GRAM STAIN REAGENT TEST KIT
FOR THE DIFFERENTIAL STAINING OF BACTERIA IN PRIMARY SPECIMENS AND FROM CULTURE
INTENDED USE
The Curtiss Gram Stain Reagents and kits are recommended for
the differential staining bacteria in primary specimens and from
culture.
SUMMARY AND EXPLANATION
The Gram stain is well established as an important aid in the
differentiation and identification of isolated microorganisms. A
correctly performed and interpreted Gram stain of certain clinical
specimens also can be a rapid source of presumptive diagnostic
information. Numerous studies and reports have established the
value and importance of this technique in the examination of
sputum and transtracheal specimens, cerebrospinal and other
normally sterile body fluids, uncontaminated abscess fluids and
specimens from cerebrospinal and other normally sterile body
fluids, uncontaminated abscess fluids and specimens from soft
tissue infections, and exudates from the male urethra,1-6 The gram
stain examination of uncentrifuged urine also is a procedure recommended to provide rapid preliminary information. 7 Gram stain
examinations should not be used, however, as a substitute for
complete and careful culture studies.
The basic principle of the differential Gram stain was discovered
serendipitously by Christian Gram in 1883.8 Since then, this process has been exhaustively investigated and refined.8-11 In Practice,
a thin smear of bacterial cells is stained with crystal violet, then
treated with an iodine-containing mordant to increase or intensify
binding of this primary stain. A decolorizing solution of alcohol (or
acetone or a combination thereof) is used to remove the crystal
violet from cells which bind to it weakly, and a saffranin counter
stain is used to provide a color contrast in those cells which were
decolorized. In a properly stained smear, gram-positive cells
appear blue to purple and gram-negative cells appear pink to red.
PRINCIPLES OF THE PROCEDURE
A variety of mechanisms have been proposed to explain the Gram
reaction, but its underlying basis is still incompletely understood.
There are a variety of physio-clinical differences between grampositive and gram-negative cell walls12 and cell wall permeability
may be involved in the differentially retention of the crystal violet
iodine complex.13 Gram reactivity also may depend upon or be
influenced by the presence or absence of specific chemical compounds in the bacterial cell wall. Gram-positive cells can be made
to stain gram-negative by extraction of a variety of substances,
including nucleoprotein, ribonucleic acid, and polysaccharide. 14
REAGENTS
Formula:
Curtiss Gram Crystal Violet Solution: Gram Primary Stain,
approximately 0.4% crystal violet in an aqueous alcohol solution.
Curtiss Gram Iodine Solution: Approximately 0.33% elemaental
iodine in 0.66% aqueous potassium iodine solution.
Curtiss Gram Decolorizer Solution: Gram Stain Decolorizer,
denaturedethyl alcohol and acetone, approximately three parts to
one part, respectively.
Curtiss Gram Safranin Solution: Counterstain, approximately
0.25% safranin in 20% ethyl alcohol.
PRECAUTIONS: For In Vitro Diagnostic Use
As with all techniques involving pathogenic and potentially pathogenic microorganisms, established aseptic practices should be
consistently applied throughout this procedure.
These reagents are harmful or fatal if swallowed and can cause
eye irritation if contact is made. In the event of eye contact, flush
eyes with an eye wash system or tap water for 15 minutes.
Gram decolorizer solution is flammable and its vapors may be
harmful, use in a well ventilated area away from open flame.
Storage Instructions: On receipt, store at 15º-30ºC. The expiration
date is for product in unopened bottles stored as directed. Do not
open until ready to use.
Product Deterioration. Some precipitation may occur in the
crystal violet solution upon prolonged storage. If this appears to
affect the quality of the staining, with the dispensing closure open,
briefly warm the bottle or quantities, dispensed into the bottle 37ºC
water bath, then close and shake until the precipitate is dissolved.
The Gram Iodine Solution deteriorates especially when exposed to
light and/or heat. Solution remain stable until expiration date on the
bottle. It should be discarded when color loss becomes significant
or when adequate results are no longer obtained (see “User Quality
Control”).
SPECIMEN COLLECTION AND PREPARATION
The Gram Stain may be performed on smears prepared from
clinical specimens or samples containing missed flora or pure
cultures or on smears of microbial growth from laboratory cultures.
PROCEDURE
Materials Not Provided:
1.
Clean microscope slide
2.
Inoculating loop or needle or swabs
3.
Water or saline
4.
Bunsen burner
5.
Blotting Paper
6.
Microscope
INSTRUCTIONS FOR PERFORMING THE GRAM STAIN
1.
All solutions are used as presented.
2.
Prepare a thin, evenly distributed smear of the material to
be Gram-stained, Note: in very thick smears, decolorixation
cannot be adequately evaluated or controlled: very thin
smears are easily over-decolorized.
3.
Clinical specimens and bacteria growing in broth culture can
be applied directly to a microscope slide with a swab or
inoculating loop.
4.
Growth from bacterial colonies can be picked with an
inoculating loop or needle and emulsified in a drop of saline
or water placed on the slide.
5.
Aloe the smear to completely air-dry without heat, and the
heat-fix quickly passing the slide through the (low) flame of
a Bunsen burner 2-3 times (the slide should be just hot to
the touch for correct smear fixation; if exposed to excess
heat, bacteria may not stain properly).
6.
Cover the smear with crystal violet solution and allow the
stain to act approximately one minute.
7.
Remove excess stain by briefly rinsing the slide with tap
water.
8.
Shake off the excess water, flood the smear with Iodine
Solution, and allow to stand for one minute or longer.
9.
Rinse off the iodine solution with tap water and carefully
apply decolorizer solution just until no more color is being
washed from the smear. Quickly rinse off any remaining
decolorizer with tap water. Note: Do not Over-Decolorize.
10. Shake off any excess water and apply the safranin for
approximately one minute.
11. Wash slide in tap water, blot dry, and examine microscopically.
User Quality Control: As a test of both reagent integrity and correct
reading and staining technique, the daily performance of quality
control is recommended. This is especially important when clinical
specimens are being examined to provide presumptive diagnostic
information or a guide for antimicrobial therapy. Overnight 18-24 (h)
cultures of Escherichia coli (gram-negative) and Staphylococcus
aureaus (gram-positive) are suitable control organisms. More
subtle deficiencies in reagent quality and techniques can be detected by the use of weakly reactive bacteria such as Bacillus
subtitis (gram-positive) and Moraxella (Branhamella) catarrhalis
(gram-negative)15
RESULTS
When the differential Gram procedure is performed correctly,
organisms which retain the primary stain-mordant complex will
appear microscopically blue to purple and are termed “grampositive”, organisms which are decolorized and therefore take up
the counterstain, microscopically will appear pink to red and are
termed “gram-negative”.
LIMITATIONS OF THE PROCEDURE
Gram reactivity is not an absolute characteristic and is influenced
by several factors. The age of a culture affects its degree of Gram
positivity, in general, cells from young actively-growing cultures
retain the crystal violet-iodine complex more avidly than cells from
older cultures. Although the optimum age for staining may vary
from species to species.16 It is general practice to examine 18-24-h
cultures. When experience or special conditions dictate, younger or
older cultures also should be tested. Different species of bacteria
may not be equally sensitive to deleterious effects of heat-fixation,
but over-heating, because it may cause gram-positive bacteria to
stain gram-negative, should be avoided. AQntimicrobial agents also
may make gram-positive organisms more susceptible to decolorization17.18 this fact should be kept in mind when examining clinical
specimens, especially from treated patients. The Gram stain should
be used only to provide supplementary diagnosis or taxonomic
information; it is not intended, and should not be used, as a substitute for more comprehensive tests.
REFERENCES
1.
Spengler, M.S., G.T.Rodeheaver, L. Righter, M.T.Egerton,
and R.F.Edlich, 1978. The Gram Stain-The most important
test of infection. J.Am.College Emergency Physicians, 7:434
-438.
2.
Washington, J.A. 1979. Use and Abuse of the Gram-Stained
smear. Clin.Microbiol.Newsl. 1:4-5.
3.
Riles,K., M.E.Levinson and D.Kaye. 1975 Transtrachael
aspiration in pulmonary infection. Arch.Interm,Med. 133:453458.
4.
Heineman, H.S., J.K.Chawla and W.M.Lofton. 1977. Misinformation from sputum cultures without microscopic examination. J.Clin.Microbiol, 6:518-527.
5.
Marshall, K.A., M.T.Egerton and G.T.Rodeheaver,
1976.Quantitative Mirobiology: ites application to hand
injuries. Am.J.Surg. 131:730-733.
6.
Jacobs, N.F., and S.J.Kraus, 1975. Gonococcal and nongonococcal urethritis in men. Clinical and laboratory differentiation.Ann.Interm.Med. 82:7-12.
7.
Barry, A.L.,P.B.Smityh and M.Turck. 1975. Cumitech 2,
Laboratory diagnosis of urinary tract infections. Coordinating
ed., T.L.Gavan. American Society for Microbiology, Washington, D.C.
8.
Bartholomew,J. T.Mittwer. 1952. The Gram Stain.
Bacteriol.Rev.16:1-28.
9.
Mittwer, T., J.W.Bartholomew and B.J.Kallman. 1950. The
Mechanism of the Gram Reaction, II. Stain Technol. 25:169179.
10. Bartholomew, J.W. and T.Mittwer. 1951. The Mechanism of
the Gram reaction, III. Stain Technol. 26:231-240.
11. Hendrickson, D.A., and M.M.Krenz. 1991. Reagents and
Stains, p.1289-1314. In A.Balows, W.J.Hausler, Jr.,
K.L.Hermann, H.D.Isenberg and H.J.Shadomy (ed), Manual
of Clinical Biology, 5th ed. American Soc. For Microbiol.,
Wash. D.C.
12. Lamanna, C., and M.F.Mallette. 1965. Basic bacteriology,
3rd ed., p.162-176. The Williams & Wilkins Co., Baltimore.
13. Salton, M.R.J., 1964. The Bacterial cell wall, p.29-36.
14.
15.
16.
17.
18.
Elsevier, Amsterdam.
Biswas, B.B., P.S.Basu and M.K.Pal. 1970. Gram Staining
and its molecular mechanism. Int. Rev. Cytol 29:1-27.
Russell, R.L. 1974. Quality Control in the Microbiology
laboratory, p.866. In E.H.Lennette, E.H.Spaulding and
J.P.Truant (ed), Manual of Clinical Microbiology, 2nd. Ed.
American Society for Microbiology, Washington, D.C.
Hucker, G.J., and H.J.Conn. 1927. Further studies on the
methods of Gram Staining. N.Y. (Geneva) Agr.Exp.
Tech.Bull. 128:1-34.
Dufrenoy, J., and R.Pratt. 1947. Cytochemical mechanisms
of penicillin action. J.Bacteriol. 54:283-289.
Catlin, B.W. 1975. Cellular elongation under the influence of
antibacterial agents: way to differentiate coccobacilli from
cocci. J.Clin.Microbiol. 1:102-105.
WARRANTY
Curtiss makes no warranty of any kind, either expressed or implied,
except that the material from which its products are made are of
standard quality. If any materials are defected, Curtiss will provide a
replacement product. Buyer assumes all risk and liability resulting
from the use of this product. There is no warranty of merchantability
of this product, or of the fitness of the product for any purpose.
Curtiss shall not be liable for any damages, including special or
consequential damage, or expense arising directly or indirectly from
the use of this product.