Lab Report: Biological Staining Techniques for Bacterial Classification

Categories: Biology

Report, Pages 5 (1020 words)

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Abstract

The purpose of this study was to employ various biological staining techniques on two samples of bacteria to investigate whether these methods could aid in the identification and observation of different sizes, shapes, and types of bacteria, thereby enhancing their classification. We prepared wet mounts of two bacterial samples and stained one with methylene blue and the other with a Gram stain. After microscopic examination, we concluded that the Gram stain was the most effective method for clearly discerning variations in bacteria types, while methylene blue staining offered rapid but less detailed results.

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Introduction

The primary objective of this experiment was to overcome the challenge of visualizing and classifying prokaryotic bacterial cells with a microscope. Accurate identification of different types of bacteria and their structures is crucial, not only for a fundamental understanding of life's building blocks but also for applications in medical laboratories and healthcare settings. The initial step in our experiment involved using a methylene blue simple stain to visualize the shapes of bacteria.

Bacterial cells exhibit three primary shapes: spirilla, cocci, and bacilli (Acharya, 2019). Beyond simple staining, Gram staining is employed to characterize cell membrane properties, providing a more specific classification of bacteria. Gram staining involves a series of steps, including a primary stain, mordant, decolorization, and a secondary stain, allowing differentiation between Gram-positive and Gram-negative stains. Gram-positive stains appear blue or violet, whereas Gram-negative stains appear red or pink (Bruckner, 2016).

Utilizing methylene blue as a simple stain to dye bacterial cells proved to be the most effective method for identifying and differentiating bacterial shapes and types.

The bacterial samples were obtained from two sources: the lab computer's surface and Grace's belly button. Prior to the experiment, both bacterial samples were incubated for forty-eight hours to allow colonies to develop. We selected these two samples due to the presence of various microorganisms on the petri dishes, expecting that they would yield the best results. Therefore, our hypothesis was that by employing different biological staining techniques on these two bacterial samples, we could identify and observe variations in size, shape, and type of bacteria.

Methods and Materials

On September 16, 2019, we conducted a bacterial lab experiment using samples obtained from the lab computer's surface and Grace's belly button. The following steps were undertaken:

We placed two drops of deionized water on a glass slide and added a bacteria sample from the lab computer.
After creating a wet mount on the slide, we heated it on a hot plate for approximately one minute to stimulate bacterial growth.
We flooded the slide with methylene blue stain for one minute and subsequently rinsed it with water for one minute.
The slide was dried using bibulous paper, and we proceeded to examine the specimen under the microscope.
For the bacteria sample from Grace's belly button, we followed similar steps:
We placed two drops of deionized water on a glass slide and added the belly button bacteria sample.
After creating a wet mount on the slide, we flooded it with a crystal violet solution for twenty seconds.
Following this, we rinsed the slide with water, flooded it with an iodine solution for one minute, and applied a decolorizer solution for approximately twenty seconds.
Lastly, we applied the safranin counterstain, washed the slide with water, and dried it with bibulous paper before examining the specimen under the microscope.

Results

The results of the simple staining technique were distinctive upon observation. At 4X total magnification, the stained sample exhibited a geometric shape resembling folded paper. Upon increasing the magnification to 40X, numerous small, cocci (round) shaped bacteria became visible, surrounding the geometric-shaped bacteria on the slide. The edges of the bacteria were stained a darker shade of blue, while the central areas appeared lighter blue (see Table 1 and Figure 1).

Table 1: Simple Stain Observations
Magnification	Microscopic Observations
4X	Geometric shape resembling folded paper
40X	Small, cocci-shaped bacteria with darker edges and lighter central areas

For the Gram staining technique at 40X total magnification, the sample appeared very dark red, indicating a Gram-negative stain. Within the irregular and complex-shaped bacteria, small cocci-shaped specks were observed. In some regions of the slide, these circular specks were more dispersed, allowing for the identification of individual bacteria and separate colonies (see Table 2 and Figure 2).

Table 2: Gram Stain Observations
Magnification	Microscopic Observations
40X	Dark red color indicative of Gram-negative stain, small cocci-shaped specks within complex-shaped bacteria

Discussion

The results of our experiment demonstrate that both methylene blue simple staining and Gram staining are effective techniques for visualizing and classifying bacteria. However, they offer different advantages and levels of detail in bacterial identification.

Methylene blue simple staining allowed us to quickly identify the shapes of bacteria. At 40X magnification, we observed small, cocci-shaped bacteria with distinct staining patterns, where the edges were darker, and the central areas were lighter. This staining technique is efficient for a preliminary assessment of bacterial morphology. However, it may not provide sufficient detail for precise bacterial classification.

In contrast, Gram staining provided more specific information about bacterial properties. The observation of a very dark red color at 40X magnification indicated a Gram-negative stain. We also noticed small cocci-shaped specks within complex-shaped bacteria, allowing us to discern individual bacteria and separate colonies. Gram staining is valuable for differentiating between Gram-positive and Gram-negative bacteria, aiding in the categorization of bacterial strains.

Our choice of bacterial samples from both the lab computer's surface and Grace's belly button provided diverse microorganisms, contributing to a more comprehensive evaluation of staining techniques. The diversity of microorganisms present in these samples likely contributed to the variation in staining patterns observed.

In conclusion, the selection of staining technique depends on the specific objectives of bacterial analysis. Methylene blue simple staining is ideal for quickly identifying bacterial shapes, making it suitable for initial observations. On the other hand, Gram staining offers more detailed information about bacterial properties and is particularly valuable for distinguishing between Gram-positive and Gram-negative bacteria. Future studies could explore additional staining techniques or focus on specific bacterial strains to further enhance bacterial classification and identification.