RE: [Histonet] growing gram pos and neg

From:"Monfils, Paul"

In the past I have made such gram controls, but I preferred to obtain the organisms already grown, rather than trying to grow them myself.  I ordered cultures of gram+ and gram- organisms, grown on broth, from a biological supply company.  Then I just spun them down to concentrate the organisms, injected the concentrate into a piece of lung, and dropped the tissue into formalin.  Push the needle almost all the way through the piece of tissue, then inject continuously as you withdraw it.  I used lung because there is plenty of microscopic open space into which the broth can be injected, without doing any great damage to the tissue structure.  Injecting into a more solid tissue like liver doesn't work well because there is nowhere for the injected fluid to go. A space has to be torn open by the pressure of injection.  I would usually inject two different gram+ organisms, mixed together, into one piece of lung.  Then two gram- organisms mixed, into another piece of lung. After fixation I would cut the injected tissue into small cubes, and embed a gram+ cube and a gram- cube side by side in a block. For gram+ I liked to use a Bacillus, such as B. cereus or B. subtilus, mixed with a Staphylococcus like S. epidermidis. This provides two different shapes of organisms, both gram+.  For gram- I used Escherichia coli mixed with Neisseria subflava. My source for the organisms was  The cost is less than $10 per culture and you can make enough good control blocks to last forever.

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