|Cat. no. Z302||Q-Slide™ Gram||5 slides/box|
Hardy Diagnostics Q-Slide™ Gram control slides are used to qualify reagents and the method used in gram stain procedures.
The gram stain is critical to the identification of organisms in clinical and industrial specimens. Q-Slide™ Gram control slides are used to ensure the quality of reagents used as well as the efficacy of staining procedures. Methanol fixed smears of Staphylococcus aureus and Escherichia coli serve as the positive and negative controls. The remainder of the slide is marked with a grid to allow for the simultaneous staining of up to four unknown specimens in parallel with the positive and negative control.
The gram stain differentiates bacteria on the basis of their cell wall structure. Gram-positive organisms have a very thick peptidoglycan layer in their cell wall compared to that of gram-negative organisms. Crystal violet stain complexes with the mordant, Gram's iodine and becomes integrated within the peptidoglycan layer. When the smear is decolorized, the crystal violet/iodine complex is washed out of the gram-negative cell wall, but the complex remains integrated within the thicker peptidoglycan layer of the gram-positive organisms. Counterstain is used to give a visible color to the gram-negative cells that were decolorized. Safranin is the most common counterstain used, but carbolfuchsin can be used for faintly staining organisms.
STORAGE AND SHELF LIFE
Upon receipt store at 15-30ºC. in the original container. Do not use if there are any signs of deterioration. Protect from moisture and dust.
1. Prepare smears of unknown organisms in the empty quadrants on the slides. Avoid making smears that are too thick or too thin, either extreme will make the stain difficult to interpret.
2. Fix the unknown smears using one of the two methods below.
After the smear has air dried, pass the slide through a flame or hold next to an incinerator for 5 to 10 seconds. Take care not to overheat the slides. Allow slide to cool prior to staining.
Air dry the smear, and place a few drops of methanol over the smear for 1 minute. Drain off excess without rinsing. Allow to air dry.
Follow the manufacturer's instructions for performing the stain. The following is provided as a reminder.
1. Flood fixed smear with crystal violet stain. Allow to sit for 30 seconds.
2. Drain crystal violet and gently rinse with tap water.*
3. Remove excess water and flood smear with Gram's Iodine. The Gram's Iodine should sit for 30 seconds.
4. Gently rinse with tap water.
5. Decolorize by running the decolorizer over the smear until the runoff becomes clear. The amount of time required for decolorization will vary with the thickness of smear and the type of decolorizer used.
6. Gently rinse away excess decolorizer.*
7. Flood slide with Safranin**, allow stain to set for 30 seconds.
8. Briefly rinse, allow to air dry.
1. Using a low power objective, locate and focus on the cells.
2. Use the oil immersion lens (100x) to determine gram reaction, cell morphology and arrangement.
* Excessive rinsing in these steps could result in the loss of dye in the gram-positive cell walls.
** Alternatively, basic fuchsin can be used as the counterstain for weakly staining gram-negative organisms.
INTERPRETATION OF RESULTS
If the gram stain procedure has been performed properly and the reagents are fresh, the Staphylococcus aureus (ATCC ® 25923) in the positive control well should appear as a purple to lavender (gram-positive) cocci. The negative control, Escherichia coli (ATCC ® 25922) should appear as a pink to red (gram-negative) rod.
LIMITATIONS OF THE PROCEDURE
Cultures should be 18-24 hours old for gram staining procedures.
MATERIALS REQUIRED BUT NOT PROVIDED
Standard microbiological equipment such as microscopes, inoculating loops, incinerators, gram stain reagents, methanol, etc., are not provided.
Appearance of the positive and negative wells when stained according to standard gram stain procedures:
ATCC ® 25923
|Gram-positive (blue/purple) cocci in pairs or clusters|
ATCC ® 25922
|Gram-negative (pink/red) bacilli|
Q-Slide™ Gram control slide should have a methanol fixed smear of Staphylococcus aureus in the positive control well and Escherichia coli in the negative control well.
Q-Slide™ Gram (Cat. no. Z302). Showing the two known QC circles and four test specimen squares.
1. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.
2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.
ATCC is a registered trademark of the American Type Culture Collection.