Simple And Gram Staining 492k5

Simple and Gram Staining of Bacteria And Aseptic transfer of microorganisms and proper handling of bacteria By Bright Belinda Afra 3/04/2010 Microbiology lab: 303 Thursdays 8:00-10:50

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Introduction The process and understanding of simple gram staining has been introduced in this lab experiment. Simple stains use basic dyes which are positively charged. These positive dyes interact with the slightly negatively charged bacterial cell wall therefore lending the color of the dye to the cell wall. Gram staining is one of the most important techniques in microbiology that helps identify bacteria. The Gram stain is a technique used to classify bacteria in which a bacterial specimen is first stained with crystal violet, then treated with and iodine solution, decolorized with alcohol and counterstained with safranin. Bacteria are classified as gram positive and gram negative based on their cell wall properties. Gram positive cells have a thick peptidoglycan cell wall which is able to retain the crystal violet iodine complex that occurs during chemical staining. However, gram negative have a thin peptiodoglycan cell wall, which is covered with the outer membrane. The results in the experiment will differentiate the correct name of the bacteria and show which bacteria is gram positive and gram negative. In the Aseptic transfer of microorganisms and proper handling of bacteria this particular laboratory bacteria, must be cultured in order to facilitate identification and to examine their growth and metabolism. In order to keep bacteria alive they must be inoculated into various forms of culture media. When doing inoculations, unwanted microbes should not be introduced or contaminated, into the media. Aseptic technique is used to prevent contamination. In transferring of bacteria all cultures should be sterilized such as broth. Broth cultures provides large numbers of bacteria in a small space and are easily transported (Transfer of Bacteria Aseptic Technique worksheet). Agar slants are test tubes containing solid culture media that provides a solid growth surface. Agar deep is often used to grow bacteria that prefer less oxygen than is present on the surface of the medium. Transferring and inoculation are usually performed with a sterile, heat- resistant. When a wire is bent at the end into a loop it’s called inoculating loop; when straight it is an inoculating needle (streak plate technique). Objectives In the experiment, we will prepare a bacterial smear, and heat fixation to understand bacterial morphology by doing the simple staining procedures and also gram staining. These two procedures will give us the ability to distinguish bacteria based on the gram reaction, size, shape and arrangements.

Materials and Methods Burner Loop Microscope Bacterial Cultures: Escherichia coli (agar slant tube); and Bacillus cereus (agar plate) 2

Clean glass slide Permanent marker Crystal violet Gram’s iodine 95% ethyl alcohol Safranin Bibulous paper In performing the experiment for Aseptic technique was performed and two bacteria cultures were given A and B. Tube A was Escherichia coli and tube B was Bacillus cereus. One was aseptically introduced to the agar plate (A) by zig zag and the other bacterium was introduced by the agar plate (B) by a four quadrant streaking technique. The plate and the tube was both inoculated threw incubation at 37 degrees Celsius for 18-24 hours for growth. The plates were then kept refrigerated at 4 degrees Celsius so the bacteria can stay alive without growth. The growth was then observed on the agar plate and agar tube In performing the experiment for simple and gram staining, a clean glass slide was obtained, waving the slide over the open flame of a burner a couple times so the remaining dust and stains were removed which is called heat fixation. A marker was used to draw two dime sized circles on the back of the glass slide and labeled “A” and “B” for each specimen. Two loopfuls of phosphate buffered saline was added to each circle. The Bacillus cereus was spread evenly in the circles to make then smears (on the opposite side of the circles). The loop was flame, and then we waited until it cooled down. Escherichia coli culture from its agar slant was spread in circle B. The smears was air dried and went through a process called heat fixation, which consist of flaming the slides 2-3 times. After the smears and heat fixation, simple staining was conducted. A drop of crystal violet was added and then spread on both slides for 60 seconds. The CV was carefully rinsed off with running tap water. The bibulous paper was used to absorb the remaining water on the slide. The slide was air dried, after the slide was dried we examined the slide under a microscope in 10X, 40X and 100X (oil immersion) lens to identify the arrangement and shape of the specimens. The gram staining procedures were conducted by repeating the process of smear preparation and heat fixation. The CV was used to cover both specimens A and B and used as the primary stain to bind the cell membrane. After CV was rinsed off, a drop of Gram’s Iodine was added over the CV smears and stayed for 20 seconds. The iodine was then rinsed off with running tap water. The smear was submerged with 95% ethyl alcohol for 10 seconds. The alcohol was rinsed off quickly and then a drop of safranin was added to each specimen for 45 seconds. The safranin was rinsed off with running tap water and then dried with bibulous paper. The slides were examined under the microscope to determine the gram reaction of the gram negative and gram positive bacteria.

Discussion I have concluded that the procedures were conducted properly and successful. In the simple stain of Escherichia coli and Bacillus cereus bacteria, slide 1 and 2 had a spherical shape 3

structure which is known as cocci and a crystal/violet purple pigment. In the gram staining of Escherichia coli and Bacillus cereus bacteria, slide 1 Bacillus cereus had a round shape structure and a pigment of pinkish purple, pink or light pink all resulting in a gram negative bacterium. Slide 2 Bacillus cereus, had a rod shape (bacillus) structure and a purple or purplish red pigment which resulted in a positive gram stain. Gram reaction help demonstrate the structural differences of gram negative and gram positive bacteria. Gram negative bacteria are the bacteria that do not retain crystal violet dye. In the gram stain test gram negative bacteria has a red or pink color. Gram positive bacteria are those that are stained violet, which can retain the crystal violet stain due to the high amount of peptidoglycan in the cell wall. Results Bacillus cereus- gram positive, rod shape bacterium (purple color) Escherichia coli- gram negative, rod shape bacterium (pink color) See data attached

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