7. Gram Stain and KOH Test Introduction Gram stain procedure is the most used staining procedure in pathology labs. It is fast, inexpensive and informative; however, to exclude the possibility of false positive and false negative results, a number of precautions must be taken. Old cultures should not be used because degraded cell walls in dying Gram (+) cells give a false negative result. Thick smears, over- or undercolorization will give false results. A known sample is usually included as a control of the staining procedure. Gram stain is use to stain bacteria as a first step for their identification. Gram stain differentiates between two types of cell wall. Bacteria with cell walls containing a large layer of peptidoglycan and no lipopolysaccharide are Gram positive. Bacteria with cell walls containing a thin layer of peptidoglycans and an outer cell wall containing lipopolysaccharides are Gram negative. Mycobacteria, e.g. the causative agent of tuberculosis, and Nocardia, do not stain well with Gram stain and require a special stain called acid fast. The exact mechanism of the Gram stain is not fully understood and does not seem to be directly connected to the actual structure of the cell wall. This vexing fact does not detract from the usefulness of the staining procedure itself! Here is a great site that summarizes the technique and use of Gram stain in pathology. An easy technique (Ryu 1938) also characterizes the Gram stain reaction to a large extent. We use this test in our lab to complement the results of the Gram test. It is not a replacement for the commonly used Gram stain. We will test the same bacteria that you stained in the preceding exercise: Escherichia coli, Staphylococcus epidermidis, and Bacillus megaterium. Neisseria sicca is an example of a Gram negative coccus and was not used in the Simple Stains. We do not use Candida in this exercise.
Relationship to Class Instruction: Chapters 3 and 10. Lab report due on: _______________________________________
Gram stain of Staphylococcus epidermidis and Escherichia coli. 1,000x
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