APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1982, p. 992-993 Vol. 44, No.

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0099-2240/82/100992-02$02.00/0
Copyright C 1982, American Society for Microbiology

Nonstaining (KOH) Method for Determination of Gram

Reactions of Marine Bacteriat
JOHN D. BUCKS
Mote Marine Laboratory, Sarasota, Florida 33577
Received 6 May 1982/Accepted 15 June 1982

A rapid nonstaining (KOH) method for the determination of the Gram reactions
of bacteria is described, and its application to marine isolates is discussed. All
gram-positive and gram-negative results obtained by Gram staining were con-
firmed by the KOH method. Gram-variable bacteria produced equivocal results.

A century ago, Hans Christian Gram devel-
oped a staining procedure which enabled the
separation of most commonly encountered bac-
teria into two groups: gram positive and gram
negative. Today, Gram staining remains the
most important differential technique applied to
bacteria (2); in fact, for 8 of the 19 major
groupings of bacteria (1), staining is required as
a primary aid to identification. Although many
biochemical features of diagnostic bacteriology
have been adapted for increased time-cost effi-
ciency, the Gram stain technique is essentially
the same now as it was in 1884. Premixed stains
can be purchased, but the procedure is still
relatively time consuming, costly, and often
messy, and reagents must be replaced periodi-
cally.
Approximately 40 years ago, a simple and
rapid nonstaining (KOH) method for the deter-
mination of the Gram reaction appeared in the
Japanese literature (3). The method was de-
scribed once again and used for 69 strains of
bacteria encountered in veterinary microbiology
clinics (3). The efficacy of the KOH method was
confirmed further with 22 bacteria of importance
in the brewing industry (6). Quality control for
the traditional Gram stain procedure was
achieved with 100 clinical isolates (4), and, to
keep costs down, the KOH method has been
suggested for routine use in hospitals (5). In this
study, I extended the usefulness of the KOH
technique to the characterization of a large col-
lection of marine bacteria. I hope to remind the
teaching and research community of or intro-
duce it to the effectiveness of the method.
To perform the test, place a drop of 3%
aqueous KOH on a slide. If one prefers, 10 ,ul
t Contribution no. 149 from the University of Connecticut
Marine Research Laboratory.
t Present address: Department of Marine Sciences and
Marine Sciences Institute, Marine Research Laboratory, The
University of Connecticut, Noank, CT 06340.
992
from an automatic pipette is adequate. In prac-
tice, as many as 8 to 10 tests per slide can be
done conveniently. Using a sterile loop, transfer
a visible amount of bacterial growth from an
agar culture to the drop of KOH. Mix the cells
and KOH thoroughly on the slide, constantly
stirring over an area about 1.5 cm in diameter. If
the bacterium-KOH suspension becomes mark-
edly viscid or gels within 5 to 60 s, the isolate is
gram negative. If no gelling is observed, the
isolate is gram positive. The best way to deter-
mine viscosity is to raise the loop about 1 cm
from the slide. If an obvious stringiness is pres-
ent, then the culture is gram negative.
In this study, 400 isolates were tested by both
the KOH method and conventional staining; for
the latter, a commercial stain kit (Difco Labora-
tories) was used. Bacteria were isolated from
seawater and a wide variety of fish. All cultures
were streaked to ensure purity and maintained
on slants of marine agar (Difco) or tryptic soy
agar (Difco). Staining and the KOH procedure
were performed at the same time with slant
cultures ranging in age from several hours to
several weeks.
Using the stain procedure on 18- to 24-h
cultures, I found 81% of the cultures to be gram
negative, 11% to be gram positive, and 8% to be
gram variable, i.e., having both purple and pink
cells. All gram-positive and gram-negative re-
sults obtained by staining were confirmed by the
KOH technique. Gram-variable bacteria gave
equivocal results. In previous studies, only Flu-
harty and Packard (3) encountered a bacterium
found to be gram variable by staining, and it was
found to be gram negative by the KOH proce-
dure. A total of 30 organisms were found to be
gram variable by staining in the present study;
when tested by the KOH method, 18 (60%) were
found to be gram negative, and 12 (40%) were
found to be gram positive. Six contaminated
cultures contained initially both gram-positive
and gram-negative bacteria and gelled with
VOL. 44, 1982
KOH (i.e., responded as gram negative). This
pattern has been noted earlier (3, 6).
In one study (3), the results for young (16- to
20-h) and older (2- to 10-month) cultures ob-
tained by staining and the KOH method were
found to be identical. In the current study, 4-
week-old cultures of recently obtained Bacillus,
Staphylococcus, and Streptococcus isolates
held at room temperature never showed any
gelling. These strains remained gram positive by
the KOH method, whereas staining revealed
mixtures of pink and purple cells after a few
days.
The advantages of time, ease, and cost of the
KOH technique are obvious. In a study in
progress on the bacteria used in these experi-
ments that were isolated from sharks and other
fish, it was necessary to routinely separate
gram-negative, oxidase-negative rods from the
other bacteria for subsequent identification by
rapid diagnostic tests for members of the Entero-
bacteriaceae. The KOH technique, together
with a wet-mount preparation for observing
morphology and cytochrome oxidase test strips,
provided an efficient and rapid screening proce-
dure.
The gel reaction is apparent; generally there is
no confusion regarding stringiness, although
some cultures are slow to react, and the reaction
is not obvious if too few cells are used. Holding
the slide at an appropriate angle against a dark
background aids observation. The KOH tech-
nique does have some disadvantages. It does not
by itself enable any notation of cell morphology.
However, additional characteristics necessary
for eventual identification (e.g., motility) would
furnish this. The culture sample necessary for
the KOH test is bigger than that needed for
Gram staining, although a large colony and a
small drop of KOH will function if necessary.
The KOH procedure does not enable the detec-
tion of gram-variable bacteria: as shown above,
these bacteria are seen as gram positive or gram
negative. The gram-variable cultures used in this
study were members of the coryneform group
(Corynebacterium and Arthrobacter). Several
isolates of both genera were found to be gram
positive by the KOH method, whereas some
NOTES 993
gelled weakly (gram-negative reaction). A larger
collection of gram-variable bacteria should be
studied to clarify this anomaly further. It is clear
that pure cultures are required for reliable obser-
vations; the KOH method will not enable the
detection of contamination in unknown cultures.
The present study did not include a detailed
parallel comparison of staining results with
KOH method results for cultures over time;
however, no discrepancies among cultures of
different ages were noted. There seems to be no
evidence in this report or elsewhere (3) suggest-
ing that the KOH technique cannot be used for
cultures up to at least several weeks old.
The above results show that the KOH method
enabled the accurate determination of the Gram
reactions of 92% of a large collection of marine
bacteria. The procedure can be used to efficient-
ly and rapidly characterize a wide variety of
isolates encountered in both clinical and nonma-
rine environmental material. Early instruction
should continue to stress the staining procedure
as it relates to cell chemistry and should intro-
duce the KOH technique as an appropriate
example of modern, economically practical mi-
crobiology.
This work was supported by the Mote Marine Laboratory,
Sarasota, Fla.
I thank William H. Taft, Director, Mote Marine Laboratory,
for providing space and facilities during a sabbatical leave. I
am grateful to Mary Parks for tracing several literature cita-
tions.
LITERATURE CITED
1. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's
manual of determinative bacteriology, 8th ed. The Williams
& Wilkins Co., Baltimore.
2. Doetsch, R. N. 1981. Determinative methods of light mi-
croscopy, p. 21-33. In P. Gerhardt (ed.), Manual of meth-
ods for general bacteriology. American Society for Micro-
biology, Washington, D.C.
3. Fluharty, D. M., and W. L. Packard. 1967. Differentiation
of Gram-positive and Gram-negative bacteria without
staining. Am. J. Vet. Clin. Pathol. 1:31-35.
4. Kohn, F. S., and S. A. Henneman. 1977. Novel quality
assurance procedure for the Gram stain. J. Am. Med.
Technol. 39:20-21.
5. Kuhn, P. J. 1981. Practical ideas for cost containment in
microbiology. Med. Lab. Observ. 13:73-86.
6. Lin, Y. 1980. Use of potassium hydroxide technique for the
differentiation of Gram-positive and Gram-negative bacte-
ria. Brew. Dig. 55:36-37.