So, I came up with this silly idea to compare water samples from 4 locations for their bacterial content, in addition I wanted to determine if they were gram negative or gram positive bugs. I ended up invalidating the ditch water due to some stupidity on my part, however, that left my grease trap water, my tap water, and some water I nabbed from the Gulf of Mexico (at the Bay St. Louis beach). As soon as I rinsed the first stain I pretty much already knew what I was going to find in the gulf and tap water because the slides became almost instantly spotless. Whereas the grease trap water (I already knew it had bacteria - but didn't know if it was + or -) was visibly stained.
The image below is where I determined the bacteria in the grease trap water are gram negative bacilli. It should be noted that G- bacteria are famous for eliciting an immune response, particularly in humans (for example, E. Coli and Salmonella are G- bacilli among many).
(1000x mag, oil immersion, with crappy camera)
For size comparison, a single strand of hair usually has a diameter of 20 to 180 μm in dia. These bacteria are 1-2 μm in dia.
Slide Sets and Results
Slide Set A - Ditch Water
Invalidated Samples
Slide Set B - Grease Trap Water
Bacteria: Safranin (red) stained Gram Negative bacteria (2-3 μm) in vast numbers. Image above.
Other findings: 2 Rotifers. 1 unidentified circular mass with 8 arm-like extensions (possible contracted contractile vacoule from a paramecium).
Slide Set C - Gull of Mexico Water
Bacteria: None.
Other findings: 3 small protozoa. Some algea.
Slide Set D - Tap Water
Bacteria: None.
Other findings: Several particles of dirt.
Protocol Used
Crystal Violet (CV - .05%) Flood 1 Min
Rinse - 5 sec (bottled water)
Grams Iodine - (GI) Flood 1 Min
Rinse - 5 sec (bottled water)
Acetone Alcohol Decolorizer - (AA) Rinse 1-2 seconds
Rinse - 5 sec (bottled water)
Safranin (SAF .05%) - Flood 1 min
Rinse - 5 sec (bottled water)
Hypothesis
This, being my very first try at Gram Staining, was my project hypothesis.
The Gram Positive (G+) bacteria, if found, should be bluish/purple in color, since the CV become lodged behind the peptidoglycan of the large outer cell wall following the bond with iodine and the dehydration of the outer cell wall due to the alcohol. The Gram Negative (G-) bacteria, if found, should be redish in color, since the SAF stain can not overpower the CV stained G+ bacteria, but will bind to the lipopolysaccharides of the double walled remaining bacteria that lost their CV stain during alcohol decolorization. Note there are exceptions to Gram Staining that would require further testing with growth mediums, and subsequent analysis in order to identify. However, generally speaking, this is the predominate expected result for most bacteria.
Note to self - locate new standing water samples in various areas around town, and don't mess it up.