EXAMPLES OF GRAM-POSITIVE & GRAM-NEGATIVE BACTERIA
Gram-Positive Bacteria : Actinomyces, Bacillus spp., Clostridium spp., Corynebacterium diptheriae, Gardnerella, Lactobacillus, Mycoplasma, Nocardia, Staphylococcus aureus, Staphylococcus epidermidis and other Staphylococcus spp. Streptococcus pneumoniae, Sterptococcus pyogenes, Streptomyces etc.
Gram-Negative Bacteria : Escherichia coli (E. coli), Salmonella typhi, Salmonella paratyphi and other Salmonella species. Shigella dysentriae, Klebsiella pneumoniae, Pseudomonas, Moraxella, Helicobacter pylori, Stenotrophomonas, Legionella etc.
DEHYDRATION OF TISSUES – TECHNIQUE OF REMOVING WATER FROM THE TISSUES
INTRODUCTION TO DEHYDRATION OF TISSUES
Dehydration of tissues is the important process because of the paraffin, in which the tissues are embedded, is not miscible with water and does not penetrate the tissue effectively. Hence water in the tissue should be removed before embedding; this process is called as dehydration. Tissues are generally soft after fixation and thus for satisfactory staining and the right degree of hardening, the tissue is treated with the dehydrating agent.
THE IDEAL TECHNIQUE OF DEHYDRATION
Water is extracted from the tissues by passing them through the graded alcohol as 70%, 80%, 90% and then Absolute alcohol. This technique prevents the mechanical damage to delicate intracellular structures, which may occur due to the rapid passage of water from the cells to the dehydrating medium.
VARIATIONS ARE APPLICABLE
The dehydrating schedule varies as per the fixative used in the process of Fixation. In case of Carnoy’s fluid and other alcoholic fixatives, the tissues are directly transferred to 90% or 95% or even Absolute alcohol.
Sometimes for soft tissues like an embryo, the first stage of 50% alcohol is used followed by a graded treatment of the alcohol to prevent tissue shrinkage.
Tissues fixed in Susa fluid are transferred to 90% alcohol because lower grades of alcohol may cause swelling of the fibrous tissues.
VARIOUS DEHYDRATING AGENTS
1.) Ethanol: Ethyl alcohol or Ethanol is a clear, colorless, inflammable liquid with a pleasant odor, is hydrophilic and therefore miscible with water in all concentrations, as well as with many organic solvents.
2.) Methylated Spirit: It is also known as denatured alcohol, with a pronounced odor. It consists of ethanol to which a portion of methanol has been added to render it unfit for consumption.
3.) Methanol: This is a Clear, colorless, inflammable fluid with a characteristic unpleasant odor, miscible with water, ethanol and organic solvents and is highly poisonous.
4.) Isopropyl alcohol: It is miscible with water, ethanol and organic solvents.
5.) Acetone: It is a clear inflammable fluid with a characteristic pungent odor and rarely used as the dehydrating agent.
6.) Dioxane: It is a colorless liquid with a faintly sweet odor and is a good dehydrating as well as the clearing agent.
Here is the list of commonly used dehydrating agents their advantages and disadvantages:
| S. No. | DEHYDRATING AGENTS | ADVANTAGES | DISADVANTAGES |
|---|---|---|---|
| 1.) | ETHYL ALCOHOL (ETHANOL) | i.) Non-Toxic |
ii.) Miscible with water
iii.) No shrinkage when graded treatment is employed.
iv.) Ideal for Delicate tissues|i.) It may cause hardening of tissues when left for longer period in absolute alcohol.
ii.) Highly inflammable.|
|2.)|ISOPROPYL ALCOHOL|i.) Excellent substitute of Ethanol.
ii.) Miscible with water and organic solvents.|i.) Cannot be used when Celloidin embedding have to be done.
ii.) Highly inflammable.|
|3.)|ACETONE|i.) Rapid dehydration within 1 hr.
ii.) Miscible with water & organic solvents.|i.) Extremely volatile.
ii.) Highly inflammable.|
|4.)|DIOXANE|i.) It is a both Dehydrating and Clearing agent.
ii.) Miscible with Water, Alcohol, Xylene and Paraffin.|i.) Over hardens the tissue if left for longer period.
ii.) It is Expensive, Toxic and Odorous.|
The most commonly used Dehydrating agent is Ethanol because of its reliability, cost-effectiveness and excellent dehydration properties.
QUALITY CHECK FOR ABSOLUTE ALCOHOL
To ensure that the final bath of the alcohol containing the absolute alcohol or ethanol is free of water, a layer of Anhydrous copper sulfate which should be approx. 2-2-5 cm thick, covered by filter paper to prevent the contamination, is employed in dehydrating vessel. This technique prolongs the life of Alcohol and the Anhydrous copper sulfate (white color) turns blue when the alcohol gets diluted, indicates that the alcohol has reached the reliability threshold and need to be changed to ensure the proper dehydration of the tissues.