Gram Positive & Gram Negative Bacteria, Artificial
Validation Stain: Gram, Brown-Hopps
Other Applicable Stains: Gram, Brown-Brenn and Gram-Twort
(Not in human tissue.) Organisms artificially introduced into a rat organ (e.g., lung) through a non-infective process.
Tissue: Positive staining rat lung and negative staining human lung.
Fixation: Formalin 10%, Phosphate Buffered (Part 1090).
Section/Glass: Paraffin sections cut at 4 microns on Superfrost™ Plus slides.
Quality Control Stain: Brown-Hopps quality control stained slide(s) included.
Reactivity: Guaranteed product specific reactivity for one year from date of receipt. Revalidate after one year to verify continued reactivity.
Storage: 15-30°C in a light deprived and humidity controlled environment.
Intended Use: To verify histological techniques and reagent reactivity.
Before using unstained control slides, review the enclosed stained slide(s) to ensure that this tissue source is acceptable for testing needs.
CONTROL SLIDE VALIDATION:
|With Gram, Brown-Hopps Stain Kit:||Part 9124A||Individual Stain Solution|
|Solution A:||Crystal Violet Stain 1%, Aqueous, Brown-Hopps||250 ml||Part 1041|
|Solution B:||Iodine, Gram, Aqueous||250 ml||Part 1140|
|Solution C:||Basic Fuchsin Stain 0.25%, Aqueous||250 ml||Part 1011|
|Solution D:||Gallego Solution||250 ml||Part 1098|
|Solution E:||Picric Acid-Acetone 0.05%||250 ml||Part 13351|
|Solution F:||Acetone-Xylene 1:1||250 ml||Part 10015|
|Acetone, ACS||Part 10014|
Newcomer Supply Gram Positive & Gram Negative Bacteria, Artificial Control Slides are for the positive histochemical staining of gram positive and gram negative bacteria in the same tissue section. Escherichia coli and Staphylococcus aureus purchased from Remel Microbiology Products is used to produce the positive control tissue.
NEWCOMER SUPPLY VALIDATION PROCEDURE:
- Heat dry sections in oven according to your laboratory protocol.
- Deparaffinize sections thoroughly in three changes of xylene, 3 minutes each. Hydrate through two changes each of 100% and 95% ethyl alcohols, 10 dips each. Wash well with distilled water.
- See Procedure Notes #1 and #2.
- Stain in Solution A: Crystal Violet Stain 1%, Aqueous, Brown-Hopps for 2 minutes.
- Rinse well in distilled water, ensuring excess stain is removed.
- Mordant in Solution B: Iodine, Gram, Aqueous for 5 minutes.
- Sections should turn black.
- Rinse well in running tap water, ensuring excess iodine is removed.
- Blot excess water from slide; decolorize one slide at a time in Acetone, ACS (Part 10014) until blue color stops running; 1-2 dips.
- Sections should be very light gray in color.
- Quickly rinse in running tap water to remove excess Acetone.
- Stain in Solution C: Basic Fuchsin Stain 0.25%, Aqueous; 5 minutes.
- Rinse well in running tap water.
- Differentiate in Solution D: Gallego Solution for 5 minutes.
- Rinse in running tap water. Blot water off slide(s), but not to dryness.
- Proceed with Steps #13 to #16 one slide at a time.
- Dip quickly in Acetone, ACS (Part 10014); 1-2 dips.
- Dip directly in Solution E: Picric Acid-Acetone 0.05%; 3-10 dips.
- Dip quickly in Solution F: Acetone-Xylene 1:1; 5 dips.
- Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium.
|Gram positive bacteria||Blue/violet|
|Gram negative bacteria||Red|
|Nonreactive lung||Negative for gram positive/negative bacteria|
- Drain slides after each step to prevent solution carry over.
- Do not allow sections to dry out at any point during procedure.
- If using a xylene substitute, closely follow the manufacturer’s recommendations for deparaffinization and clearing steps.
- Brown, Robert C., and Howard C. Hopps. “Staining of Bacteria in Tissue Sections: A Reliable Gram Stain Method.” American Journal of Clinical Pathology 60.2 (1973): 234-240.
- Carson, Freida L., and Christa Hladik Cappellano. Histotechnology: A Self-instructional Text. 4th ed. Chicago: ASCP Press, 2015. 222-224.
- Modifications developed by Newcomer Supply Laboratory.