Gram-Positive and Gram-Negative Bacteria

The isolation of gram positive and gram-negative bacteria from a soil sample is a procedure that is done in the laboratory. The procedure begins by first assembling all the requirements, materials and tools to be used. While doing the procedure time factor is particularly crucial. That is all processes have to be monitored closely to the end of the procedure. Gram staining is an interesting procedure that causes the microorganisms to either stain with the crystal violet stain or safranin red stain. In the end, the bacteria can be differentiated as either gram positive or gram negative.

Introduction

Bacteria are living organisms. They can be either animals or plants. Bacteria grow and can mature to a size of about one cell thick. They are living organisms that exist in groups where they cling together in millions referred to as colonies. Most bacteria depend on sugars, proteins, and vitamins to live. Bacteria also require salts for survival such as calcium, magnesium, iron etc. Bacteria also have different colors depending on the bacteria that are in the colony. For instance, there are green bacteria, which synthesize their own energy by the use of light.

Bacteria are living organisms that have specific conditions   that they require in order to grow. These conditions differ from one form of bacteria to another. These conditions include temperature, pH, oxygen concentration, osmotic pressure, and other factors that are determined by the environment. Bacteria can grow in media. This is a mixture of nutrients, which are necessary for the growth of an organism. There are several types cultures that are fit for primary culture or secondary cultures. In this paper, I shall discuss the procedure of isolating bacteria from the soil (Lee 2009).

Objective

The main objective in this experiment is to isolate bacteria from soil. As we have seen in the introduction that there are bacteria that live in the soil. Hence, through this experiment the bacteria in the soil shall be isolated. In this experiment, the bacteria shall be isolated by the use of culture technique.

The agar is made from a TSA powder that is already made. The powder contains all the necessary requirements that will enhance a substantial growth of any bacteria that is in the sample.

40 grams of the TSA powder was suspended in 1 liter of clean water then was thoroughly mixed. In order to dissolve the powder well, the mixture is subjected to heat. The mixture is left to boil for about 1 minute, to enhance complete dissolving of the mixture. Once the mixture has cooled, it is then autoclaved to ensure sterility for 15 minutes at 121%u030A C. The mixture is later transferred into the agar plates and left to solidify.

Making the primary culture

The test-tube to be used in the experiment was labeled with the name and the lab number that is specific to the experiment. The test-tube contained 5ml of Trypticase Soy Broth. An amount of the soil taken for the experiment was put into the test-tube. The test-tube was then shaken to suspend any bacteria that was in  the soil. The test-tube was then put under 25%u030A C (this is the room temperature) for about 30minutes to settle the soil.

In another set up, the soil was obtained to be inoculated on the agar plates. A little soil was taken and put on the TSA agar plates. Using a sterile inoculum, the student did an excellent T-streak on the surface of the agar. These plates were then incubated at a temperature of 30%u030A C. later after 48 hours the same plates were transferred into the 4%u030A C for some days.

Method

A little specimen was taken from the culture that had been refrigerated. From this, a little portion of the colonies that were set was taken and spread evenly on one of the slides. The slide was air-dried. After five minutes, the slide was flooded with crystal violet stain that was left to stand for 1minute before being washed off in the sink. Again, the slide was flooded with iodine solution that was also washed away after I minute. (The slide this point was colored blue). The rinsing was only done for 5 seconds.

After the second washing, the slide was decolorized by using ethanol. Ethanol was added drop by drop so as to minimize chances of decolorizing the bacteria. Hence, ethanol was added drop by drop until no more blue was coming from the slide. Then safranin was added to the slide and left to absorb for 1 minute. After the 1 minute, the excess stain was drained and by the use of a blotting paper  the glass slide was dried without rubbing the slide. The slide was then viewed under the microscope.

On another slide that is also labeled but with a different tag, the same procedure was repeated. On this slide, a sterile inoculum was used to pick on a different colony that had colony characteristics of gram negative bacteria. This was spread on the slide and the procedure repeated.

The final slide that was dry was examined under the microscope to verify the gram staining.

Discussion

The experiment above is often done in the laboratories in times of isolation and identification of bacteria or other microorganism from a given specimen. In this experiment, the specimen used was a sample extracted from a garden. The sample was sent to the laboratory while sealed in the specimen bottle. The reason as to why the specimen bottle has to be well sealed is to minimize the chances of contamination of the sample even by the person transporting the sample to the laboratory.

Once the sample has reached the laboratory, inoculation processes have to begin  as soon as possible while the microbes are  still viable. In this experiment, the samples were inoculated in  the TSA agar. This media is known as a media that is supplementary. Hence, in this media all microbes that are present in the sample will grow. In this regard, the students were able to harvest a variety of bacteria. Once the bacteria is grown after 48 hours of incubation, the culture plates can be set aside for further testing or be refrigerated incase the culture are to be used late. Refrigeration at 4 degrees C minimizes the growth of the bacteria (Vasanthakumari 2007).

After the primary culture, the colonies obtained are again sub-cultured in order to obtain a pure gram positive or gram negative culture. Hence, it is essential for the person culturing to ensure he or she does the gram staining. Gram staining is a technique that is used in the laboratories as a way of identifying bacteria. Thus, the process is done and the specimen to be used is taken from the  colonies which have grown on the primary cultures.

By use of a sterile needle, the colonies are obtained from the primary culture plates and spread on a sterile glass slide. This is then dried and set for gram staining.  Crystal blue was used in order to stain the gram-positive strains of bacteria. Iodine is the fixative in this experiment. Once iodine is put on the slide, it ensures that the crystal blue stain sticks on the bacteria. Then ethanol is also poured on to the slide. This alcohol decolorizes the crystal blue. That is if there are gram negative strains, they lose the crystal blue color. Later, Safranin red is used on the slide. Safranin red the best reagent that can be used to permanently stain the gram-positive bacteria.

Once the Bacteria have stained appropriately, then one can use oil immersion and the microscope to view the gram staining. From this experiment, a person is able to identify which colony is gram positive and gram negative. Coupled with the morphological characteristics then a person can make a pure culture isolate of a gram positive or gram-negative bacteria.

Conclusion

Gram staining is an efficient technique that can be used in the laboratory in order to identify bacteria types from a given sample. Coupled with the modern culture techniques all the culture and media procedures can be done with precision. In this case, precision is important because once a person has missed out on the bacteria to be identified then all other processes to be done after are false and invalid. In addition, it is important to ensure cleanliness and minimize any habits that may result in to contamination of the samples. Cultures that are well done can be used in determination of disease causing agent in medical laboratories.