Lab 4: Bacterial Staining PDF

Lab (4)  Bacteria are colourless and highly transparent structures, therefore, when they are observed under a microscope they are opaque or nearly invisible.  To visualize bacteria different type of staining methods are used to give color to them. What is the Aim of Staining? 1. To visua...

Lab (4)  Bacteria are colourless and highly transparent structures, therefore, when they are observed under a microscope they are opaque or nearly invisible.  To visualize bacteria different type of staining methods are used to give color to them. What is the Aim of Staining? 1. To visualize bacteria. 2. To study bacteria morphology ( size, shape and arrangement) 3. Differentiate between Gram +ve and Gram –ve bacteria. 4. To visualize bacteria structure ( capsule, flagella).  Types of Staining Techniques Simple Staining Differential Staining Use two stains Use a single stain Identification Visualization of For visualization bacteria shape structure and arrangement Gram stain Acid fast stain  Types of Stain Acidic Basic +Ve charge -Ve charge Example: Methylene blue Example: India ink, nigrosin Safranin Crystal violet  Which type of stain usually used to stain the bacterial cell and why? The bacterial cell wall has a net charge of negative, Basic stains have positively ,which causes them to become attracted to the negative charges on the surface of the bacterial cells. This enables the cell to retain the dye.  Smear Preparation  Bacterial smear consists of a thin film of bacterial culture.  For the preparation of smear, we need to perform the following steps : 1. Take a clean glass slide and label it. 2. A. For agar culture , add a drop of distilled water at the centre of the glass slide, Then transfers a small amount of bacterial colony with inoculating loop on the glass slide, Spread the bacterial suspension thinly over the slide. B. For broth culture, insert the Sterilized loop into the culture tube and take out a loopful of cultured broth, Spread the bacterial suspension thinly over the slide. 3. Air dry the smear. 4. Fixing the smear: Gently heat the slide by holding above the blue portion of the Bunsen flame 3 times.  Why we must perform heat fixing, because: Heat fixing helps in the fixation of bacteria to the glass slide and kill the bacteria.  Simple Staining Direct Indirect Using basic stain Using Acidic stain, the negatively charged of the acidic dye is repelled by the negatively charged bacterial cell. Therefore the background will be stained and the cell will remain colourless.  Practical part 1. Direct simple staining procedure  Name of bacteria Shape Arrangement Staphylococcus aureus Cocci Cluster Escherichia coli Short Single bacilli Bacillus cereus Long chain Bacilli  Differential Staining Gram staining Acid Fast staining Differentiate the Mycobacteria Is used to differentiate the bacterial species from the other bacterial cells by staining the Cell wall and groups based on the staining and distinguish two major groups of cell wall differences. bacteria that are gram-positive and gram-negative.  How Does Gram Staining Work  Gram positive cell wall has thicker peptidoglycan layer as compared to Gram negative cell wall.  Gram negative cell wall contains a thick layer of lipopolysaccharide in their cell wall which is absent in Gram positive bacteria.  Practical part Gram Staining procedure Gram staining Staphylococcus aureus Bacillus cereus Gram reaction : G +ve Gram reaction : G +ve Escherichia coli Gram reaction : G - ve  Acid Fast staining (Ziehl-Neelsen method )  Examples of acid-fast bacteria: Mycobacterium tuberculosis, M. leprae  Acid-fast organisms are characterized by wax cell wall, and complex lipids. So they require a special staining technique.