Youtor
Grams Staining
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This video is all about the Gram staining which is an important topic for the young Microbiologists and Laboratory technicians. Please let me know if anything from the video need to be corrected. • Please watch my video on the bacterial cell wall composition by following the link below. • • Structure of Bacteria | Part 3 | The ... • Script from the video is here for your convenience. • Gram staining is a technique, used to differentiate two major groups of bacteria based on their cell wall composition, called gram positive, and gram negative bacteria. • The technique was named after Hans Christian Gram who developed this method in 1884. • Gram staining is a preliminary test in the bacterial identification process. It plays an important role in clinical microbiology. • It helps the medical professionals in the diagnosis of infectious diseases directly from the clinical sample. • As the cell wall of gram positive bacteria is completely different from the gram negative bacteria, it is important for them to understand the gram nature to provide the appropriate treatment for the infection. • The widely accepted theory is based on the differences in the cell wall composition, between the gram positive and gram negative bacteria. • To understand the cell wall composition better, please do watch my video on the Structure of Cell wall by following the link in the description. • When crystal violet and Iodine are added to the smear, both will penetrate through the cell wall, and form a large crystal violet- Iodine complex within the inner and outer layers of gram positive and gram negative bacteria. • The cell wall of gram negative bacteria is thin and made of one or two layers of peptidoglycan. In addition to this, it has got an outer Lipo polysaccharide layer surrounding the cell wall. • When decolourizer like alcohol or acetone is added, the outer Lipo polysaccharide layer will be completely dissolved, leaving the thin peptidoglycan layer exposed. • The effect of alcohol makes the peptidoglycan layer become perforated. • So, in the decolourization step, the gram negative bacteria fail to retain the CV-I complex, and become colourless, as the complex is washed away. • On contrary to this, the thick and multi-layered peptidoglycan in gram positive bacteria, will be dehydrated by the addition of alcohol. • Because of the thick peptidoglycan layer, and dehydration by the alcohol treatment, the CV-I complex gets trapped in the cell. • Therefore, after the decolourization step, the gram positive bacteria appear purple in colour and the gram negative bacteria become colourless. • In the last step, when a counter stain like Safranin is added, the gram negative bacteria easily adsorb the stain and gives pink colour to the cells. • And, the gram positive bacteria remain purple in colour due to the retention of CV-I complex, which is darker than Safranin. • Although the gram staining is used as primary test in the identification process, this method will not be able to identify the bacteria to the species level. • It will be used in combination of other modern and traditional identification tests. • The CV-I complex may get lost from the gram positive bacteria, due to over decolourization, which might lead to misinterpretation. • There were evidences, where some old gram positive cultures were not able to retain purple colour and therefore observed as gram negative. • • Gram's Staining Procedure
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