One way to classify bacteria is by examining its morphology. Bacteria appear in many different shapes. We will examine the three basic shapes but note that others exist. 3 basic shapes: Bacilli (singular is bacillus), are rod-shaped. Cocci (singular is coccus), are spherical-shaped. Spirilla ( singular is spirillum), are spiral-shaped. Bacterial cells can appear as single cells or can be found in arrangements. Some of these are: Chains (prefix strepto-) Clusters (prefix staphylo-) Pairs (prefix diplo-) 2

In 1884 Hans Christian Gram developed a staining procedure known as Gram Staining. The composition of the cell wall of bacteria vary among species. Due to this difference, bacteria can be divided into two groups; Gram positive and Gram negative. Gram positive bacteria have thick peptidoglycan cell walls and retain a purple color when stained with crystal violet. Gram negative bacteria have a thin peptidoglycan inner wall and outer wall made of carbohydrates, proteins and lipids. These cells retain the red color when stained with safranin. Before anything, you need to make the heat-fix sample. 3

Obtain a slide and add one drop of distilled water (dH2O) and place in a staining tray Sterilize a loop Staining tray slide drop of H2O 4

With the loop transfer bacteria to your slide and mix it with the drop of dH₂O. Sterilize the loop once done. Heat-fix the slide by passing the slide over an open flame several times. This evaporates the water leaving the bacteria sample dry and fixed on the slide. Bacteria colony loop slide 5

Put your slide in the staining rack. Flood the sample with crystal violet (CV) for 1 minute. Wash away the excess CV using dH₂O. Flood the sample with iodine for 1 minute. Rinse off the iodine with dH₂O. 6

Decolorize with ethanol. Rinse slide with dH₂O. Ethanol must be added drop by drop to a slanted slide. Stop when first clear drop is seen (about 4 drops). The ethanol washes out the thin layer of protein/lipids if it is gram negative, causing it to lose the CV. If it is a gram +, the peptoglycan layer is so thick that it will most likely retain most of it, retaining the CV impregnated in it. 7

Flood smear with safranin (counterstain) for 1 minute. Only decolorized cells will stain red. Gram negative will stain red, gram positive remain purple. Rinse slide off with dH₂O and blot slide dry between sheets of bibulous paper. Slide can be examined using the microscope. Here is a 3 minute video of the staining process. 8

9 Do you recognize this bacteria?

10 Each round dot is an individual bacteria, notice it shows in pairs. Due to the color it is a Gram negative. Bacteria type: Coccus Diplococcus sp. 1000x

11 Do you recognize this bacteria?

12 Notice each individual bacteria is elongated and the colony shows a clumped form. Due to the color this is a gram positive bacteria Bacteria type: Bacillus Staphylobacillus sp. 1000x

13 Do you recognize this bacteria?

The End 14 Notice the individual bacteria has a long, curved shape. Due to its color it is a Gram negative Bacteria type: Spirilla Spirillum sp. 1000x