Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Patent
1996-06-12
1998-10-27
Tsang, Cecilia
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
435 4, 435 29, 435810, 8 9411, C12Q 104
Patent
active
058276807
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a reagent and a staining method for bacteria. More particularly, the present invention relates to a Gram staining reagent combining a decolorizer and a counter-stain and a three reagent Gram staining method.
BACKGROUND ART
The Gram stain is a well known and widely used microbiological technique used for classifying and identifying bacteria. In hospitals and clinical laboratories, the Gram stain is one of the most frequently performed microbiological diagnostic tests. The Gram stain essentially divides the bacterial world into two distinct classifications: Gram positive bacteria and Gram negative bacteria. Separation into these two classes is based on differences in the structure of the bacterial walls.
The conventional and most commonly used method of the Gram staining technique requires the use of four separate reagents. The reagents consist of a primary staining solution usually containing crystal violet dye, a mordant solution usually containing an iodine-potassium iodide solution, a decolorizing agent usually a solution of acetone and alcohol, and a counter-staining solution usually containing safranin. The conventional method involves the steps of: (1) treating a slide having bacteria affixed upon it with the principal stain, waiting 30-60 seconds; (2) rinsing the slide with water; (3) treating the slide with the mordant solution, waiting 30-60 seconds; (4) rinsing slide with water; (5) decolorizing the slide with acetone and alcohol solution, waiting 30-60 seconds; (6) rinsing the slide with water; (7) counter-staining the slide with the safranin or fuchsin solution for 30-60 seconds and; (8) rinsing the slide with water. Only after these steps are completed can the bacteria affixed to the slide be observed and analyzed to determine Gram status. Typically, Gram positive bacteria will appear purple in color, while Gram negative bacteria will appear reddish-pink in color.
It should be obvious from the above description of the conventional method of Gram staining that the conventional method of Gram staining is a very involved, time consuming, and labor intensive process. Each additional step introduces the possibility of error which can lead to an incorrect or inconclusive identification of the bacteria. A Gram stain which results in an inconclusive or incorrect identification of a bacterial specimen must either be repeated or have other diagnostic tests performed to more positively identify the bacterial specimen. In either case, the initiation of effective treatment is delayed when conclusive bacterial identification is not obtained.
For years, technicians using the conventional Gram staining method have been plagued by these inconsistent results. Much of the inconsistency is directly attributable to the conventional method itself. For instance, the separate decolorizing step is problematic in that it is difficult to achieve the proper amount of decolorization of the bacteria. In other words, decolorization, the step of removing the fixed crystal violet stain from the Gram negative organisms, is prone to inconsistency. The inconsistency stems from difficulties associated with the amount of decolorizing solvent applied to bacteria and the amount of time the decolorizing solvent is allowed to contact the bacteria. If the decolorizing solvent is applied in large volumes or is applied too vigorously, the bacteria in the sample may be under-stained, thereby potentially yielding an inconsistent or incorrect result i.e., a truly Gram positive bacterium may appear to be a Gram negative bacterium. If, on the other hand, a bacterial sample is not adequately decolorized, the bacteria in the sample may be over-stained, thereby potentially yielding an inconsistent or incorrect result, i.e., a truly Gram negative bacterium may appear to be a Gram positive bacterium.
Using the conventional four reagent Gram staining method, certain bacteria may appear as to be both Gram positive and Gram negative. These bacteria are referred to in the art as Gram variable bacteri
REFERENCES:
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Romero et al., J. Clin. Microbiol. , vol. 26, No. 7, pp. 1378-1382, Jul. 1988.
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Meszaros Amy
Strenkoski Leon F.
Difco Laboratories
Mohamed Abdel A.
Tsang Cecilia
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