Bacterial Smear Preparation

LABORATORY EXERCISE 8-A: Preparation of Bacterial Smear and Simple Staining

I. INTRODUCTION
Bacterial smears are prepared for the purpose of viewing microorganisms under the microscope. 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. A bacterial smear is a dried preparation of bacterial cells on a glass slide. Smears may be made from a dry culture (from a slant, stab or plate), or from a wet/liquid culture. In preparing bacterial smears, the following qualities must be observed: 1. The bacteria must be spread evenly over the slide and be concentrated enough to be located, but dispersed enough so that their natural arrangement can be seen. Ideally a single cell layer will be achieved; dense areas on a slide is often not useful for examination. 2. The bacteria must be adequately fixed to the slide so they don’t wash off during normal washing procedures in staining. 3. The bacteria must not be deformed so that their original morphology can be observed.
Most bacteria have no color, so they generate little contrast in the microscopic field. Therefore, to see bacteria with the microscope, it is neccessaryto apply color by using a staining reagent. Once stained, the bacteria may be observed and studied with respect to their morphology (shape, size, and arrangement).
Stains are solutions that contain a solute called a chromophore dissolved in a solvent. A chromophore is the color possessing portion of the solution and is therefore responsible for the stains color. Bacterial cells usually have a negative surface charge, meaning that a positively charged stain is needed to stain the surface of the cell. Most stains are basic and have a positively charged chromophore. When the stain is applied, there is an attraction between the negatively charged cell surface and the positively charged chromophore, leading to the surface of the cell taking on the color of the stain. When only one stain is applied, this is called a simple stain. The most common stain used for smears (and wet mounts) is Methylene Blue. Other stains such as IKI, crystal violet, and safranin may also be used. After preparing the smear, the stain is applied for a specified amount of time. In simple staining, it is important that the stain is allowed to penetrate the cell wall. Usually this is accomplished within one minute.
II. EXPERIMENTAL PROCEDURE
Preparation of Bacterial Smears
A. In 2 clean slides, place two loops of S. aureus liquid bacterial growth or one needle of solid bacterial growth.
B. If a solid bacterial growth is used, add one drop of water, then, mix completely.
C. Spread the specimen to cover about half of the slide area using a heat sterilized inoculating loop.
D. Pass the slide over the flame of the alcohol lamp and wait for it to dry.
E. Prepare 2 more bacterial smears using B. subtilis. Slides should be properly labelled with the organism and the type of stain to be used.
Simple Staining
A. Once the slides have cooled, place them on the staining rack over the sink.
B. Flood a smear of S. aureus and a smear of B. subtilis with methylene blue for 1 minute.
C. Gently wash of the methylene blue dye using a distilled water wash bottle or from the running tap.
D. Flood the 2 remaining smears with carbol fuchsin for 1 minute.
E. Gently wash of the carbol fuchsin dye using a distilled water wash bottle or from the running tap.
F. Examine all stained smears under oil immersion objectives.
III. RESULTS B. subtilis stained with methylene blueDescription: rod-shaped, in singles or chains | B. subtilis stained with carbol fuchsinDescription: rod-shaped, in singles or chains | S. aureus stained with methylene blueDescription: round, in singles or clusters | S. aureus stained with carbol fuchsinDescription: round, in singles or clusters |

IV. DISCUSSION AND CONCLUSION
A simple stain is application to a smear of a single dye that colors the cell uniformly. The purpose of a simple stain is to highlight the entire population of microorganisms. A stain is a procedure, the dye and smear used are components of that procedure. Most dyes are salts in which one of the ions is colored, it reflects light of a specific wave length. This ion is called a chromophore and it will specifically bind to a cell or a structural part of the cell. Methylene blue, for example, is methylene blue chloride (MBCl) which, when ionized, becomes: MB+ (chromophore) + Cl-. The chromophore attaches to negatively charged particles enabling such structures to be stained. If the color-bearing ion (chromophore) is positively charged, the stain is basic (it has given up a negative charge). If the chromophore is negatively charged it is acidic (it has given up a positive charge). When methylene blue is used to stain bacteria, the entire bacterial cell is colored blue; this is the essence of a simple stain procedure.

Five basic dyes will be used in our laboratory: BASIC DYES | ABSORBANCE MAXIMA (NM) | * Crystal Violet | 590 | * Safranin | 530 | * Carbol Fuchsin | 544 | * Methylene Blue | 665 | * Malachite Green | 621 | ACIDIC DYE | | * Eosin | 525 |
Through the use of simple stains, we were able to appreviate the morphology of S. aureus and B. subtilis. Staphylococcus aureus are round or coccus-shaped bacteria which occur singly, in pairs, or irregular clusters. Bacillus subtilis, on the other hand, are rod-shaped bacteria which occur singly, or in chains.
V. RESEARCH QUESTIONS 1. What is the purpose of staining? * To make cells or microbes visible for morphological observation * To be able to defferentiate and classify bacteria into categories * To enhance contrast and highlight structures under the study of interest
2. How are stains able to impart color to the bacteria? Explain the mechanism involved. Stains impart color on bacteria based on their ionization. Basic dyes, having positively charged groups bind to negatively chared particles, while acidic dyes, having negatively chared groups bind to positively charged structures.
3. What is the principle of simple staining? Simple staining makes use of a single stain and is primarily used for visualization of morphology which includes: shape (cocci, bacilli, spirilli), size, and arrangement (singles, chains, clusters, pairs, tetrads).
VI. JOURNAL ARTICLE VII. REFERENCES * Smear Preparation and Simple Stain http://sarahredd.com/biol260/lab/smearprep.pdf * Bacterial Staining http://www.eplantscience.com/botanical_biotechnology_biology_chemistry/biotechnology_methods/microbiology/bacterial_staining.php * Preparation of Bacterial Smear and Simple Staining Technique http://samples.jbpub.com/9780763795573/95573_CH04.pdf * Preparing a Smear and Simple Staining http://facstaff.gpc.edu/~ddonald/biolab/smearstain.htm * Bacterial Smears and Simple Stains http://www.highlands.edu/academics/divisions/scipe/biology/labs/rome/bacterial_smears_and_stains.htm * Microbiology Techniques http://www2.hendrix.edu/biology/CellWeb/Techniques/microstain.html * Simple Staining http://www.microbiologyinpictures.com/staphylococcus%20aureus.html

References: * Smear Preparation and Simple Stain http://sarahredd.com/biol260/lab/smearprep.pdf * Bacterial Staining http://www.eplantscience.com/botanical_biotechnology_biology_chemistry/biotechnology_methods/microbiology/bacterial_staining.php * Preparation of Bacterial Smear and Simple Staining Technique http://samples.jbpub.com/9780763795573/95573_CH04.pdf * Preparing a Smear and Simple Staining http://facstaff.gpc.edu/~ddonald/biolab/smearstain.htm * Bacterial Smears and Simple Stains http://www.highlands.edu/academics/divisions/scipe/biology/labs/rome/bacterial_smears_and_stains.htm * Microbiology Techniques http://www2.hendrix.edu/biology/CellWeb/Techniques/microstain.html * Simple Staining http://www.microbiologyinpictures.com/staphylococcus%20aureus.html