Stains And Staining Techniques In Microbiology Pdf

stains and staining techniques in microbiology pdf

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Staining Methods – Simple Staining, Negative Staining, Gram’s Staining and Acid-Fast Staining

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Full Name Comment goes here. Are you sure you want to Yes No. Barnali Sarkar. Show More. No Downloads. Views Total views. Actions Shares. No notes for slide. Staining Techniques in Microbiology 1. They are also colorless for the most part.

In order to visualize them to study their structure, shape and other structural characteristics, it becomes necessary to make them more easily visible. Ex: Haematoxillin, methylene blue, crystal violet, gention violet. Method: -Aseptically a small sample of the culture is spread over a slide surface. Do not overheat. Alternatively, flood smear with methanol for 1 minute.

Stain for seconds and wash well with water. Over-staining must be avoided, as this is an intense stain, and prolonged application colours the cell protoplasm in addition to nuclei and bacteria. He later published this procedure in At the time, Dr. Gram was studying lung tissue sections from patients who had died of pneumonia.

Gram described how he was able to visualize what we now call Staphylococcus, Streptococcus, Bacillus, and Clostridia in various histological sections. Interestingly, Dr. Gram did not actually use safranin as a counter stain in the original procedure Gram negative cells would be colorless.

He instead recommended using Bismarck brown as a counter stain to enable tissue cell nuclei to be visualized. This causes decreasing of the space between the molecules causing the cell wall to trap the crystal violet iodine complex within the cell. Hence the Gram positive bacteria do not get decolorized and retain primary dye appearing violet.

Also, Gram positive bacteria have more acidic protoplasm and hence bind to the basic dye more firmly. As a result, the dye-iodine complex is not retained within the cell and permeates out of it during the process of decolourisation. Hence when a counter stain is added, they take up the colour of the stain and appear pink. In Gram positive bacteria, there are "wall teichoic acids". As well, between the cell wall and cell membrane, there is a "membrane teichoic acid".

Stain with crystal violet solution, 2 min. Rinse in tap water. Iodine solution, 2 min. Wash in tap water. Counter stain in neutral red, 3 min. Result: Gram-positive organisms, fibrin, some fungi, keratohyalin, and keratin - purple Gram — negative organisms -red. Wash off with water, blot and dry. Fix and stain as described. Some of them have improved the method, others include some minor technical variants of no value.

Any final result is the outcome of the interaction of all of the possible variables. Decolourizer is Absolute Alcohol. Counter stain is Neutral Red. Decolourizer is Iodine-Acetone.

Decolourizer is —Aniline-Xylol. Weigert stain is used to stain tissue sections. Rapid presumptive diagnosis of diseases such as Bacterial meningitis. Selection of Empirical antibiotics based on Gram stain finding. Selection of suitable culture media based on Gram stain finding. Counting of bacteria. The degree of acid fastness varies in different bacteria. The tubercle bacilli resist the decolorizing action of acid-alcohol which confers acid fastness to the bacteria.

The non-acid fast bacilli readily absorb the colour of the counter stain appearing blue, while the acid fast cells retain the red colour of primary stain. Counterstain with Methylene Blue. Make a smear. Air Dry. Heat Fix. Steam for 5 minutes.

Flood slide with acid alcohol leave 15 seconds. Rinse slide and let it dry. Use oil immersion objective to view. Smears of sputum should be thin. Do not use blotting paper.

Tubercle bacilli are seen as yellow luminous rods in a dark field. When detected under low power, the morphology of the bacilli is confirmed with an oil-immersion objective. Lipid granules also stain red, appearing like small spherical spores. Lipid granules are unstained. Two dyes, one acidic and one basic, are used to stain the background and the cell wall, respectively.

Note: The smear should not be heat fixed. Allow it to stain for minutes. Cryptococcus neoformans india i The smaller ones, e. They are best observed in unstained wet films under the dark ground microscope where their bright appearance and motility draw attention to them. The classical methods of Fontana for films and Leavaditi for sections. Touch a loopful of water onto the edge of a colony and let motile bacteria swim into it. The bacterial suspension is thus prepared with a minimum of agitation, which would detach the flagella.

Examine with the microscope after standing min at ambient temperature. It contains both positively and negatively charged imparts different colors to different components.

The original stain has now been replaced by various modifications which are easier to use and give better results.

Differential Staining Techniques

The microscope is a very important tool in microbiology, but there are limitations when it comes to using one to observe cells in general and bacterial cells in particular. Two of the most important concerns are resolution and contrast. Contrast, however, can be improved by either using a different type of optical system, such as phase contrast or a differential interference contrast microscope, or by staining the cells or the background with a chromogenic dye that not only adds contrast, but gives them a color as well. There are many different stains and staining procedures used in microbiology. Some involve a single stain and just a few steps, while others use multiple stains and a more complicated procedure. Before you can begin the staining procedure, the cells have to be mounted smeared and fixed onto a glass slide.

Microorganisms or microbes are organisms which can only be individually seen by microscopy. Many do not cause disease in humans and act as normal colonizers of human hosts. Complex interactions between pathogens, which are capable of causing diseases, the host and the environment lead to clinical infections. Pathogens fall into five main groups:. With the advent of new and broader spectrum antibiotics, improved environmental hygiene, and advances in microbiological techniques it was widely expected that the need for diagnosis of infectious agents in tissue would diminish in importance.

In their natural state, most of the cells and microorganisms that we observe under the microscope lack color and contrast. This makes it difficult, if not impossible, to detect important cellular structures and their distinguishing characteristics without artificially treating specimens. We have already alluded to certain techniques involving stains and fluorescent dyes, and in this section we will discuss specific techniques for sample preparation in greater detail. Indeed, numerous methods have been developed to identify specific microbes, cellular structures, DNA sequences, or indicators of infection in tissue samples, under the microscope. Here, we will focus on the most clinically relevant techniques. In clinical settings, light microscopes are the most commonly used microscopes. There are two basic types of preparation used to view specimens with a light microscope: wet mounts and fixed specimens.

2014, Number 1

Important information such as shape and degree of motility can be obtained by observation of living bacteria with the phase contrast or dark field microscope. However bacteria are routinely stained with different dyes in order to reveal different properties and to enhance contrast for viewing with conventional bright field microscopy. A number of stains have been developed to distinguish spores, nuclear bodies, capsules, and characteristics of the cell wall. The staining methods we will use kill the bacteria, reducing the risk of infection by pathogenic organisms.

Staining Methods – Simple Staining, Negative Staining, Gram’s Staining and Acid-Fast Staining

Show Caption Hide Anthrax gram stain. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process. Gram staining involves three processes: staining with a water-soluble dye called crystal violet, decolorization, and counterstaining, usually with safanin.

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PDF | The correct identification of micro organisms is of fundamental importance Some staining techniques, such as the Gram and Ziel Neelsen stains, although (e.g. agriculture, clinical microbiology and food production).

Traditional stains and modern techniques for demonstrating microorganisms in histology

Show Credits Hide This Giemsa stained micrograph depicts an example of a slightly acidic slide that yielded a pink colored resultant stain. The micrograph shows malarial cells. Photo courtesy of the Public Health Image Library.


Armen M.


Uses an acidic stain: Congo red or Nigrosin and a basic stain: crystal violet or safranin.

Galatee G.


Stain – Majority of the stains used for staining bacteria are of the basic type as ➢A simple staining technique that stains the bacterial cells in a single color.