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  Section: Biotechnology Methods » Microbiology
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Bacterial Staining

  The Microscopy
  The Bright Field Microscope
  Introduction to the Microscope and Comparison of Sizes and Shapes of Microorganisms
  Cell Size Measurements: Ocular and Stage Micrometers
  Measuring Depth
  Measuring Area
  Cell Count by Hemocytometer or Measuring Volume
  Measurement of Cell Organelles
  Use of Darkfield Illumination
  The Phase Contrast Microscope
  The Inverted Phase Microscope
  Aseptic Technique and Transfer of Microorganisms
  Control of Microorganisms by using Physical Agents
  Control of Microorganisms by using Disinfectants and Antiseptics
  Control of Microorganisms by using Antimicrobial Chemotherapy
  Isolation of Pure Cultures from a Mixed Population
  Bacterial Staining
  Direct Stain and Indirect Stain
  Gram Stain and Capsule Stain
  Endospore Staining and Bacterial Motility
  Enumeration of Microorganisms
  Biochemical Test for Identification of Bacteria
  Triple Sugar Iron Test
  Starch Hydrolysis Test (II Method)
  Gelatin Hydrolysis Test
  Catalase Test
  Oxidase Test
  IMVIC Test
  Extraction of Bacterial DNA
  Medically Significant Gram–Positive Cocci (GPC)
  Protozoans, Fungi, and Animal Parasites
  The Fungi, Part 1–The Yeasts
  Performance Objectives
  The Fungi, Part 2—The Molds
  Viruses: The Bacteriophages
  Serology, Part 1–Direct Serologic Testing
  Serology, Part 2–Indirect Serologic Testing

Visualization of microorganisms in the living state is very difficult, not just because they are minute, but because they are transparent and almost colorless when suspended in an aqueous medium.

To study their properties and divide microorganisms into specific groups for diagnostic purposes, biological stains and staining procedures, in conjunction with light microscopy, have become major tools in microbiology. Chemically, a stain may be defined as an organic compound containing a benzene ring plus a chromophore and an auxochrome.

Stains are of 2 types:
  1. Acidic stains e.g., picric acid
  2. Basic stains e.g., methylene blue.
Types of staining techniques:
  1. Simple staining (use of a single stain): This type of staining is used for visualization of morphological shape (cocci, bacilli, and spirilli) and arrangement (chains, clusters, pairs, and tetrads).
  2. Differential staining: (use of 2 contrasting stains) It is divided into two groups:
    1. Separation into groups, Gram stain and acid-fast stain.
    2. Visualization of structures, Flagella stain, apsule stain, spore stain,
      nuclear stain.
Staining Microbes
  • Microbes are invisible to the naked eye and are difficult to see and identify, even when using a microscope. Staining microbes makes them easier to observe and reveals the presence of microscopic structures, because charged portions of the stain bind to specific macromolecules within the structures. A simple stain displays the microorganisms, and a differential stain displays the chemical differences in cellular structures, including the cell wall and cell membrane, because the macromolecules within the structure bind to different components of the stain.

  • An example of this differential staining is seen in staining used for blood smears.

  • Staining white blood cells with a differential stain displays the difference between the 5 white blood cell types: basophils, eosinophils, neutrophils, monocytes, and lymphocytes. The intracellular granules of basophils stain dark blue because of their affinity to basic portion of the stain. Basophil means basic loving. On the other hand, the eosinophil (acid loving) stains red as a result of the intracellular granule’s affinity for the acidic portion of the stain. Treatment of microbial diseases depends upon the correct identification of microorganisms and relies upon the ability to identify specific internal structures. Bacterial cells are commonly stained with a differential stain called the Gram stain and protozoal cells with the trichrome stain, in order to reveal the internal structural differences and to identify other organisms. Properly preparing slides for staining is important to ensure good results. Remember, you cannot see the material you are working with, so you must develop good technique based upon principles.

  • Always start with clean slides, using lens paper to clean them. Slides can be made from direct clinical material (a wound, sputum, knee fluid, the throat, etc.), broth cultures, and solid media cultures. The first principle is that some fluid is needed to emulsify the material if it is dry; however, too much fluid may make the microbes hard to find. Slides from clinical cultures are usually placed directly on the slide without the addition of water, as are slides from broth cultures. Slides from solid media require water to emulsify and separate the individual bacterial cells for better observation, but a single drop of water is usually adequate. Next, the material must be attached to the slide so it doesn’t wash off with the staining process. The second principle involves fixing the slide using either a chemical fixative or heat. In this lab, a heat-fixing tray will be used.

  • This lab will use the principles and techniques above to make and stain bacterial slides, using a differential staining technique called the Gram stain. Initially, 3 stock cultures (known types) of bacteria will be stained, and then the 3 isolated unknown microbes from the environmental cultures will be stained and examined. The environmental culture will contain a variety of bacteria and possibly some fungi. Bacterial cells can be observed for shape (rod, coccus, or spirilla) and arrangement (in chains, clusters, etc.). Arrangements of cells are best observed from clinical and broth cultures because the emulsification process disrupts the natural arrangement from colonies “picked” from solid media.

  • 3 isolation plates
  • Original environmental broth culture
  • Original TSA plate

Stock Cultures
  • S. epidermidis,
  • E. coli.,
  • Bacillus sp.
  • Slides, transfer loops

  • Gram stain reagents
  • Crystal violet
  • Iodine
  • Decolorizer—acetone-ethanol
  • Safranin

Preparing the Smears
  1. Collect broth and pure subcultures and observe the colony morphology: Ask the instructor to critique your isolation technique. This will be very important in later labs. Practice the isolation technique on a new plate if you need some more experience.
  2. Preparing a smear: There are 3 steps to prepare a smear for staining. Remember to use aseptic technique and flame the loop before and after each use.
    1. Preparation of the slide—Clean and dry the slide thoroughly to remove oils.
    2. Preparation of the smear—From the broth culture, use the loop to spread 1 or 2 drops of specimen in the center of the slide, spreading it until it is approximately the size of a nickel. When making a smear from solid media cultures, start by putting a very small drop of water in the center of the slide and then mix a loopful of bacteria from the surface of solid media in the water, spreading it out to the size of a nickel.
    3. Fixation—The point of fixation is to attach the organisms and cells to the slide without disrupting them. In this class, we will use an electric fixing tray that will dry and fix the smears in one step. Slides must be completely dry and fixed before staining, or they will wash off.

Staining the Smears
  1. In the beginning, it is wise to make a single broth slide and a single solid medium slide, and then stain and observe these before making the other slides. This allows you to alter your technique if the results are not optimal.
  2. Begin with the known culture smears (S. epidermidis, E. coli., or Bacillus sp.). Place the smear on the staining rack over the sink.
  3. Cover the smear area with the crystal violet stain, leave it for 15 seconds, and then rinse the slide with a gentle stream of water.
  4. Apply Gram’s iodine, covering the smear completely for 15 seconds, and then rinse.
  5. Using Gram’s decolorizer, apply it a drop at a time to the smear area until no more color leaves the area. Quickly rinse with water to stop the decolorizing process.
  6. Apply safranin to the smear for 15 seconds, then rinse with water.
  7. Allow the smear to air dry or place it on the drying and fixation tray.

Simple Staining
Perform the simple staining procedure to compare morphological shapes and arrangements of bacterial cells.

In simple staining, the bacterial smear is stained with a single reagent. Basic stains with a positively charged chromogen are preferred, because bacterial nucleic acids and certain cell wall components carry a negative charge that strongly attracts and binds to the cationic chromogen. The purpose of simple staining is to elucidate the morphology and arrangement of bacterial cells. The most commonly used basic stains are methylene blue, crystal violet, and carbol fuchsin.

  • Cultures: 24-hours nutrient agar slant culture of E.coli and Bacillus cereus, and a 24-hour nutrient broth culture of S. aureus.
  • Reagents: Methylene blue, crystal violet, and carbol fuchsin.

  1. Prepare separate bacterial smears of the organisms. All smears must be heat fixed prior to staining.
  2. Place a slide on the staining tray and flood the smear with one of the indicated stains, using the appropriate exposure time for each carbol fuchsin, 15 to 30 seconds, crystal violet, 20 to 60 seconds, and methylene blue, 1 to 2 minutes.
  3. Wash the smear with tap water to remove excess stain. During this step, hold the slide parallel to the stream of water. This way, you can reduce the loss of organisms from the preparation.
  4. Using bibulous paper, blot dry, but do not wipe the slide.
  5. Repeat this procedure with the remaining 2 organisms, using a different stain for each.
  6. Examine all stained slides under oil immersion.

Negative Staining
Negative staining requires the use of an acidic stain such as nigrosin. The acidic stain, with its negatively charged chromogen, will not penetrate the cells because of the negative charge on the surface of bacteria. Therefore, the unstained cells are easily discernible against the colored background.

The practical application of the negative staining is 2-fold: first, since heat fixation is not required and the cells are not subjected to the distorting effects of the chemicals and geat, their natural size and shape can be seen. Second, it is possible to observe bacteria that are difficult to stain, such as some spirille.

Cultures: 24-hour agar slant cultures of Micrococcus luteus, Bacillus cereus, and Aquaspirillum itersonii.

  1. Place a small drop of nigrosin close to one end of a clean slide.
  2. Using sterile technique, place a loopful of inoculum from the Micrococcus luteus culture in the drops of nigrosin and mix.
  3. With the edge of a second slide, held at a 30° angle and placed in front of the bacterial suspension. Push the mixture to form a thin smear.
  4. Air dry. Do not heat fix the slide.
  5. Repeat steps 1 to 4 for slide preparations of Bacillus cerus and Aquaspirillum itersonii.
  6. Examine the slide under oil immersion.


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