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Carolina Macero

In my experience as a bacteriologist, the most common errors are: 1) when inflammatory response and abundant leukocytes achieve an effect of pink background where it is difficult to distinguish the Gram negative bacteria, especially if they are scarce, and therefore not will report 2) when from a bacterial suspension problem, where there are no cells to guide us, we made the mistake of overstain or decolorear and 3) setting heat smear from liquid, which usually leads to a part of the sample is lost in the wash while she underwent Gram stain, which makes us report it wrongly. Recommendations: 1) make thin smears if the sample is purulent or revise the microscope the edges of the smear, where we see the separate leukocytes from each other, 2) in a sheet make three circles, and in the center circle to place the problem bacterial suspension, in the circle on the right side to place a Gram positive control strain, and left circle a Gram negative and thus proceed to color and 3) choose fixed with methanol samples from liquid as it prevents the smear off with water by withdrawing crystal violet.

taoufik djerboua

-for clinical specimen i prefer to start with methylen-blue staining i think it's easier to read. If its "positive" for bacteria i complete with a Gram staining.
-thick samples should be diluted in 0.9% NaCl water before smearing. Nevertheless fushin sticks a lot thats why methylene blue is preferable to start with for me.
-for cultured samples whether its a Gram or Ziehl staining i often use and advise others to make a "control smear". In Gram staining i mix a colony of a known Gram negative isolate with a colony of a known Gram positive isolate on the same smear. It helps verify the quality of the methode and of the reagents and makes the smears a lot easier to read.

Dr Sourav Maiti

What I do is using a mixture of a Gram positive cocci and a Gram negative rod on the same slide marked as control and at the same time both smears (control and test) are flooded with staining procedures as per protocol followed. As per control, the rods must stain pink and the cocci should stain purple. If it's not then staining is wrong.
However in clinical setting with experience we can get clues like pus cell nuclei should stain pink, any spore bearing bacillus should be Gram positive.

Dr Sourav Maiti

One must check quality of the batch of stains and the performance of the technician too to have good Gram stain. Smears must be of optimum thickness and proper timing should also be maintained. But most importantly the microscopist must have dedication and interpretative open minds to decode the mysteries....!!!!

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