Out of 28 unknown microorganisms, slant 6O was randomly selected and subjected to testing to identify its species. Seventeen of the unknown bacteria were classified as Gram-negative bacilli: Corynebacterium pseudodiphtheriticum, Enterobacter aerogenes, Escherichia coli, Flavobacterium capsulatum, Helicobacter pylori, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus hauseri, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Alcaligenes faecalis, Rhizobium radiobacter, Salmonella typhi, Serratia marcescens, Shigella flexneri, or Yersinia pseudotuberculosis.
Ten of the unknown bacteria were classified as Gram-positive bacilli: Bacillus cereus, Bacillus circulars, Bacillus coagulans, Bacillus megatherium, Bacillus subtilis, Bacillus thuringiensis, Brevibacillus brevis, Geobacillus stearothermophilus, Lysinibacillus sphaericus, Clostridium perfringens.
Lastly, there were also 11 Gram-positive cocci microbes: Enterococcus faecalis (formerly Streptococcus), Streptococcus lactis (now known as Lactococcus), Streptococcus mutants, Streptococcus pneumoniae, Micrococcus (Kocuria) roses, Sarcina lutea (Micrococcus luteus), Sporosarcina ureae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Rhodococcus rhodochrous.
Procedure: A Gram stain was performed to identify whether the unknown 6O is a Gram-positive or Gram-negative bacterium and determine its shape. The Gram stain was performed as stated in Laboratory Exercises In Microbiology, 8th Ed. pages 45-49 (Harley, 2011). A minor adjustment was made in the process of decolorization with crystal violet, reducing the time to 20 seconds to prevent over-decolorization.
Purpose: To identify an unknown microbe, a Gram stain is the first procedure used to determine whether the bacteria is Gram-negative or Gram-positive. This process eliminates a substantial amount of microbes and provides the cell shape.
Gram-positive cells do not decolorize with alcohol, while Gram-negative cells do. This allows Gram-positive bacteria to appear purple and Gram-negative bacteria to appear pink after staining. A Gram-positive cell has a thick peptidoglycan cell wall that can retain the crystal violet stain during staining, while a Gram-negative cell has only a thin layer of peptidoglycan.
Data: The Gram stain procedure resulted in microbe 6O having a clear view of long purple rods.
Analysis: The Gram stain determined 6O as a Gram-positive bacillus, eliminating thirty microbes: Corynebacterium pseudodiphtheriticum, Enterobacter aerogenes, Escherichia coli, Flavobacter capsulatum, Helicobacter pylori, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus hauseri, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Alcaligenes faecalis, Rhizobium radiobacter, Salmonella typhi, Serratia marcescens, Shigella flexneri, Yersinia pseudotuberculosis, Erwinia persicinus, Serratia liquefaciens, Enterococcus faecalis, Streptococcus lactis, Streptococcus mutans, Streptococcus pneumoniae, Micrococcus roseus, Sarcina lutea, Sporosarcina ureae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Rhodococcus rhodochrous.
Ten possible microorganisms remained, Gram-positive Bacilli: Bacillus cereus, Bacillus circulars, Bacillus coagulans, Bacillus megatherium, Bacillus subtilis, Bacillus thuringiensis, Brevibacillus brevis, Geobacillus stereothermophilus, Lysinibacillus sphaericus, Clostridium perfringens (Bergey’s, 1984).
Error: Many errors can occur when performing a Gram stain. Some Gram-positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-variable). When sterilizing the inoculating loop, if it is not cooled to the proper temperature, the microbe can be killed. Finally, do not blot the slide vigorously with bibulous paper, or the Gram stain can be rubbed off (Harley, 2011).
