Gram staining was developed by Christian Gram in the 1800’s, a Danish bacteriologist. (Smith and Hussey, 2005) It was the first differential staining technique and most common used in microbiology. Furthermore, bacteria are transparent and cannot be seen through the microscope. For that reason, Gram staining is an important tool for distinguishing between two main types of bacteria Gram-positive and Gram-negative. The Gram stain differentiates the Gram positive and gram-negative on the basis of their cell wall structure. (Menard, et al. 20150)
Most bacteria gram positive or gram negative but they are a few gram variable bacteria and very small bacteria without a cell wall that do not have a gram reaction. For the purpose of this lab we are focusing on the two main types Gram + and Gram-bacteria. Bacteria are prokaryotes that have a cell wall; they are classified as bacillus (rod-shaped), Coccus (spherical) and Spirillum (Spiral) (Menard, et al. , 2015). Therefore, in this lab we will examine be the staining of the cells walls the different shapes and characteristics of the gram + and gram – bacteria.
Furthermore, gram staining is widely used in the health care to identify the cause of a bacterial infection; detecting whether or not the infection is caused by a gram + or gram – bacteria. Thus, allowing health professionals to prescribe appropriate antibiotic for adequate treatment. In this Lab we will be using a series of dyes that will result in some bacteria staining purple and others staining pink. The bacteria that stains purple are gram-positive and the bacteria that stains pink is the gram-negative bacteria.
Gram-positive bacteria has a thicker double layer of cell wall peptidoglycan molecules, and Gram-negative bacteria have a cell wall with only a thin layer of peptidoglycan molecules. Therefore, gram stain differential stain uses two dyes to differentiate between the two basic cell wall types. The differential stain has four components; a primary stain, a mordant that sets the stain, a decolorizing agent to remove the primary stain, and a counter stain used to color the cells that lost the primary stain in the technique gram+ and gram- slides are heat fixed and prepared for staining.
Then the application of the primary stain- crystal violet which is what gives it the purple color Followed by an lodine mordant to rinse the primary stain and set the crystal violet inside the peptidoglycan cell walls of the gram+. The cell is than washed with decolorizer Alcohol, the gram positive cell will remain purple and gram negative cells dye will rinses out of the thin layer of peptidoglycan gram- cell wall and remain colorless. The last step is the counter stain safranin is added to color the gram- bacteria pink or they would remain colorless and you wouldn’t be able to see them.
Hypothesis The purposes of this lab is to examine how the Gram Stain techniques are uses by microbiologists to observe the morphological characteristics of bacteria. How will the gram stain techniques show morphology of a bacteria cell? Furthermore, after using the Gram Stain Techniques the gram positive bacteria will turn purple and the gram negative pink showing the difference in each bacteria characteristics. Method one The first step, we used in preparing the bacterial smear on the glass slide was to label it on the frosted side up with C. f. (C. freundii) or S. . (S. aureus); then we placed a water drop in the center of the clean slide.
In the next step, we aseptically transfer bacterial colonies, using the Bunsen burner and inoculating loop to properly sterilize before selecting and transferring it to the media. A minute amount of a colony bacteria was selected from an agar slant tube and place into the drop of water on the glass slide. In the third step, we gently emulsify the organism in the drop of liquid spreading the mixture over the entire surface of slide to produce an even thin film of bacterial smear.
That is to say, thickly applied specimen stain does not penetrate the cell walls easily, making it difficult to interpret the morphology. However, a thin film would produce faster drying, and separation of colonies making it easy for interpretation. The fourth step, we allow the smear to air dry completely. This step takes the most time, because excess water on the glass can prolong air drying time, destroy the cells, or cause the glass to explode in the flames. Then, in the fifth step we heat-fix the slide after it has air dried by passing the slide through the upper part of a flame two or three times.
Being careful not overheat the bacterial cell walls or incinerating them before staining. Thus, heat-fixing dried smears helps the bacterial cells adhere to the glass, kills them, and makes them more easily stained because their proteins become coagulated. Finally, smear was ready to be stained and we move on to the second method in gram staining Method two In method two we first place the prepared and heat-fixed smear on the staining rack, that we prepared during method one. In the second step we flood the smear with Crystal Violet for 60 seconds.
Crystal violet is the primary stain that penetrates the cell wall and gives it the purple color. In the third step we used a clothespin to pick up the slide and tilting it at a 45 degree angle pouring the stain off; while gently rinsing the excess stain off with a stream of water from the plastic water battle. We continue to rinsing until the water ran clear of crystal violet. Next, in the fourth step we removed the clothespin, place the smear back on the staining rack and flood it with lodine.
Iodine was followed as a mordant that forms a crystal-violet iodine complex; that affixed it to the thick peptidoglycan layers and remains in the gram positive cell wall even after decolorizing. In step five we repeated step four raising with water until all excess iodine was clear. Next, step six was the most critical step. Hold the slide at a 45 degree angle dripping a few drops of the alcohol acetone decolorizer so the solution trickles down the slide and over the smear. Stopping when the solvent is no longer colored as it flows over the slide, between 5 and 15 seconds or as soon as it runs clear, immediately rinsing with water.
Further delay will cause excess de-colorization in the gram positive cells, and defeat the purpose of staining. For example, during the lab we got to see the difference of what the cells looked like when it got excess de-colorization. On one of the students slide you could see a lighter purple with white spaces in the walls of the S. aureus cells that made it difficult to recognize the color, shapes and size. Another student that had the same S. aureus bacteria had a darker purple cell walls making it easy to recognize.
In step seven, we remove the clothespin, place the smear on the staining rack and flood with Safranin for 60 seconds. Safranin is a counter stain use to color the thin peptidoglycan walls of the gram-negatives pink that the de-colorizer striped of crystal violet. This allow us to be able to see them or they would be colorless. Then, in step eight gently rinsing the excess stain with a stream of water from the plastic water bottle, rinsing until the water runs clear. Lastly, we prepared the slide for the microscopic examination by gently blotting the slide dry in a tablet of bibulous paper.
When slide was completely dry we proceeded to examine the result of the gram staining Discussion/Analysis In lab, my bench was assigned to stain the Citrobacter freundii bacteria to gram stain and we obtained the proper staining results for this bacteria. Thus, receiving a pinkish reddish color appearance on the heat smear glass slide, under the microscope on 10x objective lens. However, full observation of the shape and arrangements of the cells was more visible under the 100x objective oil immersion lens, noticing the bacilli rod shaped cells.
C. reundii was supposed to stain pinkish reddish, because after the decolorizing with alcohol the purple crystal violet washed out the thin walls of the gram negative cells. (Figure: 1 step 3). Adding the counter stain Safranin give the Citrobacter freundii pinkish color, the color result of a gram negative cell. Citrobacter freundii are facultative anaerobic, gram-negative bacilli commonly found in water, soil, food and the intestinal tracts of animals and humans. The groups of patients that are at greatest risk of acquiring Citobacter infections are immunocompromised patient and neonates.
Citobacter can cause a wide spectrum of infection in humans, such a urinary tract, respiratory tract, wounds, bone, peritoneum, endocardium, meninges and bloodstream. A positive result of Citrobacter freundii bacteria can be confirm only from a culture in a laboratory for proper diagnosis and treatment. When C. freundii was tested for antimicrobial agents on Mueller Hinton agar by the standard disc diffusion method for susceptibility test, Amoxicillin, Ampicillin and Carbenicillin c. freundii higher resistance to these antibiotics.
In some studies aminoglycosides, fluoroquinolones and Cephems and Carbpenems have the highest in vitro antimicrobial activities against C. freundii. Conclusion In completion of the gram staining lab activity, we learned the how to prepare the bacterial smear on the glass slide, heat fixing the glass slide and gram staining a bacterial slide. After using the gram Stain Techniques the gram positive bacteria turned purple and the gram negative pink showing the difference in each bacteria characteristics.
Then, observing the morphological characteristics of bacteria, under a brightfield microscope, of the Citrobacter freundii a gram positive bacteria and the Staphylococcus aureus a gram negative bacteria. We also learned what can go wrong when using gram staining that could produce errors in the testing. Hence, witnessing the different results that occurred when too much decolorizer is added or heat fixing the glass slide incorrectly. Learning the gram staining techniques have helped us understand how bacteria can be identify for proper treatment and for future studies of bacterial infections.
