Bacterial Morphology and Arrangements

• Staining a bacterial specimen makes it easier to observe under a brightfield light microscope and gives it different colors

Morphology & Arrangements

• Morphology refers to the shape of a cell, while arrangements refer to how cells are positioned in relation to one another
• Circular or round cells indicate cocci morphology
• Rod-shaped cells indicate bacilli morphology
• S-shaped or spiral-shaped cells indicate spirillum morphology
• Individual cells not touching end-to-end are single in arrangement
• Two cells touching end-to-end, or a pair of cells touching end-to-end, are in diplo arrangement
• A chain of cells touching end-to-end is in strepto arrangement
• A grape-like cluster of circular/round cells is in staphylo arrangement
• Circular/round cells arranged in a square are in tetrad arrangement
• Circular/round cells arranged in a 3-D cube are in sarcinae arrangement
• Morphology and arrangement should not be merged as one single term
  • Staphylococcus cannot be used as an arrangement
• Staphylo is the correct arrangement term

Bacterial Cells

• Most bacterial cell walls are negatively charged
• Positive chemicals are attracted to the cell while the cells repel negatively charged chemicals

Stains and Chromophores

• Stains are chemicals containing a colored ion, called a chromophore
• The chromophore of a stain may be either positively or negatively charged

Types of Stains

• There are two types of stains: basic and acidic
• Basic stains have a positively charged chromophore
  • They stain negatively charged bacterial cells, leaving the background white
  • Examples include methylene blue, crystal violet, safranin
• Acidic stains have a negatively charged chromophore
  • They do not stain bacterial cells, instead, they stain the background
  • Nigrosin is an example of an acidic stain

Simple Staining Techniques

• A simple staining technique uses only one dye
• The purpose is to identify the shape, size, and arrangement of bacterial cells
• There are two types: Direct Stain and Negative Stain
• Direct staining involves staining the bacteria using basic dyes
• Negative Staining involves staining the background using acidic dyes

Types of Staining Techniques

• Simple staining uses one stain to determine cell size, shape, and arrangement
• Differential staining uses more than one stain and is used as a classification tool or to view structures like endospores

Smear Preparation

• A smear is a thin film of bacterial cells placed on a slide
• Smear preparation for direct stains is different from that used for negative stains
• For liquid media, at least 2-3 loopfuls of bacteria should be used
  • The loop should be flamed between each loopful of bacteria
• For solid media, half a drop of sterile water should be placed on the slide

Heat Fixing

• Should only be utilized when basic stains are used
• Gently heating it kills the bacterial cells without incinerating them
  • It also helps the cells adhere to the slide surface
  • The slide should only be passed through the flame two to three times
• Smear must be completely dry before heat-fixing

Negative Stain Technique

• For cultures on solid media, add half a drop of water before adding a small drop of nigrosin to one end of the slide
• For cultures in broth media, mix a loopful of the culture into the drop of nigrosin to one end of the slide
• Use the end edge of another slide to spread out the drop across the length of the slide
• The slide should be allowed to air-dry, and should not be heat-fixed

Alternative Negative Stain Techniques

• Add a small drop of nigrosin to the middle of the slide, mix in the bacteria, then gently heat-fix the slide
• For liquid cultures, add a small drop of nigrosine to the middle of the slide, mix in 2-3 loopfuls of bacteria, then gently heat-fix the slide

Things To Do

• Do a Direct Stain with 1 solid and 1 liquid culture
• Do a Negative Stain with 1 liquid or 1 solid culture
• Streak B. subtilis and S. epidermidis on a NA plate for the next lab (Gram Staining)
• Use a very small amount of solid bacteria and 2-3 loopfuls of bacteria for liquid cultures for smears
• Use the small dropper bottle for all your staining procedures
• Store your slide inside the class slide box if you are not able to complete the lab procedure during the class
• Used slides should be placed inside the disinfectant pan after use
• The staining tray should be poured down the sink and rinse with water after use
• Store the microscope correctly