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Use of Gram Stain kit with Reagents differentation

Gram Stain Kit methylene blue solution Reagents Scytek Laboratories

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2 replies to this topic

#1 peterson1



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Posted 01 February 2015 - 03:38 AM

Gram Stains is typically used to categorize bacteria into 2 groups (gram positive or negative) due to the differences in their cell wall structure. Generally, gram positive bacteria, such as those found in yogurt (Lactobacillus and Streptococcus) are less pathogenic. Their cell walls can be disrupted with penicillin or with lysozyme (an enzyme that our cells are able to produce). Gram negative bacteria, such as E.coli, Helicobacter, and other bacteria of the stomach, are more pathogenic. Gram negative bacterial cell walls are composed of lipopolysaccharides that can be an endotoxin (toxic shock syndrome). Gram stain experiments are another type of a multi-step staining and de-staining experiment.


0.1M Sodium Acetate Buffer, pH4.2:

Sodium acetate, trihydrate (MW 136.1) ------ 1.36 g

Distilled water ----------------------------------- 100 ml

Mix to dissolve and adjust pH to 4.2 using concentrated glacial acetic acid

Methyl Green Solution (0.5%)

Methyl green (ethyl violet free from Sigma) ---- 0.5 g

0.1M Sodium acetate buffer, pH4.2 ------------- 100 ml

Mix to dissolve.

Staining Pattern


Suggested Use

Counterstaining for immunohistochemistry or other special stains.

Staining Procedure

1.  Sections to distilled water after IHC.

2.  Stain in methyl green solution for 5 minutes at room temperature (60 C may produce slightly stronger stain).

3.  Rinse in distilled water (sections will look blue).

4.  Dehydrate quickly through 95% alcohol (10 dips, sections turn green), 2 changes of 100% alcohol (10 dips each) (alcohol used for dehydration removes some of the stain).

5.  Clear in xylene or xylene substitute.

6.  Mount with resinous mounting medium.

Edited by peterson1, 01 February 2015 - 03:39 AM.

#2 Srilatha Dharmavaram

Srilatha Dharmavaram


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Posted 01 February 2015 - 11:22 PM

Gram-positive bacteria are definitely pathogenic, and as compared to Gram-negative E coli (non-pathogenic) much more so; organisms such as Mycobacterium (Gram positive) are definitely quite pathogenic.

#3 Phil Geis

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Posted 03 February 2015 - 01:15 PM

Of course there are pathogens in both groups(Gran negatives including Yersinia pestis, Salmonella and Shigella spp. etc. as well as speciifc strains of E. coli - O157) and very few of either are direct pathogens

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