Tag: #gramstaining

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).

BASICS OF A MICROBIOLOGY LAB

Microbiology is the study of microbes i.e. the organisms which we can’t see with the naked eyes. Although many microorganisms are beneficial for the human use, some are pathogenic also which causes diseases. Clinical Microbiological Laboratory is concerned with finding of those infectious, pathogenic microbes.

MATERIALS USED IN MICROBIOLOGY LAB
Laminar flow hood, Incubator, Autoclave, Refrigerator, Bunsen Burner, Wire loop, Petri plates, Glass slides, Weighing balance, Media plates, Sensitivity disks, Staining rack, Microscope, Bio safety Cabinet, Centrifuge etc.

INTRODUCTION TO DIFFERENT MEDIA
Some of the media used in the microbiology lab are :

  1. MacCONKEY AGAR : It is the selective and differential media used for the isolation of Gram-negative Bacteria. This media can be used for differentiating Lactose fermenting and Non-lactose fermenting bacteria.
  2. BLOOD AGAR : It is the enriched media for the growth of bacteria such as streptococci.
  3. CHOCOLATE AGAR : It is the lysed Blood Agar. The only difference in blood agar and chocolate agar is that in blood agar RBCs are lysed. This enriched media is suitable for the growth of bacteria that are unable to grow on Blood Agar.
  4. THIOSULFATE CITRATE BILE SALT AGAR (TCBS) : It is the selective as well as differential media for the growth of vibrio cholerae , a causative organism for cholera.

GRAM STAINING
Gram staining is the process for differentiating Gram positive and Gram negative bacteria. When the whole procedure of gram stain is followed and the slide is observed under the microscope, Gram positive bacteria appear Violet in color and Gram negative bacteria appear Pink in color.
For the gram staining we need Glass slide, Normal Saline, Inoculating loop, Bunsen burner, Crystal Violet, Gram’s Iodine, Acetone, Safranine.

PROCESS :

  1. The isolated colony of the microorganism is taken and in the drop of normal saline on the glass slide the colony is mixed with the help of inoculating loop to make a smear. The prepared smear is heat fixed.
  2. A staining rack is taken and on the smear, Crystal Violet is added. After 1 minute, the stain was removed by washing the slide in running water.
  3. After that, Gram’s Iodine is added on the smear as a decolorizing agent which is again washed after 1 minute under the running tap water.
  4. The next step is to add Acetone on the smear which is added in the hand to hand process.
  5. After the decolorisation is done, Safranine is added on the smear which is also washed after 1 minute.
  6. The glass slide is then air dried and observed under the microscope.

RESULTS :
It was observed under the microscope that the Gram positive bacteria appear Violet in color due to Crystal Violet stain whereas Gram negative bacteria appear Pink in color due to safranine.

TESTS ANALYZED
The tests analyzed in the microbiology section of the laboratory are basically the culture and sensitivities tests of urine, stool, sputum, pus swab etc.
The basic procedure of performing all the tests are :

  1. First of all, all the tests are performed inside the laminar flow hood.
  2. The samples collected from the patients and the media plates are kept inside the laminar flow.
  3. The inoculating wire loop is heat sterilized and with the help of it, the samples are cultured or streaked on the media plates.
  4. After inoculation, the cultured media plates are incubated for 24 hours (48 hours if necessary) for allowing the growth of bacteria.
  5. After the growth, staining is done or sensitivities are checked according to the requirement by the doctor.
  6. The report is prepared for the patient.