SIMPLE STAIN
LEARNING OBJECTIVES
Properly make a bacterial smear for staining
Properly perform the simple stain technique
Identify morphology of bacteria
Identify arrangement of bacteria
MCCCD OFFICIAL COURSE COMPETENCIES
Utilize aseptic technique for safe handling of microorganisms
Apply various laboratory techniques to identify types of microorganisms
Identify structural characteristics of the major groups of microorganisms
Compare and contrast prokaryotic cell and eukaryotic cell
Compare and contrast the physiology and biochemistry of the various groups of microorganisms
MATERIALS
Stock cultures:
Slant culture of Staphylococcus epidermidis (Gram-positive cocci) 6 / 2 lab sections for day two of Gram stain
Slant culture of Escherichia coli (Gram-negative bacilli) 6 / 2 lab sections for day two Gram stain
Equipment:
“E” slide and “S” slide prepared in the streak plate lab exercise
Microscope slides
Deionized water dropper bottle
Inoculating loop
Bibulous paper
Lens wipes
Stain container
Slide warmer
Bacticinerator
Microscope
Immersion oil
Stains:
Crystal violet
Safranin
BACTERIA ALBUM
Unstained bacteria are nearly transparent. To see them with the microscope we often use chemical compounds called stains. Staining bacteria improves the ability to see small, colorless bacteria. To understand how staining works, it will be helpful to know a little about the physical and chemical nature of stains. Stains are generally salts in which one of the ions is colored. A salt is a compound composed of a positively charged ion and a negatively charged ion. For example, the stain methylene blue is the salt methylene blue chloride which will dissociate in water into a positively charged methylene blue ion which is blue in color and a negatively charged chloride ion which is colorless.
Stains may be divided into two groups: basic and acidic. If the color portion of the stain resides in the positive ion, it is called a basic stain. Some examples of basic stains include methylene blue, crystal violet, and safranin. If the color portion of the stain is in the negative ion, it is called an acidic stain. Some examples of acidic stains include nigrosin, congo red and eosin.
Because of its chemical nature, the cytoplasm of all bacterial cells has a slight negative charge when growing in a medium of near neutral pH. Therefore, when using a basic stain (positively charged), the positively charged color portion of the stain combines with the negatively charged bacterial cytoplasm and the organism becomes directly stained. An acidic stain (negatively charged), due to its chemical nature, reacts differently. Since the color portion of the stain is on the negative ion, it will not readily combine with the negatively charged bacterial cytoplasm. Instead, it forms a deposit around the organism, leaving the organism itself colorless. Since the organism is seen indirectly, this type of staining is called a negative stain.
In a simple stain, the bacteria are stained with one positively charged stain, staining the all bacteria the same color. Simple stains provide basic information about size, morphology (shape), and arrangement.
PRE-ASSESSMENT
