Gram stain technique
Created | Updated Jan 28, 2002
Gram staining is a technique carried out in microbiology to classify a bacterium into one of two groups: Gram positive or Gram negative. It was developed by Hans Christian Gram, a Danish physician, in 1884. Those bacteria that stain purple after treatment are Gram positive, and those that stain red are Gram negative. This is useful since certain antibiotics that cure Gram negative infections have no effect on Gram positive infections (and vice versa), so it is good to know which kind of bacteria is infecting you, so that you can attain the right treatment. The following is a method for carrying out a Gram stain. Unfortunately, most of the equipment required is not available to the average hitchhiker, and can only be found in a science lab. Therefore, suggestions for improvisions that can be found around the household have been included in sqaure brackets.
EQUIPMENT
-18 to 24 hour liquid culture of bacterium [bowl of hot porridge left out overnight]
-Bunsen burner [gas stove]
-innoculating loop [paper clip bent into a small loop at one end]
-microscope slide [broken piece of picture frame/glasses/light bulb]
-Gram's crystal violet [blackcurrant Ribena]
-Gram's iodine [antiseptic iodine from medicine cupboard]
-30% acetone alcohol [Smirnoff Red Vodka]
-Gram's safranin [red dye - diluted ketchup may work]
-microscope [there is no good substitute for this - even a magnifying glass is no good. A cheap children's junior microscope kit will do]
-distilled water [Evian]
METHOD
1. Put a drop of distilled water on the slide. Dip the loop into the bacterial culture, and smear the loop on the slide.
2. The bacteria now need to be heat-fixed to the slide. To do this, the water must be evaporated - this is quite boring to wait for, even more mind-boggingly dull than watching water boil. The slide may be passed through a bunsen flame for a few seconds to speed up evaporation, then allowed to cool. Take care not to let the slide get too hot - this can be tested by touching the slide to the back of your other hand. If your hand jumps away, or if your reflexes are slow and you start to smell hair burning, the slide needs to be allowed to cool before being passed through the flame again.
3. Once all water has evaporated, the slide can be stained. These chemicals will also stain your clothes, so it is worth wearing a lab coat [or that shirt and jeans you used to paint the fence in]. Flood the slide with crystal violet, leave for 1-2 minutes, then rinse with distilled water.
4. Flood the slide with iodine, leave for one minute, rinse the slide with water. Shake or blot lightly, but not to dryness.
5. Add a few drops of alcohol, leave for 5-10 seconds, and rinse thouroughly with water.
6. Finally, flood the slide with safranin, leave for one minute, and rinse with water [the viscosity of your ketchup may or may not make this step difficult, but take care not to rub the slide since this will remove the bacteria]. Blot the slide dry, being careful not to rub it.
The slide is now ready for examination under the microscope. Gram negative bacteria, such as E. coli, will show up as red. Gram positive bacteria will show up as purple. Gram negative bacteria are resistant to penicillin, and you could get quite ill from taking that antibiotic if you have a Gram negative infection. To prevent further infections, remember to wash your hands before eating.
EQUIPMENT
-18 to 24 hour liquid culture of bacterium [bowl of hot porridge left out overnight]
-Bunsen burner [gas stove]
-innoculating loop [paper clip bent into a small loop at one end]
-microscope slide [broken piece of picture frame/glasses/light bulb]
-Gram's crystal violet [blackcurrant Ribena]
-Gram's iodine [antiseptic iodine from medicine cupboard]
-30% acetone alcohol [Smirnoff Red Vodka]
-Gram's safranin [red dye - diluted ketchup may work]
-microscope [there is no good substitute for this - even a magnifying glass is no good. A cheap children's junior microscope kit will do]
-distilled water [Evian]
METHOD
1. Put a drop of distilled water on the slide. Dip the loop into the bacterial culture, and smear the loop on the slide.
2. The bacteria now need to be heat-fixed to the slide. To do this, the water must be evaporated - this is quite boring to wait for, even more mind-boggingly dull than watching water boil. The slide may be passed through a bunsen flame for a few seconds to speed up evaporation, then allowed to cool. Take care not to let the slide get too hot - this can be tested by touching the slide to the back of your other hand. If your hand jumps away, or if your reflexes are slow and you start to smell hair burning, the slide needs to be allowed to cool before being passed through the flame again.
3. Once all water has evaporated, the slide can be stained. These chemicals will also stain your clothes, so it is worth wearing a lab coat [or that shirt and jeans you used to paint the fence in]. Flood the slide with crystal violet, leave for 1-2 minutes, then rinse with distilled water.
4. Flood the slide with iodine, leave for one minute, rinse the slide with water. Shake or blot lightly, but not to dryness.
5. Add a few drops of alcohol, leave for 5-10 seconds, and rinse thouroughly with water.
6. Finally, flood the slide with safranin, leave for one minute, and rinse with water [the viscosity of your ketchup may or may not make this step difficult, but take care not to rub the slide since this will remove the bacteria]. Blot the slide dry, being careful not to rub it.
The slide is now ready for examination under the microscope. Gram negative bacteria, such as E. coli, will show up as red. Gram positive bacteria will show up as purple. Gram negative bacteria are resistant to penicillin, and you could get quite ill from taking that antibiotic if you have a Gram negative infection. To prevent further infections, remember to wash your hands before eating.
