What is the difference between simple stain and gram staining

These structures are produced by a few genera of Gram-positive bacteria, almost all bacilli, in response to adverse environmental conditions. Two common bacteria that produce endospores are Bacillus or Clostridum. Both live primarily in soil and as symbionts of plants and animals, and produce endospores to survive in an environment that change rapidly and often. The process of endosporulation the formation of endospores involves several stages. After the bacterial cell replicates its DNA, layers of peptidoglycan and protein are produced to surround the genetic material.

Once fully formed, the endospore is released from the cell and may sit dormant for days, weeks, or years. When more favorable environmental conditions prevail, endospores germinate and return to active duty as vegetative cells.

Mature endospores are highly resistant to environmental conditions such as heat and chemicals and this permits survival of the bacterial species for very long periods. Endospores formed millions of years ago have been successfully brought back to life, simply by providing them with water and food.

Because the endospore coat is highly resistant to staining, a special method was developed to make them easier to see with a brightfield microscope. This method, called the endospore stain , uses either heat or long exposure time to entice the endospores to take up the primary stain, usually a water soluble dye such as malachite green since endospores are permeable to water.

Following a decolorization step which removes the dye from the vegetative cells in the smear, the counterstain safranin is applied to provide color and contrast. When stained by this method, the endospores are green, and the vegetative cells stain pink, as shown in Figure 7.

Although endospores themselves are resistant to the Gram stain technique, bacterial cells captured in the process of creating these structures can be stained. In this case, the endospores are seen as clear oval or spherical areas within the stained cell. Endospores can also be directly observed in cells by using phase contrast microscopy, as shown in Figure 8. Because many differential staining methods require several steps and take a long time to complete, we will not be performing all of the differential staining methods discussed above.

Pre-stained slides will be used to visualize bacterial capsules, metachromatic granules, and acid-fast bacilli. Obtain one slide of each of the three bacteria listed in the table below. Your environmental isolate may have one or more of these cellular features, and learning to recognize them will aid in identification. These should all be viewed using the oil immersion objective lens. All staining procedures should be done over a sink. The Gram stain procedure will be demonstrated, and an overview is provided in Table 1.

A volunteer from your lab bench should obtain cultures of the bacteria you will be using in this lab, as directed by your instructor. One of the cultures will be a Gram positive bacterium, and the other will be Gram negative. Below, write the names of the bacteria you will be using, along with the BSL for each culture:.

Obtain two glass slides, and prepare a smear of each of the two bacterial cultures, one per slide, as demonstrated. Stain both smears using the Gram stain method. Observe the slides with a light microscope at 1,X and record your observations in the table below.

Heat fix the smear and Gram stain it. You should be able to determine the Gram stain reaction, cellular morphology and arrangement of BOTH bacteria in this mixed smear. Your instructor may ask to see this slide and offer constructive commentary. Only a few genera of bacteria produce endospores and nearly all of them are Gram-positive bacilli. Most notable are Bacillus and Clostridium species, which naturally live in soil and are common contaminants on surfaces.

The growth of Clostridium spp. Endospore-forming bacteria are distinct from other groups of Gram positive bacilli and distinguishable by their endospores. After staining, endospores typically appear as light green oval or spherical structures, which may be seen either within or outside of the vegetative cells, which appear pink.

The shape and location of the endospores inside the bacterial cells, along with whether the sporangium is either distending D or not distending ND the sides of the cell, are important characteristics that aid in differentiating among species see Figure 9. A volunteer from your lab bench should obtain bacterial cultures for endospore staining, as directed by your instructor. Note that these will all be species of Bacillus.

Prepare smears and stain each using the endospore staining technique. Observe the slides and note the shape and location of the endospore and the appearance of the sporangium swollen or not swollen in the table below:.

For more information, visit coronavirus. Not only are most bacteria very small, they are also very clear and difficult to view under a microscope without first staining. You must firmly attach your bacteria to a glass slide before you can stain them.

There are two important things to consider when preparing a slide for staining:. You will be preparing slides for staining from both broth and agar surfaces. While the goals are the same for both, evenly and lightly dispersed cells firmly adhered to the slide surface, the techniques are slightly different. Staining is as much art as science. It will undoubtedly take you several tries before you are successful.

Be careful of aerosols when transferring bacteria from your loop to the slide. The loop is very flexible and it is easy to zing off a loop-full of organisms. Do not assume your organism is dead. Heat or methanol fixation is not guaranteed to kill the organism. Dispose of your completed slides in the disinfectant bucket at your bench.

You are striving for a light suspension of cells that will leave a faint cloudy deposit on your slide. You have lots of room on your slide; use it!

It helps to initially draw a circle on the bottom of the slide so you know where to look for your smear. It is very easy to get confused which side of the slide your smear is on. Be sure to label the far edge of the slide. Do this consistently on the same end of the slide to help orient your slide.

Be patient and take the time to let your slide air dry before proceeding with adhering it to the slide. If your slide is wet and you heat fix it, the bacteria will boil and the cellular morphology will be lost. If your slide is wet and fix it in methanol, it will most likely wash off the slide. Smears that are too thick will most likely wash off the slide regardless of the fixation method.

Broth cultures are usually easier to work with because the cells are already diluted in the broth. Be sure to carefully mix the culture tube to suspend the bacteria in the broth. You can scoop a lot of organisms off with your loop. You may want to use an inoculating needle to transfer your organism to the slide.

Be sure to use sterile water to dilute your samples. Regular tap water or the de-ionized water in your rinse bottles are often contaminated with bacteria. The fixation procedure is the same regardless of smear source, plate or broth. There are two methods of adhering your bacteria to the slide, heat fixation or methanol fixation.

Heat fixing is only used with BSL1 organisms. The organisms we will be working with are BSL2, so you will need to use the methanol fixation technique. Heat fixing the slide can create aerosols and with BSL2 organisms, we need to prevent this as much as possible.

Methanol fixation causes fewer changes in cellular morphology and creates no aerosols. Simple stains are just that - add one stain to a fixed smear slide, let it sit, rinse it off, let it dry, and view. It is a quick procedure for determining the presence and morphology of bacteria in clinical samples such as stool and discharges.

Methylene blue is used to determine the morphology of fusiform and spirochetes in oral infections. It is also the stain of choice for identifying the metachromatic granules in Corynebacterium diphtheriae. The granules will stain a distinctly deeper blue than the surrounding blue bacteria. Other species of Corynebacterium do not have the metachromatic granules. Any basic dyes, such as methylene blue, crystal violet, malachite green, or safranin work well. Basic cationic or positively charged dyes bind to negatively charged components in the cell membrane and cytoplasm.

Staining is part art and part science. There are no hard and fast rules for staining and rinsing times. The times listed are suggestions that usually work well. You will need to experiment with what works for the bacteria you have and the techniques you use. It is essential that you record exactly what you do and the results you observe in your lab book. It would be useful for each lab bench member to pick a different stain so you can see what they all look like. Negative stains are even simpler than simple stains because you do not have to make a smear.

A drop of cells is spread on a slide and viewed without fixation. The stain is a suspension of carbon, found in India ink or nigrosin. The carbon particles are negatively-charged, as is the cell membrane. A simple stain that stains the background but leaves the bacteria unstained. Of what value is a simple stain?

Determining cell morphology, size, and arrangement. Simple stain can make cell parts show up that would usually be naked to the eye.

Kym Jemlikhanov Pundit. Why is time important in simple staining? Basic dyes are more successful in staining bacteria than acid dyes because basic dyes have positive charges and the bacterial cell walls are negative, so they attract. Time is important because it creates a contrast between the bacteria and the stain. If you over or under stain you won't be able to see bacteria. Magaly Slattery Teacher. What is the difference between positive and negative staining?

In this technique, the background is stained, leaving the actual specimen untouched, and thus visible. This contrasts with ' positive staining ', in which the actual specimen is stained.

For bright field microscopy, negative staining is typically performed using a black ink fluid such as nigrosin and India ink. Yonaida Schiebel Supporter. What is gram variability? Definition of gram - variable. Andries Bucha Supporter. What is the function of Decolorizer in Gram staining? A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it.

The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria. Eniko Sobe Supporter. Is the negative stain simple or differential? Methylene blue is a simple stain that colors cells blue.

In a negative staining technique, a negatively charged stain colors the background, leaving the cells light colored and unstained. The resulting difference in color can be used to distinguish different cell types. Ayoube Beiras Beginner. What is a differential stain name two examples? A differential stain is one in which two dyes are used. Ghassan Saddik Beginner. How do you do Gram staining? Place slide with heat fixed smear on staining tray.

Gently flood smear with crystal violet and let stand for 1 minute. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.

Gently flood the smear with Gram's iodine and let stand for 1 minute. Lohitzune Fortini Beginner.