Tag: #alcohols

GRAM STAINING

Bacteria can be classified into two different categories i.e. gram-positive bacteria and gram-negative bacteria. To differentiate the type of bacteria present in any collected sample there is a technique known as GRAM STAINING OR GRAM STAIN. It is sometimes also known as gram’s method. This technique is a different step process which can easily distinguish and classify between different types of bacteria. This was named after the great scientist Hans Christian Gram.
Gram Staining method differentiates bacteria on the basis of their physical and chemical structure of cell wall. They are stained with different reagents and are observed in different colors when seen under the microscope. It is due to the fact that gram positive bacteria have a thick layer of peptidoglycan which allows it to retain the primary stain which is crystal violet and thus they appear purple whereas on the other hand, gram-positive bacteria have thin peptidoglycan cell wall and thus only retain the secondary or counter stain which is Safranin and thus they appear slightly pinkish when observed under microscope. Gram staining is the basic technique which is widely used in the microbiology labs to distinguish between both the types of bacteria. It provides a great help to microbiologists to perform their clinical tasks. If any infection is suspected in the patient then after the collection of sample, the gram staining is done and the type of bacteria is observed.
Gram staining is completed in the various steps –

  1. Fixation of clinical materials i.e. the sample collected are being fixed on the glass slide by first making a smear using water and then that smear is either heat fixed or methanol fixed. It is a first and a very important step. Methanol fixation was later discovered keeping in mind its advantage of not destroying the morphology of host cell, as well as bacteria present in that. It is majorly used for the testing of blood samples collected from patients.
  2. Application of primary stain i.e. crystal violet. Primary stain means the first stain which is applied onto the fixed smear which stains all the calls purple or blue.
  3. To distinguish the slide is then washed off in a gentle and indirect stream of water for 2 seconds which removes the extra stain that is not absorbed by the cells.
  4. The next step involves the application of a mordant i.e. Iodine solution. This forms a complex with crystal violet due to which all the cells start appearing blue.
  5. Again the slide is washed for 2 seconds to remove extra stain.
  6. Addition of a decolorizing agent is the next step which will ultimately remove the excess stain which bacteria has not absorbed. The decolorizer contains the combination of acetone and alcohol. In this step, the gram positive bacteria continue appearing violet or blue in color whereas gram negative bacteria start appearing colorless.
  7. Application of counter stain or secondary stain i.e. safranin is the next step. It should let remain on the slide for 30 second to 1 minute. This will stain all the colorless gram negative bacteria pink and gram positive bacteria remains blue in color.
  8. Again the slide is washed off in a gentle stream of water.
  9. The prepared stained slide is then observed under a microscope using immersion oil (for observing under 100x).

CHEMICAL AGENTS IN MICROBIAL CONTROL

The chemical agents are mostly employed in disinfection and antisepsis. The proper use of these agents is essential to laboratory and hospital safety. Many disinfectants are available and each has its own advantages and disadvantages, but ideally the disinfectant must be effective against a wide variety of infectious agents. The disinfectant must be stable upon storage, odorless, or with pleasant order, soluble in water and lipids for penetration into microorganisms, and have a low surface tension through that it can enter cracks in surfaces.

  1. Phenols
    In 1867, Joseph Lister employed it to reduce the risk of infection during operations and phenol was the first widely used antiseptic and disinfectant. Today phenol and phenolics such as cresols, xylenols, and orthophenylphenol are used as disinfectants in laboratories and hospitals. Lysol is made of a mixture of phenolics which is commercially available disinfectant. They act by denaturing proteins and disrupting cell membranes.
  2. Alcohols
    Alcohols are the most widely used disinfectant and antiseptic. They are bactericidal and fungicidal but not sporicidal. Ethanol and isopropanol are the two most popular alcohol germicides. Small instruments like thermometers can be disinfected by soaking them for 10 to 15 minutes in alcohol solutions. 70% ethanol is more effective than 95% as water is needed for proteins to coagulate.
  3. Halogens
    Halogens exist as diatomic molecules in the free state and form salt like compounds with sodium and most other metals. Iodine and chlorine are the most important antimicrobial agents. Spores can be destroyed at higher concentration. Iodine is often applied as tincture of iodine, 2% or more iodine in a water-ethanol solution of potassium iodide. Skin scars result and sometimes iodine allergies can result.
    Chlorine is mostly used as a disinfectant for municipal water supplies and swimming pools and also employed in dairy and food industry. It may be applied as chlorine gas, sodium hypochlorite or calcium hypochlorite, all of which yield hypochlorous acid and then atomic oxygen.
  4. Heavy metals
    Heavy metals such as mercury, silver, arsenic, zinc and copper were used as germicides and these have nit been most recently superseded by other less toxic and more effective germicides. A 1% solution of silver nitrate if often added to the eyes of infants to prevent ophthalmic gonorrhea but now erythromycin is used instead of silver nitrate because it is more effective. Silver sulfadiazine is used on burns. Copper sulphate is an effective algicide in lakes and swimming pools. The action of these heavy metals is mostly on the proteins, and they combine often with their sulfhydryl groups, and inactivate them. They may also precipitate cell proteins.
  5. Quaternary ammonium compounds
    Detergents are organic molecules that serve as wetting agents and emulsifiers and are amphipathic in nature and hence solubilize otherwise insoluble residues and are very effective cleansing agents and are efficient from soaps, which are derived from fats.
    Only cationic detergent are effective disinfectants characterized by positively charged quaternary nitrogen and a long hydrophobic aliphatic chain. They are mostly used as disinfectants for food utensils and small instruments and as skin antiseptics.
  6. Sterilizing gases
    Gases may also be used as sterilizing agents in order to sterilize many heat-sensitive items such as disposable petri dishes and many syringes, heat-lung machine components, sutures, etc. Ethylene oxide gas is used for this purpose as it readily penetrates packing materials, even plastic wraps and is both microbicidal and sporicidal and kills by combining with cell proteins.
  7. Hydrogen peroxide
    Hydrogen peroxide effects our direct and indirect actions of oxygen as it forms hydroxyl free radical which is highly toxic and reactive to cells. As an antiseptic, 3% hydrogen peroxide serves a variety of needs including skin and wound cleansing, bedsore care and mouth washing. When it is applied to a wound, the enzyme catalase in the tissue decomposes the hydrogen peroxide into water and free oxygen. The oxygen causes the wound tissues to bubble and the bubbling removes microorganism mechanically.
  8. Acids and alkalis
    Aqueous solutions of ammonium hydroxide remain a common component of detergent, cleanser and deodorizers. Organic acids are widely used in food preservatives because they prevent spore germination and bacterial and fungal growth. Acetic acid in the form of vinegar is a picking agents that inhibits bacterial growth, propionic acid is commonly incorporated into breads and cakes to retard molds, benzoic acid and sorbic acids are added to beverages, syrups to inhibit yeasts.