Staining Techniques and Aseptic Technique/Streak Plates

In most microbial experiments, the inoculation and culturing measures are important because they assess the consistency of the specimens required for the experiment. As a result, it is important to remove potential sources of infection that could result in the release of unwanted microbes. The aseptic (sterile) technique refers to the method of culture of pure or uncontaminated bacteria in the laboratory for experiments. The aseptic approach requires the use of sterile media and tools. Three specific aseptic methods may be used to ensure sterility of the devices to be used (Pommerville, 2010). The methods may include steam or autoclave sterilization, sterilizing the mouth of the containers, and steam sterilization. The purpose of the present laboratory report is to outline the guidelines necessary for carrying out aseptic technique with bacteria.

Procedure

Broth to Broth Transfer

Two tubes of nutrients are obtained and labeled name initials, date and “EC” for Escherichia coli or “C” for control.

The wire loop is sterilized until red hot and then allowed to cool for a few seconds. The wire loop should not be cooled by blowing or shaking. The loop should be held at 45° to avoid burning

The mouth of the test tube containing E. Coli is sterilized by passing it through the flame of Bunsen burner.

A loop full of broth is then obtained from the test tube after which the test tube is sterilized again by passing it through the Bunsen burner flame.

The mouth of the EC test tube is a flame for sterilization after which loop containing the culture is inserted into the test tube and swirled few a few seconds. The loop is then removed and flamed for sterilization.

The same procedure is repeated for the controlled ‘C’ test tube.

Simple Stain

Introduction

Staining is carried out to increase the visibility of bacterial cells that have the same refractive index as that of water. Stains are utilized to improve the contrast between the cells and the background with the dyes being the chemicals that are commonly used to characterize the morphological shapes of bacteria. The common forms of bacteria include rod-shaped cells (bacilli), round-shaped cells (cocci), and spiral-shaped cells (spirilla). Simple staining technique uses a single basic dye to obtain the contrast between the background and the bacterial cells under experiment. On the other hand, differential method utilizes a combination of different colors to differentiate between cells with different chemical properties.

Procedure

Preparation of Bacterial Smear

Drawing two cycles on the bottom of slides and labeling the frosted end TOP

Spreading E. coli broth culture aseptically in one cycle and one loopful on the other.

Heat fix the slide

Simple Stain

Methylene Blue dye is added to the heat-fixed slide

The slide is after that observed under microscope

Endospore Stain

Introduction

Organisms use various techniques and forms to survive in hostile environments. One such form is an endospore that is produced by members of the Bacillus and Clostridium genera. The endospores are thus regarded as a definitive feature of Bacillus and Clostridium (Johnson & Case, 2015). The bacteria can, however, be identified based on other characteristics as well. Staining in Bacillus and Clostridium is challenging as the endospores are highly resistant to stains. Steaming of a stain into the wall and cell of the endospores is thus carried out to enhance the visibility of the cells. The purpose of the present laboratory exercise is to conduct the staining process of the endospores.

Procedure

A bacterial smear is prepared and covered with a bibulous paper.

The slide is then steamed with Malachite Green for 7‐10 minutes on a steamer

The slide is then removed allowed to cool and rinse with water after which counterstaining is achieved Safranin for 1 minute.

The slide is rinsed, blotted and observed under a microscope.

The Gram Stain

Introduction

The Gram stain is the most widely utilized staining technique in bacteriology. The technique is a differential staining method as it involves a complex procedure with a combination of different dyes. The bacteria under Gram stain are distinguished based on the peptidoglycan in the cell wall with Gram-positive bacteria having thick layers of cell peptidoglycan (90% cell wall). The Gram-negative bacteria have 10% cell wall. Four basic steps are involved in the performance of the Gram Stain on samples that entails the addition of crystal violet, mordant (Gram's iodine), acetone/alcohol and safranin for counterstaining. Alcohol or acetone is applied in the procedure to enable decolorization (Pommerville, 2010). The theory involved in the process is that the crystal violet enters the peptidoglycan with mordant acting as a binding agent to the crystal violet leading to the formation of the violet-iodine complex. Peptidoglycan holds the violet-iodine complex in Gram-positive samples preventing decolorization. On the other hand, the violet-iodine complex is mainly taken by the lipid layer that is soluble hence the stain is washed away. The purpose of the experiment is to outline the guideline for Gram stain laboratory test.

Procedure

Smears of Gram positive and Gram negative bacteria are prepared and heat fixed by passing through a flame three times

Heat-fixed smears are then placed in a staining jar containing crystal violet for one minute and then rinsed to remove excess stain, excess water is removed by tilting the slide

The slide is then placed in a staining jar containing mordant for one minute and rinsed followed by decolorization for 10-12 minutes

Rinsing is immediately using the previously described rinsing procedure and the slide placed in a jar containing counterstaining safranin.

Rinsing is done after that and the slide placed on a piece of bibulous paper and observed under a microscope (100x magnification).

References

Johnson, T. R., & Case, C. L. (2015). Laboratory experiments in microbiology. Pearson.

Pommerville, J. C. (2010). Alcamo's laboratory fundamentals of microbiology. Jones & Bartlett Publishers.