Question

Perform a Gram stain on a portion of one wellisolated colony. Record its Gram reaction and cell morphology, arrangement and size on the Data Sheet under Preliminary Observations. If the isolate is a Gram-negative rod, transfer a portion of the same colony to a Trypticase Soy Agar slant (or another suitable growth medium as available in your lab) and incubate it at its optimum temperature. This is your pure culture to be used as a source of organisms for further testing. Complete the record of your isolation procedure on the Data Sheet.

Short answer

Perform a Gram stain, identify Gram-negative rods, transfer to Trypticase Soy Agar, incubate at optimal temperature, and complete records.

Step-by-step solution

- Prepare the Glass Slide

Begin by cleaning a glass slide thoroughly with soap and water, rinsing well, and handling by the edges or with gloves to avoid contamination.

- Obtain a Sample

Use a sterile inoculating loop to pick up a small portion of the well-isolated colony. Smear the sample onto the center of the slide in a thin layer.

- Fix the Sample

Allow the smear to air dry. Once dry, pass the slide through a flame quickly two or three times to fix the bacteria to the slide.

- Stain with Crystal Violet

Flood the smear with Crystal Violet stain and let it sit for 1 minute. Rinse the slide gently with water.

- Apply Iodine Solution

Flood the slide with iodine solution to fix the crystal violet dye and let it sit for 1 minute. Rinse again with water.

- Decolorize with Alcohol

Pour alcohol or acetone gently over the surface of the slide for 10-20 seconds to decolorize. Rinse immediately with water.

- Counterstain with Safranin

Flood the slide with safranin dye and let it sit for 1 minute. Rinse the slide and then gently blot it dry with bibulous paper.

- Observe Under the Microscope

Examine the stained slide under a microscope using the oil immersion objective lens. Record the Gram reaction (purple for Gram-positive, pink for Gram-negative), cell morphology, arrangement, and size.

- Inoculate Trypticase Soy Agar Slant

If the isolate is a Gram-negative rod, use a sterile inoculating loop to transfer a portion of the same colony to a Trypticase Soy Agar slant.

- Incubate the Pure Culture

Incubate the slant at the optimum temperature for the specific organism. Make sure to label it clearly.

- Record the Isolation Procedure

Complete the records on your Data Sheet, including the Gram reaction, morphology, arrangement, and the steps taken for isolation.

Key concepts

Bacterial Staining

Bacterial staining is a crucial technique in microbiology for observing microorganisms and differentiating them based on their cellular structures. The Gram stain is one of the most widely used methods. It helps identify bacteria as Gram-positive or Gram-negative based on their cell wall composition. This classification is vital since it influences the choice of antibiotics to treat infections.

In the Gram stain process, you start by creating a smear of bacteria on a slide, fixing it with heat, and then applying a series of dyes. Initially, Crystal Violet is used to stain all cells. After rinsing, iodine solution is added as a mordant to fix the dye. Decolorizing with alcohol removes the dye from Gram-negative cells but not from Gram-positive cells, thanks to their thicker peptidoglycan layer. Finally, a counterstain (safranin) is added, making Gram-negative cells visible in pink or red, while Gram-positive remain purple.

This multi-step process is straightforward when broken down but requires careful execution to ensure accurate results. The staining steps reveal key features that allow scientists to identify and study microbial life efficiently.

Cell Morphology

Cell morphology refers to the shape, size, and arrangement of bacterial cells. Recognizing these characteristics helps in the identification and classification of bacteria. Common shapes include cocci (spherical), bacilli (rod-shaped), and spirilla (spiral-shaped).

When you perform a Gram stain and observe the bacteria under a microscope, you can see not only the color indicating Gram reaction but also their shape and arrangement. For example, staphylococci appear in clusters, while streptococci form chains. Bacilli can be seen singly or in pairs.

Understanding cell morphology assists in diagnosing infections and determining the appropriate course of treatment. Accurate identification is a cornerstone of microbiology and is essential for effective medical interventions.

Pure Culture

A pure culture is a laboratory culture that contains a single species of microorganism. The goal is to isolate one type of bacteria from a mixture so that it can be studied without interference from other species.

After performing a Gram stain and identifying bacterial characteristics, the next step is to obtain a pure culture. For instance, if you identify a Gram-negative rod, you transfer a portion of that bacteria to a growth medium like Trypticase Soy Agar slant. Incubating this at the optimal temperature will encourage the growth of that specific microorganism.

Maintaining a pure culture is crucial for studying the physiology, genetics, and biochemistry of microorganisms. It allows scientists to perform accurate tests and research without contamination. Ensuring purity involves meticulous technique and consistent monitoring. This practice is vital for the development of new antibiotics and understanding microbial behavior.