Jul 19, 2020
Quick-Hot-Gram-Cromotropo QHGC lugol solution (Agudelo-Lopez SP and Montoya-Palacio MN, 2004)
- Javier Antonio Benavides1
- 1Universidad Nacional de Colombia Sede Palmira
External link: https://doi.org/10.1371/journal.pntd.0008734
Protocol Citation: Javier Antonio Benavides 2020. Quick-Hot-Gram-Cromotropo QHGC lugol solution (Agudelo-Lopez SP and Montoya-Palacio MN, 2004). protocols.io https://dx.doi.org/10.17504/protocols.io.bf8fjrtn
Manuscript citation:
Peña-Quistial MG, Benavides-Montaño JA, Duque NJR, Benavides-Montaño GA (2020) Prevalence and associated risk factors of Intestinal parasites in rural high-mountain communities of the Valle del Cauca—Colombia. PLoS Negl Trop Dis 14(10): e0008734. doi: 10.1371/journal.pntd.0008734
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: May 11, 2020
Last Modified: July 19, 2020
Protocol Integer ID: 36839
Keywords: standard gram procedure the aniline dye, iodine complex of gram, aniline dye, iodine complex, iodine in the lugol, negative microorganisms the dye, complex with iodine, positive microorganisms the dye, iodine solution, iodine, dye, red dye, cromotropo qhgc lugol solution, concentration of iodine, potassium iodide, iodine loss, decolorizing agent, gram negative bacteria, disinfectant for emergency disinfection, problem of iodine loss, reagent for starch detection, emergency disinfection, bacteria, standard gram procedure, microorganism, using polyvinylpyrrolidine, negative bacteria, aqueous solution, negative microorganism, cell tissue of microorganism, positive microorganism, positive bacteria
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Abstract
Lugol's solution is an aqueous solution which contains iodine (I2) and potassium iodide (KI) in a ratio of 1:2
Lugol's iodine solution is used for Gram staining and can be used as an antiseptic and disinfectant for emergency disinfection of drinking water, and as a reagent for starch detection in routine laboratory and other medical tests.
In the standard Gram procedure the aniline dyes in the cell tissue of microorganisms form a red dye-iodine complex when exposed to iodine. In Gram-positive microorganisms the dye-iodine complex cannot subsequently be dissolved from the cells by decolorizing agents such as alcohol or acetone. The cells remain dark blue in color. In Gram-negative microorganisms the dye-iodine complex is dissolved and the cell turns red as a result of counterstaining.
Lugol's solution is required to form the dye - iodine complex of Gram-positive bacteria. Stock solutions of I2- KI in water are unstable. Iodine can be lost by evaporation and that can influence the result of the staining as well as affecting the working conditions in that area. Stainless steel parts of staining instruments can also be affected by the iodine in the Lugol's solution. If the concentration of iodine in the Lugol's solution decreases the bacterial smears may become more susceptible to decolourization. Therefore, I2- KI solutions should always be stored in completely closed bottles.
The problem of iodine loss can be overcome by using polyvinylpyrrolidine (PVP) in the I2- KI solution. PVP forms a complex with iodine. This complex is more stable and has a longer shelf life. The results of the material treated with Lugol's solution stabilized with PVP will give identical results with regard to differentiation of Gram-positive and Gram negative bacteria compared to the standard Lugol's solution.
Both Lugol's solutions are available - the standard type and the stabilized type. Suitable protocols are available on the internet or on demand.
Materials
Cristal violate
| Cristal violete | 0.5 g | |
| Oxalate | 0.8g | |
| Metanol | 20ml | |
| Destilated water | 100 ml | |
Troubleshooting
Homogenize the sample well with a wooden toothpick in the container that contains it
Make a grade spread of approximately 2 cm long by 2 cm wide, sliding the stick impregnated with fecal matter onto a new degreased, dry, or used but clean and scratch-free slide.
Remove excess stool with a clean, chopped wooden toothpick and repeat this procedure with another clean chopped toothpick, until the spread is as thin as possible and with the least amount of lumps of stool
Let to dry at room temperature
Fix the plate in absolute methanol for 5 min
5m
Let it dry at room temperature to proceed with staining or store it in a box wrapped in absorbent paper until staining.
Cover the spread with Crystal violet for 1 min
1m
Wash under running water to remove excess dye
Cover with an acid alcohol solution for 3 min until discolored
3m
Go through 95% ethanol for 1 min
1m
Visualize under a microscope with immersion oil at 100X
Permanent fixation use xylene for 1 min
1m
Let to dry and cover with a cover slide and Entellan®