Practical Microbiology 18-22/11/2018 University of Sulaimani college of Pharmacy Year2 Lab. 4: Bacterial smear and Staining Before staining and observing a microbe under a microscope, a smear must be prepared. The goal of smear preparation is to place an appropriate concentration of cells on a slide and then cement them there so that they do not wash off during the subsequent staining procedure. A.Preparation of bacterial smear: Principle: Smear preparation technique consists of spreading small volume of sample on a slide and air drying the film before staining and microscopy. Bacterial smears must be prepared prior to any of the staining techniques. Step I: Preparation of the glass slide: Clean, grease free slides are needed for smear preparation. Step 2: Labeling of slides: -Proper labelling of the slide is essential. -Every slide should be labelled clearly. Step 3: Preparation of smear: -An evenly spread smear should be prepared. -Avoid thick and dense smear because thick smear prevent light penetration to visualize the morphology of cell. A good smear is one that, when dried, appears as a thin whitish layer or film. Different techniques are used for smear preparation depending upon culture media: i. Broth cultures (liquid medium): Resuspend the culture by tapping the tube with your finger. Depending on the size of the loop, one or two loopfuls should be applied to the center of the slide with a sterile inoculating loop and spread evenly. Set the smears on the laboratory table and allow to air-dry ii. Culture plates (Solid medium): -Organisms cultured in a solid medium produce thick, dense surface growth and are not amenable to direct transfer to the glass slide. -These cultures must be diluted by placing one or two loopfuls of water on the center of the slide in which the cells will be emulsified. -Transfer of the cells requires the use of a sterile inoculating loop or a needle. -Only the tip of the loop or needle should touch the culture to prevent the transfer of too many cells. 1
-Suspension is accomplished by spreading the cells in a circular motion in the drop of water with the loop or needle. This helps to avoid cell clumping. -The finished smear should occupy an area about the size of a nickel and should appear as a translucent, or semitransparent, confluent whitish film. Step4: Air dry Smear should be allowed to dry completely at room temperature at safe place Step 5: Fixation of smear: *The purpose of fixation of smear is to release of sticky proteins from the cell surface of the bacteria, to kill the microbes, fix the microbe cells to the slide and prevent their removal during washing steps *Smears are fixed by heat, alcohol and occasionally by other chemical. i. Heat fixation -After smear is air dried completely, rapidly pass the 3-4 times through flame of Bunsen burner or sprit lamp. -Avoid too much heating. -After heat fix, allow the smear to cool before staining. ii. Alcohol fixation: Allow smear to air dry completely Fix the smear with one or two drops of 70% alcohol, and leave it for 2 minutes until the alcohol dries up. Steps of microbial smear preparation: 1.Handle a clean slide by its edge, label the target place at the bottom side o the slide by drawing a circle with a diameter about 2 cm using a marker. 2. Sterile the loop until reaching the red heat. 3. If the bacterial culture was broth, shake the culture and transfer loopful of broth to the center of the slide and spread over the target circle. While if the bacteria were grown on solid medium, place loopful of water on the slide then transfer inoculums to the water and homogenize the smear. 4. Sterile the loop. 5. Leave the smear to dry at room temperature (by air). 6. After drying, pass the slide over the flame to fix it. (Avoid prolonged heating of the slide). Note: It is preferred that the microbial culture used in the staining process is a recent one, since the old one gives false results. 2
B.Staining of bacterial smear: Microbial Staining: giving colour to microbes, because microbes are colourless and highly transparent structures. Staining: process in which microbes are stained. Stains/dyes: organic compounds which carries either positive charges or negative charges or both, one of them carries the color of the stain, this is called ( chromophore ). Basic requirements for staining: 1. Clean slide. 2. Specific stain 3. Bacteria to be stained. 4. Inoculating loops- to transfer bacterial suspension to slide. 5. Bunsen burner to sterilise inoculating loops before and after smear preparation. Types of Stains: 1. According to charge: A. Basic dyes (cationic dye): these dyes are positively charged because the the chromophore is the positive ion, so they attract to the bacterial cell wall which is negatively charged. e.g : methylene blue, safranine, crystal violet, malachite green. 3
B. Acidic dyes (anionic dyes): these dyes are negatively charged because the the chromophore is the negative ion, so they not attract to the bacterial cell wall which is negatively charged, therefore they do not stain the cell, this process is called " negative staining " e.g : nigrocin, india ink, erocin, rose Bengal. These stains do not penetrate the bacterial cell wall but they make the background around the cells dark or opaque, so these stains show the shape and size of cells and the extracellular structures such as capsule or flagella and spore. C. Neutral dyes: stain with both charges. 2. According to purpose of use: 1. Simple stains. 2. Differential stains. 3. Special stains. 1. Simple stains: In this process only one stain is used for staining,,differentiation among bacteria is impossible- The most commonly used stains for simple staining are crystal violet, methylene blue, carbol fuchsin. This method of staining is useful determining basic morphology and the presence or absence of certain kinds of granules. Steps of simple staining: 1. Prepare a fixed smear. 2. Stain the smear with crystal violet by putting a couple of the stain drops and let for 1 min. 3. Wash off by tap water gently; leave it to dry at room temperature (by air). 4. Put one drop of cedar oil on the fixed smear, examine directly under oil immersion lens. 2. Differential staining: In this process, more than one stain and some chemical solutions are used. Differentiation among bacteria is possible. Eg. Acid-fast staining and Gram s staining. Gram s staining is the most important stain in bacteriology named according to Hans Christian Gram " in 1884. It is a differential stain which distinguishes between gram positive and gram negative bacteria depending on the difference in the cell wall structure. The procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain reaction. Gram-negative bacteria are decolorized by the alcohol, losing the color of the primary stain, purple. Grampositive bacteria are not decolorized by alcohol and will remain as purple. After decolorization step, a counterstain is used to impart a pink color to the decolorized gram-negative organisms. Importance of a Gram Stain: The Gram stain is a very important preliminary step in the initial characterization and classification of bacteria. It is also a key procedure in the identification of bacteria based on staining characteristics, enabling the bacteria to be examined using a light microscope. The bacteria present in an unstained smear are invisible when viewed using a light microscope. 4
Once stained, the morphology and arrangement of the bacteria may be observed as well. Furthermore, it is also an important step in the screening of infectious agents in clinical specimens such as direct smears from a patient. The Gram stain procedure enables bacteria to retain color of the stains, based on the differences in the chemical and physical properties of the cell wall. 1. Gram positive bacteria: Stain dark purple due to retaining the primary dye called Crystal Violet in the cell wall. Example: Staphylococcus aureus Fig: Gram positive bacteria 2. Gram negative bacteria: Stain red or pink due to retaining the counter staining dye called Safranin. Example: Escherichia coli Fig: Gram negative bacteria Gram Stain Mechanism: Gram Positive Cell Wall: Gram-positive bacteria have a thick mesh-like cell wall which is made up of peptidoglycan (50-90% of cell wall), which stains purple. The thick peptidoglycan layer of Gram-positive organisms allows these organisms to retain the crystal violet-iodine complex and stains the cells as purple. Lipoteichoic acid (LTA) is another major constituent of the cell wall of Gram-positive bacteria which is embedded in the peptidoglycan layer. 5
Gram Negative Cell Wall: Gram-negative bacteria have a thinner layer of peptidoglycan (10% of the cell wall) and lose the crystal violet-iodine complex during decolorization with the alcohol rinse, but retain the counter stain Safranin, thus appearing reddish or pink. They also have an additional outer membrane which contains lipids, which is separated from the cell wall by means of periplasmic space. Requirements gram staining reagents: 1. Crystal violet Primary stain 2. Gram s iodine- mordant/fixative 3. Acetone (95%)- decoloriser 4. Safranine:counter stain Gram staining procedure : Step 1: prepare a smear Step 2: Cover the smear with crystal violet and let stand for 1 minutes then Briefly wash off the stain with water. Step 3: Flood the smear with Iodine solution and let stand for 1 minute. Step 4: Pour off the iodine and flood the smear with 95% ethyl alcohol for 10 to 20 seconds then rinse with water. Step 5: Cover the smear with safranin for 1 to 2 minutes. Step 6: Rinse, dry and examine the slide under oil immersion lens to see : Either Gram positive bacteria (appear purple) Or Gram negative bacteria (appear red) 6
Factors affecting the efficiency of Gram staining process: 1. Thickness of bacterial smear. 2. Concentration and purity of the solutions and reagents. 3. The nature and age of bacterial culture. 4. The amount of washing water. Acid-fast stain (Zehiel-Nelseen method) Acid-fast staining is another example of a differential stain used in Bacteriology. It divides bacteria into two groups: acid fast-and non acid fast. Members of the genus Mycobacterium are acid-fast in nature. Like the Gram reaction, the acid-fastness is also called cell wall dependent. Mycobacteria have a high lipid content, especially mycolic acid, in their cell wall. The ordinary aniline dye solutions cannot penetrate the mycobacterial cell wall. 3. Special staining: these are specialised staining methods to demonstrate certain bacterial components. Example: Capsule staining, Spore staining and flagella staining. Thanks Chiman H. Fattah MSc.Medical Microbiology/Mycology 7