Carson Family carson at
Tue Aug 12 07:29:39 EST 1997

Tom Frey <TomFrey at> wrote:

A simpler method (but not new) for Gram differentiation is the KOH
1. Make 3% w/v potassium hydroxide in distilled water
2. Place a drop of KOH on a glass slide
3. Take a good loopful of young culture from an agar plate
4. Emulsify the colony in the KOH
5. Interpretation:
    Gram positive = watery suspension (just like suspening the colony
in water)
    Gram negative = viscous suspension, definite strand seen adhering
to the loop when withdrawn from the drop
6. Rationale:
    Gram negative - KOH digests the cell wall and releases the cell
content; the observed vicosity is DNA/RNA. To obtain this effect it is
important to use a young culture that contains cells with high levels
of nucleic acids.
    Gram positive - the cell wall is KOH resistant and there is no
release of nucleic acids.
7. Cautions: The test is reliable but must be performed on young
cultures. Some Bacillus species are Gram variable by staining but give
a true reaction by the KOH test; there are some species and/or strains
of Bacillus that give a false reaction by the KOH method.

Although the KOH method may lack some of the elan of a molecular
biology technique it is quick, easy, reliable and does not require any
expensive equipment or highly trained staff........

Jeremy Carson

>Ivano de Filippis wrote:
>> Do you know any new methodology to differentiate gram positive from gram
>> negative bacteria.  Sometimes it is very difficult to se the difference on
>> the microscope. I have  an older technique, but I would like to see  more.
>> Thanks.
>I believe that Molecular Probes ( has a kit for gram +/-
>that uses a fluorescent lectin.  They show data using flow cytometery
>and the same kit may be useful for other fluorescence detection
>methods.  Caveat emptor: I have no connection to Probes but do work for
>a flow company, and I have not used the kit.
>Tom Frey

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