Gram Staining Lab Report

Introduction

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
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 Iodine 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
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 Iodine. 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