Bacterial Staining PDF

# Bacterial Staining ## A. Bacterial Staining Visualization of microorganisms in the living state is quite difficult, not only because they are minute, but also because they are transparent. To study their properties and divide the microorganisms into groups for more identification is accomplished...

# Bacterial Staining ## A. Bacterial Staining Visualization of microorganisms in the living state is quite difficult, not only because they are minute, but also because they are transparent. To study their properties and divide the microorganisms into groups for more identification is accomplished by applying staining procedures. - Chemically, stain (dye) have a colored portion with an electrical charge allowing the stain to bind with to macromolecular cellular components such as proteins or nucleic acids of the bacteria. There are two major types of stains: - **Acidic stains:** are anionic, the colored part of the stain has a negative charge and therefore has a strong affinity for the positive constituents of the cell. Proteins, for example, are positively charged will accept the color because of the binding of the positive charge of the protein to the negatively charged portion of the stain. - **Basic stains:** are cationic, because the colored portion of the stain have positive charge and therefore has a strong affinity to the negative constituents of the cell. Nucleic acids, as an example of negatively charged cell component that will bind to the positive portion of the cationic stain. ## Types of staining techniques | Type | Description | |---|---| | Simple Staining | Use of a single stain. | | Differential Staining | Use of two contrasting stains separated by a decolorizing agent. | | | | | Simple Staining | For visualization of morphological shape & arrangement | | Differential Staining | Identification | | | Visualization of structure | | Differential Staining | Gram stain | | Differential Staining | Acid fast stain | | Differential Staining | Spore stain | | Differential Staining | Capsule stain | ## B. Bacterial Staining Procedures ### a) Preparation of bacterial smear Bacterial smear must be prepared prior of any of the staining technique. Although not difficult however, it needs adequate care. The critical step in smear preparation is the heat fixation. Bacteria will wash away unless it is fixed on the glass slide and this is done through air-heat fixation. ### b) Bacterial Simple Staining In simple staining, the bacterial smear is stained with single reagent. Basic stains are preferred, because bacterial nucleic acids and other cell wall components carrying negatively charged. The purpose of simple staining is to observe the morphology and the arrangement of the bacterial cells. The most commonly used basic stains are methylene blue and crystal violet. ## Overview of a bacterial staining procedure A bacterial smear is prepared by first spreading the culture in a thin film over a slide, then drying it in air, and finally passing the slide through a flame to fix it. Once the smear is prepared, the slide is flooded with a stain, rinsed, dried, and examined under a microscope. ### c) Gram stain principle The most important differential stain used in bacteriology is Gram stain, it divides bacterial cells into two major groups: 1) Gram-positive (blue color) and 2) Gram-negative (red color). It is an important tool for classification and differentiation of microorganisms. The gram stain reaction is based on the differences in the composition of the bacterial cell wall. Gram positive cells have thick peptidoglycan layer, while in gram negative the layer of peptidoglycan is much thinner and surrounded by lipid containing layer. ## Figure 4: Gram-stained bacterial cells | Gram-Negative Bacteria | Gram-Positive Bacteria | |---|---| | Image of Gram-Negative Bacteria | Image of Gram-Positive Bacteria | ## Figure 5: Gram-positive cell wall and Gram-negative cell wall **Gram-positive cell wall:** - Teichoic acid - Peptidoglycan - Plasma membrane - Cytosol - Polysaccharide - Protein **Gram-negative cell wall:** - Braun's lipoprotein - Porins - O-specific side chain - Lipopolysaccharides - Outer membrane - Periplasmic space and peptidoglycan - Plasma membrane - Cytosol - Phospholipid ### d) Gram stain procedures The gram stain uses four different reagents: 1) **Primary stain:** crystal violet is used to stain all cells purple. 2) **Mordant:** Gram's iodine is used as a killing agent and mordant, that means increasing the cell affinity for stain. It does this by binding to the primary stain, thus forming insoluble complex called crystal-violet- iodine complex that increase the color of the stain and the bacterial cells appear purple black color. 3) **Decolorizing agent:** Ethyl Alcohol 95% function as lipid solvent and act on the lipid containing layer in the Gram negative bacteria, thus the crystal-violet- iodine complex can be easily removed from the cell and become colorless. 4) **Counterstain:** Safranin, is a final agent used to stain the colorless bacterial cells with pink color. ## Figure 6: steps involved in the gram stain procedure | 1 | 2 | 3 | 4 | |---|---|---|---| | Image depicting the step of application of crystal violet | Image depicting the step of application of iodine | Image depicting the step of alcohol wash | Image depicting the step of application of safranin | | Application of crystal violet | Application of iodine | Alcohol wash | Application of safranin | # Bacterial Culture Purification Microorganisms are transferred from one medium to another by subculturing. This technique is of basic importance and is used routinely in preparing and maintaining stock cultures and in microbiological test procedures. ## Part A: Isolation of discrete colonies from a mixed culture The techniques commonly used in isolating discrete colonies depend on the reduction of organism number. The decrease in the population size ensures that individual cells will be sufficiently far apart on the surface of the agar medium to separate the different species. The following are techniques that can be used to accomplish this necessary dilution: ### 1) Streak-plate method Its rapid qualitative, dilution method involves spreading a loopful of culture over the surface of an agar plate. The technique is schematically illustrated in **Figure 1**. **Note:** This method only works if the spreading tool (usually an inoculating loop) is resterilized after each of steps 1-4. **Figure 1:** A) Four way streak plate technique, B) a photograph shows the plate after incubation. - Image depicting a four-way streak plate technique. - Image depicting a photograph showing a streak plate after incubation. ### 2) Spread plate technique (previously discussed) ### 3) Pour plate technique The technique requires a serial dilution of the mixed culture. The diluted inoculum is then added to a molten agar medium in a petri dish, mix and allowed to solidify (**Figure 2**). **Figure 2:** Pour plate technique. - Image depicting step 1 of pouring plate technique: bacterial sample mixed with warm agar. - Image depicting step 2 of pouring plate technique: sample poured onto a sterile plate - Image depicting step 3 of pouring plate technique: sample swirled to mix, allowed to solidify. - Image depicting step 4 of pouring plate technique: plate is then incubated until bacterial colonies grow. ## Part B: Bacterial culture characteristics When grown on a variety of media, microorganisms will exhibit differences in the macroscopic appearance of their growth. These differences called cultural characteristics, are used as a basis for separating microorganisms into taxonomic groups. The cultural characteristics are determined by culturing the organisms on nutrient agar plates and slants, and in nutrient broth. The patterns of growth are described and illustrated in **Figure 3**. On nutrient agar plates, colony size and pigmentation (color of the colony), are also included as main characteristics of identification, for example, the size of the colony could be referred to as pinpoint, small, moderate, or large. **Figure 3:** **Image depicting bacterial colonies with different cultural characteristics.**