; crystal violet stain was applied above microorganisms. We applied it because this stain provides to be observed readily. Then iodine solution was applied on microorganisms. Iodine solution incorporates with crystal violet and crystal violet iodine complex (CV-I) occurs within the cell, and cells stain violet colour. Then microorganisms were decolourized with alcohol. These cells have cell wall and the cell wall contain lipid. This lipid layer is affected by alcohol. In addition; if cell is gram positive, cell wall dehydrates and shrinkage of pores occurs on cell wall, permeability of cell decreases and cells appear violet colour. If cell is gram negative, extend of pores occurs and permeability of cell increases and cells appear colourless. After then; when safranine is added, gram positive bacteria appear violet colour, that is; bacteria do not affect from safranine, gram negative bacteria appear red colour with effect of safranine. 

In our experiment, Bacillus subtilis was observed as purple or dark pink colour, therefore it was gram positive. Escherichia coli was observed as red colour, therefore it was gram negative. As a result; experiment results were compared with original results and original results came seasonably with our experiment results.

Laboratory‎ > Smear Preparation

SMEAR PREPARATION

The preparation of a smear is required for many laboratory procedures, including the Gram-stain. The purpose of making a smear is to fix the bacteria onto the slide and to prevent the sample from being lost during a staining procedure. A smear can be prepared from a solid or broth medium. Below are some guidelines for preparing a smear for a Gram-stain.