The Gram Stain Kit (unstabilized iodine) is for staining bacteria from cultures or clinical specimens by the differential four step Gram Stain method. Gram-positive and Gram-negative bacteria are both stained by the crystal violet stain. Addition of the iodine leads to the formation of a crystal violet-iodine complex within the cell wall. The decolorizer extracts lipid from the cell wall of Gram-negative bacteria, thereby increasing the porosity of the cell wall and allowing the crystal violet-iodine complex to diffuse from the cell. At the same time, Gram-positive bacteria are dehydrated, causing a decrease in cell wall porosity and trapping the crystal violet-iodine complex within the cell. Due to the increase in porosity, the safranin counterstain permeates the cell wall of the Gram-negative bacteria.
Storage Conditions: Store at controlled room temperature (15-30°C) with bottles tightly sealed. The Gram Iodine Working Solution (Kit with Unstabilized Iodine) is stable for three months after mixing. The Gram Stain Kit with Stabilized Iodine is stable through assigned expiration date. *If precipitate forms in the Crystal Violet Solution, warm gently to 37ºC and shake well to re-dissolve.
Specimen Collection & Preparation: Liquid Media: Spread the bacteria suspension on a clean glass slide using a sterilized wire loop and allow to air dry. Heat-fix the smear by passing the slide through a flame three times and allow to cool. Solid Media: Place a loopful of deionized water on a clean glass slide. Touch a sterilized wire loop to a colony and suspend the cells in the water droplet; spread the suspension on the slide and allow to air dry. Heat-fix the smear by passing the slide through a flame three times and allow to cool. Patient: Obtain the patient specimen using a sterile wire loop, pipette or swab. Smear the specimen on a clean glass slide and allow to air dry. Heat-fix the smear by passing the slide through a flame three times and allow to cool.
Gram Iodine Preparation (ES801 Gram Stain Kit – Unstabilized Iodine) Add the Gram Iodine concentrate to the Gram Iodine diluent and mix well. This solution should be discarded after three months from preparation date.
Materials Provided: One 8oz. bottle each crystal violet, decolorizer, safranin, and 4oz. unstabilized iodine concentrate 8oz. dispensing bottle and diluent included to mix Iodine working solution A detailed product insert and convenient spout caps also included
Materials Required & Not Provided: DI Water Microscope slides Staining Rack Forceps Immersion oil
Optional Materials: Gram Stain Control Slides – Two areas containing Gram positive and Gram negative organisms with additional 8 areas for the application of unknown samples. ESCS-140 10/box ESCS-141 50/box
Procedure: 1. Place a prepared slide on a stain rack over sink. 2. Flood the prepared slide with Crystal Violet Stain and allow to stand for one minute. Rinse the slide gently with DI water. 3. Flood the slide with Gram Iodine Working Solution and allow to stand for one minute. (When using stabilized iodine, flood the slide with the solution and allow to stand for approximately two minutes). Rinse the slide gently with DI water. 4. Holding the slide vertically, gently rinse the slide with Decolorizer Solution for approximately ten seconds or until the decolorizer runs clear form the slide. Rinse the slide gently with DI water. 5. Flood the slide with Safranin Stain and allow to stand for one minute. Rinse the slide gently with DI water and blot the slide dry with absorbent paper. Examine the slide under oil immersion.
Results: Gram-positive bacteria are stained purple and Gram-negative are stained pink to red.
Limitations of Procedure: Application of excessive heat during fixation of smear may rupture bacterial cell walls and affect the Gram staining results. Treatment with antibacterial drugs may cause some Gram-positive bacteria to appear Gram-negative.
Quality Control: Performance of the Gram Stain Kit may be controlled using Gram Stain Control Slides or by following this procedure to test each kit component: Stain a prepared slide of Staphylococcus aureus from an 18 to 24-hour culture with Crystal Violet Stain allowing to stand for one minute and rising with DI water. Gram positive purple cocci should be observed; if not, discard this solution. Stain a prepared slide of Escherichia coli from an 18 to 24-hour culture with Safranin Stain allowing to stand for one minute and rising with DI water. Gram-negative pink rods should be observed; if not, discard this solution. Following the normal Gram staining procedure, stain a prepared slide of S.aureus from an 18 to 24-hour culture. Gram-positive purple cocci should be observed; if not, discard the Gram Iodine Working Solution.