GRAM STAIN KITS AND REAGENTS

Cat. no. C008 Crystal Violet 8oz bottle
Cat. no. C128 Crystal Violet One gallon
Cat. no. C008A Advanced Crystal Violet™ 8oz bottle
Cat. no. C128A Advanced Crystal Violet™ One gallon
Cat. no. I008 Iodine, Stabilized Gram's 8oz bottle
Cat. no. I128 Iodine, Stabilized Gram's One gallon
Cat. no. I008N Iodine, Non-stabilized Gram's 8oz bottle
Cat. no. I128N Iodine, Non-stabilized Gram's One gallon
Cat. no. D008S Decolorizer, Slow (25% Acetone) 8oz bottle
Cat. no. D128S Decolorizer, Slow (25% Acetone) One gallon
Cat. no. D008 Decolorizer, Intermediate (50% Acetone) 8oz bottle
Cat. no. D128 Decolorizer, Intermediate (50% Acetone) One gallon
Cat. no. D008F Decolorizer, Fast (75% Acetone) 8oz bottle
Cat. no. D128F Decolorizer, Fast (75% Acetone) One gallon
Cat. no. 100B128 Decolorizer, Very Fast (100% Acetone) One gallon
Cat. no. S008 Safranin 8oz bottle
Cat. no. S128 Safranin One gallon
Cat. no. S008A Advanced Counterstain™ 8oz bottle
Cat. no. S128A Advanced Counterstain™ One gallon
Cat. no. GK400 Gram Stain Set (with Stabilized Iodine) 4 x 8oz bottles
Cat. no. GK400N Gram Stain Set (with Non-Stabilized Iodine) 4 x 8oz bottles
Cat. no. GK400A Gram Stain Kit Advanced™,
(with Advanced Crystal Violet™ and Advanced Counterstain™)
4 x 8oz bottles
Cat. no. BF008 Basic Fuchsin 8oz bottle

INTENDED USE

Hardy Diagnostics Gram Stain Kits and Reagents are used to stain microorganisms from cultures or specimens by the differential Gram method.

SUMMARY AND PRINCIPLES

In 1883, Karl Friedlander investigated differential staining of bacterial cells in tissue. The following year, Christian Gram published a detailed account of Friedlander's staining procedure. Various modifications of the original Gram's procedure have been proposed over the years. Nearly all clinically important bacteria can be detected using this method, the only exceptions being those organisms that exist almost exclusively within host cells (e.g. chlamydia), those that lack a cell wall (e.g. mycoplasma and ureaplasma), and those of insufficient dimensions to be resolved by light microscopy (e.g. spirochetes).

The Gram Stain is one of the most basic of all bacteriologic tests and allows bacteria to be grouped according to their staining reaction, an invaluable aid in bacterial identification. The difference in staining reaction between gram-positive and negative bacteria is attributed to the distinctive chemical and physical structure of the cell walls of these organisms. This differential stain is usually described as being related to the higher lipid content of gram-negative bacterial cell walls. Some suggest it is instead due to a reaction with a magnesium ribonucleate-protein complex.

Crystal Violet is taken up equally well by both gram-positive and negative bacterial cell walls. A crystal violet iodine complex is formed within the cell of each type of bacteria upon addition of iodine. When a decolorizer is applied, lipids are extracted from the cell walls of gram-negative bacteria. Lipid extraction causes an increase in cell wall permeability and results in the loss of the dye complex. The effect of decolorizer on gram-positive bacteria is dehydration. This decreases the cell wall permeability and increases the retention of the crystal violet iodine complex. The result is gram-positive bacteria appearing violet due to retention of the crystal violet iodine complex, and gram-negative bacteria appearing pink red due to the staining from the Safranin counterstain.

Both Hardy Diagnostics Stabilized and Non-stabilized Gram's Iodine are premixed and ready to use. Stabilized Iodine is thicker and requires a longer decolorization step or a "faster" decolorizer (see below). Stabilized Iodine is less sensitive to light and has a longer shelf life. Non-stabilized Iodine has a shorter shelf life, is thinner and decolorizes more quickly than Stabilized Iodine.

Hardy Diagnostics recommends using Intermediate Decolorizer (Cat. no. D008, 50% Acetone) or Fast Decolorizer (Cat. no. D008F, 75% Acetone) with Stabilized Iodine. Our Gram Stain Kit with Stabilized Iodine (Cat. no. GK400 and GK400A) includes Intermediate Decolorizer and Fast Decolorizer, respectively.

Our Slow Speed Decolorizer (Cat. no. D008S, 25% Acetone) is recommended for use with the Non-stabilized Iodine. Our Gram Stain Kit with Non-stabilized Iodine (Cat. no. GK400N) includes Slow Decolorizer.

Hardy Diagnostics Gram Stain Kit Advanced™ (Cat. no. GK400A) is comprised of Advanced Crystal Violet™, Stabilized Iodine, Fast Decolorizer and Advanced Counterstain™. Advanced Crystal Violet™ has been shown to provide superior, consistent and brighter staining of gram-positive organisms, especially those which decolorize easily. Advanced Counterstain™, a stronger counterstain than Safranin, will stain the gram-negative organisms a very deep red. Contrast between gram-positive organisms and gram-negative organisms in a mixed field is greatly enhanced. The advanced system helps to guard against accidental over-decolorization, thus reducing the possibility of mistaking a gram-positive organism for a gram-negative one.

STORAGE AND SHELF LIFE

Storage: Upon receipt store products at 15-30ºC. away from direct light. Products should not be used if there are any signs of deterioration (contamination, discloration) or if the expiration date has passed. Products are light and temperature sensitive; protect from light, excessive heat, moisture, freezing, and exposure to air.

PRECAUTIONS

Some components of the Gram Stain(s) are poisonous and may be harmful or fatal if swallowed. Avoid contact with eyes, skin, or clothing. Avoid breathing the fumes or vapors. Use only in an adequately ventilated room. Wear protective gloves and wash thoroughly after use.

Acetone and alcohol are flammable; therefore, keep away from heat, flames or sparks.

FIRST AID: In case of eye or skin contact, immediately flush thoroughly with plenty of water for at least 15 minutes. If swallowed, call a physician, hospital or poison control center immediately. Induce vomiting if swallowed. Never give anything by mouth to an unconscious person.

PROCEDURE

Specimen Preparation:

1. Apply the test specimen to a clean glass slide in a manner that will yield a thin, uniform smear. Emulsify colonies from an 18-24 hour culture in sterile saline (Cat. no. R45) if necessary to obtain the proper density.

2. Fix smear to slide using one of the following fixation techniques:

A. Heat fix by passing slide through a low flame 2-3 times until all moisture has evaporated. Alternatively, slide may be placed on a heat block (56ºC.) for several minutes. Allow slide to cool to room temperature before staining.
Note: Do not overheat slide. Excessive heating will cause atypical staining.

B. Methanol fix slide by flooding with absolute methanol (95%) for 1-2 minutes, then tilt the slide to drain off methanol and allow to air dry.
Note: For proper fixation, absolute methanol should be stored in brown screw-capped bottles and the working supply should be replenished every two weeks.

Note: Methanol fixation is superior to heat fixation for 5 reasons:
1. It preserves bacterial and human cell morphology.
2. It preserves red blood cells, which makes it especially useful with bloody specimens.
3. It provides greater control over the decolorization process, because organisms fixed with methanol are more resistant to decolorization.
4. It prevents liquid specimens from washing off the slide.
5. It leaves a clearer background.

Staining Procedure:

1. Cover slide with Crystal Violet Reagent for one minute.

2. Rinse slide with deionized or tap water.

3. Cover slide with Iodine Reagent for one minute.

4. Gently rinse the slide with deionized or tap water and allow to drain.

5. Tilt the slide and flood with a few drops of Decolorizer until no violet color runs off. This will usually take 10 seconds or less. Do not over decolorize.

6. Rinse slide gently with deionized or tap water.

7. Cover slide with Safranin, Advanced Counterstain™, or Basic Fuchsin counterstain for one minute.

8. Rinse slide gently with deionized or tap water. The rinse water in this step should be slightly pink. Do not wash excessively.

9. Allow slide to drain and air dry, or gently dry with lintless bibulous paper (Cat. no. 28511007) or paper towel.

10. Examine slide under oil immersion lens (1,000X magnification).

Note: Use caution so that slides are not over decolorized, causing gram-positive bacteria to appear gram-negative.

INTERPRETATION OF RESULTS

Bacteria appearing dark blue to violet in color after staining are considered gram-positive. Bacteria appearing pink to red in color are gram-negative.

LIMITATIONS

The Gram Stain provides preliminary identification information only and is not a substitute for cultural studies of the specimen.

Pitfalls in the Gram stain include both inherent limitations and technical errors. The Gram stain will not detect organisms which exist within host cells (e.g., Chlamydia spp.), organisms with no cell wall (e.g., Mycoplasma spp. and Ureaplasma spp.), and organisms too small to be seen with light microscopy (e.g., spirochetes). Mycobacteria usually will not stain, and Legionella spp. stain only when taken directly from culture. Gram-negative bacteria that stain poorly with safranin include Campylobacter spp., Legionella spp., Bacteroides spp., Fusobacterium spp., and Brucella spp.

Certain conditions are known to damage the cell wall, causing gram-positive bacteria to falsely appear gram-negative or gram-variable. These include antibiotic treatment, cultures more than 48 hours old, inflammatory responses in the host, and autolytic enzymes (e.g., S. pneumoniae). To minimize ambiguous results, specimens should be collected before the patient begins antibiotic therapy. Also, Gram stains should be performed on colonies taken from culture media that do not contain antibiotics, preferably on colonies that are 18-24 hours old.

Finally, correct interpretation of Gram stains requires a theoretical background of bacteria and their morphology, because improper technique or suboptimal reagents can cause unreliable results. Errors in technique which can alter Gram stain results include the following:

• Fixation with excessive heat alters cell morphology and makes organisms more susceptible to over- decolorization.

• Low concentrations of crystal violet make gram-positive organisms more susceptible to over-decolorization.

• Insufficient exposure to iodine and lack of available iodine can prevent crystal violet from bonding firmly with the cell wall, thus making gram-positive organisms more susceptible to over-decolorization. To ensure reliable Gram stain results, only fresh iodine should be used.

Prolonged decolorization, especially with acetone, can cause gram-positive bacteria to appear gram-negative. Insufficient decolorization can make gram-negative organisms falsely appear gram-positive.

• Insufficient counterstaining can fail to stain gram-negative bacteria and background material, whereas excessive counterstaining will leach the crystal violet-iodine complex from gram-positive bacteria and stain them with safranin, thus making them falsely appear gram-negative.

• Prolonged washing between any of the steps can cause over-decolorization.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiology supplies and equipment such as microscope slides, methanol, control slides (Cat. no. Z302), bacteriological loops, swabs, blotting paper, microscopes, oil immersion lens, immersion oil, etc., are not provided.

QUALITY CONTROL

Test Organisms Reaction
Staphylococcus aureus
ATCC ® 25923
Gram-positive cocci, violet
Escherichia coli
ATCC ® 25922
Gram-negative rods, pink to red

User Quality Control

PHYSICAL APPEARANCE

Gram Stain Set

Gram Stain Set, with Stabilized Iodine (Cat. no. GK400).

REFERENCES

1. Manual of Microbiological Methods, p. 15-18. 1957. McGraw-Hill.

2. Stain Tech.; 37:139. 1962.

3. Microbiology, 2nd ed., p. 320. 1966.

4. Principles of Bacteriology and Immunology , 5th ed. Vol. I, p. 43. 1964.

5. Mangels, J.I., M.E. Cox and L.H. Lindley. 1984. Methanol fixation. An alternative to heat-fixation of smear. Diag. Microbiol. Infect. Dis. ; 2:129-137.

6. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

7. Commission on Laboratory Accreditation, Laboratory Accreditation Program Microbiology Checklist. College of American Pathologists. Rev. 9/30/2004.

8. Centers for Medicare and Medicaid, Appendix C, Survey Procedures and Interpretive Guidelines for Laboratories and Laboratory Services. Subpart K - Quality System for Non-Waived Testing. 493;1200-1265. www.cms.hhs.gov/clia.

ATCC is a registered trademark of the American Type Culture Collection.

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