Lab 2: Microscopy and Gram staining

Introduction

Gram-type differentiation is an important analytical tool in classifying bacteria. Gram-staining is a technique that utilizes light-microscopy and a procedure of dyes to determine Gram-type.
The aim of this lab is to introduce the authors to basic laboratory procedures by performing Gram-staining and size determination by light microscopy to assign three given names to three unidentified samples of microorganisms.  Two of the three species of microorganisms were bacteria, Escherichia coli and Staphylococcus epidermis, the third was yeast, Saccharomyces cerevisiae.

Method

The method was carried out as per lab instructions.

Calibration of microscope reticule

The graduated microscope eye piece reticular was calibrated to a 1:1 ratio with the graduated microscope slide, which displayed 100 lines per mm, giving the measure 1µm between lines of the graduated microscope eye piece reticule.

Gram-staining

Gram-staining utilizes two basic dyes called Crystal Violet and Saffranin. Both molecules have positively charged groups which binds to negatively charged molecules in bacteria. The thick peptidoglycan layer of Gram-positive acts as a permeable barrier, keeping the Crystal violet dye and Iodine mordant mixture inside the cell after rinsing with ethanol, keeping it blue, while the same mixture is washed out of the Gram-negative bacteria. Saffranin is then added to bind with the Gram-negative bacteria, coloring them pink. (\citealt{Willey2017}, p. 59-60) 

Results and discussion