Gram Stain Protocols and Microscopic Measurement of Organisms - Lab Report Example

“GRAM STAIN”

Introduction:

The aim was the experiment was to assess how bacteria differ from each other by subjecting gram stain through a physical and chemical process. We exposed the bacteria to staining through violet and other simple procedures to determine at the end if its gram positive or gram negative or manifest the characteristics of both. By understanding the types of the bacteria and how they react to the environment helps to have an in-depth understanding of the experiment and at the same time dealing with the bacteria in their natural form. Hence, differential staining helps to differentiate different types of bacteria.

Principles:

In bacteriology, Gram stain is the most useful and commonly differentia stain used. Knowledge about the differential staining helps one to differentiate between the gram negative and gram positive bacteria easily. The critical step while dying the lens into crystal violet is called primary stain. During this phase, iodine is mordant as it catalyzes the reaction between the bacterial cell and the dye to ensure the dye bounds itself to the cells or strongly stained. To stain at a later stage, we shall use a light microscope (x100).

Materials:

• Slides
• Lens paper and lens cleaner
• Inoculating loop
• Bunsen burner
• Bibulous paper
• Microscope
• Crystal’s violet, Gram’s iodine (2g potassium iodide in 300 ml distilled water + 1g iodine crystals), isopropanol-acetone mixture (3:1 v/v) and safranin solution or 95% ethanol.
• Cultures of formalinized (1ml of concentrated formalin per 10 ml of culture) staphylococcus aureus, Escherichia coli, and a mixture of S.aureus and E.coli.

Procedure:

• We first put on gloves to protect us from contaminated by any bacteria on our skin as they say safety comes first.
• We then took lens paper and cleaned it using lens cleaner to prevent scratch or from getting to the normal tissues on the lens. After that, we indicated our names below the lens just opposite the side bacteria were.
• We then used inoculating loop apply the bacteria on. We later used it to destroy the bacteria by placing it above the Bunsen burner three times and then cleaned it on the bottom surface to see the bacteria.
• As the experiment proceeded, other procedures were adopted to apply the bacteria. For instance, the crystal violet which had been kept for 30 seconds and rinsed for 5 seconds as to be used at a later stage.
• Then the other procedure was to apply the Gram’s iodine mordant for 1 minute and then rinse it for 5 seconds. We also used 95% of the ethanol for 15-30 seconds and then clean it for 5 seconds.
• For every step, it was mandatory to rinse for 5 seconds, but the last procedure was counterstain safranin for 60-80 seconds. After the final stage, we flush it using distilled water but gentile to avoid removing the bacteria which were necessary for the examination using the oil immersion microscope.
• In the end, we were expected to observe either gram negative as manifested by pink to red color or gram-positive by seeing color change from blue to purple

Conclusion:

Through the experiment, we were able to distinguish between gram-positive and gram-negative bacteria. It was an incredible exposure to the science of differential staining. We also learned about bacterial smear through the process of crystal violet, Gram's iodine, and 95% of ethanol and safranin solution. The results had deep purple/ blue for gram-positive and pink/red for gram negative. E-coli is known as gram-negative and staphylococcus as gram-positive.

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“Bright-Field Light Microscope And

Microscopic Measurement of Organisms”

Introduction:

It is essential to know the measurements of each organism. In biotechnology, these kinds of things are central and should be recognized in each lab while working with bacteria and other samples through the microscope. By knowing the ocular and stage micrometer space during the measuring, the samples make it less complicated to understand what we are doing precisely in each experiment. In this experiment, we measure in metric units for microorganisms.

Principles:

Bright-field light microscope is an instrument that magnifies images using two lens systems.

The magnification occurs the objective lens. The objective lenses have the low-power (10X), high dry(40X), and oil immersion(90X,100x). Depending on the microorganism and how big/small or if the liquid is in the process we use one of these three.

Materials:

• Lens paper and lens cleaner
• Ocular micrometer
• Compound microscope
• Immersion oil
• Stage micrometer
• Prepared stained slides of several types of bacteria: Proteus, Vulgaris, Spirillum

Procedure:

• The microscope was turned on, and then lens cleaned using lens paper and also the glass slides.
• We then put the slide under the stage clip and then adjusted the slide until the bacteria spot was open in the stage.
• We started by observing separately the ocular micrometer followed by the stage.
• Then after that, we fixed the 4X in position and focused it with condenser to read the measurements. During this time, the ocular micrometer was parallel to the stage micrometer.
• After we had focused the ocular and stage micrometer at first objective 4X, we checked the distance in millimeter by observing the spaces in the stage micrometer with a given number of spaces on the ocular micrometer.
• We repeated the same steps with 10X and 40X.
• After we were through with the calculations, we removed the slide and switched off the microscope and put everything into position.

Calculation:

4X

1 ocular micrometer space= 25 stage space

1 stage micrometer space = 0.01 mm/µm (the smallest space on stage micrometer).

1 ocular micrometer = 25 X 0.01 = 0.25 mm

10X

1 ocular micrometer = 10 stage space

1 stage micrometer space = 0.01 mm/µm (the smallest space on stage micrometer).

1 ocular micrometer = 10 X 0.01 = 0.1 mm

40X

1 ocular micrometer = 3 stage

1 stage micrometer space = 0.01 mm/µm (the smallest space on stage micrometer).

1 ocular micrometer = 0.03 mm

Conclusion:

From the experiment, we have exposed ourselves on how to measure samples using a microscope that uses objective lenses and focusing the ocular micrometer parallel to the stage micrometer. The experiment revealed that the stage micrometer was stable and the smallest space was always 0.01 mm/µm. I also observed that based on the sample the objective lenses ones uses varies. For instance, if a bacteria is tiny then one can use zoomed in more from the objective lenses and zoom it

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“The Hanging Drop Slide and Bacterial Motility”

Introduction:

Bacterial motility and hanging drop slides help one to acknowledge every bacteria and their environment. In some cases, some bacteria don’t have any form of motion, and they are referred to as non-motile. When these bacteria are located in aqueous solution, they move erratically by a means called Brownian. On the other hand, motility bacteria have four modes of movements which are: “bending-type motion, Flagella motion, corkscrew motion, and gliding motion.”

Materials:

• Bacterial culture
• Microscope or phase-contrast microscope
• Bunsen burner
• Petroleum jelly (Vaseline)
• Lens paper and lens cleaner
• Pipette tips
• Immersion oil
• Clean depression slides and coverslips
• Inoculating loop

Procedure:

• We used a tip to separate small ring of Vaseline around the concavity of depression slide that was in the clear depression slide
• We then placed a small drop of bacteria culture using an inoculating loop in the center of depression slide and covered it with a cover slip.
• We then turned the hanging drop slide over and put it on the stage of the microscope to have the drop on the light hole
• After that, we examined the drop under the low power and focused on the drop. Immersion oil was used for 100x objective to observe the bacteria movements accurately.
• After the examination, we discarded the coverslips and removed the depression slide.

Conclusion:

This experiment was fascinating, as I was able to see the movement of bacteria through the microscope. Even though ti was not that easy, but it was beneficial. I know, next time it will be easy.