As we know bacteria comes in various classes and are distinguished by their shapes, chemical configuration, source of energy either sunlight or chemicals, nutritional requisites and biochemical actions. We have a patient that may be infected with an unknown bacterial infection; we’ve collected a sputum sample and will be testing for one of the following genera: Bacillus, Escherichia and Mycoplasma. In order to determine which one of the three may be the source of the infection we have to recognize the characteristics of each one and the staining protocol needed to identify them. All three of the above mentioned genera are considered to be members of the Prokaryotic cell family which includes bacteria. They have certain identifiable features that distinguishes them from Eukaryotes such as: “Their DNA is not enclosed within a membrane” and “they usually divide by binary fusion, organelles are not encompassed within the membrane and the cell walls usually consist of complex polysaccharides peptidoglycan” (Tortora, 2013).
When researching the differences among the three genera’s each have specific characteristics that distinguishes one from another; Mycoplasma are the smallest members of the prokaryote family, due to their size they lack cell walls or have a scant amount of wall material, but they are known to thrive and reproduce outside the living host cells. The plasma membranes of this bacteria contain lipids (sterols) to protect them from lysis (Tortora, 2013). Bacillus are rod shaped bacteria that are usually presented as singular (single bacilli); however when they divide into pairs they’re called Diplobacilli and when appearing as a chain it’s known as streptobacilli; there’s also bacillus that resemble cocci and are referred to as cocobacilli. Escherichia also known as E. coli is a rod shaped bacteria consisting of “flagellar protein” known as H- antigens to help differentiate the various servoar within the E. coli strain. In order to continue with our investigation as to which genera is causing the infection we must determine the appropriate staining method for each bacteria listed above.
Let’s begin with Mycoplasma because this genera has a very thin or lacks a cell wall we will consider this a “Gram- positive” bacteria. If mycoplasma retains its structure, size and capsules that’s an indication of the type of bacteria causing the infection in the patient. Bacillus are part of the prokaryotic cell family containing a thin layer of peptidoglycan; however they are spore forming; this trait helps to identify them as “gram-positive”. Step (1) in the staining process is adding the sputum to the slide, step (2) is applying heat to the sample, step (3) adding crystal-violet dye which turns the cytoplasm cells purple and applying mordant to the specimen, step (4) wash with de-coloring solution (alcohol) which causes the peptidoglycan to become more impermeable crystal –violet and iodine, step (5) counterstain is absorbed and is masked by the primary purple dye that was previously absorbed by the “gram-positive” cells (pg. 86). It’s during this step when the microbes are identified.
While the bacillus is in the aging process some cells die and give the appearance of gram-negative bacteria; these are known as “gram-variable” (Tortora, 2013). Escherichia (E.coli) these rod shaped microorganisms have a thick outer membrane; however they are non-spore forming; therefore they are considered “gram-negative” cells. The steps in the staining of “gram-negative” cells are as follows: Step (1) in the staining process is adding the sputum to the slide, step (2) is applying heat to the sample to allow the sample to remain on the slide, step (3) adding crystal-violet dye which turns the cytoplasm cells purple then apply mordant to the specimen, step (4) wash with de-coloring solution (alcohol) which allows the crystal violet-iodine to diffuse through holes in the peptidoglycan layer of the cell, step (5) add the counterstain which turns the specimen red/pink this process helps to identify the microbes; as they lack color (Tortora, 2013). Without color we would be unable to identify the microorganisms creating the infections created by “gram-negative” bacteria because they are color-less.
Tortora, Funke, Case: Microbiology an Introduction 2013 (11th edition).