The carbon particles are negatively-charged, as is the cell membrane. The background looks black or sepia colored and the cells remain clear, since they repel the dye. Some positively charged inclusion bodies, such as sulfur, may stain. This stain gives accurate information on cell morphology and capsule presence because the cells are not fixed. Cell size appears slightly larger because any extracellular coatings or secretions on the outside of the cell membrane also do not stain.
Negative stains are useful for rapid determination of the presence of Cryptococcus neformans , the causative agent of cryptococcisis, in cerebral spinal fluid. This technique is also used when you stain for endospores and capsules. Just as in preparing a smear, you only need a small amount of organism. It is also important not use too much nigrosin. If it is too thick, the background will have a cracked appearance similar to mud puddles drying in the sun.
You want to get a light film. Your instructor will demonstrate this technique for you. Nigrosin comes off the slide and onto your oil immersion lens very easily. Be sure to thoroughly clean your oil lens when you are finished.
Then clean it again. Once it dries on the lens it is very difficult to remove and will impair your ability and the other micro students using that scope to see clearly out of the lens. The Gram stain is the most common differential stain used in microbiology. Differential stains use more than one dye.
The unique cellular components of the bacteria will determine how they will react to the different dyes. The Gram stain procedure has been basically unchanged since it was first developed in Almost all bacteria can be divided into two groups, Gram negative or Gram positive.
A few bacteria are gram variable. Trichomonas , Strongyloides , some fungi, and some protozoa cysts also have a Gram reaction. Very small bacteria or bacteria without a cell wall, such as Treponema , Mycoplasma , Chlamydia , or Rickettsia do not have a gram reaction. The characterization of any new bacteria must include their gram reaction.
Typically a differential stain has four components; the primary stain, a mordant that sets the stain, a decolorizing agent to remove the primary stain, and a counter stain.
In the Gram stain, the primary stain is crystal violet. This gives the cell an intense purple color. The mordant, iodine, forms a complex with the crystal violet inside the cell wall. Gram positive cells will retain the dye complex and remain purple. The dye rinses out in gram negative cells. The large iodine-crystal violet complex is retained within the cell walls of gram positive cells because of the molecular structure of the many layers of peptidoglycan in the cell wall.
There are lots of cross-linked teichoic acids and the iodine-dye complex cannot physically get out. There is also less lipid in the membrane and the decolorizing agent cannot get to it as well.
Gram negative cells have an outer membrane and only one layer of peptidoglycan, with more lipid. The crystal violet dye is easily washed out. The accuracy of the Gram stain is dependent on the integrity of the bacterial cell wall. There are a variety of things that can influence the cell wall integrity; old cells i. Under these conditions, gram positive cells will come out as gram-negative. If you de-colorize too long, Gram-positive cells will look like Gram-negative cells.
Conversely, if you do not decolorize enough, Gram-negatives will look like Gram-positives. The only way you can trust your results it to always run a known Gram-positive and a known Gram-negative on the same slide. If they stain as predicted you can be pretty sure the result of your unknown sample is reliable. The Gram staining takes practice to get right. Do not expect to get a good Gram stain on your first try.
It is a good idea to hold your slide with a clothespin; your gloves will get pretty psychedelic as will everything you touch! The Congo Red Capsule stain is a modification of the nigrosin negative stain you may have done previously.
The bacteria take up the congo red dye and the background is stained then with acid fuchsin dye. The capsule or slime layers, highly hydrated polymers, exclude both dyes. The background will appear blue, the bacterial cells will appear pink, and the clear halos are the capsules. Clinically, the capsules of some highly pathogenic bacteria i.
The bacteria are suspended in the antisera and then mixed with methlyene blue. In the antisera staining procedure, the bacteria will appear blue surrounded by a clear halo and then surrounded by a thin blue line where the antisera have attached to the capsule. This technique has been used in many areas such as cytology, hematology, oncology, histology, virology, serology, microbiology, cell biology, and immunochemistry.
One of the key pieces of equipment for preparing a slide for cell staining is cytology centrifuge cytocentrifuge such as cytospin. However, many small labs do not have this expensive equipment and its accessory, cytoclips also expensive relatively , which makes them difficult to study cell cytology. Here we present an alternative method for preparing a slide and cell staining in the absence of a cytocentrifuge and cytoclips.
This method is based on the principle that a regular cell centrifuge can be used to concentrate cells harvested from cell culture and then deposit the concentrated cell suspension to a slide evenly by using a cell spreader, followed by cell staining.
The method presented is simple, rapid, economic, and efficient. This method may also avoid a possible change in cell morphology induced by cytocentrifuge. In a basic research lab, cell morphology study, in a standard protocol, involves three steps: 1 cell culture and treatment, 2 preparation of slides, and 3 cell staining and visualization.
The preparation of the slides usually uses cytoclips or called slide clips to hold the slide with a slide funnel cytology funnel together, so that cell suspension can be dropped into the funnel. Then, the slide-funnel complex is set for centrifugation in a centrifuge especially designed for slides cytocentrifuge [ 1 — 5 ].
The purpose of the centrifugation is to concentrate and deposit cells from the funnel to the slide evenly relatively. After centrifugation, the slides are immersed in several bottles that contain either stains or water for staining and washing steps, one by one, in a specific consecutive order. Since a cytocentrifuge and metal cytoclips are relatively expensive, these prevent many small labs or individual researchers from working in the area of cell cytology.
In addition, the cytospin-induced changes in cell morphology have been reported and cytospin smears should be evaluated with caution [ 2 ].
Here we present a simple and efficient alternative method for preparing cell slides and staining in the absence of a cytocentrifuge and cytoclips. This method is based on the principle that a regular cell centrifuge all labs dealing with cell culture have this equipment can also be used to concentrate cells harvested from cell culture and then deposit the concentrated cell suspension to a slide evenly by using a cell spreader, after which the slides are ready for staining. This method avoids cytospin-induced possible change in cell morphology.
The staining procedure we used also bypasses several steps used in a standard protocol. Our results demonstrated that this new method is simple, efficient, and economic. The slides made by this method have the same applications in cytology study as the one prepared from a cytocentrifuge but avoid possible changes in cell morphology induced by cytospin method.
We have used these noncytospin slides to observe Phorbol myristate acetate- PMA- induced differentiation and Bay induced apoptosis in several human myeloid leukemia cells after staining with different stain reagents. All the media and sera were purchased from Life Technologies, Inc. Gaithersburg, MD. The control cells were treated with PMA or Bay solvent. The glass spreader was made from glass transfer pipette over an alcohol burner for few seconds to minutes.
The slide was rinsed briefly with small amounts of tap water, after which one small drop of mounting medium was added to the slide and covered with a coverslip. Therefore, these cells need to be concentrated before applying them to a slide. In the absence of a cytology centrifuge, we used regular cell centrifuge to concentrate cells harvested from the culture see Materials and Methods. Subsequently, the drops of the concentrated cells were dropped to the central area of the slide with a plastic transfer pipette, after which a glass spreader was used to move cells around to form a thin and even layer.
Figure 1 shows an image of the glass spreader during the process of applying cells to a slide. In order to test whether the slides made by using this noncytocentrifuge method and the glass spreader are good enough for cytology study, three types of human myeloid leukemia cells were applied for preparation of slides and two types of stain methods were used for staining.
The first cell line is TF-1a. Our previous experiments have demonstrated that TF-1a cells are able to respond to PMA and can be induced to macrophage-like differentiation [ 6 ]. This cell line is a good model to study cell differentiation. As shown in Figure 2 , cells treated with Giemsa a show good contrast.
At the sink, pick up the bottle of the slide cleansing liquid , pouring some of the thick suspension onto the slide. Use your finger to spread the suspension over each side of the slide, and then wash WELL with tap water. Dry the slide.
You will make smears using 2 different types of cultures, a broth and an agar culture. Label the 2 slides with the names of your bacteria. Shaking the culture first, aseptically transfer a drop of the broth culture using an inoculating loop to a clean slide. From the agar plate culture, remove a small inoculum use only a small amount of large colonies with a loop and suspend it in a small drop of distilled water.
Mix the culture well into the water drop. Label 2 slides Staph and E. Spread the smear suspensions over the glass slide so that it forms a thin layer and it is the size of nickel or larger. Let the slide air-dry —totally. This step facilitates the fixing of the smear to the slide.
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